Analysis and Characterization of the Extracellular Vesicles Released in Non-Cancer Diseases Using Matrix-Assisted Laser Desorption Ionization/Mass Spectrometry.

The extracellular vesicles (EVs) released by cells play a crucial role in intercellular communications and interactions. The direct shedding of EVs from the plasma membrane represents a fundamental pathway for the transfer of properties and information between cells. These vesicles are classified based on their origin, biogenesis, size, content, surface markers, and functional features, encompassing a variety of bioactive molecules that reflect the physiological state and cell type of origin. Such molecules include lipids, nucleic acids, and proteins. Research efforts aimed at comprehending EVs, including the development of strategies for their isolation, purification, and characterization, have led to the discovery of new biomarkers. These biomarkers are proving invaluable for diagnosing diseases, monitoring disease progression, understanding treatment responses, especially in oncology, and addressing metabolic, neurological, infectious disorders, as well as advancing vaccine development. Matrix-Assisted Laser Desorption Ionization (MALDI)/Mass Spectrometry (MS) stands out as a leading tool for the analysis and characterization of EVs and their cargo. This technique offers inherent advantages such as a high throughput, minimal sample consumption, rapid and cost-effective analysis, and user-friendly operation. This review is mainly focused on the primary applications of MALDI-time-of-flight (TOF)/MS in the analysis and characterization of extracellular vesicles associated with non-cancerous diseases and pathogens that infect humans, animals, and plants.

Supercritical Fluid Extraction of Bioactive Components from Apple Peels and Their Modulation of Complex I Activity in Isolated Mitochondria.

Supercritical fluid extraction (SFE) was used to extract bioactive compounds from apple (Malus domestica) peel waste from three different Italian cultivars. The bioactive fractions were extracted applying a temperature of 60 °C and a pressure of 250 bar for 15 min with 20% ethanol as co-solvent, at a flow rate of 2 mL/min. The total polyphenol (TP), anthocyanin (TA), ascorbic acid (AA), and antioxidant activity contents (TACs) were measured, while chromatographic analyses were performed to highlight the differences between the extracts. The Stark cultivar had the highest levels of polyphenols, anthocyanins, and ascorbic acid, while the Royal Gala cultivar showed the highest total antioxidant activity. SFE extracts were then tested for their effect on the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) activity on mitochondria isolated from human embryonic kidney cells (HEK239). The Stark extract showed the most positive response in terms of NADH oxidation. The results obtained in this work highlight the potential of apple peel waste as a source of functional phytocompounds and suggest that Stark cultivar extracts may be exploited for pharmacological applications. This study supports the circular bioeconomy by promoting the use of waste products as a valuable resource.

Effect of a Composite Alginate/Grape Pomace Extract Packaging Material for Improving Meat Storage.

The development of food packaging materials that reduce the production of plastic, preserving at the same time the quality of food, is a topic of great interest today for the scientific community. Therefore, this article aims to report the effectiveness of an eco-friendly packaging material based on alginic acid and grape pomace extract from Vitis vinifera L. (winemaking by-products) for storing red meat in a domestic refrigerator. Specifically, biogenic amines are considered "sentinels" of the putrefactive processes, and their presence was thus monitored. For this purpose, an experimental analytical protocol based on the use of solid-phase microextraction coupled with gas chromatography-mass spectrometry was developed during this work for the determination of six biogenic amines (butylamine, cadaverine, isobutylamine, isopentylamine, putrescine, and tyramine). Moreover, by combining the analytical results with those of pH and weight loss measurements, differential scanning calorimetry, and microbiological analysis, it was proved that the studied materials could be proposed as an alternative packaging material for storing foods of animal origin, thus lowering the environmental impact according to sustainability principles.

Realizing Eco-Friendly Water-Resistant Sodium-Alginate-Based Films Blended with a Polyphenolic Aqueous Extract from Grape Pomace Waste for Potential Food Packaging Applications.

