N-acetylglucosamine supplementation fails to bypass the critical acetylation of glucosamine-6-phosphate required for Toxoplasma gondii replication and invasion.

The cell surface of Toxoplasma gondii is rich in glycoconjugates which hold diverse and vital functions in the lytic cycle of this obligate intracellular parasite. Additionally, the cyst wall of bradyzoites, that shields the persistent form responsible for chronic infection from the immune system, is heavily glycosylated. Formation of glycoconjugates relies on activated sugar nucleotides, such as uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). The glucosamine-phosphate-N-acetyltransferase (GNA1) generates N-acetylglucosamine-6-phosphate critical to produce UDP-GlcNAc. Here, we demonstrate that downregulation of T. gondii GNA1 results in a severe reduction of UDP-GlcNAc and a concomitant drop in glycosylphosphatidylinositols (GPIs), leading to impairment of the parasite's ability to invade and replicate in the host cell. Surprisingly, attempts to rescue this defect through exogenous GlcNAc supplementation fail to completely restore these vital functions. In depth metabolomic analyses elucidate diverse causes underlying the failed rescue: utilization of GlcNAc is inefficient under glucose-replete conditions and fails to restore UDP-GlcNAc levels in GNA1-depleted parasites. In contrast, GlcNAc-supplementation under glucose-deplete conditions fully restores UDP-GlcNAc levels but fails to rescue the defects associated with GNA1 depletion. Our results underscore the importance of glucosamine-6-phosphate acetylation in governing T. gondii replication and invasion and highlight the potential of the evolutionary divergent GNA1 in Apicomplexa as a target for the development of much-needed new therapeutic strategies.

Low-polarity untargeted metabolomic profiling as a tool to gain insight into seminal fluid.

INTRODUCTION: A decrease in sperm cell count has been observed along the last several decades, especially in the most developed regions of the world. The use of metabolomics to study the composition of the seminal fluid is a promising approach to gain access to the molecular mechanisms underlying this fact. OBJECTIVES: In the present work, we aimed at relating metabolomic profiles of young healthy men to their semen quality parameters obtained from conventional microscopic analysis. METHODS: An untargeted metabolomics approach focusing on low- to mid-polarity compounds was used to analyze a subset of seminal fluid samples from a cohort of over 2700 young healthy men. RESULTS: Our results show that a broad metabolic profiling comprising several families of compounds (including acyl-carnitines, steroids, and other lipids) can contribute to effectively distinguish samples provided by individuals exhibiting low or high absolute sperm counts. CONCLUSION: A number of metabolites involved in sexual development and function, signaling, and energy metabolism were highlighted as being distinctive of samples coming from either group, proving untargeted metabolomics as a promising tool to better understand the pathophysiological processes responsible for male fertility impairment.

Network principal component analysis: a versatile tool for the investigation of multigroup and multiblock datasets.

MOTIVATION: Complex data structures composed of different groups of observations and blocks of variables are increasingly collected in many domains, including metabolomics. Analysing these high-dimensional data constitutes a challenge, and the objective of this article is to present an original multivariate method capable of explicitly taking into account links between data tables when they involve the same observations and/or variables. For that purpose, an extension of standard principal component analysis called NetPCA was developed. RESULTS: The proposed algorithm was illustrated as an efficient solution for addressing complex multigroup and multiblock datasets. A case study involving the analysis of metabolomic data with different annotation levels and originating from a chronic kidney disease (CKD) study was used to highlight the different aspects and the additional outputs of the method compared to standard PCA. On the one hand, the model parameters allowed an efficient evaluation of each group's influence to be performed. On the other hand, the relative relevance of each block of variables to the model provided decisive information for an objective interpretation of the different metabolic annotation levels. AVAILABILITY AND IMPLEMENTATION: NetPCA is available as a Python package with NumPy dependencies. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Challenges in ESI-MS-based Untargeted Metabolomics.

Untargeted metabolomics is now widely recognized as a useful tool for exploring metabolic changes taking place in biological systems under different conditions. In this article, we aim to provide a short overview of the liquid-phase separation methods hyphenated to MS to perform untargeted metabolomics of biological samples. Each approach is complemented by up-to-date literature to guide readers, as well as practical information for avoiding or fixing some of the most frequently encountered pitfalls. This article covers mainly data acquisition, but a short discussion is provided regarding signal processing and data treatment, as well as data analysis and its biological interpretation in the context of metabolomic studies.

