1,721,063 research outputs found
Exploiting Chemical Modulation of Mitochondrial Dynamics during Melanogenesis
No full textMitochondria participate in many intracellular processes: they are known for ATP production, but also for their roles in controlling lipid metabolism, calcium cation homeostasis, metabolism, and apoptosis. To handle all these functions, they are characterized by a particular structure that dynamically changes in response to cell needs. Interactions with other organelles play a role in adaptation to intracellular changes.
In particular, the interaction between mitochondria and endoplasmic reticulum, called MERCs, has been recognized as a fundamental player in several signaling pathways, as Ca2+ transfer, phospholipid synthesis and exchange, mitochondrial fission and unfolded protein response (UPR). Consequently, MERCs impairment contributes to many diseases, such as neurodegenerative disorders, insulin resistance, and cancer. Considering this evidence, MERCs start to be considered as possible pharmacological targets. The molecules able to change the distance between the two organelles (chemical MERCs modulators) can exert their action through different mechanisms.
Aim of this work was to identify some chemical MERCs modulators and define their mechanisms of action. We performed a chemical compound screen based on high content imaging to study mitochondrial dynamics. After validation, our approach revealed five molecules able to change MERCs independently from the mitochondrial morphology.
In parallel, we developed a methodology for high throughput quantification of mitochondrial membrane potential in 2D and 3D models using the TMRM dye. By combining automated image analysis and machine learning we were able to distinguish melanoma cells from macrophages in co-culture, and to analyze the subpopulations separately. We also applied this approach to human fibroblasts, neural stem cells, spheroids, and isolated muscle fibers.
Then, we decided to apply our knowledge to a process whose molecular mechanisms are still unclear: melanogenesis, the process of melanin synthesis. Melanogenesis occurs in lysosome-related organelles called melanosomes, present within cells of the epidermis called melanocytes. Melanin is a biopolymer with pleiotropic roles in the cell, ranging from UV light photoprotection to ion chelation and ROS modulation.
Considering melanogenesis as a protective cell response from stress factors, and the pivotal role of MERCs during the stress response, we hypothesized that the interplay between mitochondria and endoplasmic reticulum could contribute to melanogenesis as well. We observed that extrinsic factors, like UV, inducing melanogenesis impact also on mitochondrial dynamics. To study melanogenesis we also designed a high throughput approach to analyze several properties of melanosomes, such as their number, intensity and morphological parameters.
In summary, this work contributes to the development of several methodologies for the study of mitochondrial biology (e.g., functionality, morphology, MERCs, interactions with melanosomes), but also new knowledge on the molecular pathways and on the pharmacology that regulate MERCs. Moreover, it also provides for the first time an analysis of the crosstalk between melanogenesis and mitochondria dynamics
Rapid high performance liquid chromatography-high resolution mass spectrometry methodology for multiple prenol lipids analysis in zebrafish embryos
No full textStudy of the Photoinduced Fate of Selected Contaminants in Surface Waters by HPLC‐HRMS
Full text linkRationale: Photoinduced transformation of contaminants of emerging concern (CECs) can occur in aquatic environment and could lead to the formation of transformation products (TPs) of greater concern than the parent compounds. For such, the fate of epoxiconazole, hymecromone, and coumarin in water was investigated by simulating photoinduced abiotic transformations to assess the toxicity of their TPs and which CEC may be of greatest concern. Methods: Heterogeneous photocatalysis with TiO2 and direct photolysis of selected CECs were exploited to simulate their TPs. The TPs were assessed by means of HPLC coupled with an Orbitrap MS analyser in ESI positive mode, while their toxicity was evaluated through a Vibrio fischeri bioluminescence assay, and ECOSAR tool. Results: The formation of numerous TPs via different photoinduced pathways was noticed (27 for epoxiconazole, 6 for coumarin, and 8 for hymecromone, some of which are in the form of structural isomers). Toxicity assessment via V. fischeri assay showed that, unlike coumarin species, epoxiconazole transformation proceeds through the formation of toxic compounds. By means of ECOSAR software, the formation of predominant more noxious TPs of epoxiconazole was proved than the parent compound for both acute and chronic toxicities. Instead, most TPs of coumarin and hymecromone generally exhibited "harmful" and "toxic" levels of acute and chronic toxicities. Conclusions: A probable structural identification was assigned to the monitored TPs via HPLC-HRMS to recognize the several transformation pathways, of which the hydroxylation reaction was predominant, and which compound may be more hazardous in the aquatic system due to its TPs. Epoxiconazole transformation brought to potentially toxic TPs, whereas photoinduced degradation of coumarin and hymecromone resulted in less hazardous TPs. The most significant aspect of this work is the ability of this overall approach to identify the formation of photoinduced TPs that are potentially more toxic than the original CEC
Multi-Analyte MS Based Investigation in Relation to the Illicit Treatment of Fish Products with Hydrogen Peroxide
Full text linkFishery products are perishable due to the action of many enzymes, both endogenous and exogenous. The latter are produced by bacteria that may contaminate the products. When fishes age, there is a massive bacteria growth that causes the appearance of off-flavor. In order to obtain “false” freshness of fishery products, an illicit treatment with hydrogen peroxide is reported to be used. Residues of hydrogen peroxide in food may be of toxicology concern. We developed two mass spectrometry based methodologies to identify and quantify molecules related to the treatment of fishes with hydrogen peroxide. With ultra-high-performance liquid chromatography–mass spectrometry (UHPLC-MS) we evaluated the concentration of trimethylamine-N-oxide (TMAO), trimethylamine (TMA), dimethylamine (DMA), and cadaverine (CAD) in fish products. After evaluating LOQ, we measured and validated the lower limits of quantification (LLOQs as first levels of calibration curves) values of 50 (TMAO), 70 (TMA), 45 (DMA), and 40 (CAD) ng/mL. A high ratio between TMAO and TMA species indicated the freshness of the food. With a GC-MS method we confirmed the illicit treatment measuring the levels of H2O2 after an analytical reaction with anisole to give 2-hydroxyanisole as a marker. This latter product was detected in the headspace of the homogenized sample with simplification of the work-up. A LLOQ of 50 ng/mL was checked and validated. When fish products were whitened and refreshed with hydrogen peroxide, the detected amount of the product 2-hydroxyanisole could be very important, (larger than 100 mg/kg). The developed analytical methods were suitable to detect the illicit management of fishery products with hydrogen peroxide; they resulted as sensitive, selective, and robust
Caviar versus brill eggs: A novel high performance liquid chromatography‐mass spectrometry application for evaluating cosmetic ingredients composition
No full textIn this study, raw materials, sturgeon and brill roe, were analyzed and compared as regard their use in cosmetic products. Recently, expensive caviar was largely used in beauty creams as lipids source. We compared lipids from caviar and brill eggs to propose a technological alternative. The fatty acids amount was determined with a new GC-MS method. The triacylglycerols were identified and characterized by HPLC coupled with high resolution MS (HRMS). Both techniques showed a higher content of fatty acids and triacylglycerols in caviar respect to brill eggs. Some fatty acids, such as linoleic and linolenic acids, which are recognized to enhance skin metabolism, are absent, or show very low concentration, in brill eggs respect to sturgeon ones. It was possible to conclude that brill eggs are not a proper substitute of caviar in the formulation of cosmetic. Practical application: The developed analytical techniques provided a satisfactory analytical characterization of the raw materials. Although the sample preparation for GC-MS analysis is quite time consuming due to the derivatization of fatty acids, the one for triacylglycerols analyzed by HPLC-HRMS is very fast, and involves the use of a small amount of organic solvent. Fatty acids and triacylglycerols were identified and characterized. Comparison between Acipenser trasmontanus (caviar) and Scophthalmus rhombus eggs as active ingredient in beauty cream. A fatty acid quantitation and triacylglycerols quantitation, and characterization methods are showed. GC-MS and HPLC-HRMS are satisfactory tool to provide a full description of the studied raw materials