Water-resistant and environmentally friendly sodium-alginate-based films have been investigated to develop functional materials to extend the food's shelf-life. A water-stable alginate-based film was prepared, employing both the internal and external gelation approach in the presence of CaCl2. To apply this film to food packaging and thus preserve food quality, the aim of this work is to perform a chemical and physical characterization of the proposed materials, evidencing the main features and stability under different work conditions. Water contact angle measurements showed a value of 65°, suggesting an important reduced hydrophilic character of the obtained alginate films due to the novel CaCl2-induced compacted polymer network. The film's stability was thus checked through swelling measurements in water after varying pH, temperature, and ionic strength. The film was stable at high temperatures and not pH-responsive. Only highly concentrated salt-based solutions negatively affected the proposed packaging, causing a large swelling. Furthermore, a water-based polyphenolic extract from grape (Vitis vinifera L.) pomace waste was embedded inside the films in different amounts in order to confer additional properties. The extract's polyphenolic content (evaluated from HPLC/MS-MS measurements) endowed the films' UV-light screening and enhanced antioxidant properties. These important findings suggest the additional potential role of these films in protecting food from light deterioration. The stability of these hybrid films was also checked by observation, as the polyphenols' presence did not largely alter the alginate network that occurred yet was water-resistant under the described work conditions.

Identification of a characteristic VOCs pattern in the exhaled breath of post-COVID subjects: are metabolic alterations induced by the infection still detectable?

SARS-CoV-2 is expected to cause metabolic alterations due to viral replication and the host immune response resulting in increase of cytokine secretion and cytolytic activity. The present prospective observational study is addressed at exploring the potentialities of breath analysis in discrimination between patients with a documented previous history of symptomatic SARS-CoV-2 infection and, at the moment of the enrollment, exhibiting a negative nasopharyngeal swab and acquired immunity (post-COVID) and healthy subjects with no evidence of previous SARS-CoV-2 infection (no-COVID). The main purpose is to understand if traces of metabolic alterations induced during the acute phase of the infection are still detectable after negativization, in the form of a characteristic volatile organic compound (VOC) pattern. An overall number of 60 volunteers aged between 25 and 70 years were enrolled in the study (post-COVID: n.30; no-COVID: n. 30), according to well-determined criteria. Breath and ambient air samples were collected by means of an automated sampling system (Mistral) and analyzed by thermal desorption-gas chromatography-mass spectrometry (TD-GC/MS). Statistical tests (Wilcoxon/Kruskal-Wallis test) and multivariate data analysis (principal component analysis (PCA), linear discriminant analysis) were performed on data sets. Among all compounds detected (76 VOCs in 90% of breath samples), 5 VOCs (1-propanol, isopropanol, 2-(2-butoxyethoxy)ethanol, propanal and 4-(1,1-dimethylpropyl)phenol) showed abundances in breath samples collected from post-COVID subjects significantly different with respect to those collected from no-COVID group (Wilcoxon/Kruskal-Wallis test,p-values <0.05). Although not completely satisfactory separation between the groups was obtained, variables showing significant differences between the two groups and higher loadings for PCA are recognized biomarkers of COVID-19, according to previous studies in literature. Therefore, based on the outcomes obtained, traces of metabolic alterations induced by SARS-CoV-2 infection are still detectable after negativization. This evidence raises questions about the eligibility of post-COVID subjects in observational studies addressed at the detection of COVID-19. (Ethical Committee Registration number: 120/AG/11).

MALDI-TOF/MS Analysis of Non-Invasive Human Urine and Saliva Samples for the Identification of New Cancer Biomarkers.

Cancer represents a group of heterogeneous diseases that are a leading global cause of death. Even though mortality has decreased in the past thirty years for different reasons, most patients are still diagnosed at the advanced stage, with limited therapeutic choices and poor outcomes. Moreover, the majority of cancers are detected using invasive painful methods, such as endoscopic biopsy, making the development of non-invasive or minimally invasive methods for the discovery and fast detection of specific biomarkers a crucial need. Among body fluids, a valuable non-invasive alternative to tissue biopsy, the most accessible and least invasive are undoubtedly urine and saliva. They are easily retrievable complex fluids containing a large variety of endogenous compounds that may provide information on the physiological condition of the body. The combined analysis of these fluids with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS), a reliable and easy-to-use instrumentation that provides information with relatively simple sample pretreatments, could represent the ideal option to rapidly achieve fast early stage diagnosis of tumors and their real-time monitoring. On this basis, the present review summarizes the recently reported applications relevant to the MALDI analysis of human urine and saliva samples.

