To Örebro University

Sleep loss and insufficient sleep are risk factors for cardiometabolic diseases, but data on how insufficient sleep contributes to these diseases are scarce. These questions were addressed using two approaches: an experimental, partial sleep restriction study (14 cases and 7 control subjects) with objective verification of sleep amount, and two independent epidemiological cohorts (altogether 2739 individuals) with questions of sleep insufficiency. In both approaches, blood transcriptome and serum metabolome were analysed. Sleep loss decreased the expression of genes encoding cholesterol transporters and increased expression in pathways involved in inflammatory responses in both paradigms. Metabolomic analyses revealed lower circulating large HDL in the population cohorts among subjects reporting insufficient sleep, while circulating LDL decreased in the experimental sleep restriction study. These findings suggest that prolonged sleep deprivation modifies inflammatory and cholesterol pathways at the level of gene expression and serum lipoproteins, inducing changes toward potentially higher risk for cardiometabolic diseases.

Several small molecule biomarkers have been reported in the literature for prediction and diagnosis of (pre)diabetes, its co-morbidities, and complications. Here, we report the development and validation of a novel, quantitative method for the determination of a selected panel of 34 metabolite biomarkers from human plasma. We selected a panel of metabolites indicative of various clinically-relevant pathogenic stages of diabetes. We combined these candidate biomarkers into a single ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method and optimized it, prioritizing simplicity of sample preparation and time needed for analysis, enabling high-throughput analysis in clinical laboratory settings. We validated the method in terms of limits of detection (LOD) and quantitation (LOQ), linearity (R²), and intra- and inter-day repeatability of each metabolite. The method's performance was demonstrated in the analysis of selected samples from a diabetes cohort study. Metabolite levels were associated with clinical measurements and kidney complications in type 1 diabetes (T1D) patients. Specifically, both amino acids and amino acid-related analytes, as well as specific bile acids, were associated with macro-albuminuria. Additionally, specific bile acids were associated with glycemic control, anti-hypertensive medication, statin medication, and clinical lipid measurements. The developed analytical method is suitable for robust determination of selected plasma metabolites in the diabetes clinic.

This study has followed a birth cohort for over 20 years to find factors associated with neurodevelopmental disorder (ND) diagnosis. Detailed, early-life longitudinal questionnaires captured infection and antibiotic events, stress, prenatal factors, family history, and more. Biomarkers including cord serum metabolome and lipidome, human leukocyte antigen (HLA) genotype, infant microbiota, and stool metabolome were assessed. Among the 16,440 Swedish children followed across time, 1,197 developed an ND. Significant associations emerged for future ND diagnosis in general and for specific ND subtypes, spanning intellectual disability, speech disorder, attention-deficit/hyperactivity disorder, and autism. This investigation revealed microbiome connections to future diagnosis as well as early emerging mood and gastrointestinal problems. The findings suggest links to immunodysregulation and metabolism, compounded by stress, early-life infection, and antibiotics. The convergence of infant biomarkers and risk factors in this prospective, longitudinal study on a large-scale population establishes a foundation for early-life prediction and intervention in neurodevelopment.

Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure.

PFAS are ubiquitous industrial chemicals with known adverse health effects, particularly on the liver. The liver, being a vital metabolic organ, is susceptible to PFAS-induced metabolic dysregulation, leading to conditions such as hepatotoxicity and metabolic disturbances. In this study, we investigated the phenotypic and metabolic responses of PFAS exposure using two hepatocyte models, HepG2 (male cell line) and HepaRG (female cell line), aiming to define phenotypic alterations, and metabolic disturbances at the metabolite and pathway levels. The PFAS mixture composition was selected based on epidemiological data, covering a broad concentration spectrum observed in diverse human populations. Phenotypic profiling by Cell Painting assay disclosed predominant effects of PFAS exposure on mitochondrial structure and function in both cell models as well as effects on F-actin, Golgi apparatus, and plasma membrane-associated measures. We employed comprehensive metabolic characterization using liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS). We observed dose-dependent changes in the metabolic profiles, particularly in lipid, steroid, amino acid and sugar and carbohydrate metabolism in both cells as well as in cell media, with HepaRG cell line showing a stronger metabolic response. In cells, most of the bile acids, acylcarnitines and free fatty acids showed downregulation, while medium-chain fatty acids and carnosine were upregulated, while the cell media showed different response especially in relation to the bile acids in HepaRG cell media. Importantly, we observed also nonmonotonic response for several phenotypic features and metabolites. On the pathway level, PFAS exposure was also associated with pathways indicating oxidative stress and inflammatory responses. Taken together, our findings on PFAS-induced phenotypic and metabolic disruptions in hepatocytes shed light on potential mechanisms contributing to the broader comprehension of PFAS-related health risks.

Lipids have many important biological roles, such as energy storage sources, structural components of plasma membranes and as intermediates in metabolic and signaling pathways. Lipid metabolism is under tight homeostatic control, exhibiting spatial and dynamic complexity at multiple levels. Consequently, lipid-related disturbances play important roles in the pathogenesis of most of the common diseases. Lipidomics, defined as the study of lipidomes in biological systems, has emerged as a rapidly-growing field. Due to the chemical and functional diversity of lipids, the application of a systems biology approach is essential if one is to address lipid functionality at different physiological levels. In parallel with analytical advances to measure lipids in biological matrices, the field of computational lipidomics has been rapidly advancing, enabling modeling of lipidomes in their pathway, spatial and dynamic contexts. This review focuses on recent progress in systems biology approaches to study lipids in health and disease, with specific emphasis on methodological advances and biomedical applications.

AIMS: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function.

METHODS AND RESULTS: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to β-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of β1-adrenergic receptors.

CONCLUSIONS: Our findings suggest that cardiac glycosphingolipids are required to maintain β-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.

The human colon contains a diverse microbial population which contributes to degradation and metabolism of food components. Drug metabolism in the colon is generally poorly understood. Metabolomics techniques and in vitro colon models are now available which afford detailed characterization of drug metabolites in the context of colon metabolism. The aim of this work was to identify novel drug metabolites of Simvastatin (SV) by using an anaerobic human in vitro colon model at body temperature coupled with systems biology platform, excluding the metabolism of the host liver and intestinal epithelia. Comprehensive two-dimensional gas chromatography with a time-of-flight mass spectrometry (GC×GC-TOFMS) was used for the metabolomic analysis. Metabolites showing the most significant differences in the active faecal suspension were elucidated in reference with SV fragmentation and compared with controls: inactive suspension or buffer with SV, or with active suspension alone. Finally, time courses of selected metabolites were investigated. Our data suggest that SV is degraded by hydrolytic cleavage of methylbutanoic acid from the SV backbone. Metabolism involves demethylation of dimethylbutanoic acid, hydroxylation/dehydroxylation and β-oxidation resulting in the production of 2-hydroxyisovaleric acid (3-methyl-2-hydroxybutanoic acid), 3-hydroxybutanoic acid and lactic acid (2-hydroxypropanoic acid), and finally re-cyclisation of heptanoic acid (possibly de-esterified and cleaved methylpyranyl arm) to produce cyclohexanecarboxylic acid. Our study elucidates a pathway of colonic microbial metabolism of SV as well as demonstrates the applicability of the in vitro colon model and metabolomics to the discovery of novel drug metabolites from drug response profiles.

