The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is fundamental to increasing survival and therapeutic adjustment. In stages II–IV especially, additional indications for adjuvant therapy purposes after resection and for treatment of metastatic patients are urgently needed. We investigated whether the fatty acid (FA) and protein compositions of small extracellular vesicles (sEV) derived from the plasma of stage 0–I, II and III–IV melanoma patients (n = 38) could reflect disease stage. The subpopulation of sEV expressing CD81 EV marker (CD81sEV) was captured by an ad hoc immune affinity technique from plasma depleted of large EV. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA was detectable in patients with respect to healthy donors (HD). Moreover, a higher C18:0/C18:1 ratio, as a marker of cell membrane fluidity, distinguished early (stage 0–I) from late (III–IV) stages’ CD81sEV. Proteomics detected increases in CD14, PON1, PON3 and APOA5 exclusively in stage II CD81sEV, and RAP1B was decreased in stage III–IV CD81sEV, in comparison to HD. Our results suggest that stage dependent alterations in CD81sEV’ FA and protein composition may occur early after disease onset, strengthening the potential of circulating sEV as a source of discriminatory information for early diagnosis, prediction of metastatic behavior and following up of melanoma patients.

Edible plant and fruit-derived nanovesicles (NVs) are membrane-enclosed particles with round-shape morphology and signaling functions, which resemble mammalian cell-derived extracellular vesicles. These NVs can transmit cross-kingdom signals as they contain bioactive molecules and exert biological effects on mammalian cells. Their properties and stability in the gastrointestinal tract suggest NVs as a promising nutraceutical tool. In this study, we have demonstrated for the first time the presence of NVs in olive vegetation water (OVW), a waste by-product generated during olive oil production. Biophysical characterization by scanning electron microscopy, cryo-transmission electron microscopy, and nanoparticle tracking analysis revealed the presence in OVW of NVs having size and morphology similar to that of vesicles isolated from edible plants. Integrated lipidomic, metabolomic, and proteomic analyses showed that OVW-NVs carry a set of lipids, metabolites and proteins which have recognized antioxidant and anti-inflammatory activities. The nature of biomolecules identified in OVW-NVs suggests that these vesicles could exert beneficial effects on mammalian cells and could be used in the nutraceutical and food industries. The successful isolation of OVW-NVs and the characterization of their features strengthen the idea that agricultural waste might represent a source of NVs having features similar to NVs isolated from edible plants/fruits.

Background/Objectives: Donkey milk (DM) has been considered a valuable alternative to human and bovine counterparts as well as to infant formulas. Milk extracellular vesicles (EVs) have been proposed to influence key biological processes. The purpose of this study is to provide a comprehensive characterization of the protein composition of extracellular vesicles (EVs) by extending quantitative proteomic comparisons to EVs derived from donkey colostrum (DC) and mature donkey milk (MDM). Methods: The EVs were isolated from DC and MDM samples, characterized, and subjected to proteomic analysis using the tandem mass tag-based quantitative approach. Results: In addition to typical milk proteins and EV markers, EVs from DC and MDM both contain components associated with the immune system, immune response, or promoting tissue repair, and assisting with communication between the infant and their environment. The EVs from DC were enriched in proteins associated with protein turnover, specific defense functions, and regenerative processes. Conclusions: Overall, the results can contribute to the broader characterization of the overall protein composition of DC and MDM and might help to predict the beneficial effects of the corresponding EVs on various mammalian cells. They may also provide valuable insights for the development of novel DM-based products for food, pharmaceutical, and biotechnological applications.

