Metabolomics Simultaneously Derives Benchmark Dose Estimates and Discovers Metabolic Biotransformations in a Rat Bioassay.

Benchmark dose (BMD) modeling estimates the dose of a chemical that causes a perturbation from baseline. Transcriptional BMDs have been shown to be relatively consistent with apical end point BMDs, opening the door to using molecular BMDs to derive human health-based guidance values for chemical exposure. Metabolomics measures the responses of small-molecule endogenous metabolites to chemical exposure, complementing transcriptomics by characterizing downstream molecular phenotypes that are more closely associated with apical end points. The aim of this study was to apply BMD modeling to in vivo metabolomics data, to compare metabolic BMDs to both transcriptional and apical end point BMDs. This builds upon our previous application of transcriptomics and BMD modeling to a 5-day rat study of triphenyl phosphate (TPhP), applying metabolomics to the same archived tissues. Specifically, liver from rats exposed to five doses of TPhP was investigated using liquid chromatography-mass spectrometry and 1H nuclear magnetic resonance spectroscopy-based metabolomics. Following the application of BMDExpress2 software, 2903 endogenous metabolic features yielded viable dose-response models, confirming a perturbation to the liver metabolome. Metabolic BMD estimates were similarly sensitive to transcriptional BMDs, and more sensitive than both clinical chemistry and apical end point BMDs. Pathway analysis of the multiomics data sets revealed a major effect of TPhP exposure on cholesterol (and downstream) pathways, consistent with clinical chemistry measurements. Additionally, the transcriptomics data indicated that TPhP activated xenobiotic metabolism pathways, which was confirmed by using the underexploited capability of metabolomics to detect xenobiotic-related compounds. Eleven biotransformation products of TPhP were discovered, and their levels were highly correlated with multiple xenobiotic metabolism genes. This work provides a case study showing how metabolomics and transcriptomics can estimate mechanistically anchored points-of-departure. Furthermore, the study demonstrates how metabolomics can also discover biotransformation products, which could be of value within a regulatory setting, for example, as an enhancement of OECD Test Guideline 417 (toxicokinetics).

Bilateral remote ischemic conditioning in children: A two-center, double-blind, randomized controlled trial in young children undergoing cardiac surgery.

Objective: The study objective was to determine whether adequately delivered bilateral remote ischemic preconditioning is cardioprotective in young children undergoing surgery for 2 common congenital heart defects with or without cyanosis. Methods: We performed a prospective, double-blind, randomized controlled trial at 2 centers in the United Kingdom. Children aged 3 to 36 months undergoing tetralogy of Fallot repair or ventricular septal defect closure were randomized 1:1 to receive bilateral preconditioning or sham intervention. Participants were followed up until hospital discharge or 30 days. The primary outcome was area under the curve for high-sensitivity troponin-T in the first 24 hours after surgery, analyzed by intention-to-treat. Right atrial biopsies were obtained in selected participants. Results: Between October 2016 and December 2020, 120 eligible children were randomized to receive bilateral preconditioning (n = 60) or sham intervention (n = 60). The primary outcome, area under the curve for high-sensitivity troponin-T, was higher in the preconditioning group (mean: 70.0 ± 50.9 µg/L/h, n = 56) than in controls (mean: 55.6 ± 30.1 µg/L/h, n = 58) (mean difference, 13.2 µg/L/h; 95% CI, 0.5-25.8; P = .04). Subgroup analyses did not show a differential treatment effect by oxygen saturations (pinteraction = .25), but there was evidence of a differential effect by underlying defect (pinteraction = .04). Secondary outcomes and myocardial metabolism, quantified in atrial biopsies, were not different between randomized groups. Conclusions: Bilateral remote ischemic preconditioning does not attenuate myocardial injury in children undergoing surgical repair for congenital heart defects, and there was evidence of potential harm in unstented tetralogy of Fallot. The routine use of remote ischemic preconditioning cannot be recommended for myocardial protection during pediatric cardiac surgery.

Fast and Accurate Disulfide Bridge Detection.

