Multiple myeloma (MM) is tightly dependent on inflammatory bone marrow microenvironment. IL-17 producing CD4+ T cells (Th17) sustain MM cells growth and osteoclasts-dependent bone damage. In turn, ...Th17 differentiation relies on inflammatory stimuli. Here, we investigated the role of miR-21 in Th17-mediated MM tumor growth and bone disease. We found that early inhibition of miR-21 in naive T cells (miR-21i-T cells) impaired Th17 differentiation in vitro and abrogated Th17-mediated MM cell proliferation and osteoclasts activity. We validated these findings in NOD/SCID-g-NULL mice, intratibially injected with miR-21i-T cells and MM cells. A Pairwise RNAseq and proteome/phosphoproteome analysis in Th17 cells demonstrated that miR-21 inhibition led to upregulation of STAT-1/-5a-5b, STAT-3 impairment and redirection of Th17 to Th1/Th2 like activated/polarized cells. Our findings disclose the role of miR-21 in pathogenic Th17 activity and open the avenue to the design of miR-21-targeting strategies to counteract microenvironment dependence of MM growth and bone disease.
A method for the determination and quantification of ketosteroid hormones in meat by mass spectrometry, based on the derivatization of the carbonyl moiety of steroids by O-methylhydroxylamine, is ...presented. The quantitative assay is performed by means of multiple-reaction-monitoring (MRM) scan mode and using the corresponding labelled species, obtained by reaction with d ₃-methoxylamine, as internal standard. The accuracy of the method was established by evaluating artificially spiked samples, obtaining values in the range 90–110 %. Recovery tests were performed on blank matrix samples spiked with non-natural steroids including trenbolone and melengestrol acetate. The latter experiment revealed that the yield of the extraction processes was approximately 60 %. Good values of LOQ and LOD were achieved, making this method competitive with current hormone assay methods.
MS‐based proteomics is expanding its role as a routine tool for biological discovery. Nevertheless, the task of accurately and precisely quantifying thousands of analytes in a single experiment ...remains challenging. In this study, the diagnostic accuracy of three popular data‐dependent methods for protein relative quantification (label‐free LF, dimethyl labelling DML and tandem mass tags TMT) has been assessed using a mixed species proteome (three species) and five experimental replicates per condition. Data were produced using a quadrupole‐Orbitrap mass spectrometer and analysed using a single platform (the MaxQuant/Perseus software suite). The whole comparative analysis was repeated three times over a period of 6 months, in order to assess the consistency of the reported findings. As expected, label‐based methods reproducibly provided a lower false positives rate, whereas TMT and LF performed similarly, and significantly better than DML, in terms of proteome coverage using the same instrument time. Although parameters like proteome coverage and precision were consistent in between replicates, other parameters like sensitivity, intended as the capacity of correctly classifying true positives (regulated proteins), were found to be less reproducible, especially at challenging fold‐changes (1.5). Collectively, data suggest that an increased interest in data reproducibility would be desirable in the quantitative proteomics field.
A small library of psoralen carboxylic acids and their corresponding benzenesulfonamide derivatives were designed and synthesized to evaluate their activity and selectivity toward tumor associated ...human carbonic anhydrase (hCA) isoforms IX and XII. Both psoralen acids and sulfonamides exhibited potent inhibition of IX and XII isozymes in the nanomolar concentration range. However, psoralen acids resulted as the most selective in comparison with the corresponding benzenesulfonamide derivatives. Our data indicate that the psoralen scaffold is a promising starting point for the design of highly selective tumor associated hCA inhibitors.
Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin‐fixed and paraffin‐embedded (FFPE) tissues. This adaptation is based on ...the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC‐MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC‐MS/MS in combination with IMS can serve as useful exploratory tools.
This work compares the composition at different temperatures of gaseous phase of bergamot essential oil at equilibrium with the liquid phase. A new GC–MS methodology to determine quantitatively the ...volatile aroma compounds was developed. The adopted methodology involved the direct injection of headspace gas into injection port of GC–MS system and of known amounts of the corresponding authentic volatile compounds. The methodology was validated. This study showed that gaseous phase composition is different from that of the liquid phase at equilibrium with it.
The pathogenesis of impaired healing within pressure ulcers remains poorly characterized and rarely examined. We describe the results of a pilot study that applies matrix‐assisted laser ...desorption/ionization imaging mass spectrometry technology for direct tissue analysis to evaluate proteomic signatures ranging from 2 to 20 kDa and phospholipids from 300–1,200 Da in focal regions within the wound microenvironment. Distinguishing molecular differences were apparent between upper vs. lower regions of ulcers and further contrasted against adjacent dermis and epidermal margins using protein profiles, ion density maps, principal component analysis and significant analysis of microarrays. Several proteins previously uncharacterized in pressure ulcers, the α‐defensins (human neutrophil peptide HNP‐1, ‐2, ‐3), are potential markers indicating whether the wound status is improving or being prolonged in a deleterious, chronic state. Thymosin β4 appears to be a favorable protein marker showing higher relative levels in adjacent dermis and maturing areas of the wound bed. Lipidomic examination revealed the presence of major lipid classes: glycerophosphocholines, glycerophosphoglycerols, glycerophosphoinositols, and triacylglycerols. Our pilot data examined from either a global perspective using proteomic or lipidomic signatures or as individual distributions reveal that imaging mass spectrometry technology can be effectively used for discovery and spatial mapping of molecular disturbances within the microenvironment of chronic wounds.
•Quantitative analysis of resveratrol in red wine.•Paper Spray Mass Spectrometry for analytical determination.•Rapid purification step by SPE cartridge.
A rapid analytical approach for the assay of ...resveratrol in red wines, based on Paper Spray Mass Spectrometry (PS-MS) and Multiple Reaction Monitoring (MRM) is described. The assay involves the use of the stable isotope dilution method. The analytical parameters calculated analyzing fortified samples confirm the reliability of the proposed approach, with accuracy values about 100%, and LOD and LOQ values calculated at 0.5 and 0.8μg/mL, respectively. Furthermore, both the recovery, which was quantitative for the analyte, and the reproducibility (RSD%), checked on different days on the same wine, always below 7%, highlighted the consistency of the methodology.
The 3-hydroxy-3-methylglutaryl neohesperidosides of hesperetin (brutieridin, 1) and naringenin (melitidin, 2) were isolated and detected from the fruits of bergamot (Citrus bergamia). The structures ...of these compounds were determined by spectroscopic and chemical methods.
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