Steatosis-induced proteins adducts with lipid peroxidation products and nuclear electrophilic stress in hepatocytes

AbstractAccumulating evidence suggests that fatty livers are particularly more susceptible to several pathological conditions, including hepatic inflammation, cirrhosis and liver cancer. However the exact mechanism of such susceptibility is still largely obscure. The current study aimed to elucidate the effect of hepatocytes lipid accumulation on the nuclear electrophilic stress. Accumulation of intracellular lipids was significantly increased in HepG2 cells incubated with fatty acid (FA) complex (1mM, 2:1 oleic and palmitic acids). In FA-treated cells, lipid droplets were localized around the nucleus and seemed to induce mechanical force, leading to the disruption of the nucleus morphology. Level of reactive oxygen species (ROS) was significantly increased in FA-loaded cells and was further augmented by treatment with moderate stressor (CoCl2). Increased ROS resulted in formation of reactive carbonyls (aldehydes and ketones, derived from lipid peroxidation) with a strong perinuclear accumulation. Mass-spectroscopy analysis indicated that lipid accumulation per-se can results in modification of nuclear protein by reactive lipid peroxidation products (oxoLPP). 235 Modified proteins involved in transcription regulation, splicing, protein synthesis and degradation, DNA repair and lipid metabolism were identified uniquely in FA-treated cells. These findings suggest that steatosis can affect nuclear redox state, and induce modifications of nuclear proteins by reactive oxoLPP accumulated in the perinuclear space upon FA-treatment

Computational solutions in redox lipidomics – Current strategies and future perspectives

Abstract The high chemical diversity of lipids allows them to perform multiple biological functions ranging from serving as structural building blocks of biological membranes to regulation of metabolism and signal transduction. In addition to the native lipidome, lipid species derived from enzymatic and non-enzymatic modifications (the epilipidome) make the overall picture even more complex, as their functions are still largely unknown. Oxidized lipids represent the fraction of epilipidome which has attracted high scientific attention due to their apparent involvement in the onset and development of numerous human disorders. Development of high-throughput analytical methods such as liquid chromatography coupled on-line to mass spectrometry provides the possibility to address epilipidome diversity in complex biological samples. However, the main bottleneck of redox lipidomics, the branch of lipidomics dealing with the characterization of oxidized lipids, remains the lack of optimal computational tools for robust, accurate and specific identification of already discovered and yet unknown modified lipids. Here we discuss the main principles of high-throughput identification of lipids and their modified forms and review the main software tools currently available in redox lipidomics. Different levels of confidence for software assisted identification of redox lipidome are defined and necessary steps toward optimal computational solutions are proposed

BioPAN: a web-based tool to explore mammalian lipidome metabolic pathways on LIPID MAPS.

Lipidomics increasingly describes the quantification using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues

Evaluation of air oxidized PAPC: A multi laboratory study by LC-MS/MS

Oxidized LDL (oxLDL) has been shown to play a crucial role in the onset and development of cardiovascular disorders. The study of oxLDL, as an initiator of inflammatory cascades, led to the discovery of a variety of oxidized phospholipids (oxPLs) responsible for pro-inflammatory actions. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) is frequently used by the scientific community as a representative oxPL mixture to study the biological effects of oxidized lipids, due to the high abundance of PAPC in human tissues and the biological activities of oxidized arachidonic acids derivatives. Most studies focusing on oxPAPC effects rely on in-house prepared mixtures of oxidized species obtained by exposing PAPC to air oxidation. Here, we described a multi-laboratory evaluation of the compounds in oxPAPC by LC-MS/MS, focusing on the identification and relative quantification of the lipid peroxidation products (LPPs) formed. PAPC was air-oxidized in four laboratories using the same protocol for 0, 48, and 72 h. It was possible to identify 55 different LPPs with unique elemental composition and characterize different structural isomeric species within these. The study showed good intra-sample reproducibility and similar qualitative patterns of oxidation, as the most abundant LPPs were essentially the same between the four laboratories. However, there were substantial differences in the extent of oxidation, i.e. the amount of LPPs relative to unmodified PAPC, at specific time points. This shows the importance of characterizing air-oxidized PAPC preparations before using them for testing biological effects of oxidized lipids, and may explain some variability of effects reported in the literature

Quality control requirements for the correct annotation of lipidomics data.