Recombinant expression of proteins, propelled by therapeutic antibodies, has evolved into a multibillion dollar industry. Essential here is the quality control assessment of critical attributes, such as sequence fidelity, proper folding, and posttranslational modifications. Errors can lead to diminished bioactivity and, in the context of therapeutic proteins, an elevated risk for immunogenicity. Over the years, many techniques were developed and applied to validate proteins in a standardized and high-throughput fashion. One parameter has, however, so far been challenging to assess. Disulfide bridges, covalent bonds linking two cysteine residues, assist in the correct folding and stability of proteins and thus have a major influence on their efficacy. Mass spectrometry promises to be an optimal technique to uncover them in a fast and accurate fashion. In this work, we present a unique combination of sample preparation, data acquisition, and analysis facilitating the rapid and accurate assessment of disulfide bridges in purified proteins. Through microwave-assisted acid hydrolysis, the proteins are digested rapidly and artifact-free into peptides, with a substantial degree of overlap over the sequence. The nonspecific nature of this procedure, however, introduces chemical background, which is efficiently removed by integrating ion mobility preceding the mass spectrometric measurement. The nonspecific nature of the digestion step additionally necessitates new developments in data analysis, for which we extended the XlinkX node in Proteome Discoverer to efficiently process the data and ensure correctness through effective false discovery rate correction. The entire workflow can be completed within 1 h, allowing for high-throughput, high-accuracy disulfide mapping.

Protein-Centric Analysis of Personalized Antibody Repertoires Using LC-MS-Based Fab-Profiling on a timsTOF.

Endogenous antibodies, or immunoglobulins (Igs), abundantly present in body fluids, represent some of the most challenging samples to analyze, largely due to the immense variability in their sequences and concentrations. It has been estimated that our body can produce billions of different Ig proteins with different isotypes, making their individual analysis seemingly impossible. However, recent advances in protein-centric proteomics using LC-MS coupled to Orbitrap mass analyzers to profile intact Fab fragments formed by selective cleavage at the IgG-hinge revealed that IgG repertoires may be less diverse, albeit unique for each donor. Serum repertoires seem to be dominated by a few hundred clones that cumulatively make up 50-95% of the total IgG content. Enabling such analyses required careful optimization of the chromatography and mass analysis, as all Fab analytes are highly alike in mass (46-51 kDa) and sequence. To extend the opportunities of this mass-spectrometry-based profiling of antibody repertoires, we here report the optimization and evaluation of an alternative MS platform, namely, the timsTOF, for antibody repertoire profiling. The timsTOF mass analyzer has gained traction in recent years for peptide-centric proteomics and found wide applicability in plasma proteomics, affinity proteomics, and HLA peptidomics, to name a few. However, for protein-centric analysis, this platform has been less explored. Here, we demonstrate that the timsTOF platform can be adapted to perform protein-centric LC-MS-based profiling of antibody repertoires. In a side-by-side comparison of the timsTOF and the Orbitrap we demonstrate that the extracted serum antibody repertoires are alike qualitatively and quantitatively, whereby in particular the sensitivity of the timsTOF platform excels. Future incorporation of advanced top-down capabilities on the timsTOF may make this platform a very valuable alternative for protein-centric proteomics and top-down proteomics and thus also for personalized antibody repertoire profiling.

Lipidome characterisation and sex-specific differences in type 1 and type 2 diabetes mellitus.

BACKGROUND: In this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state. METHODS: An untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative). We used multiple linear regression models to (1) assess the association between each lipid feature and each condition, (2) determine sex-specific differences related to diabetes, and (3) identify lipids associated with the glycaemic state by considering the prediabetes stage. The models were adjusted by sex, age, hypertension, dyslipidaemia, body mass index, glucose, smoking, systolic blood pressure, triglycerides, HDL cholesterol, LDL cholesterol, alternate Mediterranean diet score (aMED) and estimated glomerular filtration rate (eGFR); diabetes duration and glycated haemoglobin (HbA1c) were also included in the comparison between T1D and T2D. RESULTS: A total of 54 unique lipid subspecies from 15 unique lipid classes were annotated. Lysophosphatidylcholines (LPC) and ceramides (Cer) showed opposite effects in subjects with T1D and subjects with T2D, LPCs being mainly up-regulated in T1D and down-regulated in T2D, and Cer being up-regulated in T2D and down-regulated in T1D. Also, Phosphatidylcholines were clearly down-regulated in subjects with T1D. Regarding sex-specific differences, ceramides and phosphatidylcholines exhibited important diabetes-associated differences due to sex. Concerning the glycaemic state, we found a gradual increase of a panel of 1-deoxyceramides from normoglycemia to prediabetes to T2D. CONCLUSIONS: Our findings revealed an extensive disruption of lipid metabolism in both T1D and T2D. Additionally, we found sex-specific lipidome changes associated with diabetes, and lipids associated with the glycaemic state that can be linked to previously described molecular mechanisms in diabetes.

