Proteome characterization of a human urothelial cell line resistant to the bladder carcinogen 4-aminobiphenyl

<p>Abstract</p> <p>Background</p> <p>The aromatic amine 4-aminobiphenyl (4-ABP) is an environmental and occupational contaminant known to be a major etiological agent of human bladder cancer. 4-ABP metabolites are able to form DNA adducts that may induce mutations and initiate bladder carcinogenesis. Cells exposed to 4-ABP may develop resistance to the carcinogen. The aim of the present study was to detect and identify proteins whose expression is altered in the bladder carcinoma RT112 sub-lines selected for acquired resistance to 4-ABP, in order to disentangle the mechanisms.</p> <p>Results</p> <p>Differential proteome analysis of cell lysates showed an overall perturbation in cell metabolism and energy pathways in the 4-ABP-resistant human urothelial clones, with over-expression of membrane trafficking proteins such as annexin 2. The resistant clones had altered expression of many proteins linked directly (<it>i.e</it>. lamin A/C, programmed cell death 6 interacting protein) or indirectly (<it>i.e</it>. 94 kDa glucose-regulated protein, fatty acid-binding protein) to decreased apoptosis, suggesting that resistance to 4-ABP might be associated with low apoptotic activity.</p> <p>Conclusion</p> <p>Our data provide evidence that deregulation of apoptosis and membrane trafficking proteins might be strongly implicated in the selection of carcinogen resistant cells. Some of these proteins might have potential as biomarkers of resistance and cancer risk.</p

Cocaine in surface waters: a new evidence-based tool to monitor community drug abuse

BACKGROUND: Cocaine use seems to be increasing in some urban areas worldwide, but it is not straightforward to determine the real extent of this phenomenon. Trends in drug abuse are currently estimated indirectly, mainly by large-scale social, medical, and crime statistics that may be biased or too generic. We thus tested a more direct approach based on 'field' evidence of cocaine use by the general population. METHODS: Cocaine and its main urinary metabolite (benzoylecgonine, BE) were measured by mass spectrometry in water samples collected from the River Po and urban waste water treatment plants of medium-size Italian cities. Drug concentration, water flow rate, and population at each site were used to estimate local cocaine consumption. RESULTS: We showed that cocaine and BE are present, and measurable, in surface waters of populated areas. The largest Italian river, the Po, with a five-million people catchment basin, steadily carried the equivalent of about 4 kg cocaine per day. This would imply an average daily use of at least 27 ± 5 doses (100 mg each) for every 1000 young adults, an estimate that greatly exceeds official national figures. Data from waste water treatment plants serving medium-size Italian cities were consistent with this figure. CONCLUSION: This paper shows for the first time that an illicit drug, cocaine, is present in the aquatic environment, namely untreated urban waste water and a major river. We used environmental cocaine levels for estimating collective consumption of the drug, an approach with the unique potential ability to monitor local drug abuse trends in real time, while preserving the anonymity of individuals. The method tested here – in principle extendable to other drugs of abuse – might be further refined to become a standardized, objective tool for monitoring drug abuse

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5-11 December, to 17.5% (25/143 samples) in the week 12-18, to 65.9% (89/135 samples) in the week 19-25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased from one in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons. In conclusion, we designed an RT-qPCR assay capable to detect the Omicron variant, which can be successfully used for the purpose of wastewater-based epidemiology. We also described the history of the introduction and diffusion of the Omicron variant in the Italian population and territory, confirming the effectiveness of sewage monitoring as a powerful surveillance tool

The rapid spread of SARS-COV-2 Omicron variant in Italy reflected early through wastewater surveillance

The SARS-CoV-2 Omicron variant emerged in South Africa in November 2021, and has later been identified worldwide, raising serious concerns. A real-time RT-PCR assay was designed for the rapid screening of the Omicron variant, targeting characteristic mutations of the spike gene. The assay was used to test 737 sewage samples collected throughout Italy (19/21 Regions) between 11 November and 25 December 2021, with the aim of assessing the spread of the Omicron variant in the country. Positive samples were also tested with a real-time RT-PCR developed by the European Commission, Joint Research Centre (JRC), and through nested RT-PCR followed by Sanger sequencing. Overall, 115 samples tested positive for Omicron SARS-CoV-2 variant. The first occurrence was detected on 7 December, in Veneto, North Italy. Later on, the variant spread extremely fast in three weeks, with prevalence of positive wastewater samples rising from 1.0% (1/104 samples) in the week 5–11 December, to 17.5% (25/143 samples) in the week 12–18, to 65.9% (89/135 samples) in the week 19–25, in line with the increase in cases of infection with the Omicron variant observed during December in Italy. Similarly, the number of Regions/Autonomous Provinces in which the variant was detected increased fromone in the first week, to 11 in the second, and to 17 in the last one. The presence of the Omicron variant was confirmed by the JRC real-time RT-PCR in 79.1% (91/115) of the positive samples, and by Sanger sequencing in 66% (64/97) of PCR amplicons

Mass spectrometry-based characterisation of the secretome of pancreatic cancer cell lines

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

In-depth glycoproteomic characterization of γ-conglutin by high-resolution accurate mass spectrometry.

