NMR metabolite quantification of a synthetic urine sample: an inter-laboratory comparison of processing workflows.

INTRODUCTION: Absolute quantification of individual metabolites in complex biological samples is crucial in targeted metabolomic profiling. OBJECTIVES: An inter-laboratory test was performed to evaluate the impact of the NMR software, peak-area determination method (integration vs. deconvolution) and operator on quantification trueness and precision. METHODS: A synthetic urine containing 32 compounds was prepared. One site prepared the urine and calibration samples, and performed NMR acquisition. NMR spectra were acquired with two pulse sequences including water suppression used in routine analyses. The pre-processed spectra were sent to the other sites where each operator quantified the metabolites using internal referencing or external calibration, and his/her favourite in-house, open-access or commercial NMR tool. RESULTS: For 1D NMR measurements with solvent presaturation during the recovery delay (zgpr), 20 metabolites were successfully quantified by all processing strategies. Some metabolites could not be quantified by some methods. For internal referencing with TSP, only one half of the metabolites were quantified with a trueness below 5%. With peak integration and external calibration, about 90% of the metabolites were quantified with a trueness below 5%. The NMRProcFlow integration module allowed the quantification of several additional metabolites. The number of quantified metabolites and quantification trueness improved for some metabolites with deconvolution tools. Trueness and precision were not significantly different between zgpr- and NOESYpr-based spectra for about 70% of the variables. CONCLUSION: External calibration performed better than TSP internal referencing. Inter-laboratory tests are useful when choosing to better rationalize the choice of quantification tools for NMR-based metabolomic profiling and confirm the value of spectra deconvolution tools.

PeakForest: a multi-platform digital infrastructure for interoperable metabolite spectral data and metadata management.

INTRODUCTION: Accuracy of feature annotation and metabolite identification in biological samples is a key element in metabolomics research. However, the annotation process is often hampered by the lack of spectral reference data in experimental conditions, as well as logistical difficulties in the spectral data management and exchange of annotations between laboratories. OBJECTIVES: To design an open-source infrastructure allowing hosting both nuclear magnetic resonance (NMR) and mass spectra (MS), with an ergonomic Web interface and Web services to support metabolite annotation and laboratory data management. METHODS: We developed the PeakForest infrastructure, an open-source Java tool with automatic programming interfaces that can be deployed locally to organize spectral data for metabolome annotation in laboratories. Standardized operating procedures and formats were included to ensure data quality and interoperability, in line with international recommendations and FAIR principles. RESULTS: PeakForest is able to capture and store experimental spectral MS and NMR metadata as well as collect and display signal annotations. This modular system provides a structured database with inbuilt tools to curate information, browse and reuse spectral information in data treatment. PeakForest offers data formalization and centralization at the laboratory level, facilitating shared spectral data across laboratories and integration into public databases. CONCLUSION: PeakForest is a comprehensive resource which addresses a technical bottleneck, namely large-scale spectral data annotation and metabolite identification for metabolomics laboratories with multiple instruments. PeakForest databases can be used in conjunction with bespoke data analysis pipelines in the Galaxy environment, offering the opportunity to meet the evolving needs of metabolomics research. Developed and tested by the French metabolomics community, PeakForest is freely-available at https://github.com/peakforest .

Cyclodextrins Allow the Combination of Incompatible Vancomycin and Ceftazidime into an Ophthalmic Formulation for the Treatment of Bacterial Keratitis.

Ceftazidime (CZ) and vancomycin (VA) are two antibiotics used to treat bacterial keratitis. Due to their physical incompatibility (formation of a precipitate), it is not currently possible to associate both molecules in a single container for ophthalmic administration. We firstly characterized the incompatibility then investigated if 2-hydroxypropyl-beta (HPßCD) and 2-hydroxypropyl-gamma cyclodextrins (HPγCD) could prevent this incompatibility. The impact of pH on the precipitation phenomena was investigated by analysing the supernatant solution of the mixture using high performance liquid chromatography. A characterization of the inclusion of CZ with HPγCD using 1H nuclear magnetic resonance (NMR), and VA with HPßCD using 1H-NMR and a solubility diagram was performed. A design of experiment was built to determine the optimal conditions to obtain a formulation that had the lowest turbidity and particle count. Our results showed that VA and CZ form an equimolar precipitate below pH 7.3. The best formulation obtained underwent an in-vitro evaluation of its antibacterial activity. The impact of HPCDs on incompatibility has been demonstrated through the inclusion of antibiotics and especially VA. The formulation has been shown to be able to inhibit the incompatibility for pH higher than 7.3 and to possess unaltered antibacterial activity.