Volatile Organic Compounds, Indole, and Biogenic Amines Assessment in Two Mediterranean Irciniidae (Porifera, Demospongiae).

Solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) was employed for the headspace determination of the volatile organic fraction emitted by two of the most common Mediterranean demosponges, Ircinia variabilis and Sarcotragus spinosulus, and of indole and some biogenic amines released by sponges in an aqueous medium. A total of 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane and 75 µm carboxen/polydimethylsiloxane fibers were used for the headspace extraction of low molecular weight sulfur compounds from a hermetically sealed vial containing sponge fragments, while the direct immersion determination of indole and biogenic amines was performed. The biogenic amines were extracted after in-solution derivatization with isobutyl chloroformate. All analytical parameters (linearity, limits of detection, and quantification, precision, and recovery) were evaluated for indole and biogenic amines. SPME-GC-MS proved to be a reliable means of highlighting the differences between molecules released by different sponges, principally responsible for their smell. The combined approaches allowed the identification of several volatile compounds in the headspace and other molecules released by the sponges in an aqueous medium, including indole and the BAs cadaverine, histamine, isobutylamine, isopentylamine, propylamine, 2-phenylethylamine, putrescine and tryptamine. The results obtained represent a further contribution to the picture of odoriferous molecules secreted by sponges.

Determination of Commercial Animal and Vegetable Milks' Lipid Profile and Its Correlation with Cell Viability and Antioxidant Activity on Human Intestinal Caco-2 Cells.

Lipids from milk are important nutritional components, although their health effects, especially for animal milks, are still questioned. Four types of commercial milks, two semi-skimmed animal milks (bovine and goat) and two vegetable ones (soy and rice), along with their total and free lipid fractions recovered by sequential centrifugation or by ethyl acetate extraction, respectively, have been analyzed. A higher antioxidant ability, reported as Trolox equivalent antioxidant capacity, was found for all raw milks compared to that of rice. This trend was confirmed, except for soy milk, as ROS reduction in Caco-2 cells. The free lipid fraction was shown to have the highest antioxidant potential in both chemical and biological tests. Moreover, goat and soy raw milks positively regulated Caco-2 cell viability after an inflammatory stimulus. This effect was lost when their total lipid fraction was tested. Finally, only the free lipid fraction from rice milk preserved the Caco-2 viability after LPS stimulation. Our data demonstrated that the lipid profile of each milk, characterized by GC-MS analysis, could contribute to dictate its biological effects, and, although additional in vitro and in vivo studies are needed, they could support the literature re-evaluating the health effects of animal-based versus plant-based milks in the intestinal cellular model.

Determination of hydroxytyrosol and tyrosol in human urine after intake of extra virgin olive oil produced with an ultrasounds-based technology.

Extra virgin olive oil (EVOO) is a known source of antioxidants, such as phenolic compounds, useful in the prevention of non-infectious diseases (atherosclerosis, diabetes, cancer, and other diseases). In the present study, EVOO obtained using an innovative ultrasounds-based technology was found richer in total polyphenols, hydroxytyrosol and tyrosol, than EVOO obtained using a conventional mechanical technology. The urinary excretion in humans of hydroxytyrosol and tyrosol, after the administration of ultrasounds and mechanical EVOOs, respectively, was assessed and compared. The analytes were determined in urine samples, collected for 24 h, of six healthy people (3 men and 3 women, age 22-70 years and body mass index <30 kg/m2) who ingested 20 g of oil for six consecutive days. A commercial refined olive oil was also used in the study to determine the baseline excretion levels of the two metabolites. High correlation coefficients (≥0.9311) were found between the amounts of the analytes ingested daily with EVOOs and those determined in the 24-h urines. The results clearly indicated that the EVOO obtained with the ultrasound process was characterized by the highest concentration of biophenols which were consequently available in greater quantities after ingestion, indicating that it represents a high-quality product containing high levels of beneficial compounds such as biophenols readily assimilable by the human body.