PURPOSE: Syrah red grapes are used in the production of tannin-rich red wines. Tannins are high molecular weight molecules, proanthocyanidins (PAs), and poorly absorbed in the upper intestine. In this study, gut microbial metabolism of Syrah grape phenolic compounds was investigated.

METHODS: Syrah grape pericarp was subjected to an enzymatic in vitro digestion model, and red wine and grape skin PA fraction were prepared. Microbial conversion was screened using an in vitro colon model with faecal microbiota, by measurement of short-chain fatty acids by gas chromatography (GC) and microbial phenolic metabolites using GC with mass detection (GC-MS). Red wine metabolites were further profiled using two-dimensional GC mass spectrometry (GCxGC-TOFMS). In addition, the effect of PA structure and dose on conversion efficiency was investigated by GC-MS.

RESULTS: Red wine exhibited a higher degree of C1-C3 phenolic acid formation than PA fraction or grape pericarp powders. Hydroxyphenyl valeric acid (flavanols and PAs as precursors) and 3,5-dimethoxy-4-hydroxybenzoic acid (anthocyanin as a precursor) were identified from the red wine metabolite profile. In the absence of native grape pericarp or red wine matrix, the isolated PAs were found to be effective in the dose-dependent inhibition of microbial conversions and short-chain fatty acid formation.

CONCLUSIONS: Metabolite profiling was complementary to targeted analysis. The identified metabolites had biological relevance, because the structures of the metabolites resembled fragments of their grape phenolic precursors or were in agreement with literature data.

BACKGROUND: Prenatal exposure to environmental contaminants is a significant health concern because it has the potential to interfere with host metabolism, leading to adverse health effects in early childhood and later in life. Growing evidence suggests that genetic and environmental factors, as well as their interactions, play a significant role in the development of autoimmune diseases.

OBJECTIVE: In this study, we hypothesized that prenatal exposure to environmental contaminants impacts cord serum metabolome and contributes to the development of autoimmune diseases.

METHODS: We selected cord serum samples from All Babies in Southeast Sweden (ABIS) general population cohort, from infants who later developed one or more autoimmune-mediated and inflammatory diseases: celiac disease (CD), Crohn's disease (IBD), hypothyroidism (HT), juvenile idiopathic arthritis (JIA), and type 1 diabetes (T1D) (all cases, N = 62), along with matched controls (N = 268). Using integrated exposomics and metabolomics mass spectrometry (MS) based platforms, we determined the levels of environmental contaminants and metabolites.

RESULTS: Differences in exposure levels were found between the controls and those who later developed various diseases. High contaminant exposure levels were associated with changes in metabolome, including amino acids and free fatty acids. Specifically, we identified marked associations between metabolite profiles and exposure levels of deoxynivalenol (DON), bisphenol S (BPS), and specific per- and polyfluorinated substances (PFAS).

IMPACT STATEMENT: Abnormal metabolism is a common feature preceding several autoimmune and inflammatory diseases. However, few studies compared common and specific metabolic patterns preceding these diseases. Here we hypothesized that exposure to environmental contaminants impacts cord serum metabolome, which may contribute to the development of autoimmune diseases. We found differences in exposure levels between the controls and those who later developed various diseases, and importantly, on the metabolic changes associated with the exposures. High contaminant exposure levels were associated with specific changes in metabolome. Our study suggests that prenatal exposure to specific environmental contaminants alters the cord serum metabolomes, which, in turn, might increase the risk of various immune-mediated diseases.

Polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants still present in aquatic environments despite their total or partial ban. Previously, we observed that an environmentally realistic mixture of these compounds affects energy balance, growth, and reproduction in exposed zebrafish (F0), and behavior in their unexposed offspring (F1-F4). In the present work, we performed lipidomic and transcriptomic analyses on brains of zebrafish (F0-F2) from exposed and control lineages to identify molecular changes that could explain the observed phenotypes. The use of both technologies highlighted that F0 zebrafish displayed impaired mitochondrial function and lipid metabolism regulation (depletion in triacylglycerols and phospholipids) which can explain disruption of energy homeostasis. A subset of the regulated biological pathways related to energetic metabolism and neurotransmission were inherited in 12. In addition, there were increasing effects on epigenetic pathways from the F0 to the F2 generation. Altogether, we show that the effects of an environmental exposure to PCBs and PBDEs on energetic metabolism as well as neurotransmission extend over 2 generations of zebrafish, possibly due to transgenerational epigenetic inheritance.

Transgenerational effects induced by environmental stressors are a threat to ecosystems and human health. However, there is still limited observation and understanding of the potential of chemicals to influence life outcomes over several generations. In the present study, we investigated the effects of two environmental contaminants, coumarin 47 and permethrin, on exposed zebrafish (FO) and their progeny (F1-F3). Coumarin 47 is commonly found in personal care products and dyes, whereas permethrin is used as a domestic and agricultural pyrethroid insecticide/insect repellent. Zebrafish (F0) were exposed during early development until 28 days post-fertilization and their progeny (F1-F3) were bred unexposed. On one hand, the effects induced by coumarin 47 suggest no multigenerational toxicity. On the other hand, we found that behavior of zebrafish larvae was significantly affected by exposure to permethrin in F1 to F3 generations with some differences depending on the concentration. This suggests persistent alteration of the neural or neuromuscular function. In addition, lipidomic analyses showed that permethrin treatment was partially correlated with lysophosphatidylcholine levels in zebrafish, an important lipid for neurodevelopment. Overall, these results stress out one of the most widely used pyrethroids can trigger long-term, multi- and possibly transgenerational changes in the nervous system of zebrafish. These neurobehavioral changes echo the effects observed under direct exposure to high concentrations of permethrin and therefore call for more research on mechanisms underlying effect inheritance.