New insights into the conversion of electropherograms to the effective electrophoretic mobility scale.

CE-MS is increasingly gaining momentum as an analytical tool in metabolomics, due to its ability to obtain information about the most polar elements in biological samples. This has been helped by improvements of robustness in peak identification by means of mobility-scale representations of the electropherograms (mobilograms). As a necessary step toward facilitating the use of CE-MS for untargeted metabolomics data, the authors previously developed and introduced ROMANCE, a software automating mobilogram generation for large untargeted datasets through a simple and self-contained user interface. Herein, we introduce a new version of ROMANCE including new features such as compatibility with other types of data (targeted MS data and 2D UV-Vis absorption-like electropherograms), and the much needed additional flexibility in the transformation parameters (including field ramping and the use of secondary markers), more measurement conditions (depending on detection and integration modes), and most importantly tackling the issue of quantitative peak conversion. First, we present a review of the current theoretical framework with regard to peak characterization, and we develop new formulas for multiple marker peak area corrections, for anticipating peak position precision, and for assessing peak shape distortion. Then, the new version of the software is presented and validated experimentally. We contrast the multiple marker mobility transformations with previous results, finding increased peak position precision, and finally we showcase an application to actual untargeted metabolomics data.

Approaches in metabolomics for regulatory toxicology applications.

Innovative methodological approaches are needed to conduct human health and environmental risk assessments on a growing number of marketed chemicals. Metabolomics is progressively proving its value as an efficient strategy to perform toxicological evaluations of new and existing substances, and it will likely become a key tool to accelerate chemical risk assessments. However, additional guidance with widely accepted and harmonized procedures is needed before metabolomics can be routinely incorporated in decision-making for regulatory purposes. The aim of this review is to provide an overview of metabolomic strategies that have been successfully employed in toxicity assessment as well as the most promising workflows in a regulatory context. First, we provide a general view of the different steps of regulatory toxicology-oriented metabolomics. Emphasis is put on three key elements: robustness of experimental design, choice of analytical platform, and use of adapted data treatment tools. Then, examples in which metabolomics supported regulatory toxicology outputs in different scenarios are reviewed, including chemical grouping, elucidation of mechanisms of toxicity, and determination of points of departure. The overall intention is to provide insights into why and how to plan and conduct metabolomic studies for regulatory toxicology purposes.

Neuroinflammatory Response to TNFα and IL1ß Cytokines Is Accompanied by an Increase in Glycolysis in Human Astrocytes In Vitro.

Astrogliosis has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism, and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes as a function of age have been reported, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected in supporting a functional switch of astrocytes from neurotrophic to neurotoxic. This study aimed to explore the metabolic changes occurring in astrocytes during their activation. Astrocytes were derived from human ReN cell neural progenitors and characterized. They were activated by exposure to tumor necrosis factor alpha (TNFα) or interleukin 1ß (IL1ß) for 24 h. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics and extracellular flux analyses. ReN-derived astrocytes reactivity was observed by the modifications of genes and proteins linked to inflammation (cytokines, nuclear factor-kappa B (NFκB), signal transducers and activators of transcription (STATs)) and immune pathways (major histocompatibility complex (MHC) class I). Increased NFκB1, NFκB2 and STAT1 expression, together with decreased STAT3 expression, suggest an activation towards the detrimental pathway. Strong modifications of astrocyte cytoskeleton were observed, including a glial fibrillary acidic protein (GFAP) decrease. Astrogliosis was accompanied by changes in energy metabolism characterized by increased glycolysis and lactate release. Increased glycolysis is reported for the first time during human astrocyte activation. Astrocyte activation is strongly tied to energy metabolism, and a possible association between NFκB signaling and/or MHC class I pathway and glycolysis is suggested.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.

Evolution in the design of a low sheath-flow interface for CE-MS and application to biological samples.

