Short-term changes in human metabolism following a 5-h delay of the light-dark and behavioral cycle.

Experimental inversion of circadian and behavioral rhythms by 12 h adversely affects markers of metabolic health. We investigated the effects of a more modest 5-h delay in behavioral cycles. Fourteen participants completed an 8-day in-patient laboratory protocol, with controlled sleep-wake opportunities, light-dark cycles, and diet.

Single-Cell Lipidomics: An Automated and Accessible Microfluidic Workflow Validated by Capillary Sampling.

We report the first demonstration of a microfluidics-based approach to measure lipids in single living cells using widely available liquid chromatography mass spectrometry (LC-MS) instrumentation. The method enables the rapid sorting of live cells into liquid chambers formed on standard Petri dishes and their subsequent dispensing into vials for analysis using LC-MS. This approach facilitates automated sampling, data acquisition, and analysis and carries the additional advantage of chromatographic separation, aimed at reducing matrix effects present in shotgun lipidomics approaches.

Challenges in Lipidomics Biomarker Identification: Avoiding the Pitfalls and Improving Reproducibility.

Identification of features with high levels of confidence in liquid chromatography-mass spectrometry (LC-MS) lipidomics research is an essential part of biomarker discovery, but existing software platforms can give inconsistent results, even from identical spectral data. This poses a clear challenge for reproducibility in biomarker identification. In this work, we illustrate the reproducibility gap for two open-access lipidomics platforms, MS DIAL and Lipostar, finding just 14.

Single-Cell Untargeted Lipidomics Using Liquid Chromatography and Data-Dependent Acquisition after Live Cell Selection.

We report the development and validation of an untargeted single-cell lipidomics method based on microflow chromatography coupled to a data-dependent mass spectrometry method for fragmentation-based identification of lipids. Given the absence of single-cell lipid standards, we show how the methodology should be optimized and validated using a dilute cell extract. The methodology is applied to dilute pancreatic cancer and macrophage cell extracts and standards to demonstrate the sensitivity requirements for confident assignment of lipids and classification of the cell type at the single-cell level.

Meta-Analysis of COVID-19 Metabolomics Identifies Variations in Robustness of Biomarkers.

The global COVID-19 pandemic resulted in widespread harms but also rapid advances in vaccine development, diagnostic testing, and treatment. As the disease moves to endemic status, the need to identify characteristic biomarkers of the disease for diagnostics or therapeutics has lessened, but lessons can still be learned to inform biomarker research in dealing with future pathogens. In this work, we test five sets of research-derived biomarkers against an independent targeted and quantitative Liquid Chromatography-Mass Spectrometry metabolomics dataset to evaluate how robustly these proposed panels would distinguish between COVID-19-positive and negative patients in a hospital setting.

Single-Cell Lipidomics Using Analytical Flow LC-MS Characterizes the Response to Chemotherapy in Cultured Pancreatic Cancer Cells.

In this work, we demonstrate the development and first application of nanocapillary sampling followed by analytical flow liquid chromatography-mass spectrometry for single-cell lipidomics. Around 260 lipids were tentatively identified in a single cell, demonstrating remarkable sensitivity. Human pancreatic ductal adenocarcinoma cells (PANC-1) treated with the chemotherapeutic drug gemcitabine can be distinguished from controls solely on the basis of their single-cell lipid profiles.

Oxylipin secretion by human CD3 T lymphocytes is modified by the exogenous essential fatty acid ratio and life stage.

Immune function changes across the life stages; for example, senior adults exhibit a tendency towards a weaker cell-mediated immune response and a stronger inflammatory response than younger adults. This might be partly mediated by changes in oxylipin synthesis across the life course. Oxylipins are oxidation products of polyunsaturated fatty acids (PUFAs) that modulate immune function and inflammation.

Exogenous tetracosahexaenoic acid modifies the fatty acid composition of human primary T lymphocytes and Jurkat T cell leukemia cells contingent on cell type.

Tetracosahexaenoic acid (24:6ω-3) is an intermediate in the conversion of 18:3ω-3 to 22:6ω-3 in mammals. There is limited information about whether cells can assimilate and metabolize exogenous 24:6ω-3. This study compared the effect of incubation with 24:6ω-3 on the fatty acid composition of two related cell types, primary CD3 T lymphocytes and Jurkat T cell leukemia, which differ in the integrity of the polyunsaturated fatty acid (PUFA) biosynthesis pathway.

Nanocapillary sampling coupled to liquid chromatography mass spectrometry delivers single cell drug measurement and lipid fingerprints.

This work describes the development of a new approach to measure drug levels and lipid fingerprints in single living mammalian cells. Nanocapillary sampling is an approach that enables the selection and isolation of single living cells under microscope observation. Here, live single cell nanocapillary sampling is coupled to liquid chromatography for the first time.

Fatty acid composition and metabolic partitioning of α-linolenic acid are contingent on life stage in human CD3 T lymphocytes.