The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography-multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man2(Xyl) (Fuc) GlcNAc2, Man3(Xyl) (Fuc) GlcNAc2, GlcNAcMan3(Xyl) (Fuc) GlcNAc2 and GlcNAc 2Man3(Xyl) (Fuc) GlcNAc2. These carry both core β1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit

A maladaptive ER stress response triggers dysfunction in highly active muscles of mice with SELENON loss

open12noThis study was supported by a Telethon (Italy) career award (TDEZ00112T), an ERC Cariplo grant (2014-1856), a Cariplo Biomedical Science (Italy) for young scientist grant (2014-1075) to EZ and a STARS consolidator grant, AFM Telethon (France) grant (21865) to BB.Selenoprotein N (SELENON) is an endoplasmic reticulum (ER) protein whose loss of function leads to human SELENON-related myopathies. SelenoN knockout (KO) mouse limb muscles, however, are protected from the disease, and display no major alterations in muscle histology or contractile properties. Interestingly, we find that the highly active diaphragm muscle shows impaired force production, in line with the human phenotype. In addition, after repeated stimulation with a protocol which induces muscle fatigue, also hind limb muscles show altered relaxation times. Mechanistically, muscle SELENON loss alters activity-dependent calcium handling selectively impinging on the Ca 2+ uptake of the sarcoplasmic reticulum and elicits an ER stress response, including the expression of the maladaptive CHOP-induced ERO1. In SELENON-devoid models, ERO1 shifts ER redox to a more oxidised poise, and further affects Ca 2+ uptake. Importantly, CHOP ablation in SelenoN KO mice completely prevents diaphragm dysfunction, the prolonged limb muscle relaxation after fatigue, and restores Ca 2+ uptake by attenuating the induction of ERO1. These findings suggest that SELENON is part of an ER stress-dependent antioxidant response and that the CHOP/ERO1 branch of the ER stress response is a novel pathogenic mechanism underlying SELENON-related myopathies.openPozzer, Diego; Varone, Ersilia; Chernorudskiy, Alexander; Schiarea, Silvia; Missiroli, Sonia; Giorgi, Carlotta; Pinton, Paolo; Canato, Marta; Germinario, Elena; Nogara, Leonardo; Blaauw, Bert; Zito, EsterPozzer, Diego; Varone, Ersilia; Chernorudskiy, Alexander; Schiarea, Silvia; Missiroli, Sonia; Giorgi, Carlotta; Pinton, Paolo; Canato, Marta; Germinario, Elena; Nogara, Leonardo; Blaauw, Bert; Zito, Este

Representative annotated MS3 spectrum of the Pept+HexNAc product ion (m/z 1411, z=2).

<p>The Pept+HexNAc product ion derives from the MS2 fragmentation of the MH³⁺ ion (m/z 1210) of Pept_127-145 glycoform B. The corresponding MS3 spectra for the other glycoforms were identical. The fragment ions with a <i>square</i> symbol are those with the HexNAc residue still in place, while those with a <i>star</i> have undergone the neutral loss of the HexNAc residue. HexNAc was annotated here at N₁₃₁ based on evidence not related to this MS3 spectrum (see main text). Annotated fragments are within 0.6 Da from the theoretical value.</p

Schematic overview of the glycoproteomic workflow.

<p>Combinations of the following procedures were used: 1) reducing SDS-PAGE to isolate the N-glycosylated large subunit, 2) γ-conglutin proteolytic digestion in-gel (with trypsin) or in-solution (with endopeptidase GluC (V8) followed by trypsin) to generate different mixtures of peptides and N-glycopeptides; 3) N-deglycosylation of the digests with PNGase A or PNGase F to identify the N-glycosylation site, and assess the presence/absence of “core” α1-3 fucose. The digests (with or without N-deglycosylation) were then analyzed by LC–Orbitrap MS, with MS survey scans followed by data-dependent ITMS2 or targeted MSn. Mass spectral data analysis included the use of: 1) automated charge-deconvolution of high resolution-high mass accuracy spectra, and isotope envelope simulation; 2) bioinformatic tools for <i>in </i><i>silico</i> glycoform structure prediction (GlycoMod and GlycoSuiteDB), and database search (Mascot) for sequence identification of non-glycosylated or enzymatically deglycosylated peptides; (3) manual inspection of MS2 and MS3 spectra of glycopeptides for sequence annotation of their monosaccharide and amino acid components, respectively.</p

γ-Conglutin glycoform profile obtained by LC-Orbitrap MS analysis of the in-gel tryptic digest of the large subunit.

<p>The MS spectrum (averaged over the elution time of the A-E and B′-E′ glycoforms of Pept_127-165, Pept_111-165, and the non-glycosylated Pept_127-165) was charge-deconvoluted and deisotoped to show the monoisotopic MH⁺ ions.</p