Although several interfaces for CE-MS hyphenation are commercially available, the development of new versatile, simple and yet efficient and sensitive alternatives remains an important field of research. In a previous work, a simple low sheath-flow interface was developed from inexpensive parts. This interface features a design easy to build, maintain, and adapt to particular needs. The present work introduces an improved design of the previous interface. By reducing the diameter of the separation capillary and the emitter, a smaller Taylor cone is spontaneously formed, minimizing the zone dispersion while the analytes go through the interface and leading to less peak broadening associated to the ESI process. Numerical modeling allowed studying the mixing and diffusion processes taking place in the Taylor cone. The analytical performance of this new interface was tested with pharmaceutically relevant molecules and endogenous metabolites. The interface was eventually applied to the analysis of neural cell culture samples, allowing the identification of a panel of neurotransmission-related molecules. An excellent migration time repeatability was obtained (intra-day RSD <0.5% for most compounds, and <3.0% for inter-day precision). Most metabolites showed S/N ratios >10 with an injected volume of 6.7 nL of biological extract.

ROMANCE: A new software tool to improve data robustness and feature identification in CE-MS metabolomics.

The use of capillary electrophoresis coupled to mass spectrometry (CE-MS) in metabolomics remains an oddity compared to the widely adopted use of liquid chromatography. This technique is traditionally regarded as lacking the reproducibility to adequately identify metabolites by their migration times. The major reason is the variability of the velocity of the background electrolyte, mainly coming from shifts in the magnitude of the electroosmotic flow and from the suction caused by electrospray interfaces. The use of the effective electrophoretic mobility is one solution to overcome this issue as it is a characteristic feature of each compound. To date, such an approach has not been applied to metabolomics due to the complexity and size of CE-MS data obtained in such studies. In this paper, ROMANCE (RObust Metabolomic Analysis with Normalized CE) is introduced as a new software for CE-MS-based metabolomics. It allows the automated conversion of batches of CE-MS files with minimal user intervention. ROMANCE converts the x-axis of each MS file from the time into the effective mobility scale and the resulting files are already pseudo-aligned, present normalized peak areas and improved reproducibility, and can eventually follow existing metabolomic workflows. The software was developed in Scala, so it is multi-platform and computationally-efficient. It is available for download under a CC license. In this work, the versatility of ROMANCE was demonstrated by using data obtained in the same and in different laboratories, as well as its application to the analysis of human plasma samples.

Evaluation of a new low sheath-flow interface for CE-MS.

The current trend for increasing technical complexity in the field of CE-ESI-MS interfaces has incited for more accessible alternatives. In this work, a simple low sheath-flow ESI interface operating in the submicroliter nanospray regime without nebulizing gas assistance was evaluated. The use of sheath liquid enabled improving the ionization of the analytes, while the absence of nebulizing gas minimized sample dilution and loss of efficiency. After a rapid qualification, the effect of main operational parameters such as sheath liquid composition and flow rate, working distance and ESI potential was studied. Simulation of the mixing processes inside the Taylor cone proved its size to be of utmost importance in band broadening processes. As a proof of concept, the interface was eventually applied to a set of representative basic drugs analyzed by CE-TOF/MS. Limits of detection reached the 25-100 ppb range with suitable robustness and repeatability results. This design has demonstrated good performance while being simple and accessible to the user.

Activation of endogenous glucocorticoids by HSD11B1 inhibits the antitumor immune response in renal cancer.

