Montmorillonites saturated with Al3+, H+-Al3+, and K+ were titrated in H2O, acetonitrile (AN), and dimethylformamide (DMF) to investigate the acidic properties of the clay in these solvents and to evaluate the application of nonaqueous titration procedures for studies of the acidic properties of 2:1 swelling clays. Samples were titrated with tetramethylammonium hydroxide using a combination glass electrode for Potentiometric determination. Titers of base required to reach the final Potentiometric endpoints were greater in AN than in DMF and H2O. The larger titers in AN compared to H2O were attributed to pH-dependent sites for which an inflection was not observed in the latter solvent. Titration curves of Al-saturated montmorillonite in AN showed evidence of a salt-induced hydrolysis which was confirmed by titration curves of salt extracts. The hydrolysis reaction was more evident in AN than in H2O and was probably due to a disruption of the H2O structure by AN and a polarization of the H2O of solvation. The hydrolysis reaction was enhanced in the presence of excess salt due to the ion exchange of H+ from the exchange surface. These studies indicate that acidic properties of clays may be drastically altered in organic solvents. The acidic properties may be used to advantage, for example, in the determination of edge-site acidity. In addition, they have implications concerning possible chemical reactions and/or alterations of clay in organic solvents.

Muramidases constitute a superfamily of enzymes that hydrolyse peptidoglycan (PGN) from bacterial cell walls. Recently, a fungal muramidase derived from Acremonium alcalophilum has been shown to increase broiler performance when added as a feed additive. However, the underlying mechanisms of action are not yet identified. Here, we investigated the hypothesis that this muramidase can cleave PGN to muramyl dipeptide (MDP), activating nucleotide-binding oligomerisation domain-containing protein 2 (NOD2) receptors in eukaryotic cells, potentially inducing anti-inflammatory host responses. Using Micrococcus luteus as a test bacterium, it was shown that muramidase from A. alcalophilum did not display antimicrobial activity, while it could cleave fluorescently labelled PGN. It was shown that the muramidase could degrade PGN down to its minimal bioactive structure MDP by using UPLC-MS/MS. Using HEK-Blue™-hNOD2 reporter cells, it was shown that the muramidase-treated PGN degradation mixture could activate NOD2. Muramidase supplementation to broiler feed increased the duodenal goblet cell and intraepithelial lymphocyte abundance while reducing duodenal wall CD3+ T lymphocyte levels. Muramidase supplementation to broiler feed only had moderate effects on the duodenal, ileal and caecal microbiome. It was shown that the newly discovered muramidase hydrolysed PGN, resulting in MDP that activates NOD2, potentially steering the host response for improved intestinal health.

The characterisation of proteome and peptidome of adolescent mothers’ breast milk brings important information to both mother’s and infant’s health; however, it has not been investigated. Bioactive peptides derived from milk proteins have numerous functions. The bioactivity of breast milk peptides includes anti-inflammatory and antimicrobial activities and regulation of gastrointestinal function. We aimed to characterise the proteome and peptidome of mature breast milk of adolescent mothers and investigate whether it is affected by lactational period. We used a combination of electrophoretic and nano-scale LC-quadrupole time-of-flight MS/MS (nLC-Q-TOF-MS/MS) techniques and bioinformatics to explore the proteome of human skimmed milk expressed by lactating adolescents in two groups according to postpartum period (up to 3 and over 5 weeks postpartum). This is the first study that analysed the proteome of adolescent mothers’ breast milk produced during two periods of lactation using 1D-electrophoresis combined with nLC-Q-TOF-MS/MS analysis. Our results showed that the protein composition of adolescent milk varies independently of lactation stage and showed high inter-individual variation. A total of 424 proteins were identified in skimmed milk, of which 137 proteins were common to both groups. Most of the peptides found in adolescents’ breast milk were not derived from major proteins in milk. Association maps showed several interactions between groups of peptides that pointed to the relevance of breast milk peptides to neonatal defensive system.

Atranorin is a secondary metabolite found in many lichens. This compound can act as a photo-buffer, supporting its use as a marker of metabolic response to changes in light. In preliminary trials, atranorin was found to be unstable over time when in solution, potentially precluding its usefulness in this capacity. The present study tests the stability of atranorin in different extraction solvents and at different pH values over time using HPLC analysis. We found that atranorin is most stable in acetonitrile, among six tested solvents, and that the presence of strong acid or a strong base destabilizes the compound. We propose that atranorin breaks down through transesterification in methanol and ethanol until an equilibrium is reached, while a strong base breaks down atranorin through saponification and under acidic conditions, atranorin concentration significantly increases with time. Although atranorin levels were found to be stable in whole thallus extracts from fresh lichens using a leaching method, chemicals isolated using chromatographic separation showed similar breakdown to an atranorin standard. In future work on lichens atranorin should be extracted in acetonitrile or acetone without an added base or acid to yield the greatest stability and thus provide more accurate concentration values of atranorin with time using HPLC. The interactions of atranorin with acid and with chloroform need further study.

Three peptides covering the sequence regions corresponding to the first two (CspB-1), the first three (CspB-2), and the last two (CspB-3) β-strands of CspB, the major cold shock protein of Bacillus subtilis, have been synthesized and analyzed for their conformations in solution and for their precipitation behavior. The peptides are nearly insoluble in water, but highly soluble in aqueous solutions containing 50% acetonitrile (pH 4.0). Upon shifts of the solvent condition toward lower or higher acetonitrile concentrations, the peptides all form fibrils resembling those observed in amyloid associated diseases. These fibrils have been identified and characterized by electron microscopy, binding of the dye congo red, and X-ray fiber diffraction. Characterization of the peptides in solution by circular dichroism and NMR spectroscopy shows that the formation of these fibrils does not require specific preformed secondary structure in the solution state species. While the majority of the soluble fraction of each peptide is monomeric and unstructured, different types of structures including α-helical, β-sheet, and random coil conformations are observed under conditions that eventually lead to fibril formation. We conclude that the absence of tertiary contacts under solution conditions where binding interactions between peptide units are still favorable is a crucial requirement for amyloid formation. Thus, fragmentation of a sequence, like partial chemical denaturation or mutation, can enhance the capacity of specific protein sequences to form such fibrils.