Our studies aimed to explore the protein components of the matrix of human submandibular gland sialoliths. A qualitative analysis was carried out based on the filter aided sample preparation (FASP) methodology. In the protein extraction process, we evaluated the applicability of the standard demineralization step and the use of a lysis buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT). The analysis of fragmentation spectra based on the human database allowed for the identification of 254 human proteins present in the deposits. In addition, the use of multi-round search in the PEAKS Studio program against the bacterial base allowed for the identification of 393 proteins of bacterial origin present in the extract obtained from sialolith, which so far has not been carried out for this biological material. Furthermore, we successfully applied the SWATH methodology, allowing for a relative quantitative analysis of human proteins present in deposits. The obtained results correlate with the classification of sialoliths proposed by Tretiakow. The performed functional analysis allowed for the first time the selection of proteins, the levels of which differ between the tested samples, which may suggest the role of these proteins in the calcification process in different types of sialoliths. These are preliminary studies, and drawing specific conclusions requires research on a larger group, but it provides us the basis for the continuation of the work that has already begun.

The present research shows the antitumor activity of a protein-polysaccharide complex Venetin-1 obtained from the coelomic fluid of Dendrobaena veneta earthworms against A549 cancer cells. The investigations are a continuation of experiments on the antitumor activity of coelomic fluid obtained from this species. The Venetin-1 nanoparticle was obtained after thermal treatment of the coelomic fluid, separation from coelomocytes, filtration, and lyophilization. The preparation showed a selective effect on cancer cells, whereas normal cells were unaffected. Venetin-1 was effective against the lung cancer cells at doses of 31.3 and 62.5 µg/ml, and the results were imaged using light microscopy and scanning electron microscopy (SEM). The cells died mainly via the apoptosis pathway. Necrotic cells appeared sporadically in the microscopic view. SEM imaging revealed complete destruction of the A549 cells after the incubation with Venetin-1. The atomic force microscopy (AFM) analyses showed changes in the topography, peak force error images, and Young’s modulus (elasticity) of the A549 cells after the incubation with Venetin-1. The transmission electron cryomicroscopy (Cryo-TEM) analysis indicated a polymeric nature of the analyzed preparation. The samples of Venetin-1 showed a very homogeneous size profile with the microparticle size of approximately 58.23 nm. A significant decrease in Venetin-1 binding to sphingomyelin was observed. Venetin-1 lost its pore-forming activity or deactivation of the pore-forming activity occurred. This confirms the absence of hemolytic capacity of Venetin-1 towards red blood cells. The conducted analyses show the suitability of the obtained complex for biomedical research. The next step will consist in analyses of the effect of Venetin-1 on the immune system in mice.

Salivary stones, also known as sialoliths, are formed in a pathological situation in the salivary glands. So far, neither the mechanism of their formation nor the factors predisposing to their formation are known despite several hypotheses. While they do not directly threaten human life, they significantly deteriorate the patient's quality of life. Although this is not a typical research material, attempts are made to apply various analytical tools to characterise sialoliths and search for the biomarkers in their proteomes. In this work, we used mass spectrometry and SWATH-MS qualitative and quantitative analysis to investigate the composition and select proteins that may contribute to solid deposits in the salivary glands. Twenty sialoliths, previously characterized spectroscopically and divided into the following groups: calcified (CAL), lipid (LIP) and mixed (MIX), were used for the study. Proteins unique for each of the groups were found, including: for the CAL group among them, e.g. proteins from the S100 group (S100 A8/A12 and P), mucin 7 (MUC7), keratins (KRT1/2/4/5/13), elastase (ELANE) or stomatin (STOM); proteins for the LIP group - transthyretin (TTR), lactotransferrin (LTF), matrix Gla protein (MPG), submandibular gland androgen-regulated protein 3 (SMR3A); mixed stones had the fewest unique proteins. Bacterial proteins present in sialoliths have also been identified. The analysis of the results indicates the possible role of bacterial infections, disturbances in calcium metabolism and neutrophil extracellular traps (NETs) in the formation of sialoliths.

Salivary stones, also known as sialoliths, are formed in a pathological situation in the salivary glands. So far, neither the mechanism of their formation nor the factors predisposing to their formation are known despite several hypotheses. While they do not directly threaten human life, they significantly deteriorate the patient’s quality of life. Although this is not a typical research material, attempts are made to apply various analytical tools to characterise sialoliths and search for the biomarkers in their proteomes. In this work, we used mass spectrometry and SWATH-MS qualitative and quantitative analysis to investigate the composition and select proteins that may contribute to solid deposits in the salivary glands. Twenty sialoliths, previously characterized spectroscopically and divided into the following groups: calcified (CAL), lipid (LIP) and mixed (MIX), were used for the study. Proteins unique for each of the groups were found, including: for the CAL group among them, e.g. proteins from the S100 group (S100 A8/A12 and P), mucin 7 (MUC7), keratins (KRT1/2/4/5/13), elastase (ELANE) or stomatin (STOM); proteins for the LIP group—transthyretin (TTR), lactotransferrin (LTF), matrix Gla protein (MPG), submandibular gland androgen-regulated protein 3 (SMR3A); mixed stones had the fewest unique proteins. Bacterial proteins present in sialoliths have also been identified. The analysis of the results indicates the possible role of bacterial infections, disturbances in calcium metabolism and neutrophil extracellular traps (NETs) in the formation of sialoliths.