Thrombin activation of the factor XI dimer is a multistaged process for each subunit.

BACKGROUND: Factor (F)XI can be activated by proteases, including thrombin and FXIIa. The interactions of these enzymes with FXI are transient in nature and therefore difficult to study. OBJECTIVES: To identify the binding interface between thrombin and FXI and understand the dynamics underlying FXI activation. METHODS: Crosslinking mass spectrometry was used to localize the binding interface of thrombin on FXI. Molecular dynamics simulations were applied to investigate conformational changes enabling thrombin-mediated FXI activation after binding. The proposed trajectory of activation was examined with nanobody 1C10, which was previously shown to inhibit thrombin-mediated activation of FXI. RESULTS: We identified a binding interface of thrombin located on the light chain of FXI involving residue Pro520. After this initial interaction, FXI undergoes conformational changes driven by binding of thrombin to the apple 1 domain in a secondary step to allow migration toward the FXI cleavage site. The 1C10 binding site on the apple 1 domain supports this proposed trajectory of thrombin. We validated the results with known mutation sites on FXI. As Pro520 is conserved in prekallikrein (PK), we hypothesized and showed that thrombin can bind PK, even though it cannot activate PK. CONCLUSION: Our investigations show that the activation of FXI is a multistaged procedure. Thrombin first binds to Pro520 in FXI; thereafter, it migrates toward the activation site by engaging the apple 1 domain. This detailed analysis of the interaction between thrombin and FXI paves a way for future interventions for bleeding or thrombosis.

Combination of deep XLMS with deep learning reveals an ordered rearrangement and assembly of a major protein component of the vaccinia virion.

IMPORTANCE: An outstanding problem in the understanding of poxvirus biology is the molecular structure of the mature virion. Via deep learning methods combined with chemical cross-linking mass spectrometry, we have addressed the structure and assembly pathway of P4a, a key poxvirus virion core component.

MRPS36 provides a structural link in the eukaryotic 2-oxoglutarate dehydrogenase complex.

The tricarboxylic acid cycle is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive cross-linking mass spectrometry data supported by phylogenetic analyses, we provide evidence that MRPS36 is an important member of the eukaryotic OGDHC, with no prokaryotic orthologues. Comparative sequence analysis and computational structure predictions reveal that, in contrast with bacteria and archaea, eukaryotic E2o does not contain the peripheral subunit-binding domain (PSBD), for which we propose that MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC of approximately 3.45 MDa with novel mechanistic insights.

Dysregulation of Amino Acid, Lipid, and Acylpyruvate Metabolism in Idiopathic Intracranial Hypertension: A Non-targeted Case Control and Longitudinal Metabolomic Study.

Background: Idiopathic intracranial hypertension (IIH) is characterized by increased intracranial pressure occurring predominantly in women with obesity. The pathogenesis is not understood. We have applied untargeted metabolomic analysis using ultrahigh-performance liquid chromatography-mass spectrometry to characterize the cerebrospinal fluid (CSF) and serum in IIH compared to control subjects. Methods and findings: Samples were collected from IIH patients (n = 66) with active disease at baseline and again at 12 months following therapeutic weight loss. Control samples were collected from gender- and weight-matched healthy controls (n = 20). We identified annotated metabolites in CSF, formylpyruvate and maleylpyruvate/fumarylpyruvate, which were present at lower concentrations in IIH compared to control subjects and returned to values observed in controls following weight loss. These metabolites showed the opposite trend in serum at baseline. Multiple amino acid metabolic pathways and lipid classes were perturbed in serum and CSF in IIH alone. Serum lipid metabolite pathways were significantly increased in IIH. Conclusions: We observed a number of differential metabolic pathways related to amino acid, lipid, and acylpyruvate metabolism, in IIH compared to controls. These pathways were associated with clinical measures and normalized with disease remission. Perturbation of these metabolic pathways provides initial understanding of disease dysregulation in IIH.