Postprandial responses to food are complex, involving both genetic and environmental factors. We studied postprandial responses to a Big Mac meal challenge in monozygotic co-twins highly discordant for body weight. This unique design allows assessment of the contribution of obesity, independent of genetic liability. Comprehensive metabolic profiling using 3 analytical platforms was applied to fasting and postprandial serum samples from 16 healthy monozygotic twin pairs discordant for weight (body mass index difference >3 kg/m(2)). Nine concordant monozygotic pairs were examined as control pairs. Fecal samples were analyzed to assess diversity of the major bacterial groups by using 5 different validated bacterial group specific denaturing gradient gel electrophoresis methods. No differences in fecal bacterial diversity were detected when comparing co-twins discordant for weight (ANOVA, P<0.05). We found that within-pair similarity is a dominant factor in the metabolic postprandial response, independent of acquired obesity. Branched chain amino acids were increased in heavier as compared with leaner co-twins in the fasting state, but their levels converged postprandially (paired t tests, FDR q<0.05). We also found that specific bacterial groups were associated with postprandial changes of specific metabolites. Our findings underline important roles of genetic and early life factors in the regulation of postprandial metabolite levels.

SCOPE: Dysregulation of lipid homeostasis is related to multiple major healthcare problems. The aim of this study was to investigate the effects of n-3 fatty acid (FA) and polyphenol rich diets on plasma and HDL fraction lipidomic profiles in subjects at high cardiovascular risk.

METHODS AND RESULTS: Ultra performance LC coupled to quadrupole TOF/MS mass spectrometry global lipidomic profiling was applied to plasma and HDL fraction from an 8 wk randomized intervention with four isoenergetic diets, differing in their natural n-3 FA and polyphenols content, in 78 subjects with a high BMI, abdominal obesity, and at least one other feature of the metabolic syndrome. Dependency network analysis showed a different pattern of associations between lipidomics, dietary, and clinical variables after the dietary interventions. The most remarkable associations between variables were observed after the diet high in n-3 FA and polyphenols, as the inverse association between gallic acid intake and LDL cholesterol levels, which was indirectly associated with a HDL cluster exclusively comprised lysophospholipids.

CONCLUSION: This is the first human randomized controlled trial showing direct and indirect associations with lipid molecular species and clinical variables of interest in the evaluation of the metabolic syndrome after diets naturally rich in polyphenols.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950 Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each lab using a different lipidomics workflow. A total of 1527 unique lipids were measured across all laboratories, and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and inter-laboratory quality control and method validation. These analyses were performed using non-standardized laboratory-independent workflows. The consensus locations were also compared to a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

OBJECTIVE: Gut microbial products are involved in regulation of host metabolism. In human and experimental studies, we explored the potential role of hippurate, a hepatic phase 2 conjugation product of microbial benzoate, as a marker and mediator of metabolic health.

DESIGN: In 271 middle-aged non-diabetic Danish individuals, who were stratified on habitual dietary intake, we applied ¹H-nuclear magnetic resonance (NMR) spectroscopy of urine samples and shotgun-sequencing-based metagenomics of the gut microbiome to explore links between the urine level of hippurate, measures of the gut microbiome, dietary fat and markers of metabolic health. In mechanistic experiments with chronic subcutaneous infusion of hippurate to high-fat-diet-fed obese mice, we tested for causality between hippurate and metabolic phenotypes.

RESULTS: In the human study, we showed that urine hippurate positively associates with microbial gene richness and functional modules for microbial benzoate biosynthetic pathways, one of which is less prevalent in the Bacteroides 2 enterotype compared with Ruminococcaceae or Prevotella enterotypes. Through dietary stratification, we identify a subset of study participants consuming a diet rich in saturated fat in which urine hippurate concentration, independently of gene richness, accounts for links with metabolic health. In the high-fat-fed mice experiments, we demonstrate causality through chronic infusion of hippurate (20 nmol/day) resulting in improved glucose tolerance and enhanced insulin secretion.

CONCLUSION: Our human and experimental studies show that a high urine hippurate concentration is a general marker of metabolic health, and in the context of obesity induced by high-fat diets, hippurate contributes to metabolic improvements, highlighting its potential as a mediator of metabolic health.

Data processing and identification of unknown compounds in comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC x GC/TOFMS) analysis is a major challenge, particularly when large sample sets are analyzed. Herein, we present a method for efficient treatment of large data sets produced by GC x GC/TOFMS implemented as a freely available open source software package, Guineu. To handle large data sets and to efficiently utilize all the features available in the vendor software (baseline correction, mass spectral deconvolution, peak picking, integration, library search, and signal-to-noise filtering), data preprocessed by instrument software are used as a starting point for further processing. Our software affords alignment of the data, normalization, data filtering, and utilization of retention indexes in the verification of identification as well as a novel tool for automated group-type identification of the compounds. Herein, different features of the software are studied in detail and the performance of the system is verified by the analysis of a large set of standard samples as well as of a large set of authentic biological samples, including the control samples. The quantitative features of our GC x GC/TOFMS methodology are also studied to further demonstrate the method performance and the experimental results confirm the reliability of the developed procedure. The methodology has already been successfully used for the analysis of several thousand samples in the field of metabolomics.

Using dill (Anethum graveolens L.) as a model herb, we revealnovel associations between metabolite profile and sensory quality, by integrating non-target metabolomics with sensory data. Low night temperatures and exposure to UV-enriched light was used to modulate plant metabolism, thereby improving sensory quality. Plant age is a crucial factor associated with accumulation of dill ether and α-phellandrene, volatile compounds associated with dill flavour. However, sensory analysis showed that neither of these compounds has any strong association with dill taste. Rather, amino acids alanine, phenylalanine, glutamic acid, valine, and leucine increased in samples exposed to eustress and were positively associated with dill and sour taste. Increases in amino acids and organic acids changed the taste from lemon/grass to a more bitter/pungent dill-related taste. Our approach reveals a novel approach to establish links between effects of eustressors on sensory quality, and may be applicable to a broad range of crops.

Greenhouse horticulture is in its broad definition the production of plant products within, under or sheltered by structures that provide protection against biotic and/or abiotic stress. In greenhouses, horticultural crops can grow protected from infectious agents and adverse weather conditions, allowing off-season, year-round production. However, greenhouse production often comes with a trade-off, which is a skewed light environment with a lack of UV light. 

