Secretome protein signature of human pancreatic cancer stem-like cells

Emerging research has demonstrated that pancreatic ductal adenocarcinoma (PDAC) contains a sub-population of cancer stem cells (CSCs) characterized by self-renewal, anchorage-independent-growth, long-term proliferation and chemoresistance. The secretome analysis of pancreatic CSCs has not yet been performed, although it may provide insight into tumour/microenvironment interactions and intracellular processes, as well as to identify potential biomarkers. To characterize the secreted proteins of pancreatic CSCs, we performed an iTRAQ-based proteomic analysis to compare the secretomes of Panc1 cancer stem-like cells (Panc1 CSCs) and parental cell line. A total of 72 proteins were found up-/down-regulated in the conditioned medium of Panc1 CSCs. The pathway analysis revealed modulation of vital physiological pathways including glycolysis, gluconeogenesis and pentose phosphate. Through ELISA immunoassays we analysed the presence of the three proteins most highly secreted by Panc1 CSCs (ceruloplasmin, galectin-3, and MARCKS) in sera of PDAC patient. ROC curve analysis suggests ceruloplasmin as promising marker for patients negative for CA19-9.Overall, our study provides a systemic secretome analysis of pancreatic CSCs revealing a number of secreted proteins which participate in pathological conditions including cancer differentiation, invasion and metastasis. They may serve as a valuable pool of proteins from which biomarkers and therapeutic targets can be identified. Biological significance: The secretome of CSCs is a rich reservoir of biomarkers of cancer progression and molecular therapeutic targets, and thus is a topic of great interest for cancer research. The secretome analysis of pancreatic CSCs has not yet been performed. Recently, our group has demonstrated that Panc-CSCs isolated from parental cell line by using the CSC selective medium, represent a model of great importance to deepen the understanding of the biology of pancreatic adenocarcinoma. To our knowledge, this is the first proteomic study of pancreatic CSC secretome. We performed an iTRAQ-based analysis to compare the secretomes of Panc1 CSCs and Panc1 parental cell line and identified a total of 43 proteins secreted at higher level by pancreatic cancer stem cells. We found modulation of different vital physiological pathways (such as glycolysis and gluconeogenesis, pentose phosphate pathway) and the involvement of CSC secreted proteins (for example 72 kDa type IV collagenase, galectin-3, alpha-actinin-4, and MARCKS) in pathological conditions including cancer differentiation, invasion and metastasis. By ELISA verification we found that MARCKS and ceruloplasmin discriminate between controls and PDAC patients; in addition ROC curve analyses indicate that MARCKS does not have diagnostic accuracy, while ceruloplasmin could be a promising marker only for patients negative for CA19-9.We think that the findings reported in our manuscript advance the understanding of the pathways implicated in tumourigenesis, metastasis and chemoresistance of pancreatic cancer, and also identify a pool of proteins from which novel candidate diagnostic and therapeutic biomarkers could be discovered

Authentication and traceability study on barbera d\u2019asti and nizza docg wines: The role of trace-and ultra-trace elements

Barbera d\u2019Asti\u2014including Barbera d\u2019Asti superiore\u2014and Nizza are two DOCG (Denominazione di Origine Controllata e Garantita) wines produced in Piemonte (Italy) from the Barbera grape variety. Differences among them arise in the production specifications in terms of purity, ageing, and zone of production, in particular with concern to Nizza, which follows the most stringent rules, sells at three times the average price, and is considered to have the highest market value. To guarantee producers and consumers, authentication methods must be developed in order to distinguish among the different wines. As the production zones totally overlap, it is important to verify whether the distinction is possible or not according to metals content, or whether chemical markers more linked to winemaking are needed. In this work, Inductively Coupled Plasma (ICP) elemental analysis and multivariate data analysis are used to study the authentication and traceability of samples from the three designations of 2015 vintage. The results show that, as far as elemental distribution in wine is concerned, work in the cellar, rather than geographic provenance, is crucial for the possibility of distinction

A Fully Automated Online SPE-LC-MS/MS Method for the Determination of 10 Pharmaceuticals in Wastewater Samples

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

Modelling of technical, environmental, and economic evaluations of the effect of the organic loading rate in semi-continuous anaerobic digestion of pre-treated organic fraction municipal solid waste