Although immune-based therapies have revolutionized the management of cancer, novel approaches are urgently needed to improve their outcome. We investigated the role of endogenous steroids in the resistance to cancer immunotherapy, as these have strong immunomodulatory functions. Using a publicly available database, we found that the intratumoral expression of 11 beta-hydroxysteroid dehydrogenase type 1 (HSD11B1), which regenerates inactive glucocorticoids into active glucocorticoids, was associated with poor clinical outcome and correlated with immunosuppressive gene signatures in patients with renal cell carcinoma (RCC). HSD11B1 was mainly expressed in tumor-infiltrating immune myeloid cells as seen by immunohistochemistry in RCC patient samples. Using peripheral blood mononuclear cells from healthy donors or immune cells isolated from the tumor of RCC patients, we showed that the pharmacological inhibition of HSD11B1 improved the response to the immune checkpoint inhibitor anti-PD-1. In a subcutaneous mouse model of renal cancer, the combination of an HSD11B1 inhibitor with anti-PD-1 treatment increased the proportion of tumor-infiltrating dendritic cells. In an intrarenal mouse tumor model, HSD11B1 inhibition increased the survival of mice treated with anti-PD-1. In addition, inhibition of HSD11B1 sensitized renal tumors in mice to immunotherapy with resiquimod, a Toll-like receptor 7 agonist. Mechanistically, we demonstrated that HSD11B1 inhibition combined with resiquimod increased T cell-mediated cytotoxicity to tumor cells by stimulating the antigen-presenting capacity of dendritic cells. In conclusion, these results support the use of HSD11B1 inhibitors to improve the outcome of immunotherapy in renal cancer and highlight the role of the endogenous glucocorticoid metabolism in the efficacy of immunotherapy.

Investigation of several chromatographic approaches for untargeted profiling of central carbon metabolism.

Monitoring the central carbon metabolism (CCM) network using liquid chromatography/mass spectrometry (LC-MS) analysis is hampered by the diverse chemical nature of its analytes, which are extremely difficult to analyze using single chromatographic conditions. Furthermore, CCM-related compounds present non-specific adsorption on metal surfaces, causing detrimental chromatographic effects and sensitivity loss. In this study, polar reversed-phase, mixed-mode (MMC), and zwitterionic hydrophilic interaction chromatography (HILIC) featuring low-adsorption hardware were investigated towards untargeted analysis of biological samples with a focus on energy metabolism-related analytes. Best results were achieved with sulfoalkylbetaine HILIC with different supports, where polymeric option featured the highest coverage and inert hybrid silica facilitated best throughput and kinetic performance at a cost of less selectivity for small carboxylic acids. MMC demonstrated excellent performance for strongly anionic analytes such as multiresidue phosphates. The obtained experimental data also suggested that an additional hydrophilic modulation might be necessary to facilitate better resolution of carboxylic acids in zHILIC mode, as found during the application of the developed method to study the effect of two different mutations on the energy metabolism of S. aureus.

Digoxin Induces Human Astrocyte Reaction In Vitro.

Astrocyte reaction is a complex cellular process involving astrocytes in response to various types of CNS injury and a marker of neurotoxicity. It has been abundantly studied in rodents but relatively poorly in human cells due to limited access to the brain. Astrocytes play important roles in cerebral energy metabolism and are also key players in neuroinflammation. Astroglial metabolic and inflammatory changes have been reported with age, leading to the hypothesis that mitochondrial metabolism and inflammatory responses are interconnected. However, the relationship between energy metabolism and astrocyte reactivity in the context of neurotoxicity is not known. We hypothesized that changes in energy metabolism of astrocytes will be coupled to their activation by xenobiotics. Astrocyte reaction and associated energy metabolic changes were assessed by immunostaining, gene expression, proteomics, metabolomics, and extracellular flux analyses after 24 h of exposure of human ReN-derived astrocytes to digoxin (1-10 µM) or TNFα (30 ng/ml) used as a positive control. Strong astrocytic reaction was observed, accompanied by increased glycolysis at low concentrations of digoxin (0.1 and 0.5 µM) and after TNFα exposure, suggesting that increased glycolysis may be a common feature of reactive astrocytes, independent of the triggering molecule. In conclusion, whether astrocyte activation is triggered by cytokines or a xenobiotic, it is strongly tied to energy metabolism in human ReN-derived astrocytes. Increased glycolysis might be considered as an endpoint to detect astrocyte activation by potentially neurotoxic compounds in vitro. Finally, ReN-derived astrocytes may help to decipher mechanisms of neurotoxicity in ascertaining the ability of chemicals to directly target astrocytes.

Extended steroid profiling in H295R cells provides deeper insight into chemical-induced disturbances of steroidogenesis: Exemplified by prochloraz and anabolic steroids.

