Concordant inter-laboratory derived concentrations of ceramides in human plasma reference materials via authentic standards.

In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals.

Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice.

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

Multicentric investigations of the role in the disease severity of accelerated phospholipid changes in COVID-19 patient airway.

CONTEXT: The changes in host membrane phospholipids are crucial in airway infection pathogenesis. Phospholipase A2 hydrolyzes host cell membranes, producing lyso-phospholipids and free fatty acids, including arachidonic acid (AA), which contributes significantly to lung inflammation. AIM: Follow these changes and their evolution from day 1, day 3 to day 7 in airway aspirates of 89 patients with COVID-19-associated acute respiratory distress syndrome and examine whether they correlate with the severity of the disease. The patients were recruited in three French intensive care units. The analysis was conducted from admission to the intensive care unit until the end of the first week of mechanical ventilation. RESULTS: In the airway aspirates, we found significant increases in the levels of host cell phospholipids, including phosphatidyl-serine and phosphatidyl-ethanolamine, and their corresponding lyso-phospholipids. This was accompanied by increased levels of AA and its inflammatory metabolite prostaglandin E2 (PGE2). Additionally, enhanced levels of ceramides, sphingomyelin, and free cholesterol were observed in these aspirates. These lipids are known to be involved in cell death and/or apoptosis, whereas free cholesterol plays a role in virus entry and replication in host cells. However, there were no significant changes in the levels of dipalmitoyl-phosphatidylcholine, the major surfactant phospholipid. A correlation analysis revealed an association between mortality risk and levels of AA and PGE2, as well as host cell phospholipids. CONCLUSION: Our findings indicate a correlation between heightened cellular phospholipid modifications and variations in AA and PGE2 with the severity of the disease in patients. Nevertheless, there is no indication of surfactant alteration in the initial phases of the illness.

Interplatform comparison between three ion mobility techniques for human plasma lipid collision cross sections.

The implementation of ion mobility spectrometry (IMS) in liquid chromatography-high-resolution mass spectrometry (LC-HRMS) workflows has become a valuable tool for improving compound annotation in metabolomics analyses by increasing peak capacity and by adding a new molecular descriptor, the collision cross section (CCS). Although some studies reported high repeatability and reproducibility of CCS determination and only few studies reported good interplatform agreement for small molecules, standardized protocols are still missing due to the lack of reference CCS values and reference materials. We present a comparison of CCS values of approximatively one hundred lipid species either commercially available or extracted from human plasma. We used three different commercial ion mobility technologies from different laboratories, drift tube IMS (DTIMS), travelling wave IMS (TWIMS) and trapped IMS (TIMS), to evaluate both instrument repeatability and interlaboratory reproducibility. We showed that CCS discrepancies of 0.3% (average) could occur depending on the data processing software tools. Moreover, eleven CCS calibrants were evaluated yielding mean RSD below 2% for eight calibrants, ESI Low concentration tuning mix (Tune Mix) showing the lowest RSD (< 0.5%) in both ion modes. Tune Mix calibrated CCS from the three different IMS instruments proved to be well correlated and highly reproducible (R2 > 0.995 and mean RSD ≤ 1%). More than 90% of the lipid CCS had deviations of less than 1%, demonstrating high comparability between techniques, and the possibility to use the CCS as molecular descriptor. We highlighted the need of standardized procedures for calibration, data acquisition, and data processing. This work demonstrates that using harmonized analytical conditions are required for interplatform reproducibility for CCS determination of human plasma lipids.

Impact of Source Conditions on Collision Cross Section Determination by Trapped Ion Mobility Spectrometry.

Collision cross section (CCS) values determined in ion mobility-mass spectrometry (IM-MS) are increasingly employed as additional descriptors in metabolomics studies. CCS values must therefore be reproducible and the causes of deviations must be carefully known and controlled. Here, we analyzed lipid standards by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) to evaluate the effects of solvent and flow rate in flow injection analysis (FIA), as well as electrospray source parameters including nebulizer gas pressure, drying gas flow rate, and temperature, on the ion mobility and CCS values. The stability of ion mobility experiments was studied over 10 h, which established the need for a delay-time of 20 min to stabilize source parameters (mostly pressure and temperature). Modifications of electrospray source parameters induced shifts of ion mobility peaks and even the occurrence of an additional peak in the ion mobility spectra. This behavior could be essentially explained by ion-solvent cluster formation. Changes in source parameters were also found to impact CCS value measurements, resulting in deviations up to 0.8%. However, internal calibration with the Tune Mix calibrant reduced the CCS deviations to 0.1%. Thus, optimization of source parameters is essential to achieve a good desolvation of lipid ions and avoid misinterpretation of peaks in ion mobility spectra due to solvent effects. This work highlights the importance of internal calibration to ensure interoperable CCS values, usable in metabolomics annotation.

