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Abstract Lipid bi‐layered particles known as membrane vesicles (MVs), produced by Gram‐positive bacteria are a communication tool throughout the entire bacterial growth. However, the MVs characteristics may vary across all stages of maternal culture growth, leading to inconsistencies in MVs research. This, in turn, hinders their employment as nanocarriers, vaccines and other medical applications. In this study, we aimed to comprehensively characterize MVs derived from Lacticaseibacillus rhamnosus CCM7091 isolated at different growth stages: early exponential (6 h, MV6), late exponential (12 h, MV12) and late stationary phase (48 h, MV48). We observed significant differences in protein content between MV6 and MV48 (data are available via ProteomeXchange with identifier PXD041580), likely contributing to their different immunomodulatory capacities. In vitro analysis demonstrated that MV48 uptake rate by epithelial Caco‐2 cells is significantly higher and they stimulate an immune response in murine macrophages RAW 264.7 (elevated production of TNFα, IL‐6, IL‐10, NO). This correlated with increased expression of lipoteichoic acid (LTA) and enhanced TLR2 signalling in MV48, suggesting that LTA contributes to the immunomodulation. In conclusion, we showed that Lacticaseibacillus rhamnosus CCM7091‐derived MVs from the late stationary phase boost the immune response the most effectively, which pre‐destines them for therapeutical application as nanocarriers.

Abstract Ultraviolet irradiation is an effective method of virus and bacteria inactivation. The dose of UV-C light necessary for baculovirus inactivation by measurement of fluorescent GFP protein produced by baculovirus expression system after the irradiation of baculovirus culture in doses ranging from 3.5 to 42 J/m2 was determined. At a dose of 36.8 J/m2, only 0.5% of GFP-expressing cells were detected by flow cytometry and confocal microscopy. The stability of purified VP1-PCV2bCap protein produced by baculovirus expression system was analyzed after the irradiation at doses ranging from 3.5 to 19.3 J/m2. Up to the dose of 11 J/m2, no significant effect of UV-C light on the stability of VP1-PCV2bCap was detected. We observed a dose-dependent increase in VP1-PCV2bCap-specific immune response in BALB/c mice immunized by recombinant protein sterilized by irradiation in dose 11 J/m2 with no significant difference between vaccines sterilized by UV-C light and filtration. A substantial difference in the production of VP1-PCV2bCap specific IgG was observed in piglets immunized with VP1-PCV2bCap sterilized by UV-C in comparison with protein sterilized by filtration in combination with the inactivation of baculovirus by binary ethylenimine. UV-C irradiation represents an effective method for vaccine sterilization, where commonly used methods of sterilization are not possible.

Carbon-based nanomaterials (C-BNM) have recently attracted an increased attention as the materials with potential applications in industry and medicine. Bioresistance and proinflammatory potential of C-BNM is the main obstacle for their medicinal application which was documented in vivo and in vitro. However, there are still limited data especially on graphene derivatives such as graphene platelets (GP). In this work, we compared multi-walled carbon nanotubes (MWCNT) and two different types of pristine GP in their potential to activate inflammasome NLRP3 (The nod-like receptor family pyrin domain containing 3) in vitro. Our study is focused on exposure of THP-1/THP1-null cells and peripheral blood monocytes to C-BNM as representative models of canonical and alternative pathways, respectively. Although all nanomaterials were extensively accumulated in the cytoplasm, increasing doses of all C-BNM did not lead to cell death. We observed direct activation of NLRP3 via destabilization of lysosomes and release of cathepsin B into cytoplasm only in the case of MWCNTs. Direct activation of NLRP3 by both GP was statistically insignificant but could be induced by synergic action with muramyl dipeptide (MDP), as a representative molecule of the family of pathogen-associated molecular patterns (PAMPs). This study demonstrates a possible proinflammatory potential of GP and MWCNT acting through NLRP3 activation.