Human adrenocortical H295R cells have been validated by the OECD Test Guideline 456 to detect chemicals disrupting testosterone and 17ß-estradiol (estradiol) biosynthesis. This study evaluated a novel approach to detect disturbances of steroidogenesis in H295R cells, exemplified by prochloraz and five anabolic steroids. Steroid profiles were assessed by an untargeted LC-MS-based method, providing a relative quantification of 57 steroids annotated according to their accurate masses and retention times. Such a panel of steroids included several mineralocorticoids, glucocorticoids, progestins and adrenal androgens. The coverage of a high number of metabolites in this extended steroid profiling facilitated grouping of chemicals with similar effects and detecting subtler differences between chemicals. It allowed, for example, distinguishing between the effects of turinabol and oxymetholone, supposed to act similarly in a previous characterization including only nine adrenal steroids. Furthermore, the results revealed that product/substrate ratios can provide superior information on altered enzyme activities compared to individual metabolite levels. For example, the 17α-hydroxypregnenolone/pregnenolone ratio was found to be a more sensitive marker for detecting 17α-hydroxylase inhibition by prochloraz than the corresponding individual steroids. These results illustrate that chemical grouping and calculation of product/substrate ratios can provide valuable information on mode-of-action and help prioritizing further experimental work.

Evaluation of Prototype CE-MS Interfaces.

Capillary electrophoresis-mass spectrometry (CE-MS) coupling is a powerful analytical solution bringing together the separation power of CE and the wealth of chemical information afforded by MS. Nevertheless, interfaces making the hyphenation of both techniques possible have always been the subject of a quest for improvement by their users in search for more sensitive and robust setups. This fact has led to numerous technical developments and new interface designs claiming to outrival existing approaches in different aspects. Nevertheless, the task of evaluating and comparing a new interface to previous solutions is not always straightforward. Issued from our own experience in the field, we herein propose a protocol to optimize the operation parameters of a new CE-MS interface design, assess its analytical performance, and compare it to a reference interface if desired. Electrospray stability, sensitivity, reproducibility, and robustness are practically evaluated as key elements of the process.

Separation and determination of cysteine enantiomers in plasma after derivatization with 4-fluoro-7-nitrobenzofurazan.

The importance of D-amino acids in mammals associated with enantio-dependent biological functions has been increasingly highlighted. In addition to naturally occurring, D-amino acid supplementation could have a positive biological impact, including cytoprotective implications. In this context, supplementation with D-cysteine has revealed beneficial effects. Quantification of cysteine enantiomers in rodent plasma has been achieved by using 4-fluoro-7-nitrobenzofurazan derivatization of the target analytes. Cystine, the main form of cysteine in the plasma, was initially reduced to cysteine using DL-dithiothreitol. Baseline enantioseparation was then achieved in less than 3 min using a (R,R)-Whelk-O 1 stationary phase and isocratic elution using CH3OH-H2O 90:10 (v/v) with 15 mM ammonium formate (apparent pH 6.0) at 0.5 mL/min. The derivatives were then detected using negative ESI-MS in SRM mode. An external calibration was employed for D-cysteine, while L-cysteine quantification, as an endogenous analyte, was addressed using a background subtraction strategy. The method was validated. Response functions were obtained from 0 to 300 µM and from 0 to 125 µM for D-cysteine and L-cysteine, respectively. The trueness ranged from 96% to 105% for both enantiomers with repeatability and intermediate precision lower than 8% and 15% for the D-form and the endogenous L-form, respectively. The method was successfully applied for determining D- and L-cysteine in mouse plasma after D-cysteine administration.

Fluorescence Sensors Based on Hydroxycarbazole for the Determination of Neurodegeneration-Related Halide Anions.