C/EBPα Confers Dependence to Fatty Acid Anabolic Pathways and Vulnerability to Lipid Oxidative Stress-Induced Ferroptosis in FLT3-Mutant Leukemia.

Although transcription factor CCAAT-enhancer binding protein α (C/EBPα) is critical for normal and leukemic differentiation, its role in cell and metabolic homeostasis is largely unknown in cancer. Here, multiomics analyses uncovered a coordinated activation of C/EBPα and Fms-like tyrosine kinase 3 (FLT3) that increased lipid anabolism in vivo and in patients with FLT3-mutant acute myeloid leukemia (AML). Mechanistically, C/EBPα regulated the fatty acid synthase (FASN)-stearoyl-CoA desaturase (SCD) axis to promote fatty acid (FA) biosynthesis and desaturation. We further demonstrated that FLT3 or C/EBPα inactivation decreased monounsaturated FA incorporation to membrane phospholipids through SCD downregulation. Consequently, SCD inhibition enhanced susceptibility to lipid redox stress that was exploited by combining FLT3 and glutathione peroxidase 4 inhibition to trigger lipid oxidative stress, enhancing ferroptotic death of FLT3-mutant AML cells. Altogether, our study reveals a C/EBPα function in lipid homeostasis and adaptation to redox stress, and a previously unreported vulnerability of FLT3-mutant AML to ferroptosis with promising therapeutic application. SIGNIFICANCE: FLT3 mutations are found in 30% of AML cases and are actionable by tyrosine kinase inhibitors. Here, we discovered that C/EBPα regulates FA biosynthesis and protection from lipid redox stress downstream mutant-FLT3 signaling, which confers a vulnerability to ferroptosis upon FLT3 inhibition with therapeutic potential in AML. This article is highlighted in the In This Issue feature, p. 1501.

Metabolomic profiling of cardiac allografts after controlled circulatory death.

BACKGROUND: Assessment of myocardial viability during ex situ heart perfusion (ESHP) is based on the measurement of lactate concentrations. As this provides with limited information, we sought to investigate the metabolic signature associated with donation after circulatory death (DCD) and the impact of ESHP on the myocardial metabolome. METHODS: Porcine hearts were retrieved either after warm ischemia (DCD group, N = 6); after brain-stem death (BSD group, N = 6); or without DCD nor BSD (Control group, N = 6). Hearts were perfused using normothermic oxygenated blood for 240 minutes. Plasma and myocardial samples were collected respectively every 30 and 60 minutes, and analyzed by an untargeted metabolomic approach using liquid chromatography coupled to high-resolution mass spectrometry. RESULTS: Median duration of warm ischemia was 23 minutes [19-29] in DCD animals. Lactate level within myocardial biopsies was not significantly different between groups at T0 (p = 0.281), and remained stable over the 4-hour period of ESHP. More than 300 metabolites were detected in plasma and heart biopsy samples. Compared to BSD animals, metabolomics changes involving energy and nucleotide metabolisms were observed in plasma samples of DCD animals before initiation of ESHP, whereas 2 metabolites (inosine monophosphate and methylbutyrate) exhibited concentration changes in biopsy samples. Normalization of DCD metabolic profile was remarkable after 4 hours of ESHP. CONCLUSION: A specific metabolic profile was observed in DCD hearts, mainly characterized by an increased nucleotide catabolism. DCD and BSD metabolomes proved normalized during ESHP. Complementary investigations are needed to correlate these findings to cardiac performances.

Correlative radioimaging and mass spectrometry imaging: a powerful combination to study 14C-graphene oxide in vivo biodistribution.

Research on graphene based nanomaterials has flourished in the last decade due their unique properties and emerging socio-economic impact. In the context of their potential exploitation for biomedical applications, there is a growing need for the development of more efficient imaging techniques to track the fate of these materials. Herein we propose the first correlative imaging approach based on the combination of radioimaging and mass spectrometry imaging for the detection of Graphene Oxide (GO) labelled with carbon-14 in mice. In this study, 14C-graphene oxide nanoribbons were produced from the oxidative opening of 14C-carbon nanotubes, and were then intensively sonicated to provide nano-size 14C-GO flakes. After Intravenous administration in mice, 14C-GO distribution was quantified by radioimaging performed on tissue slices. On the same slices, MS-imaging provided a highly resolved distribution map of the nanomaterial based on the detection of specific radical anionic carbon clusters ranging from C2˙- to C9˙- with a base peak at m/z 72 (12C) and 74 (14C) under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. This proof of concept approach synergizes the strength of each technique and could be advantageous in the pre-clinical development of future Graphene-based biomedical applications.

