Combining intensity correlation analysis and MALDI imaging to study the distribution of flavonols and dihydrochalcones in Golden Delicious apples

Mass spectrometry-based imaging techniques applied to small molecules complement the growing research field of metabolomics and can be used to interpret many important biological processes occurring in plants. In untargeted imaging applications, chemical identification is a critical step since it cannot take advantage of separative techniques applied to neutral molecules (e.g. liquid chromatography). The use of high resolution spectrometers is of great help, but fragmentation experiments are often necessary. In many cases, the information on ion fragmentation is embedded in the data sets, because analytes break up during ionization, but the extraction of this information is not easy considering the complexity of the imaging data files. Here an approach is proposed for applying conventional untargeted MALDI (matrix-assisted laser desorption ionization) profiling and advanced data analysis to perform imaging of metabolites in apple tissues. The pipeline, based on intensity correlation analysis, is used to extract fragmentation information from untargeted, high resolution, wide range mass spectra and to reconstruct compound-specific images which can be used for interpretation purposes. The proposed approach was used to investigate the distribution of glycosylated flavonols and dihydrochalcones in Golden Delicious apples. The results indicate that the method is effective, showing a high potential for ascertaining detailed metabolite localization.     

Transport and bioactivity of cyanidin 3-glucoside into the vascular endothelium

Flavonoids are dietary components involved in decreasing oxidative stress in the vascular endothelium and thus the risk of endothelial dysfunction. However, their very low concentrations in plasma place this role in doubt. Thus, a relationship between the effective intracellular concentration of flavonoids and their bioactivity needs to be assessed. This study examined the uptake of physiological concentrations of cyanidin 3-glucoside, a widespread dietary flavonoid, into human vascular endothelial cells. Furthermore, the involvement of the membrane transporter bilitranslocase (TC No. 2.A.65.1.1) as the key underlying molecular mechanism for membrane transport was investigated by using purified anti-sequence antibodies binding at the extracellular domain of the protein. The experimental observations were carried out in isolated plasma membrane vesicles and intact endothelial cells from human endothelial cells (EA.hy926) and on an ischemia-reperfusion model in isolated rat hearts. Cyanidin 3-glucoside was transported via bilitranslocase into endothelial cells, where it acted as a powerful intracellular antioxidant and a cardioprotective agent in the reperfusion phase after ischemia. These findings suggest that dietary flavonoids, despite their limited oral bioavailability and very low postabsorption plasma concentrations, may provide protection against oxidative stress-based cardiovascular diseases. Bilitranslocase, by mediating the cellular uptake of some flavonoids, is thus a key factor in their protective activity on endothelial function.     

Activation of Ran GTPase by a Legionella Effector Promotes Microtubule Polymerization,Pathogen Vacuole Motility and Infection

The causative agent of Legionnaires'' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS) to form in phagocytes a distinct “Legionella-containing vacuole” (LCV), which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF) domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP) in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED) fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.     

Stable free radicals as ubiquitous components of red wines

A stable ESR signal, centred at g = 2.0037 ± 0.0002, characterised by a single resonance and assignable to a free radical, was found in all the bottled red wines, both commercial and experimental, that we have examined. The radical concentration was calculated to be in the range of 5–82 nM. After exposure of the wines to air for a few minutes a two fold increase of the ESR signal, followed by a slow decrease with time, was observed. The intensity of ESR signal in experimental red wines, was found to increase with the ageing of the wines and was strictly correlated to the total content of polyphenols. The formation of semiquinone radicals of polyphenols is suggested as one possible mechanism leading to the presence of stable free radicals in red wines.     

A metabolomic approach to the study of wine micro-oxygenation

Wine micro-oxygenation is a globally used treatment and its effects were studied here by analysing by untargeted LC-MS the wine metabolomic fingerprint. Eight different procedural variations, marked by the addition of oxygen (four levels) and iron (two levels) were applied to Sangiovese wine, before and after malolactic fermentation. Data analysis using supervised and unsupervised multivariate methods highlighted some known candidate biomarkers, together with a number of metabolites which had never previously been considered as possible biomarkers for wine micro-oxygenation. Various pigments and tannins were identified among the known candidate biomarkers. Additional new information was obtained suggesting a correlation between oxygen doses and metal contents and changes in the concentration of primary metabolites such as arginine, proline, tryptophan and raffinose, and secondary metabolites such as succinic acid and xanthine. Based on these findings, new hypotheses regarding the formation and reactivity of wine pigment during micro-oxygenation have been proposed. This experiment highlights the feasibility of using unbiased, untargeted metabolomic fingerprinting to improve our understanding of wine chemistry.     