Oxonium Ion-Guided Optimization of Ion Mobility-Assisted Glycoproteomics on the timsTOF Pro.

Spatial separation of ions in the gas phase, providing information about their size as collisional cross-sections, can readily be achieved through ion mobility. The timsTOF Pro (Bruker Daltonics) series combines a trapped ion mobility device with a quadrupole, collision cell, and a time-of-flight analyzer to enable the analysis of ions at great speed. Here, we show that the timsTOF Pro is capable of physically separating N-glycopeptides from nonmodified peptides and producing high-quality fragmentation spectra, both beneficial for glycoproteomics analyses of complex samples. The glycan moieties enlarge the size of glycopeptides compared with nonmodified peptides, yielding a clear cluster in the mobilogram that, next to increased dynamic range from the physical separation of glycopeptides and nonmodified peptides, can be used to make an effective selection filter for directing the mass spectrometer to analytes of interest. We designed an approach where we (1) focused on a region of interest in the ion mobilogram and (2) applied stepped collision energies to obtain informative glycopeptide tandem mass spectra on the timsTOF Pro:glyco-polygon-stepped collision energy-parallel accumulation serial fragmentation. This method was applied to selected glycoproteins, human plasma- and neutrophil-derived glycopeptides. We show that the achieved physical separation in the region of interest allows for improved extraction of information from the samples, even at shorter liquid chromatography gradients of 15 min. We validated our approach on human neutrophil and plasma samples of known makeup, in which we captured the anticipated glycan heterogeneity (paucimannose, phosphomannose, high mannose, hybrid and complex glycans) from plasma and neutrophil samples at the expected abundances. As the method is compatible with off-the-shelve data acquisition routines and data analysis software, it can readily be applied by any laboratory with a timsTOF Pro and is reproducible as demonstrated by a comparison between two laboratories.

Invariant NKT cells metabolically adapt to the acute myeloid leukaemia environment.

Acute myeloid leukaemia (AML) creates an immunosuppressive environment to conventional T cells through Arginase 2 (ARG2)-induced arginine depletion. We identify that AML blasts release the acute phase protein serum amyloid A (SAA), which acts in an autocrine manner to upregulate ARG2 expression and activity, and promote AML blast viability. Following in vitro cross-talk invariant natural killer T (iNKT) cells become activated, upregulate mitochondrial capacity, and release IFN-γ. iNKT retain their ability to proliferate and be activated despite the low arginine AML environment, due to the upregulation of Large Neutral Amino Acid Transporter-1 (LAT-1) and Argininosuccinate Synthetase 1 (ASS)-dependent amino acid pathways, resulting in AML cell death. T cell proliferation is restored in vitro and in vivo. The capacity of iNKT cells to restore antigen-specific T cell immunity was similarly demonstrated against myeloid-derived suppressor cells (MDSCs) in wild-type and Jα18-/- syngeneic lymphoma-bearing models in vivo. Thus, stimulation of iNKT cell activity has the potential as an immunotherapy against AML or as an adjunct to boost antigen-specific T cell immunotherapies in haematological or solid cancers.

Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached.

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.

An improved strategy for analysis of lipid molecules utilising a reversed phase C30 UHPLC column and scheduled MS/MS acquisition.

Measuring physiochemically diverse molecules (including lipids) which vary significantly in their concentrations poses a great analytical challenge. In untargeted lipidomics studies, reversed phase chromatography coupled with data-dependent MS/MS acquisition (DDA) is frequently applied. The optimal assay should deliver a high number of detected compounds with associated fragmentation data. In this work, we introduce novel 30 and 50 min UHPLC assays utilising lipid separation on a C30 stationary phase with a modified DDA strategy using smaller precursor m/z ranges scheduled for different lipid classes across the retention time range (defined as scheduled MS/MS). To evaluate the efficiency of the novel assays, mammalian tissue extracts (lamb liver, kidney and heart) were analysed and data were compared to a 15 min reversed phase C18 assay with multiple traditional DDA injections. The 30 min C30 assay detected double the number of detected compounds compared to the 15 min C18 assay. Applying the scheduled MS/MS DDA strategy with a single injection, a similar number of annotated lipids were reported compared to the traditional DDA strategy applied with five replicate injections on a C18 column. A longer 50 min C30 chromatographic assay did not result in an expected improvement in the chromatographic separation of overlapping isomer peaks compared to the 30 min method but did result in loss of accuracy of peak picking algorithms. We recommend the 30 min C30 assay with scheduled MS/MS acquisition as an efficient tool to analyse complex biological matrices and to annotate lipid species based on MS/MS data.