Relevance of the TRIAP1/p53 axis in colon cancer cell proliferation and adaptation to glutamine deprivation.

Human TRIAP1 (TP53-regulated inhibitor of apoptosis 1; also known as p53CSV for p53-inducible cell survival factor) is the homolog of yeast Mdm35, a well-known chaperone that interacts with the Ups/PRELI family proteins and participates in the intramitochondrial transfer of lipids for the synthesis of cardiolipin (CL) and phosphatidylethanolamine. Although recent reports indicate that TRIAP1 is a prosurvival factor abnormally overexpressed in various types of cancer, knowledge about its molecular and metabolic function in human cells is still elusive. It is therefore critical to understand the metabolic and proliferative advantages that TRIAP1 expression provides to cancer cells. Here, in a colorectal cancer cell model, we report that the expression of TRIAP1 supports cancer cell proliferation and tumorigenesis. Depletion of TRIAP1 perturbed the mitochondrial ultrastructure, without a major impact on CL levels and mitochondrial activity. TRIAP1 depletion caused extramitochondrial perturbations resulting in changes in the endoplasmic reticulum-dependent lipid homeostasis and induction of a p53-mediated stress response. Furthermore, we observed that TRIAP1 depletion conferred a robust p53-mediated resistance to the metabolic stress caused by glutamine deprivation. These findings highlight the importance of TRIAP1 in tumorigenesis and indicate that the loss of TRIAP1 has extramitochondrial consequences that could impact on the metabolic plasticity of cancer cells and their response to conditions of nutrient deprivation.

A re-calibration procedure for interoperable lipid collision cross section values measured by traveling wave ion mobility spectrometry.

Collision cross sections (CCS) have been described as relevant molecular descriptors in metabolomics and lipidomics analyses for ascertaining compound identity. Ion mobility spectrometry (IMS) allows to determine CCS with different techniques, such as drift tube ion mobility spectrometry (DTIMS), traveling wave ion mobility spectrometry (TWIMS) or trapped ion mobility spectrometry (TIMS). In contrast with DTIMS where CCS can be obtained directly with measured drift times and mathematical relationship, TWIMS and TIMS techniques require an additional step of calibration to obtain CCS values. However, literature reports significantly disparate CCS values depending on the calibrant used (often more than 10%), as no consensus has been reached to define a universal CCS reference standard or harmonized calibration procedure. Therefore, publicly available CCS databases cannot be regarded as readily interoperable and exchangeable. Here, we performed a comprehensive evaluation of 11 distinct CCS calibrants in a traveling wave ion mobility spectrometry-mass spectrometry (TWIMS-MS) instrument. We showed that, using lipids from plasma as model compounds, CCS determination drastically fluctuates from one calibrant to the other with up to 25% differences, which precludes direct CCS comparison. Using the large panel of calibration curves generated, we showed that any CCS value can be efficiently re-calibrated relatively to the calibration curve made with the widely used Tune Mix solution whatever the calibration procedure originally used. The re-calibrated CCS values for each calibrant constitute a database which allows to correct any deviation on lipid CCS values whatever the calibrant originally used. Resulting corrected CCS values from plasma lipids were thus efficiently matched to those previously reported in the literature (with deviations<2%). Therefore, this work shows that unique and comparable CCS values can be obtained upon re-calibration relatively to Tune Mix CCS values, while also paving the way for the establishment of a universal CCS database of various metabolite or lipid classes.

Mitochondrial dysfunction governs immunometabolism in leukocytes of patients with acute-on-chronic liver failure.