The environmental presence of anions of natural origin or anthropogenic origin is gradually increasing. As a tool to tackle this problem, carbazole derivatives are an attractive gateway to the development of luminescent chemosensors. Considering the different mechanisms proposed for anion recognition, the fluorescence properties and anion-binding response of several newly synthesised carbazole derivatives were studied. Potential anion sensors were designed so that they combined the native fluorescence of carbazole with the presence of hydrogen bonding donor groups in critical positions for anion recognition. These compounds were synthesised by a feasible and non-expensive procedure using palladium-promoted cyclodehydrogenation of suitable diarylamine under microwave irradiation. In comparison to the other carbazole derivatives studied, 1-hydroxycarbazole proved to be useful as a fluorescent sensor for anions, as it was able to sensitively recognise fluoride and chloride anions by establishing hydrogen bond interactions through the hydrogen atoms on the pyrrolic nitrogen and the hydroxy group. Solvent effects and excited-state proton transfer (ESPT) of the carbazole derivatives are described to discard the role of the anions as Brönsted bases on the observed fluorescence behaviour of the sensors. The anion-sensor interaction was confirmed by 1H-NMR. Molecular modelling was employed to propose a mode of recognition of the sensor in terms of complex stability and interatomic distances. 1-hydroxycarbazole was employed for the quantitation of fluoride and chloride anions in commercially available medicinal spring water and mouthwash samples.

Oxidative Stress and Extracellular Matrix Remodeling Are Signature Pathways of Extracellular Vesicles Released upon Morphine Exposure on Human Brain Microvascular Endothelial Cells.

Morphine, a commonly used antinociceptive drug in hospitals, is known to cross the blood-brain barrier (BBB) by first passing through brain endothelial cells. Despite its pain-relieving effect, morphine also has detrimental effects, such as the potential induction of redox imbalance in the brain. However, there is still insufficient evidence of these effects on the brain, particularly on the brain endothelial cells and the extracellular vesicles that they naturally release. Indeed, extracellular vesicles (EVs) are nanosized bioparticles produced by almost all cell types and are currently thought to reflect the physiological state of their parent cells. These vesicles have emerged as a promising source of biomarkers by indicating the functional or dysfunctional state of their parent cells and, thus, allowing a better understanding of the biological processes involved in an adverse state. However, there is very little information on the morphine effect on human brain microvascular endothelial cells (HBMECs), and even less on their released EVs. Therefore, the current study aimed at unraveling the detrimental mechanisms of morphine exposure (at 1, 10, 25, 50 and 100 µM) for 24 h on human brain microvascular endothelial cells as well as on their associated EVs. Isolation of EVs was carried out using an affinity-based method. Several orthogonal techniques (NTA, western blotting and proteomics analysis) were used to validate the EVs enrichment, quality and concentration. Data-independent mass spectrometry (DIA-MS)-based proteomics was applied in order to analyze the proteome modulations induced by morphine on HBMECs and EVs. We were able to quantify almost 5500 proteins in HBMECs and 1500 proteins in EVs, of which 256 and 148, respectively, were found to be differentially expressed in at least one condition. Pathway enrichment analysis revealed that the "cell adhesion and extracellular matrix remodeling" process and the "HIF1 pathway", a pathway related to oxidative stress responses, were significantly modulated upon morphine exposure in HBMECs and EVs. Altogether, the combination of proteomics and bioinformatics findings highlighted shared pathways between HBMECs exposed to morphine and their released EVs. These results put forward molecular signatures of morphine-induced toxicity in HBMECs that were also carried by EVs. Therefore, EVs could potentially be regarded as a useful tool to investigate brain endothelial cells dysfunction, and to a different extent, the BBB dysfunction in patient circulation using these "signature pathways".

Metabolomic- and Molecular Networking-Based Exploration of the Chemical Responses Induced in Citrus sinensis Leaves Inoculated with Xanthomonas citri.

Citrus canker, caused by the bacterium Xanthomonas citri subsp. citri (X. citri), is a plant disease affecting Citrus crops worldwide. However, little is known about defense compounds in Citrus. Here, we conducted a mass spectrometry-based metabolomic approach to obtain an overview of the chemical responses of Citrus leaves to X. citri infection. To facilitate result interpretation, the multivariate analyses were combined with molecular networking to identify biomarkers. Metabolite variations among untreated and X. citri-inoculated Citrus samples under greenhouse conditions highlighted induced defense biomarkers. Notably, the plant tryptophan metabolism pathway was activated, leading to the accumulation of N-methylated tryptamine derivatives. This finding was subsequently confirmed in symptomatic leaves in the field. Several tryptamine derivatives showed inhibitory effects in vitro against X. citri. This approach has enabled the identification of new chemically related biomarker groups and their dynamics in the response of Citrus leaves to Xanthomonas infection.