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.

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.

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.

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.

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.

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.

Functions of vasopressin and oxytocin in bone mass regulation.

Prior studies show that oxytocin (Oxt) and vasopressin (Avp) have opposing actions on the skeleton exerted through high-affinity G protein-coupled receptors. We explored whether Avp and Oxtr can share their receptors in the regulation of bone formation by osteoblasts. We show that the Avp receptor 1α (Avpr1α) and the Oxt receptor (Oxtr) have opposing effects on bone mass: Oxtr(-/-) mice have osteopenia, and Avpr1α(-/-) mice display a high bone mass phenotype. More notably, this high bone mass phenotype is reversed by the deletion of Oxtr in Oxtr(-/-):Avpr1α(-/-) double-mutant mice. However, although Oxtr is not indispensable for Avp action in inhibiting osteoblastogenesis and gene expression, Avp-stimulated gene expression is inhibited when the Oxtr is deleted in Avpr1α(-/-) cells. In contrast, Oxt does not interact with Avprs in vivo in a model of lactation-induced bone loss in which Oxt levels are high. Immunofluorescence microscopy of isolated nucleoplasts and Western blotting and MALDI-TOF of nuclear extracts show that Avp triggers Avpr1α localization to the nucleus. Finally, a specific Avpr2 inhibitor, tolvaptan, does not affect bone formation or bone mass, suggesting that Avpr2, which primarily functions in the kidney, does not have a significant role in bone remodeling.

Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor.

We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr-EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with ß-arrestins (Arrbs), the small GTPase Rab5, importin-ß (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors.

A Simple and Effective Mass Spectrometric Approach to Identify the Adulteration of the Mediterranean Diet Component Extra-Virgin Olive Oil with Corn Oil.

Extra virgin olive oil (EVOO) with its nutraceutical characteristics substantially contributes as a major nutrient to the health benefit of the Mediterranean diet. Unfortunately, the adulteration of EVOO with less expensive oils (e.g., peanut and corn oils), has become one of the biggest source of agricultural fraud in the European Union, with important health implications for consumers, mainly due to the introduction of seed oil-derived allergens causing, especially in children, severe food allergy phenomena. In this regard, revealing adulterations of EVOO is of fundamental importance for health care and prevention reasons, especially in children. To this aim, effective analytical methods to assess EVOO purity are necessary. Here, we propose a simple, rapid, robust and very sensitive method for non-specialized mass spectrometric laboratory, based on the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) coupled to unsupervised hierarchical clustering (UHC), principal component (PCA) and Pearson's correlation analyses, to reveal corn oil (CO) adulterations in EVOO at very low levels (down to 0.5%).

Highly selective palladium-benzothiazole carbene-catalyzed allylation of active methylene compounds under neutral conditions.

The Pd-benzothiazol-2-ylidene complex I was found to be a chemoselective catalyst for the Tsuji-Trost allylation of active methylene compounds carried out under neutral conditions and using carbonates as allylating agents. The proposed protocol consists in a simplified procedure adopting an in situ prepared catalyst from Pd2dba3 and 3-methylbenzothiazolium salt V as precursors. A comparison of the performance of benzothiazole carbene with phosphanes and an analogous imidazolium carbene ligand is also proposed.

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.

MALDI-TOF mass spectrometric determination of intact phospholipids as markers of illegal bovine milk adulteration of high-quality milk.

In the dairy industry one of the most common frauds is mixing high-value milk (sheep's and goats') with milk of lower value (cows'). This illegal practice has commercial, ethical, and serious sanitary consequences because consumers can be exposed to hidden allergens contained in the undeclared cows' milk. Here, we investigated the possibility of using matrix-assisted laser-desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS) as a rapid, sensitive, and accurate technique for detection of milk adulteration by analysis of phospholipid profiles. Lipid extracts of pure raw milk, commercial milk, and binary mixtures of cows' and goats' milk and cows' and sheep's milk (the concentrations of each milk varied from 0 % to 50 %) were analyzed with α-cyano-4-chlorocinnamic acid as matrix. The abundance ratio of the ions at m/z 703 and m/z 706 was found to be species-correlated and was used as marker of cows' milk in sheep's and goats' milk. Furthermore, the procedure could potentially be applied to cheese samples, because peaks at m/z 703 and 706 were also found in several commercial cheese samples. This approach proved to be an efficient, rapid, and inexpensive method of detecting milk fraud.

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).

Lipid fingerprinting of gram-positive lactobacilli by intact--matrix-assisted laser desorption/ionization mass spectrometry using a proton sponge based matrix.

A method of direct lipid analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) in intact membranes, without prior extraction/separation steps, is described. Here, we demonstrate the efficacy of a strong base, 1,8-bis(dimethylamino)naphthalene (DMAN; proton sponge), as a novel matrix for MALDI-time-of-flight (TOF) MS analysis of whole cell bacteria. Initially, individual acidic low-molecular-weight analytes such as standard free fatty acids and phospholipids were analyzed using DMAN as matrix. Clear negative-mode MALDI-TOF MS spectra of all analytes show only deprotonated analyte signals at a low picomole limit of detection with the complete absence of matrix-related signals. These results indicate that DMAN represents a suitable matrix for MALDI-TOF MS analysis of mixtures of complex lipids as the intact membranes of microorganisms. DMAN was successfully applied to the analysis of Lactobacillus sanfranciscensis and L. plantarum microorganisms. Different components were sensitively detected in a single spot, including 16:0, 18:2, 18:3, and 21:0 free acids, glycolipids, phosphatidylglycerols (PGs) and cardiolipins. This method might be of general application, offering the advantage of quickly gaining information about lipid components of other gram-positive bacterial membranes.

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.

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.

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.

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.