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Abstract Background Mucosal Melanomas (MM) are highly aggressive neoplasms arising from mucosal melanocytes. Current treatments offer a limited survival benefit for patients with advanced MM; moreover, the lack of pre-clinical cellular systems has significantly limited the understanding of their immunobiology. Methods Five novel cell lines were obtained from patient-derived biopsies of MM arising in the sino-nasal mucosa and designated as SN-MM1-5. The morphology, ultrastructure and melanocytic identity of SN-MM cell lines were validated by transmission electron microscopy and immunohistochemistry. Moreover, in vivo tumorigenicity of SN-MM1-5 was tested by subcutaneous injection in NOD/SCID mice. Molecular characterization of SN-MM cell lines was performed by a mass-spectrometry proteomic approach, and their sensitivity to PI3K chemical inhibitor LY294002 was validated by Akt activation, measured by pAkt(Ser473) and pAkt(Thr308) in immunoblots, and MTS assay. Results This study reports the validation and functional characterization of five newly generated SN-MM cell lines. Compared to the normal counterpart, the proteomic profile of SN-MM is consistent with transformed melanocytes showing a heterogeneous degree of melanocytic differentiation and activation of cancer-related pathways. All SN-MM cell lines resulted tumorigenic in vivo and display recurrent structural variants according to aCGH analysis. Of relevance, the microscopic analysis of the corresponding xenotransplants allowed the identification of clusters of MITF-/CDH1-/CDH2 + /ZEB1 + /CD271 + cells, supporting the existence of melanoma-initiating cells also in MM, as confirmed in clinical samples. In vitro, SN-MM cell lines were sensitive to cisplatin, but not to temozolomide. Moreover, the proteomic analysis of SN-MM cell lines revealed that RICTOR, a subunit of mTORC2 complex, is the most significantly activated upstream regulator, suggesting a relevant role for the PI3K-Akt-mTOR pathway in these neoplasms. Consistently, phosphorylation of NDRG1 and Akt activation was observed in SN-MM, the latter being constitutive and sustained by PTEN loss in SN-MM2 and SN-MM3. The cell viability impairment induced by LY294002 confirmed a functional role for the PI3K-Akt-mTOR pathway in SN-MM cell lines. Conclusions Overall, these novel and unique cellular systems represent relevant experimental tools for a better understanding of the biology of these neoplasms and, as an extension, to MM from other sites.

Fish freshness consists of complex endogenous and exogenous processes; therefore, the use of a few parameters to unravel illicit practices could be insufficient. Moreover, the development of strategies for the identification of such practices based on additives known to prevent and/or delay fish spoilage is still limited. The paper deals with the identification of the effect played by a Cafodos solution on the conservation state of sea bass at both short-term (3 h) and long-term (24 h). Controls and treated samples were characterized by a multi-omic approach involving proteomics, lipidomics, metabolomics, and metagenomics. Different parts of the fish samples were studied (muscle, skin, eye, and gills) and sampled through a non-invasive procedure based on EVA strips functionalized by ionic exchange resins. Data fusion methods were then applied to build models able to discriminate between controls and treated samples and identify the possible markers of the applied treatment. The approach was effective in the identification of the effect played by Cafodos that proved to be different in the short- and long-term and complex, involving proteins, lipids, and small molecules to a different extent.

BackgroundUrinary extracellular vesicles (uEVs) can be released by different cell types facing the urogenital tract and are involved in cellular trafficking, differentiation and survival. UEVs can be easily detected in urine and provide pathophysiological information “in vivo” without the need of a biopsy. Based on these premises, we hypothesized that uEVs proteomic profile may serve as a valuable tool in the differential characterization between Essential Hypertension (EH) and primary aldosteronism (PA).MethodsPatients with essential hypertension (EH) and PA were enrolled in the study (EH= 12, PA=24: 11 Bilateral Primary Aldosteronism subtype (BPA) and 13 Aldosterone Producing Adenoma (APA)). Clinical and biochemical parameters were available for all the subjects. UEVs were isolated from urine by ultracentrifugation and analysed by Transmission Electron Microscopy (TEM) and nanotrack particle analysis (NTA). UEVs protein content was investigated through an untargeted MS-based approach. Statistical and network analysis was performed to identify potential candidates for the identification and classification of PA.ResultsMS analysis provided more than 300 protein identifications. Exosomal markers CD9 and CD63 were detected in all samples. Several molecules characterizing EH vs PA patients as well as BPA and APA subtypes were identified after statistical elaboration and filtering of the results. In particular, some key proteins involved in water reabsorption mechanisms, such as AQP1 and AQP2, were among the best candidates for discriminating EH vs PA, as well as A1AG1 (AGP1).ConclusionThrough this proteomic approach, we identified uEVs molecular indicators that can improve PA characterization and help in the gain of insights of the pathophysiological features of this disease. In particular, PA was characterized by a reduction of AQP1 and AQP2 expression as compared with EH.

Even though cancer patients are generally considered more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, the mechanisms driving their predisposition to severe forms of coronavirus disease 2019 (COVID-19) have not yet been deciphered. Since metabolic disorders are associated with homeostatic frailty, which increases the risk of infection and cancer, we asked whether we could identify immunometabolic pathways intersecting with cancer and SARS-CoV-2 infection. Thanks to a combined flow cytometry and multiomics approach, here we show that the immunometabolic traits of COVID-19 cancer patients encompass alterations in the frequency and activation status of circulating myeloid and lymphoid subsets, and that these changes are associated with i) depletion of tryptophan and its related neuromediator tryptamine, ii) accumulation of immunosuppressive tryptophan metabolites (i.e., kynurenines), and iii) low nicotinamide adenine dinucleotide (NAD+) availability. This metabolic imbalance is accompanied by altered expression of inflammatory cytokines in peripheral blood mononuclear cells (PBMCs), with a distinctive downregulation of IL-6 and upregulation of IFNγ mRNA expression levels. Altogether, our findings indicate that cancer not only attenuates the inflammatory state in COVID-19 patients but also contributes to weakening their precarious metabolic state by interfering with NAD+-dependent immune homeostasis.