Abstract There is an increasing imperative to explore safer alternatives for pesticides due to their indiscriminate use and consequential health impacts on the environment and humans. Nanoformulations of pesticides are being developed as potential alternatives due to their beneficial properties, including enhanced solubility, targeted delivery to the site of action, improved stability and efficacy and reduced non-target effects. Nevertheless, a comprehensive assessment is necessary for these emerging nanopesticides compared to existing formulations, aiming to ascertain whether their "nano" characteristics exacerbate toxicity for non-target organisms. This study investigated the toxicity of tebuconazole (TBZ) in different formulations, including nanoformulations (poly-ε-caprolactone [PCL] and nanostructured lipid carrier [NLC] loaded with TBZ), as well as a commercial formulation, on the reproduction of the nematode Caenorhabditis elegans in both aqueous and soil matrices. Additionally, the impact of the correspondent nanocarriers without TBZ on C. elegans was examined. In water, TBZ in the form of nano and commercial formulations exhibited higher toxicity on the nematodes' reproduction than the TBZ (a.s.) attributable to higher freely dissolved concentrations of TBZ, which resulted in a toxicity order, ranging from the most to the least toxic as follows: NLC-TBZ > PCL-TBZ > commercial formulation > TBZ (a.s.). For NLC-TBZ, the excess toxicity could be clearly explained by combined toxicity of TBZ (a.s.) and nanocarriers, with the effect addition of the separate single compounds matching the observed effects of the nanoformulation. For PCL-TBZ, effects were stronger than expected from the effect addition of TBZ (a.s.) and PCL nanocarriers, potentially due to enhanced bioavailability of encapsulated TBZ in the gut of the nematodes. In soil, NLC with and without loaded TBZ showed higher toxicity than other tested compounds, while PCL nanocarriers without TBZ did not exhibit negative effects on the reproduction of C. elegans. Microcosm experiment, where long-term effects on native soil nematode fauna were tested, confirmed that TBZ-nanoformulations act via combined toxic effects of TBZ and nanocarriers. These findings contribute valuable insights to understanding nanopesticides' ecotoxicity and underscore the need for harmonized regulatory assessments to evaluate these novel formulations adequately.

Abstract High‐grade serous carcinoma of the ovary, fallopian tube and peritoneum (HGSC), the most common type of ovarian cancer, ranks among the deadliest malignancies. Many HGSC patients have excess fluid in the peritoneum called ascites. Ascites is a tumour microenvironment (TME) containing various cells, proteins and extracellular vesicles (EVs). We isolated EVs from patients’ ascites by orthogonal methods and analyzed them by mass spectrometry. We identified not only a set of ‘core ascitic EV‐associated proteins’ but also defined their subset unique to HGSC ascites. Using single‐cell RNA sequencing data, we mapped the origin of HGSC‐specific EVs to different types of cells present in ascites. Surprisingly, EVs did not come predominantly from tumour cells but from non‐malignant cell types such as macrophages and fibroblasts. Flow cytometry of ascitic cells in combination with analysis of EV protein composition in matched samples showed that analysis of cell type‐specific EV markers in HGSC has more substantial prognostic potential than analysis of ascitic cells. To conclude, we provide evidence that proteomic analysis of EVs can define the cellular composition of HGSC TME. This finding opens numerous avenues both for a better understanding of EV's role in tumour promotion/prevention and for improved HGSC diagnostics.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted great interest in novel broad-spectrum antivirals, including perylene-related compounds. In the present study, we performed a structure–activity relationship analysis of a series of perylene derivatives, which comprised a large planar perylene residue, and structurally divergent polar groups connected to the perylene core by a rigid ethynyl or thiophene linker. Most of the tested compounds did not exhibit significant cytotoxicity towards multiple cell types susceptible to SARS-CoV-2 infection, and did not change the expressions of cellular stress-related genes under normal light conditions. These compounds showed nanomolar or sub-micromolar dose-dependent anti-SARS-CoV-2 activity, and also suppressed the in vitro replication of feline coronavirus (FCoV), also termed feline infectious peritonitis virus (FIPV). Perylene compounds exhibited high affinity for liposomal and cellular membranes, and efficiently intercalated into the envelopes of SARS-CoV-2 virions, thereby blocking the viral–cell fusion machinery. Furthermore, the studied compounds were demonstrated to be potent photosensitizers, generating reactive oxygen species (ROS), and their anti-SARS-CoV-2 activities were considerably enhanced after irradiation with blue light. Our results indicated that photosensitization is the major mechanism underlying the anti-SARS-CoV-2 activity of perylene derivatives, with these compounds completely losing their antiviral potency under red light. Overall, perylene-based compounds are broad-spectrum antivirals against multiple enveloped viruses, with antiviral action based on light-induced photochemical damage (ROS-mediated, likely singlet oxygen-mediated), causing impairment of viral membrane rheology.

Perylenylethynyl derivatives have been recognized as broad-spectrum antivirals that target the lipid envelope of enveloped viruses. In this study, we present novel perylenylethynylphenols that exhibit nanomolar or submicromolar antiviral activity against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and feline infectious peritonitis virus (FIPV) in vitro. Perylenylethynylphenols incorporate into viral and cellular membranes and block the entry of the virus into the host cell. Furthermore, these compounds demonstrate an ability to generate singlet oxygen when exposed to visible light. The rate of singlet oxygen production is positively correlated with antiviral activity, confirming that the inhibition of fusion is primarily due to singlet-oxygen-induced damage to the viral envelope. The unique combination of a shape that affords affinity to the lipid bilayer and the capacity to generate singlet oxygen makes perylenylethynylphenols highly effective scaffolds against enveloped viruses. The anticoronaviral activity of perylenylethynylphenols is strictly light-dependent and disappears in the absence of daylight (under red light). Moreover, these compounds exhibit negligible cytotoxicity, highlighting their significant potential for further exploration of the precise antiviral mechanism and the broader scope and limitations of this compound class.