The ability to form spheroids under non‐adherent conditions is a well‐known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the ...present study, we tested the ability of hMSCs isolated from the vascular wall (hVW‐MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW‐MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra‐low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX‐2, OCT‐4, NANOG) were analyzed by immunocytochemistry and real‐time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki‐67 proliferation marker. Metabolomic profile was obtained with liquid chromatography–mass spectrometry. In 2D, hVW‐MSCs were spindle‐shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW‐MSCs were CD44+, CD105low, CD90low, exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki‐67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW‐MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.
hVW‐MSCs are able to form spheres, characterized by high NANOG and CD44 expression, low ki‐67 levels and slow metabolism. hVW‐MSC spheres represent an in vitro model of stem cell niche, suitable for investigating MSCs in the vascular field.
Summary
Metformin, the first drug chosen to be tested in a clinical trial aimed to target the biology of aging per se, has been clinically exploited for decades in the absence of a complete ...understanding of its therapeutic targets or chemical determinants. We here outline a systematic chemoinformatics approach to computationally predict biomolecular targets of metformin. Using several structure‐ and ligand‐based software tools and reference databases containing 1,300,000 chemical compounds and more than 9,000 binding sites protein cavities, we identified 41 putative metformin targets including several epigenetic modifiers such as the member of the H3K27me3‐specific demethylase subfamily, KDM6A/UTX. AlphaScreen and AlphaLISA assays confirmed the ability of metformin to inhibit the demethylation activity of purified KDM6A/UTX enzyme. Structural studies revealed that metformin might occupy the same set of residues involved in H3K27me3 binding and demethylation within the catalytic pocket of KDM6A/UTX. Millimolar metformin augmented global levels of H3K27me3 in cultured cells, including reversion of global loss of H3K27me3 occurring in premature aging syndromes, irrespective of mitochondrial complex I or AMPK. Pharmacological doses of metformin in drinking water or intraperitoneal injection significantly elevated the global levels of H3K27me3 in the hepatic tissue of low‐density lipoprotein receptor‐deficient mice and in the tumor tissues of highly aggressive breast cancer xenograft‐bearing mice. Moreover, nondiabetic breast cancer patients receiving oral metformin in addition to standard therapy presented an elevated level of circulating H3K27me3. Our biocomputational approach coupled to experimental validation reveals that metformin might directly regulate the biological machinery of aging by targeting core chromatin modifiers of the epigenome.
Advances in metabolomics analysis and data treatment increase the knowledge of complex biological systems. One of the most used methodologies is gas chromatography-mass spectrometry (GC-MS) due to ...its robustness, high separation efficiency, and reliable peak identification through curated databases. However, methodologies are not standardized, and the derivatization steps in GC-MS can introduce experimental errors and take considerable time, exposing the samples to degradation. Here, we propose the injection-port derivatization (IPD) methodology to increase the throughput in plasma metabolomics analysis by GC-MS. The IPD method was evaluated and optimized for different families of metabolites (organic acids, amino acids, fatty acids, sugars, sugar phosphates, etc.) in terms of residence time, injection-port temperature, and sample/derivatization reagent ratio. Finally, the method’s usefulness was validated in a study consisting of a cohort of obese patients with or without nonalcoholic steatohepatitis. Our results show a fast, reproducible, precise, and reliable method for the analysis of biological samples by GC-MS. Raw data are publicly available at MetaboLights with Study Identifier MTBLS5151.
Phenolic compounds, which are secondary plant metabolites, are considered an integral part of the human diet. Physiological properties of dietary polyphenols have come to the attention in recent ...years. Especially, proanthocyanidins (ranging from dimers to decamers) have demonstrated potential interactions with biological systems, such as antiviral, antibacterial, molluscicidal, enzyme-inhibiting, antioxidant, and radical-scavenging properties. Agroindustry produces a considerable amount of phenolic-rich sources, and the ability of polyphenolic structures to interacts with other molecules in living organisms confers their beneficial properties. Cocoa wastes and grape seeds and skin byproducts are a source of several phenolic compounds, particularly mono-, oligo-, and polymeric proanthocyanidins. The aim of this work is to compare the phenolic composition of Theobroma cacao and Vitis vinifera grape seed extracts by high pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometer and equipped with an electrospray ionization interface (HPLC-ESI-QTOF-MS) and its phenolic quantitation in order to evaluate the proanthocyanidin profile. The antioxidant capacity was measured by different methods, including electron transfer and hydrogen atom transfer-based mechanisms, and total phenolic and flavan-3-ol contents were carried out by Folin-Ciocalteu and Vanillin assays. In addition, to assess the anti-inflammatory capacity, the expression of MCP-1 in human umbilical vein endothelial cells was measured.
