An antifungal active fraction (AAF) from the coelomic fluid (CF) of the earthworm Dendrobaena veneta was isolated. The aim of the study was to analyze the antifungal activity of the AAF and to carry out chemical characterization of the fraction. The active fraction showed antifungal activity against a clinical C. albicans isolate, C. albicans ATCC 10231, and C. krusei ATCC 6258. It effectively reduced the metabolic activity of C. albicans cells and influenced their morphology after 48 hours of incubation. Scanning electron microscopy (SEM) images revealed loss of integrity of the cell wall induced by the active fraction. Calcofluor White staining showed changes in the structure of the C. albicans cell wall induced by the AAF. The fungal cells died via apoptosis and necrosis after the treatment with the studied fraction. Electrophoresis under native conditions revealed the presence of two compounds in the AAF, while SDS/PAGE gel electrophoresis showed several protein and carbohydrate compounds. The active fraction was analyzed using Raman spectroscopy, MALDI TOF/TOF, and ESI LC-MS. The Raman analysis confirmed the presence of proteins and determined their secondary structure. The MALDI TOF/TOF analysis facilitated detection of four main compounds with a mass of 7694.9 m/z, 12292.3 m/z, 21628.3 m/z, and 42923.2 m/z in the analyzed fraction. The presence of carbohydrate compounds in the preparation was confirmed by nuclear magnetic resonance (NMR) and gas chromatography (GC-MS). The ATR-FTIR spectrum of the AAF exhibited high similarity to the spectrum of egg white lysozyme. The AAF showed no endotoxicity and cytotoxicity towards normal skin fibroblasts (HSF); therefore, it can be used for the treatment of skin and mucous membrane candidiasis in the future. Given its efficient and selective action, the fraction seems to be a promising preparation with antifungal activity against C. albicans.

The results show the morphological analyses and spectroscopic studies of snow and glacier algae and their parasitic fungi in Svalbard (High Arctic). Fixed algal cells of two species, Sanguina nivaloides and Ancylonema nordenskioeldii, were imaged using light microscopy, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Fluorescence microscopy using Calcofluor white stain supported the observations of parasitic fungi on the algal cells. Images in brightfield microscopy showed chytrid-like fungi penetrating the cells of both algal species. Parasites were found to colonize the cells of A. nordenskioeldii and hypnozygotes of S. nivaloides, while no fungi infected the cyst stages of S. nivaloides. The autofluorescence analysis revealed the ability of S. nivaloides to glow when excited with different wavelengths, while A. nordenskioeldii did not fluoresce. The hypnozygotes of S. nivaloides emitted brighter fluorescence than the cysts, and the most intense luminosity was observed in the UV range. The Fourier-transform infrared spectroscopy (FTIR) and energy-dispersive X-ray spectroscopy (EDS) spectroscopic analysis showed differences in the chemical composition between samples collected from three different sites. Samples dominated by cyst cells were characterized by the presence of an abundant polysaccharide envelope.

Earthworm coelomic fluid (CF) is known as a rich source of various bioactive compounds with promising anticancer features. However, it has been demonstrated that CF affects functionality of both, cancer and normal cells. This non-selective activity causes a major problem for medical application of CF. In this study, we present the anticancer activity of the active protein-carbohydrate fraction (AF) isolated from thermally treated CF of earthworm Dendrobaena veneta. The in vitro effect of the AF was examined in human colon model including normal human colon epithelium (CCD 841 CoTr) and human colon adenocarcinoma (HT-29 and LS180) cell lines. We investigated the impact of AF on cell viability neutral red and lactate dehydrogenase assays, morphology May-Grünwald-Giemsa staining assay proliferation MTT tetrazolium salt and BrdU incorporation assays as well as cell cycle progression propidium iodide/RNase staining and the activity of human 20S proteasome the hydrolysis of AMC from a Suc-LLVY-AMC peptide substrate. Additionally, the influence of AF on apoptosis was examined in HT-29 cells by Annexin V/PI, Hoechst 33342 staining and active caspase-3 assays. Our investigation demonstrated that AF at the tested concentration range does not affect the viability and morphology of CCD 841 CoTr cells. Simultaneously, AF inhibits human 20S proteasome activity as well as significantly decreases mitochondrial metabolism, disturbs cell cycle and induces apoptosis via activation of procaspase-3 in HT-29 cancer cells. Obtained results demonstrate the antiproliferative and proapoptotic activity of AF that can be useful in developing therapeutic strategies to treat human colon cancer.

The protein–polysaccharide fraction (AAF) isolated from the coelomic fluid of the earthworm Dendrobaena veneta destroys C. albicans cells by changing their morphology, disrupting cell division, and leading to cell death. Morphological changes in C. albicans cells induced by treatment with AAF were documented using DIC, SEM, and AFM. Congo Red staining showed that the fungal wall structure was changed after incubation with AAF. The effect on C. albicans cell walls was shown by AFM analysis of the surface roughness of fungal cell walls and changes in the wall thickness were visualized using Cryo-SEM. The FTIR analysis of C. albicans cells incubated with AAF indicated attachment of protein or peptide compounds to the fungal walls. The intact LC–ESI–MS analysis allowed accurate determination of the masses of molecules present in AAF. As shown by the chromatographic study, the fraction does not cross biological membranes. The Cryo-TEM analysis of AAF demonstrated the ability of smaller subunits to combine into larger agglomerates. AAF is thermally stable, which was confirmed by Raman spectroscopy. AAF can be considered as a potential antifungal antibiotic with activity against clinical C. albicans strains.

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.