10.1038/s41467-021-24984-yNature Communications121477

Fluorescence labeling of carbonylated lipids and proteins in cells using coumarin-hydrazide

Carbonylation is a generic term which refers to reactive carbonyl groups present in biomolecules due to oxidative reactions induced by reactive oxygen species. Carbonylated proteins, lipids and nucleic acids have been intensively studied and often associated with onset or progression of oxidative stress related disorders. In order to reveal underlying carbonylation pathways and biological relevance, it is crucial to study their intracellular formation and spatial distribution. Carbonylated species are usually identified and quantified in cell lysates and body fluids after derivatization using specific chemical probes. However, spatial cellular and tissue distribution have been less often investigated. Here, we report coumarin-hydrazide, a fluorescent chemical probe for time- and cost-efficient labeling of cellular carbonyls followed by fluorescence microscopy to evaluate their intracellular formation both in time and space. The specificity of coumarin-hydrazide was confirmed in time- and dose-dependent experiments using human primary fibroblasts stressed with paraquat and compared with conventional DNPH-based immunocytochemistry. Both techniques stained carbonylated species accumulated in cytoplasm with strong perinuclear clustering. Using a complimentary array of analytical methods specificity of coumarin-hydrazide probe towards both protein- and lipid-bound carbonyls has been shown. Additionally, co-distribution of carbonylated species and oxidized phospholipids was demonstrated. Keywords: Coumarin-hydrazide, Dinitrophenyl hydrazine, Fluorescence microscopy, Protein and lipid carbonylation, Spatial distributio

A preliminary investigation into the impact of soft tissue augmentation-based periodontal phenotype modification therapy for patients exhibiting class III decompensation

Abstract Background Patients with skeletal angle Class III malocclusion usually have inadequate hard and soft tissue volume at the mandibular anterior teeth. The labial proclination at the teeth may lead to gingival recession. The purpose of this study was to explore whether periodontal phenotype modification therapy with soft tissue augmentation (PhMT-s) can prevent gingival recession in these patients. Methods Four patients with skeletal Class III malocclusion and a thin periodontal phenotype underwent surgical-orthodontic treatment. Prior to tooth movement, they underwent a minimally invasive vestibular incision with subperiosteal tunnel access combined with autogenous connective tissue grafts for periodontal phenotype modification with soft tissue augmentation (PhMT-s). The labial gingival thickness of the anterior mandibular teeth was measured at three distinct levels: at the cementoenamel junction (GT0), 3 mm apical to the CEJ (GT3), and 6 mm apical to the CEJ (GT6). These measurements were taken at baseline, three months following PhMT-s, and after tooth decompensation. Additionally, a biopsy sample was obtained from the PhMT-s site of one patient. All sections were subsequently stained using hematoxylin and eosin, Masson trichrome, Sirius Red, and immunohistochemistry. Results The thickness of the labial gingiva was increased about 0.42 to 2.00 mm after PhMT-s. At the end of pre-orthognathic surgical orthodontic treatment, the thickness of the labial gingiva was increased about − 0.14 to 1.32 mm compared to the baseline and no gingival recession occurred after the pre-orthognathic surgical orthodontic treatment. The histologic results demonstrated that the grafts obtained from the PhMT-s site exhibited increased deposition of collagen fibers. Moreover, the proportion of type III collagen increased and the grafts displayed significantly reduced positive expression of CD31 and OCN. Conclusions PhMT-s increased the thickness of the soft tissue, stabilizing the gingival margin for teeth exhibiting a thin periodontal phenotype and undergoing labial movement. This is attributed to the increased deposition of collagen fibers

LipidHunter Identifies Phospholipids by High-Throughput Processing of LC-MS and Shotgun Lipidomics Datasets

Lipids are dynamic constituents of biological systems, rapidly responding to any changes in physiological conditions. Thus, there is a large interest in lipid-derived markers for diagnostic and prognostic applications, especially in translational and systems medicine research. As lipid identification remains a bottleneck of modern untargeted lipidomics, we developed LipidHunter, a new open source software for the high-throughput identification of phospholipids in data acquired by LC-MS and shotgun experiments. LipidHunter resembles a workflow of manual spectra annotation. Lipid identification is based on MS/MS data analysis in accordance with defined fragmentation rules for each phospholipid (PL) class. The software tool matches product and neutral loss signals obtained by collision-induced dissociation to a user-defined white list of fatty acid residues and PL class-specific fragments. The identified signals are tested against elemental composition and bulk identification provided via LIPID MAPS search. Furthermore, LipidHunter provides information-rich tabular and graphical reports allowing to trace back key identification steps and perform data quality control. Thereby, 202 discrete lipid species were identified in lipid extracts from rat primary cardiomyocytes treated with a peroxynitrite donor. Their relative quantification allowed the monitoring of dynamic reconfiguration of the cellular lipidome in response to mild nitroxidative stress. LipidHunter is available free for download at https://bitbucket.org/SysMedOs/lipidhunter