Heat-induced accumulation of chloroplast protein synthesis elongation factor, EF-Tu, in winter wheat

Chloroplast protein synthesis elongation factor, EF-Tu, has been implicated in heat tolerance in maize (Zea mays). Chloroplast EF-Tu is highly conserved, and it is possible that this protein may be of importance to heat tolerance in other species including wheat (Triticum aestivum). In this study, we assessed heat tolerance and determined the relative levels of EF-Tu in mature plants (at flowering stage) of 12 cultivars of winter wheat experiencing a 16-d-long heat treatment (36/30 degrees C, day/night temperature). In addition, we also investigated the expression of EF-Tu in young plants experiencing a short-term heat shock (4h at 43 degrees C). Heat tolerance was assessed by examining the stability of thylakoid membranes, measuring chlorophyll content, and assessing plant growth traits (shoot dry mass, plant height, tiller number, and ear number). In mature plants, relative levels of EF-Tu were determined after 7 d of heat stress. High temperature-induced accumulation of EF-Tu in mature plants of all cultivars, and a group of cultivars that showed greater accumulation of EF-Tu displayed better tolerance to heat stress. Young plants of all cultivars but one did not show significant increases in the relative levels of EF-Tu. The results of the study suggest that EF-Tu protein may play a role in heat tolerance in winter wheat.     

Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.     

Quantification of cell hybridoma yields with confocal microscopy and flow cytometry

The fusion of antigen presenting and cancer cells leads to the formation of hybrid cells, which are considered a potential vaccine for treating cancer. The quality assessment of hybrid cell vaccines is crucial for the introduction of this new treatment. Flow cytometry was the method used recently, since it is faster in comparison to classical microscopy. Here we describe a rapid confocal microscopy based approach to quantify hybrid cell yields. The extent of fusion rate was determined by confocal microscopy by counting dual fluorescent cells and by measuring the area of co-localized pixels. Results of both methods showed high degree of correlation. The same samples were also analyzed by flow cytometry. Fusion rates determined with both techniques showed significant correlation. In conclusion, using confocal microscopy we developed a sensitive and a rapid method to assess the yield of hybridomas in a large number of electrofused cells.     

Pharmacoproteomics Profile in Response to Acamprosate Treatment of an Alcoholism Animal Model

Acamprosate is an FDA‐approved medication for the treatment of alcoholism that is unfortunately only effective in certain patients. Although acamprosate is known to stabilize the hyper‐glutamatergic state in alcoholism, pharmacological mechanisms of action in brain tissue remains unknown. To investigate the mechanism of acamprosate efficacy, the authors employ a pharmacoproteomics approach using an animal model of alcoholism, type 1 equilibrative nucleoside transporter (ENT1) null mice. The results demonstrate that acamprosate treatment significantly decreased both ethanol drinking and preference in ENT1 null mice compared to that of wild‐type mice. Then, to elucidate acamprosate efficacy mechanism in ENT1 null mice, the authors utilize label‐free quantification proteomics comparing both genotype and acamprosate treatment effects in the nucleus accumbens (NAc). A total of 1040 protein expression changes are identified in the NAc among 3634 total proteins detected. The proteomics and Western blot result demonstrate that acamprosate treatment decreased EAAT expression implicating stabilization of the hyper‐glutamatergic condition in ENT1 null mice. Pathway analysis suggests that acamprosate treatment in ENT1 null mice seems to rescue glutamate toxicity through restoring of RTN4 and NF‐κB medicated neuroimmune signaling compared to wild‐type mice. Overall, pharmacoproteomics approaches suggest that neuroimmune restoration is a potential efficacy mechanism in the acamprosate treatment of certain sub‐populations of alcohol dependent subjects.