Advanced Gel Permeation Chromatography system with increased loading capacity: Polycyclic aromatic hydrocarbons detection in olive oil as a case of study.

Gel permeation chromatography (GPC) is herein used as size exclusion clean-up technique for highly sensitive and straightforward detection of Polycyclic Aromatic Hydrocarbons (PAHs) in olive oil samples. An advanced chromatographic system has been developed to isolate a series of PAHs with cancerogenic potential, including PAH4 (benzo(a)pyrene BaP, benzo(a)anthracene BaA, benzo(b)fluoranthene BbF and chrysene Chry) reported in the European Regulation. The system avails of two glass chromatographic columns and a switching valve, that allow removal of interfering analytes in olive oil without resorting to any preliminary extraction process. A seven-fold increase of the loaded sample amount versus conventional chromatographic systems (1 g vs 0.150 g) has been pursued, as well as improved PAHs detection and quantification limits (LOD-LOQ for PAH4: 0.21-0.70 ng/g for BaA, 0.26-0.86 ng/g for Chry, 0.23-0.76 ng/g for BbF, 0.32-1.06 ng/g for BaP), in accordance with the continuous need of more and more reducing these limits in food analysis by the European Regulation. The protocol developed represents a highly innovative and efficient analytical method for organic pollutants in complex biological matrices as olive oil, that can have huge impact on technology for PAHs detection in food samples, being suitable for both industrial and small-scale laboratories.

Supercritical CO2 Extraction of Phytocompounds from Olive Pomace Subjected to Different Drying Methods.

Olive pomace is a semisolid by-product of olive oil production and represents a valuable source of functional phytocompounds. The valorization of agro-food chain by-products represents a key factor in reducing production costs, providing benefits related to their reuse. On this ground, we herein investigate extraction methods with supercritical carbon dioxide (SC-CO2) of functional phytocompounds from olive pomace samples subjected to two different drying methods, i.e., freeze drying and hot-air drying. Olive pomace was produced using the two most common industrial olive oil production processes, one based on the two-phase (2P) decanter and one based on the three-phase (3P) decanter. Our results show that freeze drying more efficiently preserves phytocompounds such as α-tocopherol, carotenoids, chlorophylls, and polyphenols, whereas hot-air drying does not compromise the ß-sitosterol content and the extraction of squalene is not dependent on the drying method used. Moreover, higher amounts of α-tocopherol and polyphenols were extracted from 2P olive pomace, while ß-sitosterol, chlorophylls, and carotenoids were more concentrated in 3P olive pomace. Finally, tocopherol and pigment/polyphenol fractions exerted antioxidant activity in vitro and in accelerated oxidative conditions. These results highlight the potential of olive pomace to be upcycled by extracting from it, with green methods, functional phytocompounds for reuse in food and pharmaceutical industries.

Targeting mitochondrial metabolite transporters in Penicillium expansum for reducing patulin production.

There is an increasing need of alternative treatments to control fungal infection and consequent mycotoxin accumulation in harvested fruits and vegetables. Indeed, only few biological targets of antifungal agents have been characterized and can be used for limiting fungal spread from decayed fruits/vegetables to surrounding healthy ones during storage. On this concern, a promising target of new antifungal treatments may be represented by mitochondrial proteins due to some species-specific functions played by mitochondria in fungal morphogenesis, drug resistance and virulence. One of the most studied mycotoxins is patulin produced by several species of Penicillium and Aspergillus genera. Patulin is toxic to many biological systems including bacteria, higher plants and animalia. Although precise biochemical mechanisms of patulin toxicity in humans are not completely clarified, its high presence in fresh and processed apple fruits and other apple-based products makes necessary developing a strategy for limiting its presence/accumulation. Patulin biosynthetic pathway consists of an enzymatic cascade, whose first step is represented by the synthesis of 6-methylsalicylic acid, obtained from the condensation of one acetyl-CoA molecule with three malonyl-CoA molecules. The most abundant acetyl-CoA precursor is represented by citrate produced by mitochondria. In the present investigation we report about the possibility to control patulin production through the inhibition of mitochondrial/peroxisome transporters involved in the export of acetyl-CoA precursors from mitochondria and/or peroxisomes, with specific reference to the predicted P. expansum mitochondrial Ctp1p, DTC, Sfc1p, Oac1p and peroxisomal PXN carriers.