ABSTRACT Disease resistance in plants depends on a molecular dialogue with microbes that involves many known chemical effectors, but the time course of the interaction and the influence of the environment are largely unknown. The outcome of host-pathogen interactions is thought to reflect the offensive and defensive capabilities of both players. When plants interact with Pseudomonas syringae, several well-characterized virulence factors contribute to early bacterial pathogenicity, including the type III secretion system (T3SS), which must be activated by signals from the plant and environment to allow the secretion of virulence effectors. The manner in which these signals regulate T3SS activity is still unclear. Here, we strengthen the paradigm of the plant-pathogen molecular dialogue by addressing overlooked details concerning the timing of interactions, specifically the role of plant signals and temperature on the regulation of bacterial virulence during the first few hours of the interaction. Whole-genome expression profiling after 1 h revealed that the perception of plant signals from kiwifruit or tomato extracts anticipated T3SS expression in P. syringae pv. actinidiae compared to apoplast-like conditions, facilitating more efficient effector transport in planta, as revealed by the induction of a temperature-dependent hypersensitive response in the nonhost plant Arabidopsis thaliana Columbia-0 (Col-0). Our results show that in the arms race between plants and bacteria, the temperature-dependent timing of bacterial virulence versus the induction of plant defenses is probably one of the fundamental parameters governing the outcome of the interaction. IMPORTANCE Plant diseases—their occurrence and severity—result from the impact of three factors: the host, the pathogen, and the environmental conditions, interconnected in the disease triangle. Time was further included as a fourth factor accounting for plant disease, leading to a more realistic three-dimensional disease pyramid to represent the evolution of disease over time. However, this representation still considers time only as a parameter determining when and to what extent a disease will occur, at a scale from days to months. Here, we show that time is a factor regulating the arms race between plants and pathogens, at a scale from minutes to hours, and strictly depends on environmental factors. Thus, besides the arms possessed by pathogens and plants per se, the opportunity and the timing of arms mobilization make the difference in determining the outcome of an interaction and thus the occurrence of plant disease.

Abstract The COVID-19 pandemic is still raging in most countries. Although the recent mass vaccination campaign has opened a new chapter in the battle against SARS-CoV-2, the world is still far from herd immunity. There is an urgent need to identify healthy people at high risk of contracting COVID-19, as well as supplements and nutraceuticals that can reduce the risk of infection or mitigate symptoms. In the present study, a metabolic phenotype that could protect individuals from SARS-CoV-2 infection or predispose them to developing COVID-19 was investigated. Untargeted metabolomics was performed on serum samples collected from 51 healthcare workers who were in good health at the beginning of the COVID-19 outbreak in Italy, and who were later exposed to the same risk of developing COVID-19. Half of them developed COVID-19 within three weeks of the blood collection. Our results demonstrate the presence of a specific signature associated with protection from SARS-CoV-2. Circulating monolaurin, which has well-known antiviral and antibacterial properties, was higher in protected subjects, suggesting a potential defensive role against SARS-CoV-2 infection; thus, dietary supplements could boost the immune system against this infection. In addition, our data demonstrate that people with higher levels of cholesterol are at higher risk of developing COVID-19. The present study demonstrates that metabolomics can be of great help for developing personalized medicine and for supporting public healthcare strategies. Studies with larger cohorts of subjects are necessary to confirm our findings.

The role of the microbiota–gut–brain axis in maintaining a healthy status is well recognized. In this bidirectional flux, the influence of host hormones on gut bacteria is crucial. However, data on commensal/probiotics are scarce since most reports analyzed the effects of human bioactive compounds on opportunistic strains, highlighting the risk of increased pathogenicity under stimulation. The present investigation examined the modifications induced by 5HT, a tryptophan-derived molecule abundant in the intestine, on the probiotic Enterococcus faecium NCIMB10415. Specific phenotypic modifications concerning the probiotic potential and possible effects of treated bacteria on dendritic cells were explored together with the comparative soluble proteome evaluation. Increased resistance to bile salts and ampicillin in 5HT-stimulated conditions relate with overexpression of specific proteins (among which Zn-beta-lactamases, a Zn-transport protein and a protein involved in fatty acid incorporation into the membrane). Better auto-aggregating properties and biofilm-forming aptitude are consistent with enhanced QS peptide transport. Concerning interaction with the host, E. faecium NCIMB10415 enhanced dendritic cell maturation, but no significant differences were observed between 5HT-treated and untreated bacteria; meanwhile, after 5HT exposure, some moonlight proteins possibly involved in tissue adhesion were found in higher abundance. Finally, the finding in stimulated conditions of a higher abundance of VicR, a protein involved in two-component signal transduction system (VicK/R), suggests the existence of a possible surface receptor (VicK) for 5HT sensing in the strain studied. These overall data indicate that E. faecium NCIMB10415 modifies its physiology in response to 5HT by improving bacterial interactions and resistance to stressors.

Abstract Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC.