Strengthening Peptoid Helicity through Sequence Site-Specific Positioning of Amide cis-Inducing NtBu Monomers.

The synthesis of biomimetic helical secondary structures is sought after for the construction of innovative nanomaterials and applications in medicinal chemistry such as the development of protein-protein interaction modulators. Peptoids, a sequence-defined family of oligomers, enable a peptidomimetic strategy, especially considering the easily accessible monomer diversity and peptoid helical folding propensity. However, cis-trans isomerization of the backbone tertiary amides may impair the peptoid's adoption of stable secondary structures, notably the all-cis polyproline I-like helical conformation. Here, we show that cis-inducing NtBu achiral monomers strategically positioned within chiral sequences may reinforce the degree of peptoid helicity, although with a reduced content of chiral side chains. The design principles presented here will undoubtedly help achieve more conformationally stable helical peptoids with desired functions.

A combined LC-MS and NMR approach to reveal metabolic changes in the hemolymph of honeybees infected by the gut parasite Nosema ceranae.

Nosema ceranae is an emerging and invasive gut pathogen in Apis mellifera and is considered as a factor contributing to the decline of honeybee populations. Here, we used a combined LC-MS and NMR approach to reveal the metabolomics changes in the hemolymph of honeybees infected by this obligate intracellular parasite. For metabolic profiling, hemolymph samples were collected from both uninfected and N. ceranae-infected bees at two time points, 2 days and 10 days after the experimental infection of emergent bees. Hemolymph samples were individually analyzed by LC-MS, whereas each NMR spectrum was obtained from a pool of three hemolymphs. Multivariate statistical PLS-DA models clearly showed that the age of bees was the parameter with the strongest effect on the metabolite profiles. Interestingly, a total of 15 biomarkers were accurately identified and were assigned as candidate biomarkers representative of infection alone or combined effect of age and infection. These biomarkers included carbohydrates (α/ß glucose, α/ß fructose and hexosamine), amino acids (histidine and proline), dipeptides (Glu-Thr, Cys-Cys and γ-Glu-Leu/Ile), metabolites involved in lipid metabolism (choline, glycerophosphocholine and O-phosphorylethanolamine) and a polyamine compound (spermidine). Our study demonstrated that this untargeted metabolomics-based approach may be useful for a better understanding of pathophysiological mechanisms of the honeybee infection by N. ceranae.

Quality of antiepileptic drugs in sub-Saharan Africa: A study in Gabon, Kenya, and Madagascar.

OBJECTIVE: Epilepsy is a major public health issue in low- and middle-income countries, where the availability and accessibility of quality treatment remain important issues, the severity of which may be aggravated by poor quality antiepileptic drugs (AEDs). The primary objective of this study was to measure the quality of AEDs in rural and urban areas in 3 African countries. METHODS: This cross-sectional study was carried out in Gabon, Kenya, and Madagascar. Both official and unofficial supply chains in urban and rural areas were investigated. Samples of oral AEDs were collected in areas where a patient could buy or obtain them. Pharmacological analytical procedures and Medicine Quality Assessment Reporting Guidelines were used to assess quality. RESULTS: In total, 102 batches, representing 3782 units of AEDs, were sampled. Overall, 32.3% of the tablets were of poor quality, but no significant difference was observed across sites: 26.5% in Gabon, 37.0% in Kenya, and 34.1% in Madagascar (P = .7). The highest proportions of substandard medications were found in the carbamazepine (38.7%; 95% confidence interval [CI] 21.8-57.8) and phenytoin (83.3%; 95% CI 35.8-99.5) batches, which were mainly flawed by their failure to dissolve. Sodium valproate was the AED with the poorest quality (32.1%; 95% CI 15.8-42.3). The phenobarbital (94.1%; 95% CI 80.3-99.2) and diazepam (100.0%) batches were of better quality. The prevalence of substandard quality medications increased in samples supplied by public facilities (odds ratio [OR] 9.9; 95% CI 1.2-84.1; P < .04) and manufacturers located in China (OR 119.8; 95% CI 8.7-1651.9; P < .001). The prevalence of AEDs of bad quality increased when they were stored improperly (OR 5.4; 95% CI 1.2-24.1; P < .03). SIGNIFICANCE: No counterfeiting was observed. However, inadequate AED storage conditions are likely to lead to ineffective and possibly dangerous AEDs, even when good-quality AEDs are initially imported.