Corrigendum: Chemical Ecology of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae): Behavioral and Biochemical Strategies for Intraspecific and Host Interactions.

[This corrects the article DOI: 10.3389/fphys.2019.00604.].

Development, Optimization, and Comparison of Different Sample Pre-Treatments for Simultaneous Determination of Vitamin E and Vitamin K in Vegetables.

The absence of vitamin E from the diet can lead to cardiovascular disease, cancer, cataracts, and premature aging. Vitamin K deficiency can lead to bleeding disorders. These fat-soluble vitamins are important nutritional factors that can be determined in different methods in vegetables. In this work, the simultaneous determination of α-tocopherol, α-tocopheryl acetate, phylloquinone, and menaquinone-4 by gas chromatography-mass spectrometry (GC-MS) has been optimized using both direct injection and solid phase microextraction (SPME). Three different sample pre-treatment approaches based on: (A) solid-liquid-liquid-liquid extraction (SLE-LLE), (B) SLE, and (C) SPME were then applied to extract the target analytes from vegetables samples using menaquinone as internal standard. All the procedures allowed the determination of the target analytes in onion, carrot, celery, and curly kale samples. Similar results were obtained with the three different approaches, even if the one based on SPME offers the best performance, together with a reduced use of solvent, time consumption, and experimental complexity, which makes it the preferable option for industrial applications.

Relationship between cancer tissue derived and exhaled volatile organic compound from colorectal cancer patients. Preliminary results.

New insight into the omic sciences suggests that volatile organic compounds (VOCs) contained in exhaled breath can reflect the healthy or disease state of patients, representing an attractive, promising and non-invasive method of medical investigation. This approach has recently been proposed as a new potential screening tool in colorectal cancer (CRC) patients. However, a possible correlation between the exhaled VOCs and those produced by the cancerous tissue has never been investigated. In this preliminary study, we compare the VOCs exhaled by seven patients affected by CRC with those produce by own cancer tissue and normal colonic mucosa. The VOCs contained in the exhaled breath were sampled with the ReCIVA breath sampler©, while those produced by ex-vivo human tissues weresampled by headspace solid-phase microextraction (HS-SPME) at different incubation times after surgery. In both cases, the collected VOCs were analyzed by Gas Chromatography with Mass Spectrometry (GC-MS). Benzaldehyde, benzene ethyl, benzene methyl, butanoic acid, dodecanoic acid, indole, nonanal, octanoic acid, pentanoic acid, phenol and tetradecane were the VOCs most frequently detected both in the exhaled breath and secreted by tissues. The results showed that cancer tissue and normal colonic mucosa from the same patient produced a similar VOCs pattern but with different fingerprints. In particular, the concentrations of benzaldehyde, benzene ethyl and indole were significantly different in cancer tissue respect the normal colonic mucosa. In conclusion, these preliminary data suggest the involvement of the three compounds in CRC by encouraging further investigation.

Chemical Ecology of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae): Behavioral and Biochemical Strategies for Intraspecific and Host Interactions.

This study focuses on several aspects of communication strategies adopted by adults of the Mediterranean flat-headed root-borer Capnodis tenebrionis (Coleoptera: Buprestidae). Morphological studies on the structures involved in mate recognition and acceptance revealed the presence of porous areas in the pronota in both sexes. These areas were variable in shape and size, but proportionally larger in males. The presence of chaetic, basiconic, and coeloconic sensilla in the antennae of both males and females was verified. Bioassays revealed stereotyped rituals in males and the involvement of female pronotal secretions in mate recognition and acceptance. During the mating assays, the female's pronotum was covered by a biologically inert polymeric resin (DenFilTM), which prevented males from detecting the secretions and from completing the copulation ritual. The use of the resin allowed for the collection of chemical compounds. GC-MS analysis of the resin suggested it may be used to retain compounds from insect body surfaces and revealed sex-specific chemical profiles in the cuticles. Since adult C. tenebrionis may use volatile organic compounds (VOCs) emitted from leaves or shoots, the VOC emission profiles of apricot trees were characterized. Several volatiles related to plant-insect interactions involving fruit tree species of the Rosaceae family and buprestid beetles were identified. To improve understanding of how VOCs are perceived, candidate soluble olfactory proteins involved in chemoreception (odorant-binding proteins and chemosensory proteins) were identified using tissue and sex-specific RNA-seq data. The implications for chemical identification, physiological and ecological functions in intraspecific communication and insect-host interactions are discussed and potential applications for monitoring presented.

