In our ongoing search for new bioactive fungal metabolites, two new cytochalasans were isolated from stromata of the hypoxylaceous ascomycete Hypoxylon fragiforme. Their structures were elucidated via high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. Together with 23 additional cytochalasans isolated from ascomata and mycelial cultures of different Ascomycota, they were tested on their ability to disrupt the actin cytoskeleton of mammal cells in a preliminary structure–activity relationship study. Out of all structural features, the presence of hydroxyl group at the C7 and C18 residues, as well as their stereochemistry, were determined as important factors affecting the potential to disrupt the actin cytoskeleton. Moreover, reversibility of the actin disrupting effects was tested, revealing no direct correlations between potency and reversibility in the tested compound group. Since the diverse bioactivity of cytochalasans is interesting for various applications in eukaryotes, the exact effect on eukaryotic cells will need to be determined, e.g., by follow-up studies involving medicinal chemistry and by inclusion of additional natural cytochalasans. The results are also discussed in relation to previous studies in the literature, including a recent report on the anti-Biofilm activities of essentially the same panel of compounds against the pathogenic bacterium, Staphylococcus aureus.

During the course of our ongoing work to discover new inhibitors of biofilm formation of Staphylococcus aureus from fungal sources, we observed biofilm inhibition by cytochalasans isolated from cultures of the ascomycete Hypoxylon fragiforme for the first time. Two new compounds were purified by a bioassay-guided fractionation procedure; their structures were elucidated subsequently by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). This unexpected finding prompted us to test further cytochalasans from other fungi and from commercial sources for comparison. Out of 21 cytochalasans, 13 showed significant inhibition of Staphylococcus aureus biofilm formation at subtoxic levels. These findings indicate the potential of cytochalasans as biofilm inhibitors for the first time, also because the minimum inhibitory concentrations (MIC) are independent of the anti-biofilm activities. However, cytochalasans are known to be inhibitors of actin, making some of them very toxic for eukaryotic cells. Since the chemical structures of the tested compounds were rather diverse, the inclusion of additional derivatives, as well as the evaluation of their selectivity against mammalian cells vs. the bacterium, will be necessary as next step in order to develop structure-activity relationships and identify the optimal candidates for development of an anti-biofilm agent.

Fungal stromata were recently discovered in association with charcoal and burnt soil aggregates during an archaeological survey in the Châtillon-sur-Seine forest massif. The wood and soil in the samples were dated to the medieval period (between 738 and 1411 AD). Light microscopy and scanning electron microscopy revealed that a few of the stromatal fragments still contained ascospores. Their macromorphological characters were described and secondary metabolite profiles were generated using high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS). The combination of these two data lines then allowed species identification. Most of the fragments were assigned to Hypoxylon fragiforme, the type species of the Hypoxylaceae (Xylariales). Two further species whose stromata grew on the fossil charcoal could be tentatively identified as Jackrogersella cohaerens and (more tentatively) as Hypoxylon vogesiacum. These three species are still commonly encountered in the forests of Central Europe today. Furthermore, the HPLC-HRMS data of H. fragiforme suggested the presence of unknown azaphilone dimers and of further new pigments. These archaeological compounds were compared to fresh stromata of H. fragiforme collected in Germany and subjected to the same analytical protocol. While the major components in both samples were identified as the known mitorubrin type azaphilones and orsellinic acid, the chemical structures of seven novel complex azaphilone pigments, for which we propose the trivial names rutilins C-D and fragirubrins A-E, were elucidated using spectral methods (NMR and CD spectroscopy, high resolution mass spectrometry). It appears that these pigments had indeed persisted for millennia in the fossil stromata.

A full understanding of the relationship between surface properties, protein adsorption, and immune responses is lacking but is of great interest for the design of biomaterials with desired biological profiles. In this study, polyelectrolyte multilayer (PEM) coatings with gradient changes in surface wettability were developed to shed light on how this impacts protein adsorption and immune response in the context of material biocompatibility. The analysis of immune responses by peripheral blood mononuclear cells to PEM coatings revealed an increased expression of proinflammatory cytokines tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 and the surface marker CD86 in response to the most hydrophobic coating, whereas the most hydrophilic coating resulted in a comparatively mild immune response. These findings were subsequently confirmed in a cohort of 24 donors. Cytokines were produced predominantly by monocytes with a peak after 24 h. Experiments conducted in the absence of serum indicated a contributing role of the adsorbed protein layer in the observed immune response. Mass spectrometry analysis revealed distinct protein adsorption patterns, with more inflammation-related proteins (e.g., apolipoprotein A-II) present on the most hydrophobic PEM surface, while the most abundant protein on the hydrophilic PEM (apolipoprotein A-I) was related to anti-inflammatory roles. The pathway analysis revealed alterations in the mitogen-activated protein kinase (MAPK)-signaling pathway between the most hydrophilic and the most hydrophobic coating. The results show that the acute proinflammatory response to the more hydrophobic PEM surface is associated with the adsorption of inflammation-related proteins. Thus, this study provides insights into the interplay between material wettability, protein adsorption, and inflammatory response and may act as a basis for the rational design of biomaterials.

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