Synthesis of Branched-Chain Sugars with a DHAP-Dependent Aldolase: Ketones are Electrophile Substrates of Rhamnulose-1-phosphate Aldolases.

Dihydroxyacetone phosphate (DHAP)-dependent rhamnulose aldolases display an unprecedented versatility for ketones as electrophile substrates. We selected and characterized a rhamnulose aldolase from Bacteroides thetaiotaomicron (RhuABthet) to provide a proof of concept. DHAP was added as a nucleophile to several α-hydroxylated ketones used as electrophiles. This aldol addition was stereoselective and produced branched-chain monosaccharide adducts with a tertiary alcohol moiety. Several aldols were readily obtained in good to excellent yields (from 76 to 95 %). These results contradict the general view that aldehydes are the only electrophile substrates for DHAP-dependent aldolases and provide a new C-C bond-forming enzyme for stereoselective synthesis of tertiary alcohols.

Metabolomic study of the response to cold shock in a strain of Pseudomonas syringae isolated from cloud water.

INTRODUCTION: Active microorganisms have been recently discovered in clouds, thus demonstrating the capacity of microorganisms to exist in harsh environments, including exposure to UV and oxidants, osmotic and cold shocks, etc. It is important to understand how microorganisms respond to and survive such stresses at the metabolic level. OBJECTIVES: The objective of this work is to assess metabolome modulation in a strain of Pseudomonas syringae isolated from cloud water and facing temperature downshift from 17 to 5 °C by identifying key molecules and pathways of the response/adaptation to cold shock. METHODS: Bacterial extracts from suspensions of cells grown at 17 °C and further incubated in microcosms at 5 and 17 °C to mimic cloud conditions were analysed by combining LC-MS and NMR; the results were evaluated in comparison to similar suspensions kept at constant temperature. The differences in the metabolome profiles were deciphered using multivariate statistics (PLS-DA). RESULTS: Key cold shock biomarkers were observed, including cryoprotectants (trehalose, glucose, glycerol, carnitine, glutamate), antioxidants (glutathione and carnitine) and their precursors, alkaloids (bellendine and slaframine) and metabolites involved in energy metabolism (ATP, carbohydrates). Furthermore, new short peptides (nine dipeptides and a tetrapeptide) were found that have no known function. CONCLUSIONS: This study shows that in response to cold temperatures, Pseudomonas syringae PDD-32b-74 demonstrates numerous metabolism modifications to counteract the impacts of low temperatures.

Study of sequential abiotic and biotic degradation of styrene butadiene rubber.

Styrene butadiene rubber is one of the main constituents of tire tread. During tire life, the tread material undergoes different stresses that impact its structure and chemical composition. Wear particles are then released into the environment as weathered material. To understand their fate, it is important to start with a better characterization of abiotic and biotic degradation of the elastomer material. A multi-disciplinary approach was implemented to study the photo- and thermo- degradation of non-vulcanized SBR films containing 15 w% styrene as well as their potential biodegradation by Rhodoccocus ruber and Gordonia polyisoprenivorans bacterial strains. Each ageing process leads to crosslinking reactions, much surface oxidation of the films and the production of hundreds of short chain compounds. These degradation products present a high level of unsaturation and oxidation and can be released into water to become potential substrates for microorganisms. Both strains were able to degrade from 0.2 to 1.2 % (% ThOD) of the aged SBR film after 30-day incubation while no biodegradation was observed on the pristine material. A 25-75 % decrease in the signal intensity of water extractable compounds was observed, suggesting that biomass production was linked to the consumption of low-molecular-weight degradation products. These results evidence the positive impact of abiotic degradation on the biodegradation process of styrene butadiene rubber.

Phototransformation of azoxystrobin fungicide in organic solvents. Photoisomerization vs. photodegradation.