Solid-phase microextraction and on-fiber derivatization for assessment of mammalian and vegetable milks with emphasis on the content of major phytoestrogens.

A new solvent-free method for the simultaneous determination of some major phytoestrogens (equol, enterodiol, daidzein, genistein, glycitein) in different commercial milks (cow, goat and soy-rice) was developed. After solid phase microextraction, performed by direct immersion of a 65 µm-polydimethylsiloxane-divinylbenzene fiber in diluted (1:100 with 0.2% formic acid - 30% sodium chloride) milk samples (18 °C for 20 min under stirring), a direct on-fiber silylation with N,O-bis (trimethylsilyl)trifluoroacetamide) containing 1% trimethylchlorosilane (70 °C for 20 min) was performed prior to gas chromatography-mass spectrometry analysis. Since the target compounds were determined as aglycones, the hydrolytic removal of the aglycone from the glycosides was performed. The method permitted the determination of the target analytes in all the considered milk samples as well as the detection of some major amphipathic fats indicating that the approach could potentially be applied in the future for further applications, such as milk profiling.

Lindane Bioremediation Capability of Bacteria Associated with the Demosponge Hymeniacidon perlevis.

Lindane is an organochlorine pesticide belonging to persistent organic pollutants (POPs) that has been widely used to treat agricultural pests. It is of particular concern because of its toxicity, persistence and tendency to bioaccumulate in terrestrial and aquatic ecosystems. In this context, we assessed the role of bacteria associated with the sponge Hymeniacidon perlevis in lindane degradation. Seven bacteria isolates were characterized and identified. These isolates showed a remarkable capacity to utilize lindane as a sole carbon source leading to a percentage of residual lindane ranging from 3% to 13% after 12 days of incubation with the pesticide. The lindane metabolite, 1,3-6-pentachloro-cyclohexene, was identified as result of lindane degradation and determined by gas chromatography-mass spectrometry (GC-MS). The bacteria capable of lindane degradation were identified on the basis of the phenotypic characterization by morphological, biochemical and cultural tests, completed with 16S rDNA sequence analysis, and assigned to Mameliella phaeodactyli, Pseudovibrioascidiaceicola, Oceanicaulis stylophorae, Ruegeria atlantica and to three new uncharacterized species. The results obtained are a prelude to the development of future strategies for the in situ bioremediation of lindane.

Bovine and soybean milk bioactive compounds: Effects on inflammatory response of human intestinal Caco-2 cells.

In this study the effects of commercial bovine and soybean milks and their bioactive compounds, namely genistein, daidzein and equol, on the inflammatory responses induced by lipopolysaccharide (LPS) treatment of human intestinal Caco-2 cells were examined, in terms of nitric oxide (NO) release and inducible nitric oxide synthetase (iNOS) expression. Both milks and their bioactive compounds significantly inhibited, dose-dependently, the expression of iNOS mRNA and protein, resulting in a decreased NO production. The NF-κB activation in LPS-stimulated intestinal cells was also examined. In all cases we observed that cell pre-treatment before LPS activation inhibited the IkB phosphorylation. Accordingly, quantification of bioactive compounds by solid phase microextraction coupled with liquid chromatography has shown that they were absorbed, metabolized and released by Caco-2 cells in culture media. In conclusion, we demonstrated that milks and compounds tested are able to reduce LPS-induced inflammatory responses from intestinal cells, interfering with NF-kB dependent molecular mechanisms.