BACKGROUND & AIMS: Patients with acute-on-chronic liver failure (ACLF) present a systemic hyperinflammatory response associated with increased circulating levels of small-molecule metabolites. To investigate whether these alterations reflect inadequate cell energy output, we assessed mitochondrial morphology and central metabolic pathways with emphasis on the tricarboxylic acid (TCA) cycle in peripheral leukocytes from patients with acutely decompensated (AD) cirrhosis, with and without ACLF. METHODS: The study included samples from patients with AD cirrhosis (108 without and 128 with ACLF) and 41 healthy individuals. Leukocyte mitochondrial ultrastructure was visualized by transmission electron microscopy and cytosolic and mitochondrial metabolic fluxes were determined by assessing NADH/FADH2 production from various substrates. Plasma GDF15 and FGF21 were determined by Luminex and acylcarnitines by LC-MS/MS. Gene expression was analyzed by RNA-sequencing and PCR-based glucose metabolism profiler array. RESULTS: Mitochondrial ultrastructure in patients with advanced cirrhosis was distinguished by cristae rarefication and swelling. The number of mitochondria per leukocyte was higher in patients, accompanied by a reduction in their size. Increased FGF21 and C6:0- and C8:0-carnitine predicted mortality whereas GDF15 strongly correlated with a gene set signature related to leukocyte activation. Metabolic flux analyses revealed increased energy production in mononuclear leukocytes from patients with preferential involvement of extra-mitochondrial pathways, supported by upregulated expression of genes encoding enzymes of the glycolytic and pentose phosphate pathways. In patients with ACLF, mitochondrial function analysis uncovered break-points in the TCA cycle at the isocitrate dehydrogenase and succinate dehydrogenase level, which were bridged by anaplerotic reactions involving glutaminolysis and nucleoside metabolism. CONCLUSIONS: Our findings provide evidence at the cellular, organelle and biochemical levels that severe mitochondrial dysfunction governs immunometabolism in leukocytes from patients with AD cirrhosis and ACLF. LAY SUMMARY: Patients at advanced stages of liver disease have dismal prognosis due to vital organ failures and the lack of treatment options. In this study, we report that the functioning of mitochondria, which are known as the cell powerhouse, is severely impaired in leukocytes of these patients, probably as a consequence of intense inflammation. Mitochondrial dysfunction is therefore a hallmark of advanced liver disease.

Metabolomics in the understanding and management of hepatic encephalopathy.

Metabolomics refers to the study of biological components below 1000 Daltons (Da) involved in metabolic pathways as substrates, products or effectors. According to the interconnected metabolic disturbances that have been described in the pathophysiology of hepatic encephalopathy (HE), this technique appears to be well adapted to study and better delineate the disease. This review will focus on recent advances in metabolomics in the field of HE. Thus, after a brief overview of the general principles of metabolomics, we will discuss metabolomics as a potentially efficient tool for unraveling new HE pathophysiological insights, biomarkers identification, or as a predicting tool for treatment response or outcome prognosis. Finally, we will give our vision on the prospects offered by metabolomics for improving care of HE patients.

Right Ventricle Remodeling Metabolic Signature in Experimental Pulmonary Hypertension Models of Chronic Hypoxia and Monocrotaline Exposure.

INTRODUCTION: Over time and despite optimal medical management of patients with pulmonary hypertension (PH), the right ventricle (RV) function deteriorates from an adaptive to maladaptive phenotype, leading to RV failure (RVF). Although RV function is well recognized as a prognostic factor of PH, no predictive factor of RVF episodes has been elucidated so far. We hypothesized that determining RV metabolic alterations could help to understand the mechanism link to the deterioration of RV function as well as help to identify new biomarkers of RV failure. METHODS: In the current study, we aimed to characterize the metabolic reprogramming associated with the RV remodeling phenotype during experimental PH induced by chronic-hypoxia-(CH) exposure or monocrotaline-(MCT) exposure in rats. Three weeks after PH initiation, we hemodynamically characterized PH (echocardiography and RV catheterization), and then we used an untargeted metabolomics approach based on liquid chromatography coupled to high-resolution mass spectrometry to analyze RV and LV tissues in addition to plasma samples from MCT-PH and CH-PH rat models. RESULTS: CH exposure induced adaptive RV phenotype as opposed to MCT exposure which induced maladaptive RV phenotype. We found that predominant alterations of arginine, pyrimidine, purine, and tryptophan metabolic pathways were detected on the heart (LV+RV) and plasma samples regardless of the PH model. Acetylspermidine, putrescine, guanidinoacetate RV biopsy levels, and cytosine, deoxycytidine, deoxyuridine, and plasmatic thymidine levels were correlated to RV function in the CH-PH model. It was less likely correlated in the MCT model. These pathways are well described to regulate cell proliferation, cell hypertrophy, and cardioprotection. These findings open novel research perspectives to find biomarkers for early detection of RV failure in PH.

Untargeted lipidomics uncovers lipid signatures that distinguish severe from moderate forms of acutely decompensated cirrhosis.