In some instances, the blockage of UV by greenhouse glass and plastic covers is beneficial from a commercial perspective, especially on tropical latitudes where plants can often encounter higher UV levels, which may impair plant growth and nutrient absorption (Krause et al. 1999; Verdaguer et al. 2017). On the other hand, reduced UV inside greenhouses may reduce the synthesis of metabolites associated with crop protection against biotic and abiotic stress, such as flavonoids, terpenoids and alkaloids (Yang et al. 2018). This reduction in the amount of protective compounds may not be seen as an important limitation in a protected environment, but these metabolic changes caused by reduced UV exposure may in fact negatively impact on product quality. For example, it is possible to improve of the aroma and taste of greenhouse tomato by exposing plants to low levels of supplementary UV light (Dzakovich et al. 2016).

PURPOSE OF REVIEW: This review focuses on the recent findings from lipidomics studies as related to nutrition and health research.

RECENT FINDINGS: Several lipidomics studies have investigated malnutrition, including both under- and overnutrition. Focus has been both on the early-life nutrition as well as on the impact of overfeeding later in life. Multiple studies have investigated the impact of different macronutrients in lipidome on human health, demonstrating that overfeeding with saturated fat is metabolically more harmful than overfeeding with polyunsaturated fat or carbohydrate-rich food. Diet rich in saturated fat increases the lipotoxic lipids, such as ceramides and saturated fatty-acyl-containing triacylglycerols, increasing also the low-density lipoprotein aggregation rate. In contrast, diet rich in polyunsaturated fatty acids, such as n-3 fatty acids, decreases the triacylglycerol levels, although some individuals are poor responders to n-3 supplementation.

SUMMARY: The results highlight the benefits of lipidomics in clinical nutrition research, also providing an opportunity for personalized nutrition. An area of increasing interest is the interplay of diet, gut microbiome, and metabolome, and how they together impact individuals' responses to nutritional challenges.

There is an established, link between psychosis and metabolic abnormalities, such as altered glucose metabolism and dyslipidemia, which often precede the initiation of antipsychotic treatment. It is known that obesity-associated metabolic disorders are promoted by activation of specific cannabinoid targets (endocannabinoid system (ECS)). Our recent data suggest that there is a change in the circulating lipidome at the onset of first episode psychosis (FEP). With the aim of characterizing the involvement of the central and peripheral ECSs, and their mutual associations; here, we performed a combined neuroimaging and metabolomic study in patients with FEP and healthy controls (HC). Regional brain cannabinoid receptor type 1 (CB1R) availability was quantified in two, independent samples of patients with FEP (n = 20 and n = 8) and HC (n = 20 and n = 10), by applying three-dimensional positron emission tomography, using two radiotracers, [¹¹C]MePPEP and [¹⁸F]FMPEP-d2. Ten endogenous cannabinoids or related metabolites were quantified in serum, drawn from these individuals during the same imaging session. Circulating levels of arachidonic acid and oleoylethanolamide (OEA) were reduced in FEP individuals, but not in those who were predominantly medication free. In HC, there was an inverse association between levels of circulating arachidonoyl glycerol, anandamide, OEA, and palmitoyl ethanolamide, and CB1R availability in the posterior cingulate cortex. This phenomenon was, however, not observed in FEP patients. Our data thus provide evidence of cross talk, and dysregulation between peripheral endocannabinoids and central CB1R availability in FEP.

BACKGROUND: A key clinical challenge in the management of individuals at clinical high risk for psychosis (CHR) is that it is difficult to predict their future clinical outcomes. Here, we investigated if the levels of circulating molecular lipids are related to adverse clinical outcomes in this group.

METHODS: Serum lipidomic analysis was performed in 263 CHR individuals and 51 healthy control subjects, who were then clinically monitored for up to 5 years. Machine learning was used to identify lipid profiles that discriminated between CHR and control subjects, and between subgroups of CHR subjects with distinct clinical outcomes.

RESULTS: At baseline, compared with control subjects, CHR subjects (independent of outcome) had higher levels of triacylglycerols with a low acyl carbon number and a double bond count, as well as higher levels of lipids in general. CHR subjects who subsequently developed psychosis (n = 50) were distinguished from those that did not (n = 213) on the basis of lipid profile at baseline using a model with an area under the receiver operating curve of 0.81 (95% confidence interval = 0.69-0.93). CHR subjects who became psychotic had lower levels of ether phospholipids than CHR individuals who did not (p < .01).

CONCLUSIONS: Collectively, these data suggest that lipidomic abnormalities predate the onset of psychosis and that blood lipidomic measures may be useful in predicting which CHR individuals are most likely to develop psychosis.

There is a need to rapidly detect patients with traumatic brain injury (TBI) who require head computed tomography (CT). Given the energy crisis in the brain following TBI, we hypothesized that serum metabolomics would be a useful tool for developing a set of biomarkers to determine the need for CT and to distinguish between different types of injuries observed. Logistic regression models using metabolite data from the discovery cohort (n=144, Turku, Finland) were used to distinguish between patients with traumatic intracranial findings and negative findings on head CT. The resultant models were then tested in the validation cohort (n=66, Cambridge, UK). The levels of glial fibrillary acidic protein and ubiquitin C-terminal hydrolase-L1 were also quantified in the serum from the same patients. Despite there being significant differences in the protein biomarkers in patients with TBI, the model that determined the need for a CT scan validated poorly (AUC=0.64: Cambridge patients). However, using a combination of six metabolites (two amino acids, three sugar derivatives and one ketoacid) it was possible to discriminate patients with intracranial abnormalities on CT and patients with a normal CT (AUC=0.77 in Turku patients and AUC=0.73 in Cambridge patients). Furthermore, a combination of three metabolites could distinguish between diffuse brain injuries and mass lesions (AUC=0.87 in Turku patients and AUC=0.68 in Cambridge patients). This study identifies a set of validated serum polar metabolites, which associate with the need for a CT scan. Additionally, serum metabolites can also predict the nature of the brain injury. These metabolite markers may prevent unnecessary CT scans, thus reducing the cost of diagnostics and radiation load.

Purpose: Disturbed metabolism of cholesterol and triacylglycerols (TGs) carries increased risk for coronary artery calcification (CAC). However, the exact relationship between individual lipid species and CAC remains unclear. The aim of this study was to identify disturbances in lipid profiles involved in the calcification process, in an attempt to propose potential biomarker candidates.

Patients and methods: We studied 70 patients at intermediate risk for coronary artery disease who had undergone coronary calcification assessment using computed tomography and Agatston coronary artery calcium score (CACS). Patients were divided into three groups: with no coronary calcification (NCC; CACS: 0; n=26), mild coronary calcification (MCC; CACS: 1-250; n=27), or severe coronary calcification (SCC; CACS: >250; n=17). Patients' serum samples were analyzed using liquid chromatography-mass spectrometry in an untargeted lipidomics approach.

