Equivalent carbon number-based targeted odd-chain fatty acyl lipidomics reveals triacylglycerol profiling in clinical colon cancer

Odd-chain FAs (OCFAs) are present in very low level at nearly 1% of total FAs in human plasma, and thus, their functions were usually ignored. Recent epidemiological studies have shown that OCFAs are inversely associated with a variety of disease risks. However, the contribution of OCFAs incorporated into complex lipids remains elusive. Here, we developed a targeted odd-chain fatty acyl-containing lipidomics method based on equivalent carbon number and retention time prediction. The method displayed good reproducibility and robustness as shown by peak width at half height within 0.7 min and coefficient of variation under 20%. A total number of 776 lipid species with odd-chain fatty acyl residues could be detected in the ESI mode of reverse-phase LC-MS, of which 309 lipids were further validated using multiple reaction monitoring transitions. Using this method, we quantified odd-chain fatty acyl-containing lipidome in tissues from 12 colon cancer patients, revealing the remodeling of triacylglycerol. The dynamics of odd-chain fatty acyl lipids were further consolidated by the association with genomic and proteomic features of altered catabolism of branched-chain amino acids and triacylglycerol endogenous synthesis in colon cancer. This lipidomics approach will be applicable for screening of dysregulated odd-chain fatty acyl lipids, which enriches and improves the methods for diagnosis and prognosis evaluation of cancer using lipidomics.     

Validation of a multiplexed and targeted lipidomics assay for accurate quantification of lipidomes

A major challenge of lipidomics is to determine and quantify the precise content of complex lipidomes to the exact lipid molecular species. Often, multiple methods are needed to achieve sufficient lipidomic coverage to make these determinations. Multiplexed targeted assays offer a practical alternative to enable quantitative lipidomics amenable to quality control standards within a scalable platform. Herein, we developed a multiplexed normal phase liquid chromatography-hydrophilic interaction chromatography multiple reaction monitoring method that quantifies lipid molecular species across over 20 lipid classes spanning wide polarities in a single 20-min run. Analytical challenges such as in-source fragmentation, isomer separations, and concentration dynamics were addressed to ensure confidence in selectivity, quantification, and reproducibility. Utilizing multiple MS/MS product ions per lipid species not only improved the confidence of lipid identification but also enabled the determination of relative abundances of positional isomers in samples. Lipid class-based calibration curves were applied to interpolate lipid concentrations and guide sample dilution. Analytical validation was performed following FDA Bioanalytical Method Validation Guidance for Industry. We report repeatable and robust quantitation of 900 lipid species measured in NIST-SRM-1950 plasma, with over 700 lipids achieving inter-assay variability below 25%. To demonstrate proof of concept for biomarker discovery, we analyzed plasma from mice treated with a glucosylceramide synthase inhibitor, benzoxazole 1. We observed expected reductions in glucosylceramide levels in treated animals but, more notably, identified novel lipid biomarker candidates from the plasma lipidome. These data highlight the utility of this qualified lipidomic platform for enabling biological discovery.     

Ex vivo instability of lipids in whole blood: preanalytical recommendations for clinical lipidomics studies

Reliability, robustness, and interlaboratory comparability of quantitative measurements is critical for clinical lipidomics studies. Lipids’ different ex vivo stability in blood bears the risk of misinterpretation of data. Clear recommendations for the process of blood sample collection are required. We studied by UHPLC-high resolution mass spectrometry, as part of the “Preanalytics interest group” of the International Lipidomics Society, the stability of 417 lipid species in EDTA whole blood after exposure to either 4°C, 21°C, or 30°C at six different time points (0.5 h–24 h) to cover common daily routine conditions in clinical settings. In total, >800 samples were analyzed. 325 and 288 robust lipid species resisted 24 h exposure of EDTA whole blood to 21°C or 30°C, respectively. Most significant instabilities were detected for FA, LPE, and LPC. Based on our data, we recommend cooling whole blood at once and permanent. Plasma should be separated within 4 h, unless the focus is solely on robust lipids. Lists are provided to check the ex vivo (in)stability of distinct lipids and potential biomarkers of interest in whole blood. To conclude, our results contribute to the international efforts towards reliable and comparable clinical lipidomics data paving the way to the proper diagnostic application of distinct lipid patterns or lipid profiles in the future.     

