Numerical Advances in Electrophoretic Deposition in Flow Cells

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Metabolomic and lipidomic analysis of the colorectal adenocarcinoma cell line HT29 in hypoxia and reoxygenation

The poor availability of oxygen and nutrients in malignant tumors drives the activation of various molecular responses and metabolic reprogramming in cancer cells. Hypoxic tumor regions often exhibit resistance to chemotherapy and radiotherapy. One approach to enhance cancer therapy is to indirectly increase tumor oxygen availability through targeted metabolic reprogramming. Thus, understanding the underlying metabolic changes occurring during hypoxia and reoxygenation is crucial for improving therapy efficacy. In this study, we utilized the HT29 colorectal adenocarcinoma cell line as a hypoxia–reoxygenation model to investigate central carbon and lipid metabolism. Through quantitative NMR spectroscopy and flow injection analysis – differential mobility spectroscopy—tandem mass spectrometry (FIA-DMS-MS/MS) analysis, we observed alterations in components of mitochondrial metabolism, redox status, specific lipid classes, and structural characteristics of lipids during hypoxia and up to 24 h of reoxygenation. These findings contribute to our understanding of the metabolic changes occurring during reoxygenation and provide the basis for functional studies aimed at metabolic pathways in cancer cells.Proteomic

Quantitative NMR analysis of intra-and extracellular metabolism of mammalian cells: A tutorial

MTG3 - Moleculaire genetica en pathologie van endocriene tumorenMolecular tumour pathology - and tumour genetic

Metabolic analysis of vitreous/lens and retina in wild type and retinal degeneration mice

Photoreceptors are the light-sensing cells of the retina and the major cell type affected in most inherited retinal degenerations. Different metabolic pathways sustain their high energetic demand in physiological conditions, particularly aerobic glycolysis. The principal metabolome of the mature retina has been studied, but only limited information is available on metabolic adaptations in response to key developmental events, such as eye opening. Moreover, dynamic metabolic changes due to retinal degeneration are not well understood. Here, we aimed to explore and map the ocular metabolic dynamics induced by eye opening in healthy (wild type) or Pde6b-mutant (retinal degeneration 1, Rd1) mice, in which photoreceptors degenerate shortly after eye opening. To unravel metabolic differences emerging before and after eye opening under physiological and pathophysiological conditions, we performed nuclear magnetic resonance (NMR) spectrosco-py-based metabolome analysis of wild type and Rd1 retina and vitreous/lens. We show that eye opening is accompanied by changes in the concentration of selected metabolites in the retina and by alterations in the vitreous/lens composition only in the retinal degeneration context. As such, we identify N-Acetylaspartate as a potential novel vitreous/lens marker reflecting progressive retinal degeneration. Thus, our data can help elucidating mechanisms underlying key events in retinal physiology and reveal changes occurring in pathology, while highlighting the importance of the vitreous/lens in the characterization of retinal diseases.Proteomic

Dynamic differences in dietary polyunsaturated fatty acid metabolism in sputum of COPD patients and controls

Introduction: Disturbances in onset and resolution of inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. Dietary polyunsaturated fatty acids (PUFAs) can be converted into lipid mediators here collectively named oxylipins. These include classical eicosanoids, but also pro-resolving mediators. A balanced production of pro-inflammatory and pro-resolving oxylipins is of importance for adequate inflammatory responses and subsequent return to homeostasis. Objectives: Here we investigated if PUFA metabolism is disturbed in COPD patients. Methods: Free PUFA and oxylipin levels were measured in induced sputum samples from the Bergen COPD cohort and COPD exacerbation study using liquid chromatography-mass spectrometry. Additionally, effects of whole cigarette smoke on PUFA metabolism in air-liquid interface cultures of primary bronchial epithelial cells were assessed. Results: Significantly lower levels of free alpha-linolenic acid, linoleic acid and eicosapentaenoic acid (EPA) were detected in sputum from stable COPD patients compared to controls. During acute exacerbation (AE), levels of free arachidonic acid and docosapentaenoic acid were higher than in stable COPD patients. Furthermore, levels of omega-3 EPA- and docosahexaenoic acid-derived oxylipins were lower in sputum from stable COPD patients compared to controls. Cyclooxygenase-2-converted mediators were mostly increased during AE. In vitro studies additionally showed that cigarette smoke exposure may also directly contribute to altered epithelial PUFA metabolism, and indirectly by causing airway epithelial remodelling. Conclusions: Our findings show significant differences in PUFA metabolism in COPD patients compared to controls, further changed during AE. Airway epithelial remodelling may contribute to these changes. These findings provide new insight in impaired inflammatory resolution in COPD.publishedVersio

Development of an online p38α mitogen-activated protein kinase binding assay and integration of LC–HR-MS