In the field of food control for fresh products, the identification of foods subjected to illicit conservation treatments to extend their shelf life is fundamental. Fresh fish products are particularly subjected to this type of fraud due to their high commercial value and the fact that they often have to be transported over a long distance, keeping their organoleptic characteristics unaltered. Treatments of this type involve, e.g., the bleaching of the meat and/or the momentary abatement of the microbial load, while the degradation process continues. It is therefore important to find rapid methods that allow the identification of illicit treatments. The study presented here was performed on 24 sea bass samples divided into four groups: 12 controls (stored on ice in the fridge for 3 or 24 h), and 12 treated with a Cafodos-like solution for 3 or 24 h. Muscle and skin samples were then characterized using micro-Raman spectroscopy. The data were pre-processed by smoothing and taking the first derivative and then PLS-DA models were built to identify short- and long- term effects on the fish’s muscle and skin. All the models provided the perfect classification of the samples both in fitting and cross-validation and an analysis of the bands responsible for the effects was also reported. To the best of the authors’ knowledge, this is the first time Raman spectroscopy has been applied for the identification of a Cafodos-like illicit treatment, focusing on both fish muscle and skin evaluation. The procedure could pave the way for a future application directly on the market through the use of a portable device.

Abstract The topical application of lactic acid bacteria (LAB) is recognized as a useful approach to improve skin health. This work aims to characterize by a multidisciplinary approach, the wound healing, anti-inflammatory, anti-pathogens and proteomic effects of six LAB lysates, belonging to the genus Lactobacillus. Our results demonstrated that the lysates of tested LAB stimulated the proliferation of keratinocytes, and that L. plantarum SGL 07 and L. salivarius SGL 19 accelerated the re-epithelization by inducing keratinocyte migration. The bacterial lysates also reduced the secretion of specific pro-inflammatory mediators from keratinocytes. Furthermore, viable L. salivarius SGL 19 and L. fermentum SGL 10 had anti-pathogenic effects against S. aureus and S. pyogenes, while L. brevis SGL 12 and L. paracasei SGL 04 inhibited S. aureus and S. pyogenes, respectively. The tested lactobacilli lysates also induced specific proteome modulation of the exposed keratinocytes, involving dysregulation of proteins (such as interleukin enhancer-binding factor 2 and ATP-dependent RNA helicase) and pathways (such as cytokine, NF-kB, Hedgehog, and RUNX signaling) associated with their specific wound healing and anti-inflammatory effects. This study indicates the different potential of selected lactobacilli, suggesting that they may be successfully used in the future together with conventional therapies to bring relief from skin disorders.

Biochemical investigations were carried out on the embalmed head of Nebiri (Museo Egizio, Turin; S-5109)—an 18th Dynasty Ancient Egyptian dignitary—and on the canopic jar containing his lungs (Museo Egizio, Turin; S. 5111/02) with the aim of characterizing the organ’s (lung) specific paleo-proteins and of identifying the compounds used in his embalming “recipe”. The application of a functionalized film method allowed us to perform a non-invasive sampling. Paleo-proteomics confirmed the presence of lung tissue-specific proteins (organ specific) as well as the presence of proteins linked to severe inflammation. Paleoproteomics and paleometabolomics further allowed the identification of the main components of Nebiri’s embalming recipe: animal fats and glue, balms, essential oils, aromatic plants, heated Pistacia, and coniferous resins. Both the use of Pistacia and coniferous resins in an early 18th Dynasty individual confirm Nebiri’s high social status. The technique applied offers a targeted approach to the chemical characterization of human tissues, embalming compounds, and organic materials layering in pottery. The ability of the functionalized film method to harvest all types of compounds, from macromolecules (i.e., proteins) to small molecules (i.e., organic acids) opens a new path in the study of ancient material culture; furthermore, it allows to perform untargeted analysis, which is necessary when no a priori information is available.

Corticosteroids such as Dexamethasone (DEX) are commonly licensed for therapy in meat animals due to their known pharmacological properties. However, their misuse aimed to achieve anabolic effects is often found by National Residues Control Plans. The setup of a complementary “biomarker based” methods to unveil such illicit practices is encouraged by current European legislation. In this study, the combined use of molecular and histological quantitative techniques was applied on formalin fixed paraffin embedded (FFPE) muscle samples to assess the effects of illicit DEX treatment on veal calves. A PCR array, including 28 transcriptional biomarkers related to DEX exposure, was combined with a histochemical analysis of muscle fiber. An analysis based on unsupervised (PCA) and supervised (PLS-DA and Kohonen’s SOM) methods, was applied in order to define multivariate models able to classify animals suspected of illicit treatment by DEX. According to the conventional univariate approach, a not-significant reduction in type I fibres was recorded in the DEX-treated group, and only 12 out of 28 targeted genes maintained their expected differential expression, confirming the technical limitations of a quantitative analysis on FFPE samples. However, the multivariate models developed highlighted the possibility to establish complementary screening strategies, particularly when based on transcriptional biomarkers characterised by low expression profiles.

Bacterial endophytes were isolated from nodules of pea and fava bean. The strains were identified and characterized for plant beneficial activities (phosphate solubilization, synthesis of indole acetic acid and siderophores) and salt tolerance. Based on these data, four strains of Rahnella aquatilis and three strains of Serratia plymuthica were selected. To shed light on the mechanisms underlying salt tolerance, the proteome of the two most performant strains (Ra4 and Sp2) grown in the presence or not of salt was characterized. The number of proteins expressed by the endophytes was higher in the presence of salt. The modulated proteome consisted of 302 (100 up-regulated, 202 down-regulated) and 323 (206 up-regulated, 117 down-regulated) proteins in Ra4 and Sp2, respectively. Overall, proteins involved in abiotic stress responses were up-regulated, while those involved in metabolism and flagellum structure were down-regulated. The main up-regulated proteins in Sp2 were thiol: disulfide interchange protein DsbA, required for the sulfur binding formation in periplasmic proteins, while in Ra4 corresponded to the soluble fraction of ABC transporters, having a role in compatible solute uptake. Our results demonstrated a conserved response to salt stress in the two taxonomically related species.