Results: We identified 103 lipids within the glycerolipid, glycerophospholipid, sphingolipid, and sterol lipid classes. After false discovery rate correction, phosphatidylcholine (PC)(16:0/20:4) in higher levels and PC(18:2/18:2), PC(36:3), and phosphatidylethanolamine(20:0/18:2) in lower levels were identified as correlates with SCC compared to NCC. There were no significant differences in the levels of individual TGs between the three groups; however, clustering the lipid profiles showed a trend for higher levels of saturated and monounsaturated TGs in SCC compared to NCC. There was also a trend for lower TG (49:2), TG(51:1), TG(54:5), and TG(56:8) levels in SCC compared to MCC.

Conclusion: In this study we investigated the lipidome of patients with coronary calcification. Our results suggest that the calcification process may be associated with dysfunction in autophagy. The lipidomic biomarkers revealed in this study may aid in better assessment of patients with subclinical coronary artery disease.

A vegetarian diet has been associated with a lower risk of coronary artery disease (CAD). Plasma triacylglycerols, ceramides, and phosphatidylcholines may improve prediction of recurrent coronary events. We sought to investigate effects of a lacto-ovo-vegetarian diet (VD) on plasma lipidome in CAD patients and simultaneously assess associations of plasma lipids with the extent of coronary atherosclerotic burden. We analyzed 214 plasma lipids within glycerolipid, sphingolipid, and sterol lipid classes using lipidomics from a randomized controlled, crossover trial comprising 31 CAD patients on standard medical therapy. Subjects completed a four-week intervention with VD and isocaloric meat diet (MD), separated by a four-week washout period. The VD increased levels of 11 triacylglycerols and lowered 7 triacylglycerols, 21 glycerophospholipids, cholesteryl ester (18:0), and ceramide (d18:1/16:0) compared with MD. VD increased triacylglycerols with long-chain polyunsaturated fatty acyls while decreased triacylglycerols with saturated fatty acyls, phosphatidylcholines, and sphingomyelins than MD. The Sullivan extent score (SES) exhibited on coronary angiograms were inversely associated with triacylglycerols with long-chain unsaturated fatty acyls. Phosphatidylcholines that were lower with VD were positively associated with SES and the total number of stenotic lesions. The VD favorably changed levels of several lipotoxic lipids that have previously been associated with increased risk of coronary events in CAD patients.

Anorexia nervosa (AN) is an eating disorder with a high mortality. About 95% of cases are women and it has a population prevalence of about 1%, but evidence-based treatment is lacking. The pathogenesis of AN probably involves genetics and various environmental factors, and an altered gut microbiota has been observed in individuals with AN using amplicon sequencing and relatively small cohorts. Here we investigated whether a disrupted gut microbiota contributes to AN pathogenesis. Shotgun metagenomics and metabolomics were performed on faecal and serum samples, respectively, from a cohort of 77 females with AN and 70 healthy females. Multiple bacterial taxa (for example, Clostridium species) were altered in AN and correlated with estimates of eating behaviour and mental health. The gut virome was also altered in AN including a reduction in viral-bacterial interactions. Bacterial functional modules associated with the degradation of neurotransmitters were enriched in AN and various structural variants in bacteria were linked to metabolic features of AN. Serum metabolomics revealed an increase in metabolites associated with reduced food intake (for example, indole-3-propionic acid). Causal inference analyses implied that serum bacterial metabolites are potentially mediating the impact of an altered gut microbiota on AN behaviour. Further, we performed faecal microbiota transplantation from AN cases to germ-free mice under energy-restricted feeding to mirror AN eating behaviour. We found that the reduced weight gain and induced hypothalamic and adipose tissue gene expression were related to aberrant energy metabolism and eating behaviour. Our 'omics' and mechanistic studies imply that a disruptive gut microbiome may contribute to AN pathogenesis.

Lipidomics is a rapidly-growing field which focuses on global characterization of lipids at molecular and systems levels. As small changes in the concentrations of lipids may have important physiological consequences, much attention in the field has recently been paid to more accurate quantitation and identification of lipids. Community-wide efforts have been initiated, aiming to develop best practices for lipidomic analyses and reporting of lipidomic data. Nevertheless, current approaches for comprehensive analysis of lipidomes have some inherent challenges and limitations. Additionally, there is, currently, limited knowledge concerning the impacts of various external and internal exposures on lipid levels. In this review, we discuss the recent progress in lipidomics analysis, with a primary focus on analytical approaches, as well as on the different sources of variation in quantifying lipid levels, both technical and biological.

BACKGROUND: Environmental factors are strongly implicated in late-onset of inflammatory bowel disease. Here, we investigate whether high levels of perfluoroalkyl substances are associated with (1) late-onset inflammatory bowel disease, and (2) disturbances of the bile acid pool. We further explore the effect of the specific perfluoroalkyl substance perfluorooctanoic acid on intestinal barrier function in murine tissue.

METHODS: Serum levels of perfluoroalkyl substances and bile acids were assessed by ultra-performance liquid chromatography coupled to a triple-quadrupole mass spectrometer in matched samples from patients with ulcerative colitis (n = 20) and Crohn's disease (n = 20) diagnosed at the age of ≥55 years. Age and sex-matched blood donors (n = 20), were used as healthy controls. Ex vivo Ussing chamber experiments were performed to assess the effect of perfluorooctanoic acid on ileal and colonic murine tissue (n = 9).

RESULTS: The total amount of perfluoroalkyl substances was significantly increased in patients with ulcerative colitis compared to healthy controls and patients with Crohn's disease (p < .05). Ex vivo exposure to perfluorooctanoic acid induced a significantly altered ileal and colonic barrier function. The distribution of bile acids, as well as the correlation pattern between (1) perfluoroalkyl substances and (2) bile acids, differed between patient and control groups.

DISCUSSION: Our results demonstrate that perfluoroalkyl substances levels are increased in patients with late-onset ulcerative colitis and may contribute to the disease by inducing a dysfunctional intestinal barrier.

BACKGROUND/AIM: Lipidomic and metabolomic techniques become more and more important in human health research. Recent developments in analytical techniques enable the investigation of high amounts of substances. The high numbers of metabolites and lipids that are detected with among others mass spectrometric techniques challenge in most cases the statistical processes to bring out stable and interpretable results. This study targets to use the novel non-established statistical method treelet transform (TT) to investigate high numbers of metabolites and lipids and to compare the results with the established method principal component analysis (PCA). Serum lipid and metabolite profiles are investigated regarding their association to anthropometric parameters associated to obesity.