Advanced Glycation End Products Inhibition Restores Sphingolipid Rheostat in Mice Liver and Prevents Diet-Induced Insulin Resistance
No full textChronic administration of saturated fats and fructose differently affect SREBP activity resulting in different modulation of Nrf2 and Nlrp3 inflammasome pathways in mice liver
No full textThe overconsumption of both saturated fats and fructose in the modern society has been related to the development of nonalcoholic fatty liver disease (NAFLD). However, the specific contribution of individual dietary components on the progression of NAFLD to nonalcoholic steatohepatitis (NASH) has been poorly investigated. Therefore, the aim of our study was to investigate the dissimilar effects of these two dietary components on selected proinflammatory and antioxidant pathways in the liver of C57BL/6 mice fed a standard (SD), a 45% saturated fat (HFAT) or a 60% fructose (HFRT) diet for 12 weeks. HFAT diet evoked systemic metabolic alterations and overweight, not observed in HFRT mice. However, HFRT mice had a greater hepatic triglyceride deposition with increased ratio of triacylglycerols containing the palmitic acid compared to HFAT, as assessed by liquid chromatography-mass spectrometry analysis. This effect is due to the higher activation of the SCAP/SREBP1c lipogenic pathway by HFRT feeding. In addition, we found inhibition of Keap1/Nrf2 antioxidant signaling and more robust stimulation of the Nlrp3 inflammasome pathway in the livers of HFRT-fed mice when compared with HFAT-fed mice, which is consistent with the recent finding that palmitate and SREBP1c are implicated in hepatic oxidative stress and inflammation. These effects were associated with increased hepatic inflammation, as confirmed by high expression of markers of leukocyte infiltration in the HFRT group. Thus, we hypothesize an amplifying loop among lipogenesis, palmitate, Nrf2 and Nlrp3 that leads to a higher risk of NAFLD progression to NASH in a high-fructose diet compared to a high-saturated fat intake
to monitor occupational exposure
No full textThe purpose of this study was to develop a simple, direct, multiresidue highly specific procedure to evaluate the possible surface contamination of selected antineoplastic drugs in several hospital environment sites by using wipe test sampling. 5-fluorouracil (5-FU), carboplatin (C-Pt), cyclophosphamide (CYC), cytarabine (CYT), doxorubicin (DOX), gemcitabine (GEM), ifosfamide (IFO), methotrexate (MET), and mitomycin C (MIT) belong to very different chemical classes but show good ionization properties under electrospray ionization (ESI) conditions (negative ion mode for 5-FU and positive ion mode in all other cases). HPLC (high performance liquid chromatography) coupled with HRMS (high resolution mass spectrometry) appears to be the best technique for direct analysis of these analytes, because neither derivatization nor complex extraction procedure for polar compounds in samples is requested prior the analysis. Sample preparation was limited to washing wipes with appropriate solvents. Chromatographic separation was achieved on C18 reversed phase columns. The HPLC-HRMS/MS method was validated in order to obtain robustness, sensitivity and selectivity. LLOQ (lower limit of quantitation) values provided a sensitivity good enough to evidence the presence of the drugs in a very low concentration range (<1 pg/cm(2)). The method was applied for a study of real wipe tests coming from many areas from a hospital showing some positive samples. The low quantitation limits and the high specificity due to the high resolution approach of the developed method allowed an accurate description of the working environment that can be used to define procedural rules to limit working place contamination to a minimum. Copyright (C) 2015 John Wiley & Sons, Ltd
Multi-target detection of egg-white and pig gelatin fining agents in Nebbiolo-based aged red wine by means of nanoHPLC-HRMS
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