The study concerned technical feasibility, economic profitability, and carbon footprint (CF) analysis of semicontinuous anaerobic digestion (sAD) of organic fraction of municipal solid waste (OFMSW). The research assessed the pre-treatment effect on sAD by varying organic loading rates (OLR) from 3.38 to 6.75 kgvs/m3d. Three sAD configurations were investigated: hydrodynamic-cavitated (HC-OFMSW), enzymatically pre-treated (EN-OFMSW), and non-pre-treated (AD-OFMSW). Principal Component Analysis and Supervised Kohonen's Self-Organizing Maps combined the experimental, economic, and environmental evaluations. The sAD configurations were grouped predominantly according to the OLR however, within each OLR group the configurations were clustered according to the pre-treatments. The finding highlighted that pre-treatments offset inhibition in sAD of OFMSW due to the OLR increase, being economically profitable and CF negative up to 4.50 kgvs/m3d for EN-OFMSW and to 5.40 kgvs/m3d for HCOFMSW. Whereas sAD-OFMSW remained economically and environmentally viable only up to 3.87 kgvs/m3d. HC-OFMSW reached the highest performance. In detail, for HC-OFMSW the NPV and CF ranged from 17679.30 to 43827.12 euros and from -51.08 to -407.210 kg CO2eq/1 MWh daily produced, by decreasing the OLR from 5.40 to 3.87 kgvs/m3d. These results are fundamental since pre-treatment is usually expensive due to additional energy or chemical requirements

Technical feasibility and modeling of enzymatic pre-treatments of organic fraction of municipal solid waste to improve anaerobic digestion

The study explored the combined effects of enzymatic pre-treatment and anaerobic digestion (AD) on the organic fraction of municipal solid wastes (OFMSW) through experimental and multicriteria decision-making approaches. Five enzymes (UPP2, MPCS, USC4, USE2, and A. niger) and their dosages were studied. AD parameters included two inoculum origins (waste active sludge - WAS - and cow-agricultural sludge - CAS), the substrate: inoculum (SI) ratio, and inoculum incubation time (INOC). Desirability functions were used to optimize the multiple experimental responses simultaneously by converting each of them into values from 0 (unacceptable) to 1 (completely acceptable) and then combining these into a global desirability (D). D highlighted that higher enzyme dosages, INOC, and SI, improved AD performances, with optimal DOSE (at the highest level adopted for each enzyme) and INOC (5–10 d). AD tests with the five enzymes increased CH4 production by 10–13%v/v compared to untreated OFMSW. For UPP2 and MPCS, increasing DOSE boosted the biogas production, while increasing INOC enhanced the CH4 content. MPCS reached the highest efficiency (478. 43 NL CH4/kg VS with CAS, SI = 2:1, INOC = 10 d), followed by UPP2. Furthermore, higher INOC reduced A. niger doses, increasing CH4 production by 9%v/v compared to literature, with 5–10 d INOC (452.86 NL s/kg VS with WAS, SI = 2:1)

Identification of Illicit Conservation Treatments in Fresh Fish by Micro-Raman Spectroscopy and Chemometric Methods

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

Sensitive and accurate determination of 32 PFAS in human serum using online SPE-UHPLC-HRMS

Per- and polyfluoroalkyl substances' (PFAS) extreme persistence has been linked to many adverse effects on human health including increased risk of certain cancers. This study presents the development and validation of a new, highly sensitive method for the quantification of 32 PFAS in human serum using online solid-phase extraction (SPE) coupled with ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Legacy and emerging PFAS were targeted. Main steps of sample pretreatment include protein precipitation (PP), pellet rinsing, centrifugation, preconcentration through solvent evaporation, and online SPE using a weak anion-exchange polymeric sorbent. The PP and pellet-rinsing procedures were optimized through a comprehensive exploration of solvent combinations. Following this, a pretreatment that offers the best compromise for the targeted PFAS was identified using principal component analysis. The method demonstrated excellent linearity (R2 = 0.977-0.997) with limits of quantification ranging from 8.9 to 27 ng/L, 5 to 15 times lower than previous methods. Precision (intraday 2.6-14.0 % and interday 1.3-11.0 % relative standard deviation) and accuracy (recoveries 72.7-106 %) were robust. The method was validated in accordance with ISO/IEC 17025 and successfully applied to five human serum samples, confirming its suitability for high throughput profiling of PFAS in biomonitoring studies. This method is the first to use online SPE for the simultaneous determination of a broad range of PFAS, including ether congeners such as perfluoro(2ethoxyethane) sulfonic acid and Nafion byproduct 2. Furthermore, control charts were employed to assess instrument performance during routine analysis and implement necessary actions