METHODS: From 226 participants of the EPIC (European Prospective Investigation into Cancer and Nutrition)-Potsdam study blood samples were investigated with an untargeted metabolomics approach regarding serum metabolites and lipids. Additionally, participants were surveyed anthropometrically to assess parameters of obesity, such as body mass index (BMI), waist-to-hip-ratio (WHR) and body fat mass. TT and PCA are used to generate treelet components (TCs) and factors summarizing serum metabolites and lipids in new, latent variables without too much loss of information. With partial correlations TCs and factors were associated to anthropometry under the control for relevant parameters, such as sex and age.

RESULTS: TT with metabolite variables (p=121) resulted in 5 stable and interpretable TCs explaining 18.9% of the variance within the data. PCA on the same variables generated 4 quite complex, less easily interpretable factors explaining 37.5% of the variance. TT on lipidomic data (p=353) produced 3 TCs as well as PCA on the same data resulted in 3 factors; the proportion of explained variance was 17.8% for TT and 39.8% for PCA. In both investigations TT ended up with stable components that are easier to interpret than the factors from the PCA. In general, the generated TCs and factors were similar in their structure when the factors are considered regarding the original variables loading high on them. Both TCs and factors showed associations to anthropometric measures.

CONCLUSIONS: TT is a suitable statistical method to generate summarizing, latent variables in data sets with more variables than observations. In the present investigation it resulted in similar latent variables compared to the established method of PCA. Whereby less variance is explained by the summarizing constructs of TT compared to the factors of PCA, TCs are easier to interpret. Additionally the resulting TCs are quite stable in bootstrap samples.

Psychotic disorders are associated with metabolic abnormalities including alterations in glucose and lipid metabolism. A major challenge in the treatment of psychosis is to identify patients with vulnerable metabolic profiles who may be at risk of developing cardiometabolic co-morbidities. It is established that both central and peripheral metabolic organs use lipids to control energy balance and regulate peripheral insulin sensitivity. The endocannabinoid system, implicated in the regulation of glucose and lipid metabolism, has been shown to be dysregulated in psychosis. It is currently unclear how these endocannabinoid abnormalities relate to metabolic changes in psychosis. Here we review recent research in the field of metabolic co-morbidities in psychotic disorders as well as the methods to study them and potential links to the endocannabinoid system. We also describe the bioinformatics platforms developed in the EU project METSY for the investigations of the biological etiology in patients at risk of psychosis and in first episode psychosis patients. The METSY project was established with the aim to identify and evaluate multi-modal peripheral and neuroimaging markers that may be able to predict the onset and prognosis of psychiatric and metabolic symptoms in patients at risk of developing psychosis and first episode psychosis patients. Given the intrinsic complexity and widespread role of lipid metabolism, a systems biology approach which combines molecular, structural and functional neuroimaging methods with detailed metabolic characterisation and multi-variate network analysis is essential in order to identify how lipid dysregulation may contribute to psychotic disorders. A decision support system, integrating clinical, neuropsychological and neuroimaging data, was also developed in order to aid clinical decision making in psychosis. Knowledge of common and specific mechanisms may aid the etiopathogenic understanding of psychotic and metabolic disorders, facilitate early disease detection, aid treatment selection and elucidate new targets for pharmacological treatments.

Perfluorooctanesulfonate (PFOS) is a well-known contaminant in the environment and it has shown to disrupt multiple biological pathways, particularly those related with lipid metabolism. In this study, we have studied the impact of in ovo exposure to PFOS on lipid metabolism in livers in developing chicken embryos using lipidomics for detailed characterization of the liver lipidome. We used an avian model (Gallus gallus domesticus) for in ovo treatment at two levels of PFOS. The lipid profile of the liver of the embryo was investigated by ultra-high performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry and by gas chromatography mass spectrometry. Over 170 lipids were identified, covering phospholipids, ceramides, di- and triacylglycerols, cholesterol esters and fatty acid composition of the lipids. The PFOS exposure caused dose dependent changes in the lipid levels, which included upregulation of specific phospholipids associated with the phosphatidylethanolamine N-methyltransferase (PEMT) pathway, triacylglycerols with low carbon number and double bond count as well as of lipotoxic ceramides and diacylglycerols. Our data suggest that at lower levels of exposure, mitochondrial fatty acid β-oxidation is suppressed while the peroxisomal fatty acid β -oxidation is increased. At higher doses, however, both β -oxidation pathways are upregulated.

Islet autoimmunity in children who later progress to type 1 diabetes is preceded by dysregulated serum metabolite profiles, but the origin of these metabolic changes is unknown. The gut microbiota affects host metabolism and changes in its composition contribute to several immune-mediated diseases; however, it is not known whether the gut microbiota is involved in the early metabolic disturbances in progression to type 1 diabetes. We rederived non-obese diabetic (NOD) mice as germ free to explore the potential role of the gut microbiota in the development of diabetic autoimmunity and to directly investigate whether the metabolic profiles associated with the development of type 1 diabetes can be modulated by the gut microbiota. The absence of a gut microbiota in NOD mice did not affect the overall diabetes incidence but resulted in increased insulitis and levels of interferon gamma and interleukin 12; these changes were counterbalanced by improved peripheral glucose metabolism. Furthermore, we observed a markedly increased variation in blood glucose levels in the absence of a microbiota in NOD mice that did not progress to diabetes. Additionally, germ-free NOD mice had a metabolite profile similar to that of pre-diabetic children. Our data suggest that germ-free NOD mice have reduced glycaemic control and dysregulated immunologic and metabolic responses.

BACKGROUND: Supplementation of formula with bovine milk fat globule membranes has been shown to narrow the gap in immunological and cognitive development between breast-fed and formula-fed infants.

METHOD: In a double-blinded randomized controlled trial 160 formula-fed infants received an experimental formula (EF), supplemented with bovine milk fat globule membranes, or standard formula until 6 months of age. A breast-fed reference group was recruited. Lipidomic analyses were performed on plasma and erythrocyte membranes at 6 months and on serum at 4 and 12 months of age.

RESULTS: At 6 months of age, we observed a significant separation in the plasma lipidome between the two formula groups, mostly due to differences in concentrations of sphingomyelins (SM), phosphatidylcholines (PC), and ceramides, and in the erythrocyte membrane lipidome, mostly due to SMs, PEs and PCs. Already at 4 months, a separation in the serum lipidome was evident where SMs and PCs contributed. The separation was not detected at 12 months.

