Dynamic changes in lipid membrane composition are a common response to stress, often involving shifts in key lipid molecules. Phosphatidic acid (PA), a central precursor in lipid biosynthesis, accumulates when anionic phospholipid synthesis is blocked—lipids that are typically primary targets of membrane-active antimicrobial peptides (AMPs). This raises the question of how cationic AMPs adapt to such lipid remodeling, which is especially relevant given their promise as novel therapeutics against escalating antimicrobial resistance. Their killing mechanism is often unclear. To identify ongoing processes clearly linked to bacterial cell death, six assays targeting membrane integrity and cell viability were performed alongside bactericidal measurements. These assays were conducted on Escherichia coli and a mutant depleted of anionic phospholipids, treated with the cationic peptides melittin and LL-37. Correlation of assays generated characteristic antimicrobial profiles, providing insight into the peptides’ mechanisms. LL-37 acted independently of membrane composition, while melittin showed increased activity in the absence of anionic phospholipids. This study confirmed specific interactions with PA, but their action suggests targets beyond the membrane, as bacteria remained viable during membrane disruption but failed to form colonies. Overall, these findings indicate that both peptides can effectively handle lipid remodeling and uncover processes driving bacterial cell death.

Clear Cell Sarcoma (CCS) are ultra-rare fusion-translocated soft-tissue tumours occurring mainly in young adults with poor prognosis. Their aggressiveness and resistance to conventional chemotherapy, especially in a metastatic setting, characterize these tumours. A functional drug screen consisting of 80 drugs was performed on patient derived CCS cell lines. Top candidates were validated in 3D cell culture and in vivo models, allowing comparison of drug responses among CCS cell lines, including one matched pair (MUG Lucifer cell lines) representing primary and metastatic disease from the same patient. Underlying mechanisms were evaluated with RNA Seq, fluorescence/luminescence based assays, western blot and IHC. In vitro experiments in CCS spheroids highlighted the transcription inhibitor lurbinectedin to be the best overall candidate which was further confirmed in mouse xenografts, albeit to a lesser extent than observed in vitro, especially in the metastatic CCS model. Transcriptional and functional analyses revealed heterogeneous drug responses and differences in cell death mechanisms across CCS lines, reflecting patient-specific variability. Furthermore, we explored combinational treatment of lurbinectedin with selinexor. Synergistic effects were confirmed in a novel autologous co-culture model incorporating cancer-associated fibroblasts, providing a more physiologically relevant system for drug testing. This study identified promising therapeutic avenues by highlighting key vulnerabilities in CCS, considering both inter tumour heterogeneity, tumour plasticity and the stromal influence on drug response.

Hypericin, a tumour-selective photosensitizer, has shown potential in cancer therapy, but its poor water solubility has limited clinical use. To address this, we developed a water-soluble variant called high hypericin-loaded polyvinylpyrrolidone (HHL-PVP) to enhance hypericin's applicability, particularly for treating glioblastoma, a typically terminal disease. We tested HHL-PVP in both in vitro and in vivo models, first confirming its fluorescent properties in the lab and then assessing its efficacy in more complex animal models. Using subcutaneous and orthotopic tumour mouse models, we combined HHL-PVP administration with fluorescence-guided surgery and photodynamic therapy (PDT) to target residual tumour cells. Histological analysis of both healthy and tumour tissue showed HHL-PVP's over 97 % sensitivity and 100 % specificity in distinguishing tumour tissue. In subcutaneous glioblastoma models, significant tumour necrosis and remission occurred after HHL-PVP administration and a 20-minute white light application through the skin. These results highlight HHL-PVP's effectiveness in targeting and eradicating glioblastoma cells. Our findings provide strong evidence that HHL-PVP is a promising therapeutic option for glioblastoma, with its high sensitivity, specificity, and potential for tumour remission through PDT. This approach warrants further investigation in clinical trials and could improve outcomes for a disease that has been difficult to treat.

Human cathelicidin LL‐37 derivative, the 24‐mer SAAP‐148, is highly effective in vitro in eradicating multidrug‐resistant bacteria without inducing resistance. SAAP‐148 has a high cationic charge (+11) and 46% hydrophobicity, which, once the peptide folds into an alpha helix, forms a wide hydrophobic face. This highly amphipathic nature facilitates on the one hand its insertion into the membrane's fatty acyl chain region and on the other hand it´s interaction with anionic membrane components, which aids in killing bacteria. However, the contributions of the secondary and quaternary structures have not been thoroughly investigated so far. To address this, we applied circular dichroism, NMR spectroscopy, X‐ray scattering, AlphaFold 3 protein folding software, and molecular dynamics simulations. Our results reveal that SAAP‐148 adopts a stable hexameric bundle composed of three parallel dimers, that together form a hydrophobic core of aromatic side chain residues. The hexameric structure is retained at the membrane interface, whereby, MD simulation studies indicated the formation of a fiber‐like structure in the presence of anionic membranes. This certainly seems plausible, as oligomers are stabilized by aromatic residues, and the exposure of positively charged side chains on the surface likely facilitates the transition of the peptide into fibrils on anionic membranes.