Background/Objectives: Plants release extracellularly lipid bilayer-enclosed vesicles of nanometric size that can be retrieved in their fluids. Plant-derived extracellular vesicles (PDEVs) have mostly been involved in modulating host–pathogen interaction, making them a tool for cross-kingdom communication with a key role in plant immunity. In addition, PDEVs have demonstrated promising therapeutic features, not only in terms of intrinsic nutraceutical properties but also of active molecules’ delivery. Transgenic plants have been developed for a variety of purposes, i.e., to improve their functional properties like crops, but also to produce therapeutic molecules. However, it is unclear whether transgenes can end up in PDEVs, thus making them a vehicle for their cross-kingdom diffusion into the environment. Methods: Here, we investigated the association of transgenic DNA and RNA with PDEVs secreted by tobacco (Nicotiana tabacum) engineered to express the neomycine phosphotransferase II (Npt-II) gene. PDEVs were isolated from leaf apoplastic fluid by ultracentrifugation and characterized for their morphology and size. The association of DNA and RNA was assessed by qRT-PCR and their immunomodulatory properties by assaying PDEVs-induced IL1β and IL10 on THP1 monocytes. Results: Npt-II RNA, but not DNA, could be amplified from PDEVs, whereas no differences were observed between wt and transgenic tobacco PDEVs in terms of immunomodulatory properties. Conclusions: Although a different behaviour by other types of RNAs or DNAs could still be possible, our findings indicate that in this model, PDEVs are not associated with transgenic DNA, but they can protect RNA, including transgenic RNA, from degradation, contributing to their cross-kingdom spreading.

The early detection of cutaneous melanoma, a potentially lethal cancer with rising incidence, is key to increase survival and therapeutic adjustment. Especially in stages II-IV biomarkers are urgently needed for adjuvant therapy purposes after resection and for treatment of metastatic patients. We here investigated if fatty acid (FA) and protein composition of small extracellular vesicles (sEV) deriving from plasma of 0-I, II, and III-IV stage melanoma patients (n=38) could reflect disease stage and thus function as biomarker. The subpopulation of sEV expressing CD81 (CD81sEV) was isolated by an ad hoc immune affinity technique from microvesicle-depleted plasma. Biological macromolecules were investigated by gas chromatography and mass spectrometry in CD81sEV. A higher content of FA and a decrease in Saturation Index (C18:0/C18:1), already detectable in early stages, distinguished patients’ from healthy donor CD81sEV. Proteomics (identifier PXD024434) detected an exclusive and significant increase of CD14, PON1, PON3 and APOA5 in stage II and a significant decrease of Rap1b in stage III-IV CD81sEV. The FA and proteomic stage dependent CD81sEV signature strengthens the potential of circulating sEV studies in providing discriminatory information for early diagnosis, prediction of metastatic behavior and follow up of melanoma patients.

Research on viper venom has expanded into diverse medical applications, including cancer treatment. This study investigates the potential of Vipera ammodytes venom in oncology, evaluating its cytotoxicity and chemosensitising effects on malignant melanoma cells. Proteomic analysis identified 125 proteins in the venom, with Phospholipases A2, C-type lectins, and metalloproteinases among the most abundant components. These proteins are associated with cytotoxic, anti-proliferative, and tumor-inhibiting properties. Three melanoma cell lines (M001, Me501, and A375) were used to assess venom cytotoxicity. The IC50 values demonstrated consistent venom sensitivity across cell lines (approximately 1.1 µg/mL). Combined treatment with venom and cisplatin significantly increased the cytotoxicity compared to single-agent treatments. Notably, venom enhanced the sensitivity of cisplatin in resistant cell lines (M001 and Me501), increasing cell mortality by up to 40%. The A375 cell line, inherently more sensitive to cisplatin, exhibited additional cytotoxic effects only at higher venom doses. The morphological changes observed under microscopy confirmed venom-induced cellular changes, further supporting its potential as an anti-cancer agent. The selective targeting of melanoma cells by venom components, particularly in muscle-associated metastases, suggests a unique therapeutic niche. While cisplatin was chosen for this pilot study due to its established cytotoxicity, future research will explore venom combinations with contemporary treatments such as immunotherapy and targeted therapies. Although preliminary, these findings provide a foundation for integrating venom-based strategies into advanced melanoma protocols, aiming to improve outcomes in resistant or metastatic cases.