Cholesterol metabolism: from lipidomics to immunology

Oxysterols, the oxidized forms of cholesterol or of its precursors, are formed in the first steps of cholesterol metabolism. Oxysterols have interested chemists, biologists, and physicians for many decades, but their exact biological relevance in vivo, other than as intermediates in bile acid biosynthesis, has long been debated. However, in the first quarter of this century, a role for side-chain oxysterols and their C-7 oxidized metabolites has been convincingly established in the immune system. 25-Hydroxycholesterol has been shown to be synthesized by macrophages in response to the activation of Toll-like receptors and to offer protection against microbial pathogens, whereas 7α,25-dihydroxycholesterol has been shown to act as a chemoattractant to lymphocytes expressing the G protein-coupled receptor Epstein-Barr virus-induced gene 2 and to be important in coordinating the action of B cells, T cells, and dendritic cells in secondary lymphoid tissue. There is a growing body of evidence that not only these two oxysterols but also many of their isomers are of importance to the proper function of the immune system. Here, we review recent findings related to the roles of oxysterols in immunology.     

Integration of lipidomics and metabolomics for in-depth understanding of cellular mechanism and disease progression

Mass spectrometry(MS)-based omics technologies are now widely used to profile small molecules in multiple matrices to confer comprehensive snapshots of cellular metabolic phenotypes.The metabolomes of cells,tissues,and organisms comprise a variety of molecules including lipids,amino acids,sugars,organic acids,and so on.Metabolomics mainly focus on the hydrophilic classes,while lipidomics has emerged as an independent omics owing to the complexities of the organismal lipidomes.The potential roles of lipids and small metabolites in disease pathogenesis have been widely investigated in various human diseases,but system-level understanding is largely lacking,which could be partly attributed to the insufficiency in terms of metabolite coverage and quantitation accuracy in current analytical technologies.While scientists are continuously striving to develop high-coverage omics approaches,integration of metabolomics and lipidomics is becoming an emerging approach to mechanistic investigation.Integration of metabolome and lipidome offers a complete atlas of the metabolic landscape,enabling comprehensive network analysis to identify critical metabolic drivers in disease pathology,facilitating the study of interconnection between lipids and other metabolites in disease progression.In this review,we summarize omics-based findings on the roles of lipids and metabolites in the pathogenesis of selected major diseases threatening public health.We also discuss the advantages of integrating lipidomics and metabolomics for in-depth understanding of molecular mechanism in disease pathogenesis.     

The foundations and development of lipidomics

For over a century, the importance of lipid metabolism in biology was recognized but difficult to mechanistically understand due to the lack of sensitive and robust technologies for identification and quantification of lipid molecular species. The enabling technological breakthroughs emerged in the 1980s with the development of soft ionization methods (Electrospray Ionization and Matrix Assisted Laser Desorption/Ionization) that could identify and quantify intact individual lipid molecular species. These soft ionization technologies laid the foundations for what was to be later named the field of lipidomics. Further innovative advances in multistage fragmentation, dramatic improvements in resolution and mass accuracy, and multiplexed sample analysis fueled the early growth of lipidomics through the early 1990s. The field exponentially grew through the use of a variety of strategic approaches, which included direct infusion, chromatographic separation, and charge-switch derivatization, which facilitated access to the low abundance species of the lipidome. In this Thematic Review, we provide a broad perspective of the foundations, enabling advances, and predicted future directions of growth of the lipidomics field.     

Relationship between hepatic and mitochondrial ceramides: a novel in vivo method to track ceramide synthesis

Ceramides (CERs) are key intermediate sphingolipids implicated in contributing to mitochondrial dysfunction and the development of multiple metabolic conditions. Despite the growing evidence of CER role in disease risk, kinetic methods to measure CER turnover are lacking, particularly using in vivo models. The utility of orally administered ¹³C₃, ¹⁵N l-serine, dissolved in drinking water, was tested to quantify CER 18:1/16:0 synthesis in 10-week-old male and female C57Bl/6 mice. To generate isotopic labeling curves, animals consumed either a control diet or high-fat diet (HFD; n = 24/diet) for 2 weeks and varied in the duration of the consumption of serine-labeled water (0, 1, 2, 4, 7, or 12 days; n = 4 animals/day/diet). Unlabeled and labeled hepatic and mitochondrial CERs were quantified using liquid chromatography tandem MS. Total hepatic CER content did not differ between the two diet groups, whereas total mitochondrial CERs increased with HFD feeding (60%, P < 0.001). Within hepatic and mitochondrial pools, HFD induced greater saturated CER concentrations (P < 0.05) and significantly elevated absolute turnover of 16:0 mitochondrial CER (mitochondria: 59%, P < 0.001 vs. liver: 15%, P = 0.256). The data suggest cellular redistribution of CERs because of the HFD. These data demonstrate that a 2-week HFD alters the turnover and content of mitochondrial CERs. Given the growing data on CERs contributing to hepatic mitochondrial dysfunction and the progression of multiple metabolic diseases, this method may now be used to investigate how CER turnover is altered in these conditions.     