Azoxystrobin is a systemic fungicide that has a tendency to accumulate at the surface of crop leaves or inside their cuticle where it undergoes photodegradation. Its photochemistry was investigated in n-heptane and isopropanol to mimic the polarity of wax leaves. Using analytical and kinetic data, we demonstrate that azoxystrobin (isomer E) undergoes efficient photoisomerization into the isomer Z with a quantum yield of 0.75 ± 0.08. This value is 30-fold higher than that reported in aqueous solution. The photoisomerization of isomer Z into azoxystrobin is more efficient with a chemical yield of 0.95 ± 0.1. In addition, a pseudo photostationary equilibrium is reached when the ratio [azoxystrobin]/[isomer Z] is 2.0 ± 0.1. Photodegradation also takes place from azoxystrobin (quantum yield = 0.073 ± 0.008). Photoproducts mainly arise from bond cleavage between rings and from demethylation of the ether with or without saturation of the acrylate double bond. Theoretical calculations were undertaken to investigate the photoisomerization mechanism and the solvent effect. These data show that the photochemical reactivity of azoxystrobin is enhanced when the solvent polarity decreases and thus should be significant in leaf waxes.

Assessing biodegradation of roadway particles via complementary mass spectrometry and NMR analyses.

Roadway particles (RP) that can be collected with on-vehicle system, consist of a mixture of Tire and road wear particles (TRWP) with other traffic-derived particles (exhaust or non-exhaust) and/or biogenic compounds and represent a significant source of xenobiotics, susceptible to reach the different environmental compartments. The study of the RP fate is thus a major challenge to tackle in order to understand their degradation and impact. They offer a variety of carbon sources potentially usable by microorganisms, ranging from the tire-derived plasticizers, vulcanizing agents, protective agents and their transformation products, to other traffic, road and environmental-derived contaminants. A multi-analytical approach was implemented to characterize RP and study their biodegradation. Kinetics of RP extractions were monitored during 21 days in water, methanol, acetone and chloroform to identify leaching and extractable compounds and monitor the particle composition. The results confirmed that hundreds of readily leachable chemicals can be extracted from RP directly into water according to a dynamic process with time while additional poorly soluble compounds remain in the particles. Mass spectrometry (LC-HRMS and GC-MS) allowed us to propose 296 putative compounds using an extensive rubber database. The capacity of 6 bacterial strains, belonging to Rhodococcus, Pseudomonas and Streptomyces genera, to biodegrade RP was then evaluated over 14 days of incubation. The selected strains were able to grow on RP using various substrates. Elastomer monitoring by 1H NMR revealed a significant 12 % decrease of the extractable SBR fraction when the particles were incubated with Rhodococcus ruber. After incubation, the biodegradation of 171 compounds among leachable and extractable compounds was evaluated. Fatty acids and alkanes from rubber plasticizers and paraffin waxes were the most degraded putative compounds by the six strains tested, reaching 75 % of biodegradation for some of them.

Reactive sputtering onto an ionic liquid, a new synthesis route for bismuth-based nanoparticles.

Metallic bismuth and Bi-oxyfluoride nanoparticles (NPs) are successfully synthesized by non-reactive and reactive sputtering of a Bi target onto 1-butyl-3-methylimidazolium bis(trifluoro-methylsulfonyl)imide ([BMIM][TFSI]) ionic liquid (IL). Non-reactive sputtering is realized in pure Ar plasma, where isotropic, well crystallized and dispersed Bi NPs of 3-7 nm are obtained. The diameter and the size distribution of these NPs do not significantly vary with the power, gas pressure, and sputtering time; but these sputtering parameters seem to influence the NP concentration. Then, the introduction of O2 and CF4 gases in addition to Ar enables the reaction of radicals from plasma with Bi clusters at the liquid's top surface to form Bi-oxyfluoride NPs of 3-12 nm in diameter with photocatalytic activity. Hence, the reactive sputtering onto an IL is an efficient, original and promising method for synthesizing Bi-based compound NPs. Finally, we propose a mechanism based on reactions of species from plasma at the IL surface to explain the formation of Bi-compounds by reactive sputtering.

Description of the Wild Strain Rhizobium rosettiformans DSM26376, Reclassified under Peteryoungia rosettiformans comb.nov., for Producing Glucuronan.