BACKGROUND & AIMS: Acute decompensation (AD) of cirrhosis is a heterogeneous clinical entity associated with moderate mortality. In some patients, this condition develops quickly into the more deadly acute-on-chronic liver failure (ACLF), in which other organs such as the kidneys or brain fail. The aim of this study was to characterize the blood lipidome in a large series of patients with cirrhosis and identify specific signatures associated with AD and ACLF development. METHODS: Serum untargeted lipidomics was performed in 561 patients with AD (518 without and 43 with ACLF) (discovery cohort) and in 265 patients with AD (128 without and 137 with ACLF) in whom serum samples were available to perform repeated measurements during the 28-day follow-up (validation cohort). Analyses were also performed in 78 patients with AD included in a therapeutic albumin trial (43 patients with compensated cirrhosis and 29 healthy individuals). RESULTS: The circulating lipid landscape associated with cirrhosis was characterized by a generalized suppression, which was more manifest during AD and in non-surviving patients. By computing discriminating accuracy and the variable importance projection score for each of the 223 annotated lipids, we identified a sphingomyelin fingerprint specific for AD of cirrhosis and a distinct cholesteryl ester and lysophosphatidylcholine fingerprint for ACLF. Liver dysfunction and infections were the principal net contributors to these fingerprints, which were dynamic and interchangeable between patients with AD whose condition worsened to ACLF and those who improved. Notably, blood lysophosphatidylcholine levels increased in these patients after albumin therapy. CONCLUSIONS: Our findings provide insights into the lipid landscape associated with decompensation of cirrhosis and ACLF progression and identify unique non-invasive diagnostic biomarkers of advanced cirrhosis. LAY SUMMARY: Analysis of lipids in blood from patients with advanced cirrhosis reveals a general suppression of their levels in the circulation of these patients. A specific group of lipids known as sphingomyelins are useful to distinguish between patients with compensated and decompensated cirrhosis. Another group of lipids designated cholesteryl esters further distinguishes patients with decompensated cirrhosis who are at risk of developing organ failures.

Multiplatform metabolomics for an integrative exploration of metabolic syndrome in older men.

BACKGROUND: Metabolic syndrome (MetS), a cluster of factors associated with risks of developing cardiovascular diseases, is a public health concern because of its growing prevalence. Considering the combination of concomitant components, their development and severity, MetS phenotypes are largely heterogeneous, inducing disparity in diagnosis. METHODS: A case/control study was designed within the NuAge longitudinal cohort on aging. From a 3-year follow-up of 123 stable individuals, we present a deep phenotyping approach based on a multiplatform metabolomics and lipidomics untargeted strategy to better characterize metabolic perturbations in MetS and define a comprehensive MetS signature stable over time in older men. FINDINGS: We characterize significant changes associated with MetS, involving modulations of 476 metabolites and lipids, and representing 16% of the detected serum metabolome/lipidome. These results revealed a systemic alteration of metabolism, involving various metabolic pathways (urea cycle, amino-acid, sphingo- and glycerophospholipid, and sugar metabolisms…) not only intrinsically interrelated, but also reflecting environmental factors (nutrition, microbiota, physical activity…). INTERPRETATION: These findings allowed identifying a comprehensive MetS signature, reduced to 26 metabolites for future translation into clinical applications for better diagnosing MetS. FUNDING: The NuAge Study was supported by a research grant from the Canadian Institutes of Health Research (CIHR; MOP-62842). The actual NuAge Database and Biobank, containing data and biologic samples of 1,753 NuAge participants (from the initial 1,793 participants), are supported by the Fonds de recherche du Québec (FRQ; 2020-VICO-279753), the Quebec Network for Research on Aging, a thematic network funded by the Fonds de Recherche du Québec - Santé (FRQS) and by the Merck-Frost Chair funded by La Fondation de l'Université de Sherbrooke. All metabolomics and lipidomics analyses were funded and performed within the metaboHUB French infrastructure (ANR-INBS-0010). All authors had full access to the full data in the study and accept responsibility to submit for publication.

Effect of gut microbiota on depressive-like behaviors in mice is mediated by the endocannabinoid system.