A Decade of Progress in Targeted Therapy for Advanced Thyroid Cancer: An Overview

A better understanding of the molecular aberrations prevalent in thyroid cancers had led to significant advances in the management of advanced thyroid cancer. The landscape of thyroid cancer treatment has grown rapidly. Molecular profiling is the key to identify actionable targets for treatment of advanced disease. In the past decade, there have been regulatory approvals of 9 kinase inhibitors or kinase inhibitor combinations. There are now drugs approved for all of the different types of thyroid cancers, including anaplastic thyroid cancer. However, these drugs are not curative and therefore new strategies and treatments continue to be sought.     

Establishment of a novel anti-TROP2 monoclonal antibody TrMab-29 for immunohistochemical analysis

TROP2 is a type I transmembrane glycoprotein originally identified in human trophoblast cells that is overexpressed in several types of cancer. To better understand the role of TROP2 in cancer, we herein aimed to develop a sensitive and specific anti-TROP2 monoclonal antibody (mAb) for use in flow cytometry, Western blot, and immunohistochemistry using a Cell-Based Immunization and Screening (CBIS) method. Two mice were immunized with N-terminal PA-tagged and C-terminal RAP/MAP-tagged TROP2-overexpressed Chinese hamster ovary (CHO)–K1 cells (CHO/PA-TROP2-RAP-MAP), and hybridomas showing strong signals from PA-tagged TROP2-overexpressed CHO–K1 cells (CHO/TROP2-PA) and weak-to-no signals from CHO–K1 cells were selected using flow cytometry. We demonstrated using flow cytometry that the established anti-TROP2 mAb, TrMab-29 (mouse IgG₁ kappa), detected TROP2 in MCF7 breast cancer cell line as well as CHO/TROP2-PA cells. Western blot analysis showed a 40 kDa band in lysates prepared from both CHO/TROP2-PA and MCF7 cells. Furthermore, TROP2 was strongly detected by immunohistochemical analysis using TrMab-29, indicating that TrMab-29 may be a valuable tool for the detection of TROP2 in cancer.     

A high methionine and low folate diet alters glucose homeostasis and gut microbiome

Hyperhomocysteinemia (HHcy) is considered as a risk factor for several complications, including cardiovascular and neurological disorders. A high methionine low folate (HMLF) diet chronically causes HHcy by accumulating homocysteine in the systemic circulation. Elevated Hcy level is also associated with the incidence of diabetes mellitus. However, very few studies focus on the impact of HMLF diet on glucose homeostasis, and that on gut microbiome profile. HHcy was induced by feeding C57BL/6 mice a HMLF diet for 8 weeks. The HMLF diet feeding resulted in a progressive body weight loss, and development of slight glucose intolerance and insulin resistance in HHcy mice. Notably, the HMLF diet alters the gut microbiome profile and increases the relative abundance of porphyromonadaceae family of bacteria in HHcy mice. These findings provide new insights into the roles of dysregulated glucose homeostasis and gut flora in the pathogenesis of HHcy-related complications.     

The role of cellular active stresses in shaping the zebrafish body axis

Tissue remodelling and organ shaping during morphogenesis are products of mechanical forces generated at the cellular level. These cell-scale forces can be coordinated across the tissue via information provided by biochemical and mechanical cues. Such coordination leads to the generation of complex tissue shape during morphogenesis. In this short review, we elaborate the role of cellular active stresses in vertebrate axis morphogenesis, primarily using examples from postgastrulation development of the zebrafish embryo.     

Simulating cellular galectin networks by mixing galectins in vitro reveals synergistic activity

BackgroundEven though members of the family of adhesion/growth-regulatory galectins are increasingly detected to be co-expressed, they are still being routinely tested separately. The recent discovery of heterodimer formation among galectins-1, -3, and -7 in mixtures prompts further study of their functional activities in mixtures.MethodsCell agglutination, galectin binding to cells, as well as effects on cell proliferation, onset of apoptosis and migration were determined in assays using various cell types and mixtures of galectins-1, -3, and -7.ResultsEvidence for a more than additive increases of experimental parameters was consistently obtained.ConclusionTesting galectins in mixtures simulates the situation of co-expression in situ and reveals unsuspected over-additive activities. This new insight is relevant for analyzing galectin functionality in (patho)physiological conditions.     