CONCLUSIONS: The effect of MFGM supplementation on the lipidome is likely part of the mechanisms behind the positive cognitive and immunological effects of feeding the EF previously reported in the same study population.

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure.

Damage assoiated molecular patterns (DAMPs) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. During oxidative stress, stress-derived EVs (stressEVs) were found to activate Toll-like receptor 4 (TLR4), but the activating ligands were not fully determined. Additionally, several enzymes such as 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are induced during inflammation and were suggested to promote DAMP formation. Stress-EVs were produced from HEK293 exposed to 10uM A23187 and isolated with ultracentrifugation. 20:4 lysoPI was oxidized for 10 min with 15-LO. SynEVs were prepared from phospholipids (PLs), oxidized with 15-LO and hydrolyzed with sPLA2. Activity was measured by qPCR and ELISA on wt and KO cells. Ox 20:4 lysoPI was analyzed by MS. sPLA2 activity was measured in synovial fluid from patients using fluorometric assay. K/BxN serum transfer induced arthritis model on wt and TLR4 KO mice(C57Bl/6 mice) with sPLA2-IIA injection was performed. StressEVs released after oxidative stress were found to activate TLR4with a gene profile different from agonist lipopolysaccharide. StressEVs, 15-LO oxidized synEVs, but only 15-LO oxidized lysoPLs activated cytokine expression through TLR4/MD-2.Hydroxy, hydroperoxy and keto products of 20:4 lysoPI oxidation were determined by MS and they activated the same gene pattern as stressEVs. Furthermore, sPLA2 activity, which we detected in the SF from patients, promoted formation of TLR4 agonists after 15-LO oxidation. Injection of sPLA2-IIA into mice promoted K/BxN serum induced arthritis in TLR4-dependent manner. Both 15-LO and sPLA2 are induced during inflammation, therefore these results imply the role of oxidized lysoPLs in stressEVs in promoting sterile inflammation through TLR4 signaling. The formation of TLR4 agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens (Ha VT. et al., PNAS 2020).

Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived extracellular vesicles (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4), resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A₂ (sPLA₂) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain. Hydroxy, hydroperoxy, and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry (MS), and they activated the same gene pattern as stressEVs. Extracellular PLA₂ activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA₂ promoted K/BxN serum-induced arthritis in mice, whereby ankle swelling was partially TLR4 dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA₂ enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.

Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) was applied in the quantification and identification of organic compounds in patient-matched human cerebrospinal fluid (CSF) and serum samples. Concentrations of 21 amino and hydroxyl acids varied from 0.04 to 77ng/μl in CSF and from 0.1 to 84ng/μl in serum. In total, 91 metabolites out of over 1200 detected were identified based on mass spectra and retention indices. The other metabolites were identified at the functional group level. The main metabolites detected in CSF were sugar and amino acid derivatives. The CSF and serum had clearly distinct metabolic profiles, with larger biological variation in the serum than in CSF. The GC×GC-TOFMS allowed detection and identification of several metabolites that have not been previously detected in CSF.

Sugars are important nutrients for many animals, but are also proposed to contribute to overnutrition-derived metabolic diseases in humans. Understanding the genetic factors governing dietary sugar tolerance therefore has profound biological and medical significance. Paralogous Mondo transcription factors ChREBP and MondoA, with their common binding partner Mlx, are key sensors of intracellular glucose flux in mammals. Here we report analysis of the in vivo function of Drosophila melanogaster Mlx and its binding partner Mondo (ChREBP) in respect to tolerance to dietary sugars. Larvae lacking mlx or having reduced mondo expression show strikingly reduced survival on a diet with moderate or high levels of sucrose, glucose, and fructose. mlx null mutants display widespread changes in lipid and phospholipid profiles, signs of amino acid catabolism, as well as strongly elevated circulating glucose levels. Systematic loss-of-function analysis of Mlx target genes reveals that circulating glucose levels and dietary sugar tolerance can be genetically uncoupled: Krüppel-like transcription factor Cabut and carbonyl detoxifying enzyme Aldehyde dehydrogenase type III are essential for dietary sugar tolerance, but display no influence on circulating glucose levels. On the other hand, Phosphofructokinase 2, a regulator of the glycolysis pathway, is needed for both dietary sugar tolerance and maintenance of circulating glucose homeostasis. Furthermore, we show evidence that fatty acid synthesis, which is a highly conserved Mondo-Mlx-regulated process, does not promote dietary sugar tolerance. In contrast, survival of larvae with reduced fatty acid synthase expression is sugar-dependent. Our data demonstrate that the transcriptional network regulated by Mondo-Mlx is a critical determinant of the healthful dietary spectrum allowing Drosophila to exploit sugar-rich nutrient sources.

A novel method for investigating drug release in a dynamic manner from nanoparticles including, but not limited to, biodegradable poly(lactic acid) (PLA) is reported. The PLA nanoparticles were prepared by the nanoprecipitation method. Two poorly soluble drugs, beclomethasone dipropionate (BDP) and indomethacin, were encapsulated into PLA nanoparticles, and their dissolution from the nanoparticles were followed in a dynamic way. The on-line method comprised a short column (vessel) packed with the PLA nanoparticles, on-line connected to an analytical liquid chromatographic column via a multiport switching valve equipped with two loops. The system allowed monitoring of the drug release profiles in real time, and the conditions for the drug release could be precisely controlled and easily changed. The effects of solvent composition and temperature on the rate of dissolution of the drugs from the PLA nanoparticles were investigated. The system proved to be linear for the drugs tested over the concentration range 10-3000 ng (n = 6, R(2) = 0.999 and 0.997 for indomethacin and beclomethasone, respectively) and repeatable (RSD of peak areas <0.5%). The recoveries of the dissolution study were quantitative (120 and 103% for indomethacin and beclomethasone, respectively).

Non-alcoholic fatty liver is the most common liver disease worldwide. Here, we show that the mitochondrial protein mitofusin 2 (Mfn2) protects against liver disease. Reduced Mfn2 expression was detected in liver biopsies from patients with non-alcoholic steatohepatitis (NASH). Moreover, reduced Mfn2 levels were detected in mouse models of steatosis or NASH, and its re-expression in a NASH mouse model ameliorated the disease. Liver-specific ablation of Mfn2 in mice provoked inflammation, triglyceride accumulation, fibrosis, and liver cancer. We demonstrate that Mfn2 binds phosphatidylserine (PS) and can specifically extract PS into membrane domains, favoring PS transfer to mitochondria and mitochondrial phosphatidylethanolamine (PE) synthesis. Consequently, hepatic Mfn2 deficiency reduces PS transfer and phospholipid synthesis, leading to endoplasmic reticulum (ER) stress and the development of a NASH-like phenotype and liver cancer. Ablation of Mfn2 in liver reveals that disruption of ER-mitochondrial PS transfer is a new mechanism involved in the development of liver disease.