struct: an R/Bioconductor-based framework for standardized metabolomics data analysis and beyond.

SUMMARY: Implementing and combining methods from a diverse range of R/Bioconductor packages into 'omics' data analysis workflows represents a significant challenge in terms of standardization, readability and reproducibility. Here, we present an R/Bioconductor package, named struct (Statistics in R using Class-based Templates), which defines a suite of class-based templates that allows users to develop and implement highly standardized and readable statistical analysis workflows. Struct integrates with the STATistics Ontology to ensure consistent reporting and maximizes semantic interoperability. We also present a toolbox, named structToolbox, which includes an extensive set of commonly used data analysis methods that have been implemented using struct. This toolbox can be used to build data-analysis workflows for metabolomics and other omics technologies. AVAILABILITY AND IMPLEMENTATION: struct and structToolbox are implemented in R, and are freely available from Bioconductor (http://bioconductor.org/packages/struct and http://bioconductor.org/packages/structToolbox), including documentation and vignettes. Source code is available and maintained at https://github.com/computational-metabolomics.

mzMLb: A Future-Proof Raw Mass Spectrometry Data Format Based on Standards-Compliant mzML and Optimized for Speed and Storage Requirements.

With ever-increasing amounts of data produced by mass spectrometry (MS) proteomics and metabolomics, and the sheer volume of samples now analyzed, the need for a common open format possessing both file size efficiency and faster read/write speeds has become paramount to drive the next generation of data analysis pipelines. The Proteomics Standards Initiative (PSI) has established a clear and precise extensible markup language (XML) representation for data interchange, mzML, receiving substantial uptake; nevertheless, storage and file access efficiency has not been the main focus. We propose an HDF5 file format "mzMLb" that is optimized for both read/write speed and storage of the raw mass spectrometry data. We provide an extensive validation of the write speed, random read speed, and storage size, demonstrating a flexible format that with or without compression is faster than all existing approaches in virtually all cases, while with compression is comparable in size to proprietary vendor file formats. Since our approach uniquely preserves the XML encoding of the metadata, the format implicitly supports future versions of mzML and is straightforward to implement: mzMLb's design adheres to both HDF5 and NetCDF4 standard implementations, which allows it to be easily utilized by third parties due to their widespread programming language support. A reference implementation within the established ProteoWizard toolkit is provided.

Characterization of Monophasic Solvent-Based Tissue Extractions for the Detection of Polar Metabolites and Lipids Applying Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry Clinical Metabolic Phenotyping Assays.

Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and nonpolar (lipid) metabolite levels in biological samples. This approach aims to understand disease biochemistry and identify biochemical markers of disease. Sample preparation methods must be reproducible, sensitive (high metabolite and lipid yield), and ideally rapid. We evaluated three biphasic methods for polar and nonpolar compound extraction (chloroform/methanol/water, dichloromethane/methanol/water, and methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water), and a monophasic method for nonpolar compound extraction (isopropanol/water). All methods were applied to mammalian heart, kidney, and liver tissues. Polar extracts were analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) and nonpolar extracts by C18 reversed-phase UHPLC-MS. Method reproducibility and yield were assessed using multiple annotated endogenous compounds (putatively and MS/MS annotated). Monophasic methods had the highest yield and high reproducibility for both polar (positive ion: median relative standard deviation (RSD) < 18%; negative ion: median RSD < 28%) and nonpolar (positive and negative ion: median RSD < 15%) extractions for heart, kidneys, and liver. The polar monophasic method extracted higher levels of lipid than biphasic polar extractions, and these lipids caused minimal detection suppression for other compounds during HILIC UHPLC-MS. The nonpolar monophasic method had similar or greater detection responses of all detected lipid classes compared to biphasic methods (including increased phosphatidylinositol, phosphatidylserine, and cardiolipin responses). Monophasic methods are quicker and simpler than biphasic methods and are therefore most suited for future automation.