Establishment of a novel method for the identification of fertilization defective mutants in Arabidopsis thaliana

Plant reproduction is an extremely important phenomenon, as it is strongly associated with plant genetics and early development. Additionally, foundations of the reproductive system have direct implications on plant breeding and agriculture. Investigation of the functions of male and female gametophytes is critical since their fusion is required for seed formation. Although a large number of mutants have been generated to understand the functions of male and female gametophytes, only a small number of genes required for plant fertilization have been identified to date. This is because the screening method used previously required the dissection of siliques, and fertilization-specific mutants exhibiting semi-fertility (or ∼50% fertility) were difficult to identify. Here, we report a new efficient screening method for the identification of fertilization defective mutants in Arabidopsis thaliana using vanillin staining. This method is based on the pollen tube-dependent ovule enlargement morphology (POEM) phenomenon, which generates a partial seed coat within the ovule without fertilization. Using this method, we successfully identified 23 putative fertilization defective mutants in Arabidopsis.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.     

Early Prediction of COVID-19 Patient Survival by Targeted Plasma Multi-Omics and Machine Learning

The recent surge of coronavirus disease 2019 (COVID-19) hospitalizations severely challenges healthcare systems around the globe and has increased the demand for reliable tests predictive of disease severity and mortality. Using multiplexed targeted mass spectrometry assays on a robust triple quadrupole MS setup which is available in many clinical laboratories, we determined the precise concentrations of hundreds of proteins and metabolites in plasma from hospitalized COVID-19 patients. We observed a clear distinction between COVID-19 patients and controls and, strikingly, a significant difference between survivors and nonsurvivors. With increasing length of hospitalization, the survivors’ samples showed a trend toward normal concentrations, indicating a potential sensitive readout of treatment success. Building a machine learning multi-omic model that considers the concentrations of 10 proteins and five metabolites, we could predict patient survival with 92% accuracy (area under the receiver operating characteristic curve: 0.97) on the day of hospitalization. Hence, our standardized assays represent a unique opportunity for the early stratification of hospitalized COVID-19 patients.     

Novel 13C enrichment technique reveals early turnover of DHA in peripheral tissues

The brain is rich in DHA, which plays important roles in regulating neuronal function. Recently, using compound-specific isotope analysis that takes advantage of natural differences in carbon-13 content (¹³C/¹²C ratio or δ¹³C) of the food supply, we determined the brain DHA half-life. However, because of methodological limitations, we were unable to capture DHA turnover rates in peripheral tissues. In the current study, we applied compound-specific isotope analysis via high-precision GC combustion isotope ratio mass spectrometry to determine half-lives of brain, liver, and plasma DHA in mice following a dietary switch experiment. To model DHA tissue turnover rates in peripheral tissues, we added earlier time points within the diet switch study and took advantage of natural variations in the δ¹³C-DHA of algal and fish DHA sources to maintain DHA pool sizes and used an enriched (uniformly labeled ¹³C) DHA treatment. Mice were fed a fish-DHA diet (control) for 3 months, then switched to an algal-DHA treatment diet, the ¹³C enriched-DHA treatment diet, or they stayed on the control diet for the remainder of the study time course. In mice fed the algal and ¹³C enriched-DHA diets, the brain DHA half-life was 47 and 46 days, the liver half-life was 5.6 and 7.2 days, and the plasma half-life was 4.7 and 6.4 days, respectively. By using improved methodologies, we calculated DHA turnover rates in the liver and plasma, and our study for the first time, by using an enriched DHA source (very high δ¹³C), validated its utility in diet switch studies.     

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.     

Pretreatment of outer membrane vesicle and subsequent infection with influenza virus induces a long-lasting adaptive immune response against broad subtypes of influenza virus

Influenza is an acute respiratory disease and a global health problem. Although influenza vaccines are commercially available, frequent antigenic changes in hemagglutinin might render them less effective or unavailable. We previously reported that modified outer membrane vesicle (fmOMV) provided immediate and robust protective immunity against various subtypes of influenza virus. However, the effect was transient because it was innate immunity-dependent. In this study, we investigated the effects of consecutive administration of fmOMV and influenza virus on the adaptive immune response and long-term protective immunity against influenza virus. When the mice were pretreated with fmOMV and subsequently infected with influenza virus, strong influenza-specific antibody and T cell responses were induced in both systemic and lung mucosal compartments without pathogenic symptoms. Upon the secondary viral challenge at week 4, the mice given fmOMV and influenza virus exhibited almost complete protection against homologous and heterologous viral challenge. More importantly, this strong protective immunity lasted up to 18 weeks after the first infection. These results show that pretreatment with fmOMV and subsequent infection with influenza virus efficiently induces broad and long-lasting protective immunity against various virus subtypes, suggesting a novel antiviral strategy against newly-emerging viral diseases without suitable vaccines or therapeutics.