A high-resolution screening method was developed for the p38α mitogen-activated protein kinase to detect and identify small-molecule binders. Its central role in inflammatory diseases makes this enzyme a very important drug target. The setup integrates separation by high-performance liquid chromatography with two parallel detection techniques. High-resolution mass spectrometry gives structural information to identify small molecules while an online enzyme binding detection method provides data on p38α binding. The separation step allows the individual assessment of compounds in a mixture and links affinity and structure information via the retention time. Enzyme binding detection was achieved with a competitive binding assay based on fluorescence enhancement which has a simple principle, is inexpensive, and is easy to interpret. The concentrations of p38α and the fluorescence tracer SK&F86002 were optimized as well as incubation temperature, formic acid content of the LC eluents, and the material of the incubation tubing. The latter notably improved the screening of highly lipophilic compounds. For optimization and validation purposes, the known kinase inhibitors BIRB796, TAK715, and MAPKI1 were used among others. The result is a high-quality assay with Z′ factors around 0.8, which is suitable for semi-quantitative affinity measurements and applicable to various binding modes. Furthermore, the integrated approach gives affinity data on individual compounds instead of averaged ones for mixtures

Acute phase inflammation is characterized by rapid changes in plasma/peritoneal fluid N-glycosylation in mice.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.Murine zymosan-induced peritonitis is a widely used model for studying the molecular and cellular events responsible for the initiation, persistence and/or resolution of inflammation. Among these events, it is becoming increasingly evident that changes in glycosylation of proteins, especially in the plasma and at the site of inflammation, play an important role in the inflammatory response. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)-based glycosylation profiling, we investigated the qualitative and quantitative effect of zymosan-induced peritonitis on N-glycosylation in mouse plasma and peritoneal fluid. Our results show that both N-glycomes exhibit highly similar glycosylation patterns, consisting mainly of diantennary and triantennary complex type N-glycans with high levels (>95 %) of galactosylation and sialylation (mostly NeuGc) and a medium degree of core fucosylation (30 %). Moreover, MS/MS structural analysis, assisted by linkage-specific derivatization of sialic acids, revealed the presence of O-acetylated sialic acids as well as disialylated antennae ("branching sialylation") characterized by the presence of α2-6-linked NeuGc on the GlcNAc of the NeuGcα2-3-Galβ1-3-GlcNAc terminal motif. A significant decrease of (core) fucosylation together with an increase of both α2-3-linked NeuGc and "branching sialylation" were observed in N-glycomes of mice challenged with zymosan, but not in control mice injected with PBS. Importantly, substantial changes in glycosylation were already observed 12 h after induction of peritonitis, thereby demonstrating an unexpected velocity of the biological mechanisms involved.Dutch Arthritis Association (Reumafonds) LLP-24 Innovative Medicines Initiative Joint Undertaking (IMI JU)/ 115142-2 Netherlands Genomic Initiative/93511033 info:eu-repo/grantAgreement/EC/FP7/278535info:eu-repo/grantAgreement/EC/FP7/27853

Metabolic reprogramming of mammary epithelial cells during TGF-beta-induced epithelial-to-mesenchymal transition

The cytokine transforming growth factor-beta (TGF-beta) can induce normal breast epithelial cells to take on a mesenchymal phenotype, termed epithelial-to-mesenchymal transition (EMT). While the transcriptional and proteomic changes during TGF-beta-induced EMT have been described, the metabolic rewiring that occurs in epithelial cells undergoing EMT is not well understood. Here, we quantitively analyzed the TGF-beta-induced metabolic reprogramming during EMT of non-transformed NMuMG mouse mammary gland epithelial cells using nuclear magnetic resonance (NMR) spectroscopy. We found that TGF-beta elevates glycolytic and tricarboxylic acid (TCA)-cycle activity and increases glutaminolysis. Additionally, TGF-beta affects the hexosamine pathway, arginine-proline metabolism, the cellular redox state, and strongly affects choline metabolism during EMT. TGF-beta was found to induce phosphocholine production. A kinase inhibitor RSM-93A that inhibits choline kinase alpha (CHK alpha) mitigated TGF-beta-induced changes associated with EMT, i.e., increased filamentous (F)-actin stress fiber formation and N-Cadherin mesenchymal marker expression.Cancer Signaling networks and Molecular Therapeutic

Metabolomic and lipidomic signatures associated with activation of human cDC1 (BDCA3(+)/CD141(+)) dendritic cells

Dendritic cells (DCs) bridge the connection between innate and adaptive immunity. DCs present antigens to T cells and stimulate potent cytotoxic T-cell responses. Metabolic reprogramming is critical for DC development and activation; however, metabolic adaptations and regulation in DC subsets remains largely uncharacterized. Here, we mapped metabolomic and lipidomic signatures associated with the activation phenotype of human conventional DC type 1, a DC subset specialized in cross-presentation and therefore of major importance for the stimulation of CD8(+) T cells. Our metabolomics and lipidomic analyses showed that Toll-like receptor (TLR) stimulation altered glycerolipids and amino acids in cDC1. Poly I:C or pRNA stimulation reduced triglycerides and cholesterol esters, as well as various amino acids. Moreover, TLR stimulation reduced expression of glycolysis-regulating genes and did not induce glycolysis. Conversely, cDC1 exhibited increased mitochondrial content and oxidative phosphorylation (OXPHOS) upon TLR3 or TLR7/8 stimulation. Our findings highlight the metabolic adaptations required for cDC1 maturation.Proteomic