Summary
Background
Obesity can influence hepatic mitochondrial function, and cause non‐alcoholic steatohepatitis (NASH). Diagnosis and follow‐up rely on invasive liver biopsy so blood‐based markers ...are urgently required.
Aim
To investigate whether values of circulating metabolites from energy and one‐carbon (1‐C) metabolism may: (a) reflect hepatic mitochondrial flexibility failure and (b) act as NASH biomarkers.
Methods
Patients with severe obesity undergoing bariatric surgery (n = 270) were investigated using quantitative targeted plasma metabolomics. Comparisons were with non‐obese controls without liver disease (n = 50). Obese patients with NASH (n = 53) and without NASH (n = 130) representing extreme groups of liver disease were assessed to test the diagnostic ability of the measured circulating metabolites. Paired liver biopsy and plasma samples from NASH patients were available 1 year post‐surgery and were evaluated to monitor metabolomic changes with liver damage resolution.
Results
We identified correlations between human liver metabolism and obesity. High‐plasma α‐ketoglutarate (α‐KG) and lactate concentrations in NASH patients indicating citric acid cycle replenishment via glutaminolysis might also be a crucial point in NASH onset. Plasma measurements of α‐KG, β‐hydroxybutyrate, pyruvate and oxaloacetate reduced the uncertainty in clinical diagnosis of NASH area under receiver operating characteristic curve (AUC) of 0.826 and predicted NASH resolution without ambiguity (AUC of 0.999).
Conclusion
Changes in plasma mitochondrial metabolites appear to be associated with NASH. These metabolic responses may be dynamically remodelled following resolution of liver damage through massive weight loss.
Metformin has been proposed to operate as an agonist of SIRT1, a nicotinamide adenine dinucleotide (NAD
)-dependent deacetylase that mimics most of the metabolic responses to calorie restriction. ...Herein, we present an
analysis focusing on the molecular docking and dynamic simulation of the putative interactions between metformin and SIRT1. Using eight different crystal structures of human SIRT1 protein, our computational approach was able to delineate the putative binding modes of metformin to several pockets inside and outside the central deacetylase catalytic domain. First, metformin was predicted to interact with the very same allosteric site occupied by resveratrol and other sirtuin-activating compounds (STATCs) at the amino-terminal activation domain of SIRT1. Second, metformin was predicted to interact with the NAD
binding site in a manner slightly different to that of SIRT1 inhibitors containing an indole ring. Third, metformin was predicted to interact with the C-terminal regulatory segment of SIRT1 bound to the NAD
hydrolysis product ADP-ribose, a "C-pocket"-related mechanism that appears to be essential for mechanism-based activation of SIRT1. Enzymatic assays confirmed that the net biochemical effect of metformin and other biguanides such as a phenformin was to improve the catalytic efficiency of SIRT1 operating in conditions of low NAD
. Forthcoming studies should confirm the mechanistic relevance of our computational insights into how the putative binding modes of metformin to SIRT1 could explain its ability to operate as a direct SIRT1-activating compound. These findings might have important implications for understanding how metformin might confer health benefits
maintenance of SIRT1 activity during the aging process when NAD
levels decline.
The third-generation anaplastic lymphoma tyrosine kinase inhibitor (ALK-TKI) lorlatinib has a unique side effect profile that includes hypercholesteremia and hypertriglyceridemia in >80% of lung ...cancer patients. Here, we tested the hypothesis that lorlatinib might directly promote the accumulation of cholesterol and/or triglycerides in human hepatic cells. We investigated the capacity of the hepatoprotectant silibinin to modify the lipid-modifying activity of lorlatinib. To predict clinically relevant drug−drug interactions if silibinin were used to clinically manage lorlatinib-induced hyperlipidemic effects in hepatic cells, we also explored the capacity of silibinin to interact with and block CYP3A4 activity using in silico computational descriptions and in vitro biochemical assays. A semi-targeted ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS/MS)-based lipidomic approach revealed that short-term treatment of hepatic cells with lorlatinib promotes the accumulation of numerous molecular species of cholesteryl esters and triglycerides. Silibinin treatment significantly protected the steady-state lipidome of hepatocytes against the hyperlipidemic actions of lorlatinib. Lipid staining confirmed the ability of lorlatinib to promote neutral lipid overload in hepatocytes upon long-term exposure, which was prevented by co-treatment with silibinin. Computational analyses and cell-free biochemical assays predicted a weak to moderate inhibitory activity of clinically relevant concentrations of silibinin against CYP3A4 when compared with recommended (rosuvastatin) and non-recommended (simvastatin) statins for lorlatinib-associated dyslipidemia. The elevated plasma cholesterol and triglyceride levels in lorlatinib-treated lung cancer patients might involve primary alterations in the hepatic accumulation of lipid intermediates. Silibinin could be clinically explored to reduce the undesirable hyperlipidemic activity of lorlatinib in lung cancer patients.