Assessment of human plasma and urine sample preparation for reproducible and high-throughput UHPLC-MS clinical metabolic phenotyping.

Clinical metabolic phenotyping employs metabolomics and lipidomics to detect and measure hundreds to thousands of metabolites and lipids within human samples. This approach aims to identify metabolite and lipid changes between phenotypes (e.g. disease status) that aid understanding of biochemical mechanisms driving the phenotype. Sample preparation is a critical step in clinical metabolic phenotyping: it must be reproducible and give a high extraction yield of metabolites and lipids, and in high-throughput studies it needs to be rapid. Here, we assessed the extraction of polar metabolites from human urine and polar metabolites and lipids from human plasma for analysis by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) metabolomics and lipidomics. We evaluated several monophasic (urine and plasma) and biphasic (plasma) extractions, and we also tested alterations to (a) solvent-biofluid incubation time and temperature during monophasic extraction, and (b) phase partitioning time during biphasic extraction. Extracts were analysed by three UHPLC-MS assays: (i) hydrophilic interaction chromatography (HILIC) for urine and plasma, (ii) C18 aqueous reversed phase for urine, and (iii) C18 reversed phase for plasma lipids, and the yield and reproducibility of each method was assessed. We measured UHPLC-MS injection reproducibility as well as sample preparation reproducibility to assess sample solvent composition compatibility with UHPLC-MS and to pinpoint the origin of variance within the methods. For HILIC UHPLC-MS plasma and urine analysis, monophasic 50 : 50 methanol : acetonitrile had the most detected putatively-identified polar metabolites with high method reproducibility. This method had the highest lipid yield for plasma extracts analysed by the HILIC method. If lipid removal from the plasma polar HILIC extract is required, then the biphasic methanol/chloroform/water method is recommended. For C18 (aqueous) UHPLC-MS urine analysis, 50 : 50 methanol : water had high reproducibility and yield. For C18 UHPLC-MS plasma lipidomics, monophasic 100% isopropanol had the highest detection response of all annotated lipid classes with high reproducibility. Increasing monophasic incubation time and temperature had little benefit on metabolite and lipid yield and reproducibility for all methods.

Metabolic characterisation of disturbances in the APOC3/triglyceride-rich lipoprotein pathway through sample-based recall by genotype.

INTRODUCTION: High plasma triacylglyceride levels are known to be associated with increased risk of atherosclerotic cardiovascular disease. Apolipoprotein C-III (apoC-III) is a key regulator of plasma triacylglyceride levels and is associated with hypertriglyceridemia via a number of pathways. There is consistent evidence for an association of cardiovascular events with blood apoC-III level, with support from human genetic studies of APOC3 variants. As such, apoC-III has been recognised as a potential therapeutic target for patients with severe hypertriglyceridaemia with one of the most promising apoC-III-targeting drugs, volanesorsen, having recently progressed through Phase III trials. OBJECTIVES: To exploit a rare loss of function variant in APOC3 (rs138326449) to characterise the potential long-term treatment effects of apoC-III targeting interventions on the metabolome. METHODS: In a recall-by-genotype study, 115 plasma samples were analysed by UHPLC-MS to acquire non-targeted metabolomics data. The study included samples from 57 adolescents and 33 adults. Overall, 12 985 metabolic features were tested for an association with APOC3 genotype. RESULTS: 161 uniquely annotated metabolites were found to be associated with rs138326449(APOC3). The highest proportion of associated metabolites belonged to the acyl-acyl glycerophospholipid and triacylglyceride metabolite classes. In addition to the anticipated (on-target) reduction of metabolites in the triacylglyceride and related classes, carriers of the rare variant exhibited previously unreported increases in levels of a number of metabolites from the acyl-alkyl glycerophospholipid class. CONCLUSION: Overall, our results suggest that therapies targeting apoC-III may potentially achieve a broad shift in lipid profile that favours better metabolic health.

Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity.

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies.