A novel rhamnose-rich hetero-exopolysaccharide isolated from Lactobacillus paracasei DG activates THP-1 human monocytic cells

Lactobacillus paracasei DG is a bacterial strain with recognized probiotic properties and is used in commercial probiotic products. However, the mechanisms underlying its probiotic properties are mainly unknown. In this study, we tested the hypothesis that the capability of strain DG to interact with the host is, at least partly, associated with its ability to synthesize a surface-associated exopolysaccharide (EPS). Comparative genomics revealed the presence of putative EPS gene clusters in DG genome; accordingly, EPS was isolated from the surface of the bacterium. A sample of the pure EPS from strain DG (DG-EPS), upon NMR and chemical analyses, was shown to be a novel branched hetero-EPS with a repeat unit composed of L-rhamnose, D-galactose, and N-acetyl-D-galactosamine in a ratio of 4:1:1. Subsequently, we demonstrated that the DG-EPS displays immunostimulating properties by enhancing the gene expression of the pro-inflammatory cytokines TNF-α and IL-6, and, particularly, the chemokines IL-8 and CCL20 in the THP-1 human monocytic cell line. In contrast, the expression of the cyclooxygenase enzyme COX-2 was not affected. In conclusion, the DG-EPS is a bacterial macromolecule with the potential ability to boost the immune system as either a secreted molecule released from the bacterium or as a capsular envelope on the bacterial cell wall. This study provides additional information about the mechanisms supporting the cross-talk between L. paracasei DG and the host

Characterization of Electrospray Ionization Complexity in Untargeted Metabolomic Studies.

The annotation of metabolites detected in LC-MS-based untargeted metabolomics studies routinely applies accurate m/z of the intact metabolite (MS1) as well as chromatographic retention time and MS/MS data. Electrospray ionization and transfer of ions through the mass spectrometer can result in the generation of multiple "features" derived from the same metabolite with different m/z values but the same retention time. The complexity of the different charged and neutral adducts, in-source fragments, and charge states has not been previously and deeply characterized. In this paper, we report the first large-scale characterization using publicly available data sets derived from different research groups, instrument manufacturers, LC assays, sample types, and ion modes. 271 m/z differences relating to different metabolite feature pairs were reported, and 209 were annotated. The results show a wide range of different features being observed with only a core 32 m/z differences reported in >50% of the data sets investigated. There were no patterns reporting specific m/z differences that were observed in relation to ion mode, instrument manufacturer, LC assay type, and mammalian sample type, although some m/z differences were related to study group (mammal, microbe, plant) and mobile phase composition. The results provide the metabolomics community with recommendations of adducts, in-source fragments, and charge states to apply in metabolite annotation workflows