Knowledge of the host response to the novel coronavirus SARS-CoV-2 remains limited, hindering the understanding of COVID-19 pathogenesis and the development of therapeutic strategies. During the course of a viral infection, host cells release exosomes and other extracellular vesicles carrying viral and host components that can modulate the immune response. The present study used a shotgun proteomic approach to map the host circulating exosomes’ response to SARS-CoV-2 infection. We investigated how SARS-CoV-2 infection modulates exosome content, exosomes’ involvement in disease progression, and the potential use of plasma exosomes as biomarkers of disease severity. A proteomic analysis of patient-derived exosomes identified several molecules involved in the immune response, inflammation, and activation of the coagulation and complement pathways, which are the main mechanisms of COVID-19–associated tissue damage and multiple organ dysfunctions. In addition, several potential biomarkers—such as fibrinogen, fibronectin, complement C1r subcomponent and serum amyloid P-component—were shown to have a diagnostic feature presenting an area under the curve (AUC) of almost 1. Proteins correlating with disease severity were also detected. Moreover, for the first time, we identified the presence of SARS-CoV-2 RNA in the exosomal cargo, which suggests that the virus might use the endocytosis route to spread infection. Our findings indicate circulating exosomes’ significant contribution to several processes—such as inflammation, coagulation, and immunomodulation—during SARS-CoV-2 infection. The study’s data are available via ProteomeXchange with the identifier PXD021144.

The study of the cancer secretome is gaining even more importance in cancers such as pancreatic ductal adenocarcinoma (PDAC), whose lack of recognizable symptoms and early detection assays make this type of cancer highly lethal. The wild-type p53 protein, frequently mutated in PDAC, prevents tumorigenesis by regulating a plethora of signaling pathways. The importance of the p53 tumor suppressive activity is not only primarily involved within cells to limit tumor cell proliferation but also in the extracellular space. Thus, loss of p53 has a profound impact on the secretome composition of cancer cells and marks the transition to invasiveness. Here, we demonstrate the tumor suppressive role of wild-type p53 on cancer cell secretome, showing the anti-proliferative, apoptotic and chemosensitivity effects of wild-type p53 driven conditioned medium. By using high-resolution SWATH-MS technology, we characterized the secretomes of p53-deficient and p53-expressing PDAC cells. We found a great number of secreted proteins that have known roles in cancer-related processes, 30 of which showed enhanced and 17 reduced secretion in response to p53 silencing. These results are important to advance our understanding on the link between wt-p53 and cancer microenvironment. In conclusion, this approach may detect a secreted signature specifically driven by wild-type p53 in PDAC.

Prion diseases arise from the conformational conversion of the cellular prion protein (PrPC) into a self-replicating prion isoform (PrPSc). Although this process has been studied mostly in neurons, a growing body of evidence suggests that astrocytes express PrPC and are able to replicate and accumulate PrPSc. Currently, prion diseases remain incurable, while downregulation of PrPC represents the most promising therapy due to the reduction of the substrate for prion conversion. Here we show that the astrocyte-specific genetic ablation or pharmacological inhibition of the calcium-activated phosphatase calcineurin (CaN) reduces PrPC expression in astrocytes. Immunocytochemical analysis of cultured CaN-KO astrocytes and isolation of synaptosomal compartments from the hippocampi of astrocyte-specific CaN-KO (ACN-KO) mice suggest that PrPC is downregulated both in vitro and in vivo. The downregulation occurs without affecting the glycosylation of PrPC and without alteration of its proteasomal or lysosomal degradation. Direct assessment of the protein synthesis rate and shotgun mass spectrometry proteomics analysis suggest that the reduction of PrPC is related to the impairment of global protein synthesis in CaN-KO astrocytes. When WT-PrP and PrP-D177N, a mouse homologue of a human mutation associated with the inherited prion disease fatal familial insomnia, were expressed in astrocytes, CaN-KO astrocytes showed an aberrant localization of both WT-PrP and PrP-D177N variants with predominant localization to the Golgi apparatus, suggesting that ablation of CaN affects both WT and mutant PrP proteins. These results provide new mechanistic details in relation to the regulation of PrP expression in astrocytes, suggesting the therapeutic potential of astroglial cells.

The increasing use of pharmaceuticals, their presence in the aquatic environment, and the associated toxic effects, have raised concerns in recent years. In this work, a new multi-residue analytical method was developed and validated for the determination of 10 pharmaceuticals in wastewaters using online solid-phase extraction (online SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The compounds included in the method were antineoplastics (cabazitaxel, docetaxel, doxorubicin, etoposide, irinotecan, methotrexate, paclitaxel, and topotecan), renin inhibitors (aliskiren), and antidepressants (maprotiline). The method was developed through several experiments on four online SPE cartridges, three reversed phase chromatography columns, and four combinations of mobile phase components. Under optimal conditions, very low limits of detection (LODs) of 1.30 to 10.6 ng L−1 were obtained. The method was repeatable, with relative standard deviations (RSD, %) for intraday and interday precisions ranged from 1.6 to 7.8 and from 3.3 to 13.2, respectively. Recovery values ranged from 78.4 to 111.4%, indicating the reproducibility of the method. Matrix effects were mainly presented as signal suppression, with topotecan and doxorubicin being the two most affected compounds (31.0% signal suppression). The proposed method was successfully applied to hospital effluents, detecting methotrexate (4.7–9.3 ng L−1) and maprotiline (11.2–23.1 ng L−1). Due to the shorter overall run time of 15 min, including sample preparation, and reduced sample volume (0.9 mL), this on-line SPE-LC-MS/MS method was extremely convenient and efficient in comparison to the classical off-line SPE method. The proposed method was also highly sensitive and can be used for ultratrace quantification of the studied pharmaceuticals in wastewaters, providing useful data for effective environmental monitoring.