Glucuronan is a polysaccharide composed of ß-(1,4)-linked d-glucuronic acids having intrinsic properties and biological activities recoverable in many fields of application. Currently, the description of Sinorhyzobium meliloti M5N1CS mutant bacterial strain as the sole source of glucuronan makes it relevant to the exploration of new microorganisms producing glucuronan. In this study, the Peteryoungia rosettifformans strain (Rhizobia), was identified as a wild producer of an exopolysaccharide (RhrBR46) related to glucuronan. Structural and biochemical features, using colorimetric assays, Fourier infrared spectroscopy, nuclear magnetic resonance, high pressure size exclusion chromatography coupled to multi-angle light laser scattering, and enzymatic assays allowed the characterization of a polyglucuronic acid, having a molecular mass (Mw¯) of 1.85 × 105 Da, and being partially O-acetylated at C-2 and/or C-3 positions. The concentration of Mg2+ ions in the cultivation medium has been shown to impact the structure of RhrBR46, by reducing drastically its Mw¯ (73%) and increasing its DA (10%). Comparative structural analyses between RhrBR46 and the glucuronan from Sinorhyzobium meliloti M5N1CS strain revealed differences in terms of molecular weight, degree of acetylation (DA), and the distribution of acetylation pattern. These structural divergences of RhrBR46 might contribute to singular properties or biological activities of RhrBR46, offering new perspectives of application.

Primary structural features, physicochemical and biological properties of two water-soluble polysaccharides extracted from the brown Tunisian seaweed Halopteris scoparia.

Marine algae are the most abundant resource in the marine environment and are still a promising source of bioactive compounds including hydrocolloids. This study contributes to the evaluation of the biological and biotechnological potentials of two water soluble polysaccharides, namely alginates (AHS) and fucoidan (FHS), extracted and purified from Halopteris scoparia, an abundant Tunisian brown macroalgae collected in Tunisia (Tabarka region). The total sugars, neutral monosaccharides, uronic acids, proteins, polyphenols, and sulfate groups contents were quantified for both fractions, as well as their functional groups and primary structural features by Fourier transform infrared spectroscopy, ionic and/or gas chromatography and nuclear magnetic resonance analyses. AHS and FHS showed significant anti-inflammatory (IC50 ≈ 1 mg/mL), anticoagulant (e.g., 27-61.7 for the activated partial thromboplastin time), antihyperglycemic (0.1-40 µg/mL) and anti-trypsin (IC50 ≈ 0.3-0.4 mg/mL) effects. FHS and a hydrolyzed fraction showed a very promising potential against herpes viruses (HSV-1) (IC50 < 28 µg/mL). Besides, AHS and two hydrolyzed fractions were able to stimulate the natural defenses of tomato seedlings, assessing their elicitor capacity, by increasing the activity of phenylalanine ammonia-lyase (66-422 %) but also significantly changing the polyphenol content in the leaves (121-243 %) and roots (30-104 %) of tomato plants.

Pharmacological Investigations in Traditional Utilization of Alhagi maurorum Medik. in Saharan Algeria: In Vitro Study of Anti-Inflammatory and Antihyperglycemic Activities of Water-Soluble Polysaccharides Extracted from the Seeds.

The anti-inflammatory and antihyperglycemic effects of polysaccharides extracted from Alhagi maurorum Medik. seeds, spontaneous shrub collected in Southern of Algerian Sahara were investigated. Their water extraction followed by alcoholic precipitation was conducted to obtain two water-soluble polysaccharides extracts (WSPAM1 and WSPAM2). They were characterized using Fourier transform infrared, 1H/13C Nuclear Magnetic Resonance, Gas Chromatography-Mass Spectrometry and Size Exclusion Chromatography coupled with Multi-Angle Light Scattering. The capacity of those fractions to inhibit α-amylase activity and thermally induced Bovine Serum Albumin denaturation were also investigated. WSPAM1 and WSPAM2 were galactomannans with a mannose/galactose ratio of 2.2 and 2.4, respectively. The SEC-MALLS analysis revealed that WSPAM1 had a molecular weight of 1.4 × 106 Da. The investigations highlighted antinflammatory and antihyperglycemic effects in a dose-dependant manner of WSPAM1 and WSPAM2.

Click glycoconjugation of per-azido- and alkynyl-functionalized beta-peptides built from aspartic acid.

Azide- and alkynyl-containing homo-beta(3)-peptides, of up to six residues in length, were synthesised in solution from aspartic acid. Their subsequent conjugation with monosaccharides bearing an azide or a terminal alkyne function was efficiently achieved by copper-mediated cycloadditions leading to two novel families of small glycoclusters. These compounds represent ideal tools to explore carbohydrate-mediated multivalent interactions.