Introduction: Immune function changes across the life course; the fetal immune system is characterised by tolerance while that of seniors is less able to respond effectively to antigens and is more pro-inflammatory than in younger adults. Lipids are involved centrally in immune function but there is limited information about how T cell lipid metabolism changes during the life course.

Methods And Results: We investigated whether life stage alters fatty acid composition, lipid droplet content and α-linolenic acid (18:3ω-3) metabolism in human fetal CD3 T lymphocytes and in CD3 T lymphocytes from adults (median 41 years) and seniors (median 70 years).

Multi-Omics Reveals Mechanisms of Partial Modulation of COVID-19 Dysregulation by Glucocorticoid Treatment.

Treatments for COVID-19 infections have improved dramatically since the beginning of the pandemic, and glucocorticoids have been a key tool in improving mortality rates. The UK's National Institute for Health and Care Excellence guidance is for treatment to be targeted only at those requiring oxygen supplementation, however, and the interactions between glucocorticoids and COVID-19 are not completely understood. In this work, a multi-omic analysis of 98 inpatient-recruited participants was performed by quantitative metabolomics (using targeted liquid chromatography-mass spectrometry) and data-independent acquisition proteomics.

The [ C]octanoic acid breath test for gastric emptying quantification: A focus on nutrition and modeling.

Gastric emptying (GE) is the process of food being processed by the stomach and delivered to the small intestine where nutrients such as lipids are absorbed into the blood circulation. The combination of an easy and inexpensive method to measure GE such as the CO breath test using the stable isotope [ C]octanoic acid with semi-mechanistic modeling could foster a wider application in nutritional studies to further understand the metabolic response to food. Here, we discuss the use of the [ C]octanoic acid breath test to label the solid phase of a meal, and the factors that influence GE to support mechanistic studies.

The Partitioning of Newly Assimilated Linoleic and α-Linolenic Acids Between Synthesis of Longer-Chain Polyunsaturated Fatty Acids and Hydroxyoctadecaenoic Acids Is a Putative Branch Point in T-Cell Essential Fatty Acid Metabolism.

Longer-chain polyunsaturated fatty acids (LCPUFAs) ≥20 carbons long are required for leukocyte function. These can be obtained from the diet, but there is some evidence that leukocytes can convert essential fatty acids (EFAs) into LCPUFAs. We used stable isotope tracers to investigate LCPUFA biosynthesis and the effect of different EFA substrate ratios in human T lymphocytes.

Bacterial immunogenic α-galactosylceramide identified in the murine large intestine: dependency on diet and inflammation.

The glycosphingolipid, α-galactosylceramide (αGalCer), when presented by CD1d on antigen-presenting cells, efficiently activates invariant natural killer T (NKT) cells. Thereby, it modulates immune responses against tumors, microbial and viral infections, and autoimmune diseases. Recently, the production of αGalCer by from the human gut microbiome was elucidated.

Renal globotriaosylceramide facilitates tubular albumin absorption and its inhibition protects against acute kidney injury.

To elucidate the physiologic function of renal globotriaosylceramide (Gb3/CD77), which up-to-date has been associated exclusively with Shiga toxin binding, we have analyzed renal function in Gb3-deficient mice. Gb3 synthase KO (Gb3S) mice displayed an increased renal albumin and low molecular weight protein excretion compared to WT. Gb3 localized at the brush border and within vesicular structures in WT proximal tubules and has now been shown to be closely associated with the receptor complex megalin/cubilin and with albumin uptake.

Deletion of Specific Sphingolipids in Distinct Neurons Improves Spatial Memory in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and a concomitant loss of synapses and cognitive abilities. Recently, we have proposed that an alteration of neuronal membrane lipid microdomains increases neuronal resistance toward amyloid-β stress in cultured neurons and protects from neurodegeneration in a mouse model of AD. Lipid microdomains are highly enriched in a specific subclass of glycosphingolipids, termed gangliosides.

Murine Sialidase Neu3 facilitates GM2 degradation and bypass in mouse model of Tay-Sachs disease.

Tay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal β-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by β-hexosaminidase B.

Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals.

Mammals synthesize, cell-type specifically, the diastereomeric hexosylceramides, β-galactosylceramide (GalCer) and β-glucosylceramide (GlcCer), which are involved in several diseases, such as sphingolipidosis, diabetes, chronic kidney diseases, or cancer. In contrast, , a member of the human gut microbiome, and the marine sponge, , produce α-GalCer, one of the most potent stimulators for invariant natural killer T cells. To dissect the contribution of these individual stereoisomers to pathologies, we established a novel hydrophilic interaction chromatography-based LC-MS method and separated ( > 1.

Molecular imaging of brain localization of liposomes in mice using MALDI mass spectrometry.

Phospholipids have excellent biocompatibility and are therefore often used as main components of liposomal drug carriers. In traditional bioanalytics, the in-vivo distribution of liposomal drug carriers is assessed using radiolabeled liposomal constituents. This study presents matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) as an alternative, label-free method for ex-vivo molecular imaging of liposomal drug carriers in mouse tissue.