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality among adults in developed countries. The discovery of the most common genetic alterations as well as the development of organoid models of pancreatic cancer have provided insight into the fundamental pathways driving tumor progression from a normal cell to non-invasive precursor lesion and finally to widely metastatic disease, offering new opportunities for identifying the key driver of cancer evolution. Obesity is one of the most serious public health challenges of the 21st century. Several epidemiological studies have shown the positive association between obesity and cancer-related morbidity/mortality, as well as poorer prognosis and treatment outcome. Despite strong evidence indicates a link between obesity and cancer incidence, the molecular basis of the initiating events remains largely elusive. This is mainly due to the lack of an accurate and reliable model of pancreatic carcinogenesis that mimics human obesity-associated PDAC, making data interpretation difficult and often confusing. Here we propose a feasible and manageable organoid-based preclinical tool to study the effects of obesity on pancreatic carcinogenesis. Therefore, we tracked the effects of obesity on the natural evolution of PDAC in a genetically defined transplantable model of the syngeneic murine pancreatic preneoplastic lesion (mP) and tumor (mT) derived-organoids that recapitulates the progression of human disease from early preinvasive lesions to metastatic disease. Our results suggest that organoid-derived transplant in obese mice represents a suitable system to study early steps of pancreatic carcinogenesis and supports the hypothesis that inflammation induced by obesity stimulates tumor progression and metastatization during pancreatic carcinogenesis.

Food authentication is very important to protect consumers, sellers, and producers from fraud. Although several methods have been developed using a wide range of analytical techniques, most of them require sample destruction and do not allow in situ sampling or analysis, nor reliable quantification of hundreds of molecules at the same time. To overcome these limitations, we have developed and validated a new noninvasive analytical workflow for food authentication. The method uses a functionalized strip to adsorb small molecules from the surface of the food product, followed by gas chromatography–mass spectrometry analysis of the desorbed analytes. We validated the method and applied it to the classification of five different apple varieties. Molecular concentrations obtained from the analysis of 44 apples were used to identify markers for apple cultivars or, in combination with machine learning techniques, to perform cultivar classification. The overall reproducibility of the method was very good, showing a good coefficient of variation for both targeted and untargeted analysis. The approach was able to correctly classify all samples. In addition, the method was also used to detect pesticides and the following molecules were found in almost all samples: chlorpyrifos-methyl, deltamethrin, and malathion. The proposed approach not only showed very good analytical performance, but also proved to be suitable for noninvasive food authentication and pesticide residue analysis.

The excessive consumption of antibiotics in clinical, veterinary and agricultural fields has resulted in tremendous flow of antibiotics into the environment. This has led to enormous selective pressures driving the evolution of antimicrobial resistance genes in pathogenic and commensal bacteria. In this context, the World Health Organization (WHO) has promoted research aiming to develop medical features using natural products that are often competitive with synthetic drugs in clinical performance. Fungi are considered an important source of bioactive molecules, often effective against other fungi and/or bacteria, and thus are potential candidates in the search of new antibiotics. Fruiting bodies of sixteen different fungal species of Basidiomycota were collected in the Italian Alps. The identification of fungal species was performed through Internal Transcribed Spacer (ITS) sequencing. Most species belong to genera Cortinarius, Mycena and Ramaria, whose metabolite contents has been scarcely investigated so far. The crude extracts obtained from the above mushrooms were tested for their inhibition activity against five human pathogens: Candida albicans ATCC 14053, C. glabrata ATCC 15126, Staphylococcus aureus NCTC 6571, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC 13883. Twelve crude extracts showed activity against P. aeruginosa ATCC 27853. Highest activity was shown by some Cortinarius species, as C. nanceiensis.

The aim of this study was the identification of specific proteomic profiles, related to a restored cystic fibrosis transmembrane conductance regulator (CFTR) activity in cystic fibrosis (CF) leukocytes before and after ex vivo treatment with the potentiator VX770. We used leukocytes, isolated from CF patients carrying residual function mutations and eligible for Ivacaftor therapy, and performed CFTR activity together with proteomic analyses through micro-LC–MS. Bioinformatic analyses of the results obtained revealed the downregulation of proteins belonging to the leukocyte transendothelial migration and regulation of actin cytoskeleton pathways when CFTR activity was rescued by VX770 treatment. In particular, we focused our attention on matrix metalloproteinase 9 (MMP9), because the high expression of this protease potentially contributes to parenchyma lung destruction and dysfunction in CF. Thus, the downregulation of MMP9 could represent one of the possible positive effects of VX770 in decreasing the disease progression, and a potential biomarker for the prediction of the efficacy of therapies targeting the defect of Cl− transport in CF.

Background Silkworm pupae is the main by-product of the sericulture industry with an interesting nutritional profile, especially in terms of proteins. In consideration of its possible use as a food or food ingredient in Western countries, a comparative proteomic experiment has been performed to investigate the differences of the protein profile of male and female silkworm pupae reared on mulberry leaves or on an artificial diet. Methods The nutritional profile of lyophilized silkworm pupae in terms of dry matter and ash was evaluated according to the AOAC procedures, the total nitrogen content was determined by a nitrogen analyzer and the silkworm pupae gross energy value was measured using an adiabatic calorimetric bomb. The comparative proteomic analysis was performed on male and female silkworm pupae reared on mulberry leaves or on the artificial diet. Proteins were separated by two-dimensional electrophoresis and, after a multivariate statistical analysis, the differentially expressed proteins were identified by LC-MS/MS. Results The comparative proteomic approach highlighted 47 silkworm pupae proteins differentially expressed comparing diet and gender. PCA analysis showed that seven proteins were more effective in discriminating the sex and five were more effective in discriminating the diet type. In spite of the above-mentioned differences in the silkworm pupae protein profile, no strong alteration of the pupa physiological traits have been demonstrated, suggesting a general silkworm pupae flexibility to adapt to a well-balanced artificial diet. Differences in lipid transport and metabolism were found among the experimental groups, that might have a relevant effect on the timing and on hormone secretion. This aspect may also affect silk production, as univoltine strains are the most productive. The proteomic data provided in this work, may offer a contribution in understanding also the influence of gender and farming strategy on the allergen profile of Bombyx mori, when used as food or as a food ingredient. Female silkworm pupae reared on mulberry leaves seemed to contain lower levels of known allergens than those reared in the other experimental conditions; these findings will have to be taken into account when farming B. mori for food production purposes. However, our results need to be supported by further characterization of the allergenic potential of B. mori.

Mesenchymal stromal cells (MSCs) are adult, multipotent cells of mesodermal origin representing the progenitors of all stromal tissues. MSCs possess significant and broad immunomodulatory functions affecting both adaptive and innate immune responses once MSCs are primed by the inflammatory microenvironment. Recently, the role of extracellular vesicles (EVs) in mediating the therapeutic effects of MSCs has been recognized. Nevertheless, the molecular mechanisms responsible for the immunomodulatory properties of MSC-derived EVs (MSC-EVs) are still poorly characterized. Therefore, we carried out a molecular characterization of MSC-EV content by high-throughput approaches. We analyzed miRNA and protein expression profile in cellular and vesicular compartments both in normal and inflammatory conditions. We found several proteins and miRNAs involved in immunological processes, such as MOES, LG3BP, PTX3, and S10A6 proteins, miR-155-5p, and miR-497-5p. Different in silico approaches were also performed to correlate miRNA and protein expression profile and then to evaluate the putative molecules or pathways involved in immunoregulatory properties mediated by MSC-EVs. PI3K-AKT signaling pathway and the regulation of actin cytoskeleton were identified and functionally validated in vitro as key mediators of MSC/B cell communication mediated by MSC-EVs. In conclusion, we identified different molecules and pathways responsible for immunoregulatory properties mediated by MSC-EVs, thus identifying novel therapeutic targets as safer and more useful alternatives to cell or EV-based therapeutic approaches.

Arbuscular mycorrhizal fungi (AMF) colonize the roots of most terrestrial plant species, improving plant growth, nutrient uptake and biotic/abiotic stress resistance and tolerance. Similarly, plant growth promoting bacteria (PGPB) enhance plant fitness and production. In this study, three different AMF (Funneliformis mosseae, Septoglomus viscosum, and Rhizophagus irregularis) were used in combination with three different strains of Pseudomonas sp. (19Fv1t, 5Vm1K and Pf4) to inoculate plantlets of Fragaria × ananassa var. Eliana F1. The effects of the different fungus/bacterium combinations were assessed on plant growth parameters, fruit production and quality, including health-promoting compounds. Inoculated and uninoculated plants were maintained in a greenhouse for 4 months and irrigated with a nutrient solution at two different phosphate levels. The number of flowers and fruits were recorded weekly. At harvest, fresh and dry weights of roots and shoots, mycorrhizal colonization and concentration of leaf photosynthetic pigments were measured in each plant. The following fruit parameters were recorded: pH, titratable acids, concentration of organic acids, soluble sugars, ascorbic acids, and anthocyanidins; volatile and elemental composition were also evaluated. Data were statistically analyzed by ANOVA and PCA/PCA-DA. Mycorrhizal colonization was higher in plants inoculated with R. irregularis, followed by F. mosseae and S. viscosum. In general, AMF mostly affected the parameters associated with the vegetative portion of the plant, while PGPB were especially relevant for fruit yield and quality. The plant physiological status was differentially affected by inoculations, resulting in enhanced root and shoot biomass. Inoculation with Pf4 bacterial strain increased flower and fruit production per plant and malic acid content in fruits, while decreased the pH value, regardless of the used fungus. Inoculations affected fruit nutritional quality, increasing sugar and anthocyanin concentrations, and modulated pH, malic acid, volatile compounds and elements. In the present study, we show for the first time that strawberry fruit concentration of some elements and/or volatiles can be affected by the presence of specific beneficial soil microorganisms. In addition, our results indicated that it is possible to select the best plant-microorganism combination for field applications, and improving fruit production and quality, also in terms of health promoting properties.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread to nearly every continent, registering over 1,250,000 deaths worldwide. The effects of SARS-CoV-2 on host targets remains largely limited, hampering our understanding of Coronavirus Disease 2019 (COVID-19) pathogenesis and the development of therapeutic strategies. The present study used a comprehensive untargeted metabolomic and lipidomic approach to capture the host response to SARS-CoV-2 infection. We found that several circulating lipids acted as potential biomarkers, such as phosphatidylcholine 14:0_22:6 (area under the curve (AUC) = 0.96), phosphatidylcholine 16:1_22:6 (AUC = 0.97), and phosphatidylethanolamine 18:1_20:4 (AUC = 0.94). Furthermore, triglycerides and free fatty acids, especially arachidonic acid (AUC = 0.99) and oleic acid (AUC = 0.98), were well correlated to the severity of the disease. An untargeted analysis of non-critical COVID-19 patients identified a strong alteration of lipids and a perturbation of phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, aminoacyl-tRNA degradation, arachidonic acid metabolism, and the tricarboxylic acid (TCA) cycle. The severity of the disease was characterized by the activation of gluconeogenesis and the metabolism of porphyrins, which play a crucial role in the progress of the infection. In addition, our study provided further evidence for considering phospholipase A2 (PLA2) activity as a potential key factor in the pathogenesis of COVID-19 and a possible therapeutic target. To date, the present study provides the largest untargeted metabolomics and lipidomics analysis of plasma from COVID-19 patients and control groups, identifying new mechanisms associated with the host response to COVID-19, potential plasma biomarkers, and therapeutic targets.

Oleander is a spontaneous shrub widely occurring in Mediterranean regions. Poisoning is sporadically reported in livestock, mainly due to the ingestion of leaves containing toxic cardiac glycosides (primarily oleandrin). In this study, 50 lactating Fleckvieh cows were affected after being offered a diet containing dry oleander pruning wastes accidentally mixed with fodder. Clinical examination, electrocardiogram, and blood sampling were conducted. Dead animals were necropsied, and heart, liver, kidney, spleen, and intestine were submitted to histological investigation. Oleandrin detection was performed through ultra-high performance liquid chromatography-tandem mass spectrometry in blood, serum, liver, heart, milk, and cheese samples. Severe depression, anorexia, ruminal atony, diarrhea, serous nasal discharge, tachycardia, and irregular heartbeat were the most common clinical signs. The first animal died within 48 h, and a total of 13 cows died in 4 days. Disseminated hyperemia and hemorrhages, multifocal coagulative necrosis of the cardiac muscle fibers, and severe and diffuse enteritis were suggestive of oleander poisoning. The diagnosis was confirmed by the presence of oleandrin in serum, liver, heart, milk, and cheese. Our results confirm the high toxicity of oleander in cattle and report for the first time the transfer into milk and dairy products, suggesting a potential risk for the consumers.

Background: Cellulite is a condition in which the skin has a dimpled lumpy appearance. The main causes of cellulite development, studied until now, comprehends modified sensitivity to estrogens, the damage of microvasculature present among dermis and hypodermis. The differences of adipose tissue architecture between male and female might make female more susceptible to cellulite. Adipose tissue is seen to be deeply modified during cellulite development. Our study tried to understand the overall features within and surrounding cellulite to apply the best therapeutic approach. Methods: Samples of gluteal femoral area were collected from cadavers and women who had undergone surgical treatment to remove orange peel characteristics on the skin. Samples from cadavers were employed for an accurate study of cellulite using magnetic resonance imaging at 7 Tesla and for light microscopy. Specimens from patients were employed for the proteomic analysis, which was performed using high resolution mass spectroscopy (MS). Stromal vascular fraction (SVF) was obtained from the samples, which was studied using MS and flow cytometry. Results: light and electron microscopy of the cellulite affected area showed a morphology completely different from the other usual adipose depots. In cellulite affected tissues, sweat glands associated with adipocytes were found. In particular, there were vesicles in the extracellular matrix, indicating a crosstalk between the two different components. Proteomic analysis showed that adipose tissue affected by cellulite is characterized by high degree of oxidative stress and by remodeling phenomena. Conclusions: The novel aspects of this study are the peculiar morphology of adipose tissue affected by cellulite, which could influence the surgical procedures finalized to the reduction of dimpling, based on the collagen fibers cutting. The second novel aspect is the role played by the mesenchymal stem cells isolated from stromal vascular fraction of adipose tissue affected by cellulite.

Head space (HS) solid phase microextraction (SPME) followed by gas chromatography with mass spectrometry detection (GC-MS) is the most widespread technique to study the volatile profile of honey samples. In this paper, the experimental SPME conditions were optimized by a multivariate strategy. Both sensitivity and repeatability were optimized by experimental design techniques considering three factors: extraction temperature (from 50°C to 70°C), time of exposition of the fiber (from 20 min to 60 min), and amount of salt added (from 0 to 27.50%). Each experiment was evaluated by Principal Component Analysis (PCA) that allows to take into consideration all the analytes at the same time, preserving the information about their different characteristics. Optimal extraction conditions were identified independently for signal intensity (extraction temperature: 70°C; extraction time: 60 min; salt percentage: 27.50% w/w) and repeatability (extraction temperature: 50°C; extraction time: 60 min; salt percentage: 27.50% w/w) and a final global compromise (extraction temperature: 70°C; extraction time: 60 min; salt percentage: 27.50% w/w) was also reached. Considerations about the choice of the best internal standards were also drawn. The whole optimized procedure was than applied to the analysis of a multiflower honey sample and more than 100 compounds were identified.