Cucumber (Cucumis sativus L.) belongs to Cucurbitaceae family and it is widely cultivated and consumed in many varieties for its edible fruit (Cucumber) either in fresh or processed (pickled) forms. ...It is an important part of the traditional Mediterranean diet since antiquity due to its nutritional and medicinal properties. C. sativus is a rich source of polyphenols and it is used not only as food, but also in folk medicine, health-care and cosmetology. Therefore, due to the important role that cucumber plays in those fields, we have developed a simple and rapid method to characterize the polyphenolic compounds in an extract of Cucumis sativus L. using Reversed-Phase High-Performance Liquid Chromatography coupled to Electrospray Ionization Quadropole Time-of-Flight Mass Spectrometry (RP-HPLC–ESI-Q-TOF-MS). The accuracy of mass data generated by Q-TOF-MS together with the fragmentation pattern of the full scan run of MS/MS analysis have been a useful tool to tentatively characterize 73 phenolic compounds in the extract of the studied matrix. Most of these compounds are being reported here for the first time in Cucumber, highlighting the importance of Cucurbitaceae family as a source of natural bioactive antioxidants.
Antiretroviral therapy (ART) induces persistent suppression of HIV-1 replication and gradual recovery of T-cell counts, and consequently, morbidity and mortality from HIV-related illnesses have been ...significantly reduced. However, in approximately 30% of people living with HIV (PLHIV) on ART, CD4
+
T-cell counts fail to normalize despite ART and complete suppression of HIV viral load, resulting in severe immune dysfunction, which may represent an increased risk of clinical progression to AIDS and non-AIDS events as well as increased mortality. These patients are referred to as “immune inadequate responders”, “immunodiscordant responders” or “immune nonresponders (INR)”. The molecular mechanisms underlying poor CD4
+
T-cell recovery are still unclear. In this sense, the use of omics sciences has shed light on possible factors involved in the activity and metabolic dysregulation of immune cells during the failure of CD4
+
T-cell recovery in INR. Moreover, identification of key molecules by omics approaches allows for the proposal of potential biomarkers or therapeutic targets to improve CD4
+
T-cell recovery and the quality of life of these patients. Hence, this review aimed to summarize the information obtained through different omics concerning the molecular factors and pathways associated with the INR phenotype to better understand the complexity of this immunological status in HIV infection.
Epithelial-to-mesenchymal transition (EMT) is key to tumor aggressiveness, therapy resistance, and immune escape in breast cancer. Because metabolic traits might be involved along the EMT continuum, ...we investigated whether human breast epithelial cells engineered to stably acquire a mesenchymal phenotype in non-tumorigenic and H-Ras
-driven tumorigenic backgrounds possess unique metabolic fingerprints. We profiled mitochondrial-cytosolic bioenergetic and one-carbon (1C) metabolites by metabolomic analysis, and then questioned the utilization of different mitochondrial substrates by EMT mitochondria and their sensitivity to mitochondria-centered inhibitors. "Upper" and "lower" glycolysis were the preferred glucose fluxes activated by EMT in non-tumorigenic and tumorigenic backgrounds, respectively. EMT in non-tumorigenic and tumorigenic backgrounds could be distinguished by the differential contribution of the homocysteine-methionine 1C cycle to the transsulfuration pathway. Both non-tumorigenic and tumorigenic EMT-activated cells showed elevated mitochondrial utilization of glycolysis end-products such as lactic acid, β-oxidation substrates including palmitoyl-carnitine, and tricarboxylic acid pathway substrates such as succinic acid. Notably, mitochondria in tumorigenic EMT cells distinctively exhibited a significant alteration in the electron flow intensity from succinate to mitochondrial complex III as they were highly refractory to the inhibitory effects of antimycin A and myxothiazol. Our results show that the bioenergetic/1C metabolic signature, the utilization rates of preferred mitochondrial substrates, and sensitivity to mitochondrial drugs significantly differs upon execution of EMT in non-tumorigenic and tumorigenic backgrounds, which could help to resolve the relationship between EMT, malignancy, and therapeutic resistance in breast cancer.
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