The deep terrestrial subsurface (DTS) harbours a striking diversity of microorganisms. However, systematic research on microbial metabolism, and how varying groundwater composition affects the bacterial communities and metabolites in these environments is lacking. In this study, DTS groundwater bacterial consortia from two Fennoscandian Shield sites were enriched and studied. We found that the enriched communities from the two sites consisted of distinct bacterial taxa, and alterations in the growth medium composition induced changes in cell counts. The lack of an exogenous organic carbon source (ECS) caused a notable increase in lipid metabolism in one community, while in the other, carbon starvation resulted in low overall metabolism, suggesting a dormant state. ECS supplementation increased CO2 production and SO4 2- utilisation, suggesting activation of a dissimilatory sulphate reduction pathway and sulphate-reducer-dominated total metabolism. However, both communities shared common universal metabolic features, most probably involving pathways needed for the maintenance of cell homeostasis (e.g., mevalonic acid pathway). Collectively, our findings indicate that the most important metabolites related to microbial reactions under varying growth conditions in enriched DTS communities include, but are not limited to, those linked to cell homeostasis, osmoregulation, lipid biosynthesis and degradation, dissimilatory sulphate reduction and isoprenoid production.

Untargeted mass spectrometry (MS) experiments produce complex, multidimensional data that are practically impossible to investigate manually. For this reason, computational pipelines are needed to extract relevant information from raw spectral data and convert it into a more comprehensible format. Depending on the sample type and/or goal of the study, a variety of MS platforms can be used for such analysis. MZmine is an open-source software for the processing of raw spectral data generated by different MS platforms. Examples include liquid chromatography-MS, gas chromatography-MS and MS-imaging. These data might typically be associated with various applications including metabolomics and lipidomics. Moreover, the third version of the software, described herein, supports the processing of ion mobility spectrometry (IMS) data. The present protocol provides three distinct procedures to perform feature detection and annotation of untargeted MS data produced by different instrumental setups: liquid chromatography-(IMS-)MS, gas chromatography-MS and (IMS-)MS imaging. For training purposes, example datasets are provided together with configuration batch files (i.e., list of processing steps and parameters) to allow new users to easily replicate the described workflows. Depending on the number of data files and available computing resources, we anticipate this to take between 2 and 24 h for new MZmine users and nonexperts. Within each procedure, we provide a detailed description for all processing parameters together with instructions/recommendations for their optimization. The main generated outputs are represented by aligned feature tables and fragmentation spectra lists that can be used by other third-party tools for further downstream analysis.

Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many cancers. However, the precise molecular composition of lipids in tumors remains generally poorly characterized. The aim of the present study was to analyze the global lipid profiles of breast cancer, integrate the results to protein expression, and validate the findings by functional experiments. Comprehensive lipidomics was conducted in 267 human breast tissues using ultraperformance liquid chromatography/ mass spectrometry. The products of de novo fatty acid synthesis incorporated into membrane phospholipids, such as palmitate-containing phosphatidylcholines, were increased in tumors as compared with normal breast tissues. These lipids were associated with cancer progression and patient survival, as their concentration was highest in estrogen receptor-negative and grade 3 tumors. In silico transcriptomics database was utilized in investigating the expression of lipid metabolism related genes in breast cancer, and on the basis of these results, the expression of specific proteins was studied by immunohistochemistry. Immunohistochemical analyses showed that several genes regulating lipid metabolism were highly expressed in clinical breast cancer samples and supported also the lipidomics results. Gene silencing experiments with seven genes [ACACA (acetyl-CoA carboxylase α), ELOVL1 (elongation of very long chain fatty acid-like 1), FASN (fatty acid synthase), INSIG1 (insulin-induced gene 1), SCAP (sterol regulatory element-binding protein cleavage-activating protein), SCD (stearoyl-CoA desaturase), and THRSP (thyroid hormone-responsive protein)] indicated that silencing of multiple lipid metabolism-regulating genes reduced the lipidomic profiles and viability of the breast cancer cells. Taken together, our results imply that phospholipids may have diagnostic potential as well as that modulation of their metabolism may provide therapeutic opportunities in breast cancer treatment.

Changes in cellular lipid metabolism are a common feature in most solid tumors, which occur already in early stages of the tumor progression. However, it remains unclear if the tumor-specific lipid changes can be detected at the level of systemic lipid metabolism. The objective of this study was to perform comprehensive analysis of lipids in breast cancer patient serum samples. Lipidomic profiling using an established analytical platform was performed in two cohorts of breast cancer patients receiving neoadjuvant chemotherapy. The analyses were performed for 142 patients before and after neoadjuvant chemotherapy, and the results before chemotherapy were validated in an independent cohort of 194 patients. The analyses revealed that in general the tumor characteristics are not reflected in the serum samples. However, there was an association of specific triacylglycerols (TGs) in patients' response to chemotherapy. These TGs containing mainly oleic acid (C18:1) were found in lower levels in those patients showing pathologic complete response before receiving chemotherapy. Some of these TGs were also associated with estrogen receptor status and overall or disease-free survival of the patients. The results suggest that the altered serum levels of oleic acid in breast cancer patients are associated with their response to chemotherapy.

Faecal microbiota transfer (FMT) consists of the infusion of donor faecal material into the intestine of a patient with the aim to restore a disturbed gut microbiota. In this study, it was investigated whether FMT has an effect on faecal microbial composition, its functional capacity, faecal metabolite profiles and their interactions in 16 irritable bowel syndrome (IBS) patients. Faecal samples from eight different time points before and until six months after allogenic FMT (faecal material from a healthy donor) as well as autologous FMT (own faecal material) were analysed by 16S RNA gene amplicon sequencing and gas chromatography coupled to mass spectrometry (GS-MS). The results showed that the allogenic FMT resulted in alterations in the microbial composition that were detectable up to six months, whereas after autologous FMT this was not the case. Similar results were found for the functional profiles, which were predicted from the phylogenetic sequencing data. While both allogenic FMT as well as autologous FMT did not have an effect on the faecal metabolites measured in this study, correlations between the microbial composition and the metabolites showed that the microbe-metabolite interactions seemed to be disrupted after allogenic FMT compared to autologous FMT. This shows that FMT can lead to altered interactions between the gut microbiota and its metabolites in IBS patients. Further research should investigate if and how this affects efficacy of FMT treatments.