Depression is the leading cause of disability worldwide. Recent observations have revealed an association between mood disorders and alterations of the intestinal microbiota. Here, using unpredictable chronic mild stress (UCMS) as a mouse model of depression, we show that UCMS mice display phenotypic alterations, which could be transferred from UCMS donors to naïve recipient mice by fecal microbiota transplantation. The cellular and behavioral alterations observed in recipient mice were accompanied by a decrease in the endocannabinoid (eCB) signaling due to lower peripheral levels of fatty acid precursors of eCB ligands. The adverse effects of UCMS-transferred microbiota were alleviated by selectively enhancing the central eCB or by complementation with a strain of the Lactobacilli genus. Our findings provide a mechanistic scenario for how chronic stress, diet and gut microbiota generate a pathological feed-forward loop that contributes to despair behavior via the central eCB system.

High-dose biotin restores redox balance, energy and lipid homeostasis, and axonal health in a model of adrenoleukodystrophy.

Biotin is an essential cofactor for carboxylases that regulates the energy metabolism. Recently, high-dose pharmaceutical-grade biotin (MD1003) was shown to improve clinical parameters in a subset of patients with chronic progressive multiple sclerosis. To gain insight into the mechanisms of action, we investigated the efficacy of high-dose biotin in a genetic model of chronic axonopathy caused by oxidative damage and bioenergetic failure, the Abcd1- mouse model of adrenomyeloneuropathy. High-dose biotin restored redox homeostasis driven by NRF-2, mitochondria biogenesis and ATP levels, and reversed axonal demise and locomotor impairment. Moreover, we uncovered a concerted dysregulation of the transcriptional program for lipid synthesis and degradation in the spinal cord likely driven by aberrant SREBP-1c/mTORC1signaling. This resulted in increased triglyceride levels and lipid droplets in motor neurons. High-dose biotin normalized the hyperactivation of mTORC1, thus restoring lipid homeostasis. These results shed light into the mechanism of action of high-dose biotin of relevance for neurodegenerative and metabolic disorders.

Development of a Mass Spectrometry Imaging Method for Detecting and Mapping Graphene Oxide Nanoparticles in Rodent Tissues.

Graphene-based nanoparticles are continuously being developed for biomedical applications, and their use raises concerns about their environmental and biological impact. In the literature, some imaging techniques based on fluorescence and radioimaging have been used to explore their fate in vivo. Here, we report on the use of label-free mass spectrometry and mass spectrometry imaging (MSI) for graphene oxide (GO) and reduced graphene oxide (rGO) analyses in rodent tissues. Thereby, we extend previous work by focusing on practical questions to obtain reliable and meaningful images. Specific radical anionic carbon clusters ranging from C2-• to C9-• were observed for both GO and rGO species, with a base peak at m/z 72 under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. Extension to an LDI-MSI method was then performed, thus enabling the efficient detection of GO nanoparticles in lung tissue sections of previously exposed mice. The possibility of quantifying those nanoparticles on tissue sections has also been investigated. Two different ways of building calibration curves (i.e., GO suspensions spotted on tissue sections, or added to lung tissue homogenates) were evaluated and returned similar results, with linear dynamic concentration ranges over at least 2 orders of magnitude. Moreover, intra- and inter-day precision studies have been assessed, with relative standard deviation below 25% for each concentration point of a calibration curve. In conclusion, our study confirms that LDI-MSI is a relevant approach for biodistribution studies of carbon-based nanoparticles, as quantification can be achieved, provided that nanoparticle suspension and manufacturing are carefully controlled.

Improving lipid mapping in Genome Scale Metabolic Networks using ontologies.

INTRODUCTION: To interpret metabolomic and lipidomic profiles, it is necessary to identify the metabolic reactions that connect the measured molecules. This can be achieved by putting them in the context of genome-scale metabolic network reconstructions. However, mapping experimentally measured molecules onto metabolic networks is challenging due to differences in identifiers and level of annotation between data and metabolic networks, especially for lipids. OBJECTIVES: To help linking lipids from lipidomics datasets with lipids in metabolic networks, we developed a new matching method based on the ChEBI ontology. The implementation is freely available as a python library and in MetExplore webserver. METHODS: Our matching method is more flexible than an exact identifier-based correspondence since it allows establishing a link between molecules even if a different level of precision is provided in the dataset and in the metabolic network. For instance, it can associate a generic class of lipids present in the network with the molecular species detailed in the lipidomics dataset. This mapping is based on the computation of a distance between molecules in ChEBI ontology. RESULTS: We applied our method to a chemical library (968 lipids) and an experimental dataset (32 modulated lipids) and showed that using ontology-based mapping improves and facilitates the link with genome scale metabolic networks. Beyond network mapping, the results provide ways for improvements in terms of network curation and lipidomics data annotation. CONCLUSION: This new method being generic, it can be applied to any metabolomics data and therefore improve our comprehension of metabolic modulations.