Improved copper(I)-NHC catalytic efficiency on huisgen reaction by addition of aromatic nitrogen donors.

A simple addition with a large impact: Addition of aromatic amines such as phenanthroline and 4-DMAP (4-dimethylaminopyridine) increases copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) catalytic activity of [CuCl(SIMes)] at a large range of temperatures in such a way that efficient catalysis can safely take place in hydro-alcoholic solvents (see scheme).

Metabolic modulations of Pseudomonas graminis in response to H2O2 in cloud water.

In cloud water, microorganisms are exposed to very strong stresses especially related to the presence of reactive oxygen species including H2O2 and radicals, which are the driving force of cloud chemistry. In order to understand how the bacterium Pseudomonas graminis isolated from cloud water respond to this oxidative stress, it was incubated in microcosms containing a synthetic solution of cloud water in the presence or in the absence of H2O2. P. graminis metabolome was examined by LC-MS and NMR after 50 min and after 24 hours of incubation. After 50 min, the cells were metabolizing H2O2 while this compound was still present in the medium, and it was completely biodegraded after 24 hours. Cells exposed to H2O2 had a distinct metabolome as compared to unexposed cells, revealing modulations of certain metabolic pathways in response to oxidative stress. These data indicated that the regulations observed mainly involved carbohydrate, glutathione, energy, lipid, peptides and amino-acids metabolisms. When cells had detoxified H2O2 from the medium, their metabolome was not distinguishable anymore from unexposed cells, highlighting the capacity of resilience of this bacterium. This work illustrates the interactions existing between the cloud microbial metabolome and cloud chemistry.

Structure of the RNA signal essential for translational frameshifting in HIV-1.

Many pathogenic viruses use a programmed -1 translational frameshifting mechanism to regulate synthesis of their structural and enzymatic proteins. Frameshifting is vital for viral replication. A slippery sequence bound at the ribosomal A and P sites as well as a downstream stimulatory RNA structure are essential for frameshifting. Conflicting data have been reported concerning the structure of the downstream RNA signal in human immunodeficiency virus type 1 (HIV-1). Here, the solution structure of the HIV-1 frameshifting RNA signal was solved by heteronuclear NMR spectroscopy. This structure reveals a long hairpin fold with an internal three-nucleotide bulge. The internal loop introduces a bend between the lower and upper helical regions, a structural feature often seen in frameshifting pseudoknots. The NMR structure correlates with chemical probing data. The upper stem rich in conserved G-C Watson-Crick base-pairs is highly stable, whereas the bulge region and the lower stem are more flexible.

Impact of exogenous lysolipids on sensitive and multidrug resistant K562 cells: 1H NMR studies.

The ability of lysolipids to enter into a membrane bi-layer and disturb the membrane structure was used to study the behavior of K562 erythroleukemic cells, K562 wild type (K562wt) as well as the multidrug resistant cells K562adr. Both types of cells, when analyzed by proton NMR spectroscopy exhibit the high resolution signals assigned to so-called "mobile lipid" signals, which, in most cases, are located outside the lipid bi-layer as lipid droplets. In order to perform these studies, the K562wt and K562adr cells were treated for 48h with lysophosphatidylcholine oleoyl (LPC18), lysophosphatidylcholine palmitoyl (LPC16) and L-alpha-lysophosphatidyslerine (LPS). After evaluating toxicity of lysolipids, proton NMR of whole treated cells was used to analyze the mobile lipid content. Nile red staining and fluorescence microscopy were used to detect the presence of intracellular lipid droplets. Membrane lipid asymmetry perturbation was estimated by annexin V staining with use of flow cytometry. Using fluorescence spectroscopy the functioning of P-glycoprotein (P-gp) responsible for multidrug resistance was also evaluated after the treatment with lysolipids. Lysolipids were found to be more toxic for K562wt than for K562adr cells. LPS and LPC16 produced an increased of a mobile lipid NMR signal and amount of lipid droplets in K562wt cells only. LPC18, with the lowest toxicity, has shown more intense effects on NMR spectra with a large increase of lipid NMR signal without changes in lipid droplet staining. The functioning of the P-gp pump and membrane asymmetry were not modified by any of the lysolipids used.