BACKGROUND During human placentation extravillous trophoblasts (EVTs), arising from cell column trophoblasts (CCT) invade the highly differentiated uterine mucosa, called decidua, where they erode blood vessels and replace vascular endothelial cells. Maternal platelets have been detected in intercellular gaps of CCTs but their physiological role remained unclear so far. OBJECTIVE This study aimed to elucidate the impact of platelet-derived factors on trophoblasts that are exposed to maternal platelets through erosion of decidual blood vessels. METHODS Trophoblast cell line ACH-3P spheroids were incubated either with platelet-derived factors or isolated platelets obtained from pregnant women, and afterwards subjected to RNA sequencing, and validation by qPCR, ELISA and in situ padlock hybridization. Amongst deregulated genes, LAIR2 expression was confirmed in first trimester placenta and primary trophoblast organoids. The functional role of LAIR2 in trophoblast invasion and platelet activation was studied. RESULTS Platelet-derived factors altered the transcription profile of ACH-3P spheroids, including deregulation of genes linked to embryonic development. Amongst them, LAIR2 was exclusively detected in CCTs and invaded EVTs of first trimester decidua. Histology showed extravasated maternal erythrocytes within interstitial gaps of highly invaded decidua samples, coinciding with LAIR2 positive EVTs. LAIR2 inhibited type 1 collagen -induced platelet activation and enhanced invasiveness of trophoblasts. CONCLUSION This study suggests that maternal platelet-derived factors affect the transcription profile of trophoblasts, including upregulation of LAIR2, which may be involved in fine-tuning the coagulation of maternal blood leaking from eroded decidual blood vessels and could increase the invasiveness of EVTs into the decidua through an autocrine mechanism.

Background: High hypericin-loaded polyvinylpyrrolidone (HHL-PVP) constitutes a novel approach to utilize the promising characteristics of hypericin for photodynamic diagnosis (PDD) and therapy (PDT) of brain tumors in an orally bioavailable formulation. The aim of this study was to investigate the ability of a Complementary Metal-Oxide-Semiconductor (CMOS) camera-based fluorescence imaging system to selectively visualize HHL-PVP in glioblastoma tissue even in the presence of 5-Aminolvevulinic acid (5-ALA) induced fluorescence, which is widely utilized in brain tumor surgery. Methods: We applied a previously established system with a non-hypericin specific filter for 5-ALA fluorescence visualization and a newly introduced hypericin-specific filter at 575–615 nm that transmits the spectrum of hypericin, but not 5-ALA fluorescence. Glioblastoma specimens obtained from 12 patients (11 with preoperative 5-ALA intake) were ex vivo incubated with HHL-PVP. Subsequently, fluorescence intensity and lifetime changes using both the non-hypericin specific filter and hypericin-specific filter were measured before and after HHL-PVP incubation and after subsequent rinsing. Results: While no significant differences in fluorescence signal were observed using the non-hypericin specific filter, statistically significant increases in fluorescence intensity (p = 0.001) and lifetime (p = 0.028) after HHL-PVP incubation were demonstrated using the hypericin-specific filter. In consequence, specimens treated with HHL-PVP could be identified according to the fluorescence signal with high diagnostic sensitivity (87.5%) and specificity (100%). Conclusions: Our CMOS camera-based system with a hypericin-specific filter is capable of selectively visualizing hypericin fluorescence in glioblastoma tissue after ex vivo HHL-PVP incubation. In the future, this technique could facilitate clinical investigations of HHL-PVP for PDD and PDT while maintaining the current standard of care with 5-ALA guidance.

Ex vivo human skin models are valuable tools in skin research due to their physiological relevance. Traditionally, standard cultivation is performed in a cell culture incubator with a defined temperature of 37 °C and a specific atmosphere enriched with CO2 to ensure media stability. Maintaining the model under these specific conditions limits its flexibility in assessing exposures to which the skin is exposed to in daily life, for example changes in atmospheric compositions. In this study we demonstrated that the foreskin-derived skin model can be successfully cultured at room temperature outside a CO2 incubator using a CO2-independent, serum-free media. Over a cultivation period of three days, the integrity of the tissue and the preservation of immune cells is well maintained, indicating the model’s stability and resilience under the given conditions. Exposing our Medical University of Graz – human Organotypic Skin Explant Culture (MUG-hOSEC) model to cytotoxic and inflammatory stimuli results in responses analyzable within the supernatant. Besides the common analysis of released proteins upon treatment, such as cytokines and enzymes, we have included extracellular vesicle to obtain a more comprehensive picture of cell communication.

Significance S-layer proteins (SLPs) are self-assembling, crystalline proteins coating the cell surfaces of many prokaryotes. This study presents experimental atomic resolution structures of lactobacilli SLPs, deriving functional insight into key probiotic Lactobacillus strains. The structures of SlpA and SlpX proteins highlight the domain swapping critical for SlpX integration, particularly in response to environmental stress. Two binding regions are identified as crucial for attachment of the S-layer to (lipo)teichoic acid. The structure of assembled S-layer provides a foundation for employing (designed) SLPs as a therapeutic agent in the treatment of inflammatory diseases. Additionally, it opens broad avenues for the use of SLPs in vaccine development and in crafting nanostructures with tailored properties, including those designed for targeted drug delivery.