In this paper we propose a reliable surface imaging method for the non-invasive detection of morphological changes in paintings. Usually, the evaluation and quantification of changes and defects results mostly from an optical and subjective assessment, through the comparison of the previous and subsequent state of conservation and by means of condition reports. Using quantitative Reflectance Transformation Imaging (RTI) we obtain detailed information on the geometry and morphology of the painting surface with a fast, precise and non-invasive method. Accurate and quantitative measurements of deterioration were acquired after the painting experienced artificial damage. Morphological changes were documented using normal vector images while the intensity map succeeded in highlighting, quantifying and describing the physical changes. We estimate that the technique can detect a morphological damage slightly smaller than 0.3 mm, which would be difficult to detect with the eye, considering the painting size. This non-invasive tool could be very useful, for example, to examine paintings and artwork before they travel on loan or during a restoration. The method lends itself to automated analysis of large images and datasets. Quantitative RTI thus eases the transition of extending human vision into the realm of measuring change over time.

Stem cell therapy represents a promising approach in the treatment of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). The beneficial effect of stem cells is exerted by paracrine mediators, as exosomes, suggesting a possible potential use of these extracellular vesicles as non-cell based therapy. We demonstrated that exosomes isolated from adipose stem cells (ASC) display a neuroprotective role in an in vitro model of ALS. Moreover, the internalization of ASC-exosomes by the cells was shown and the molecules and the mechanisms by which exosomes could exert their beneficial effect were addressed. We performed for the first time a comprehensive proteomic analysis of exosomes derived from murine ASC. We identified a total of 189 proteins and the shotgun proteomics analysis revealed that the exosomal proteins are mainly involved in cell adhesion and negative regulation of the apoptotic process. We correlated the protein content to the anti-apoptotic effect of exosomes observing a downregulation of pro-apoptotic proteins Bax and cleaved caspase-3 and upregulation of anti-apoptotic protein Bcl-2 α, in an in vitro model of ALS after cell treatment with exosomes. Overall, this study shows the neuroprotective effect of ASC-exosomes after their internalization and their global protein profile, that could be useful to understand how exosomes act, demonstrating that they can be employed as therapy in neurodegenerative diseases.

Abdominal aortic aneurysm (AAA) is a focal dilatation of the aorta, caused by both genetic and environmental factors. Although vascular endothelium plays a key role in AAA progression, the biological mechanisms underlying the mechanical stress involvement are only partially understood. In this study, we developed an in vitro model to characterize the role of mechanical stress as a potential trigger of endothelial deregulation in terms of inflammatory response bridging between endothelial cells (ECs), inflammatory cells, and matrix remodeling. In AAA patients, data revealed different degrees of calcification, inversely correlated with wall stretching and also with inflammation and extracellular matrix degradation. In order to study the role of mechanical stimulation, endothelial cell line (EA.hy926) has been cultured in healthy (10% strain) and pathological (5% strain) dynamic conditions using a bioreactor. In presence of tumor necrosis factor alpha (TNF-α), high levels of matrix metalloproteinase-9 (MMP-9) expression and inflammation are obtained, while mechanical stimulation significantly counteracts the TNF-α effects. Moreover, physiological deformation also plays a significant role in the control of the oxidative stress. Overall our findings indicate that, due to wall calcification, in AAA there is a significant change in terms of decreased wall stretching.

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy.

Mixtures of chemicals can have additive, synergistic or antagonistic interactions. We investigated the effects of the exposure to nickel, the organophosphate insecticide chlorpyrifos at effect concentrations (EC) of 25% and 50% and their binary mixture (Ec25 + EC25) on Dictyostelium discoideum amoebae based on lysosomal membrane stability (LMS). We treated D. discoideum with these compounds under controlled laboratory conditions and evaluated the changes in protein levels using a two-dimensional gel electrophoresis (2DE) proteomic approach. Nickel treatment at EC25 induced changes in 14 protein spots, 12 of which were down-regulated. Treatment with nickel at EC50 resulted in changes in 15 spots, 10 of which were down-regulated. Treatment with chlorpyrifos at EC25 induced changes in six spots, all of which were down-regulated; treatment with chlorpyrifos at EC50 induced changes in 13 spots, five of which were down-regulated. The mixture corresponding to EC25 of each compound induced changes in 19 spots, 13 of which were down-regulated. The data together reveal that a different protein expression signature exists for each treatment, and that only a few proteins are modulated in multiple different treatments. For a simple binary mixture, the proteomic response does not allow for the identification of each toxicant. The protein spots that showed significant differences were identified by mass spectrometry, which revealed modulations of proteins involved in metal detoxification, stress adaptation, the oxidative stress response and other cellular processes.

Today the long-term conservation of cultural heritage is a big challenge: often the artworks were subjected to unknown interventions, which eventually were found to be harmful. The noninvasive investigation of the conservation treatments to which they were subjected to is a crucial step in order to undertake the best conservation strategies. We describe here the preliminary results on a quick and direct method for the nondestructive identification of the various interventions of parchment by means of direct analysis in real time (DART) ionization and high-resolution time-of-flight mass spectrometry and chemometrics. The method has been developed for the noninvasive analysis of the Dead Sea Scrolls, one of the most important archaeological discoveries of the 20th century. In this study castor oil and glycerol parchment treatments, prepared on new parchment specimens, were investigated in order to evaluate two different types of operations. The method was able to identify both treatments. In order to investigate the effect of the ion source temperature on the mass spectra, the DART-MS analysis was also carried out at several temperatures. Due to the high sensitivity, simplicity, and no sample preparation requirement, the proposed analytical methodology could help conservators in the challenging analysis of unknown treatments in cultural heritage.