A dual-function RNA balances carbon uptake and central metabolism in Vibrio cholerae.

Bacterial small RNAs (sRNAs) are well known to modulate gene expression by base pairing with trans-encoded transcripts and are typically non-coding. However, several sRNAs have been reported to also contain an open reading frame and thus are considered dual-function RNAs. In this study, we discovered a dual-function RNA from Vibrio cholerae, called VcdRP, harboring a 29 amino acid small protein (VcdP), as well as a base-pairing sequence. Using a forward genetic screen, we identified VcdRP as a repressor of cholera toxin production and link this phenotype to the inhibition of carbon transport by the base-pairing segment of the regulator. By contrast, we demonstrate that the VcdP small protein acts downstream of carbon transport by binding to citrate synthase (GltA), the first enzyme of the citric acid cycle. Interaction of VcdP with GltA results in increased enzyme activity and together VcdR and VcdP reroute carbon metabolism. We further show that transcription of vcdRP is repressed by CRP allowing us to provide a model in which VcdRP employs two different molecular mechanisms to synchronize central metabolism in V. cholerae.

Prebiotic diet changes neural correlates of food decision-making in overweight adults: a randomised controlled within-subject cross-over trial.

OBJECTIVE: Animal studies suggest that prebiotic, plant-derived nutrients could improve homoeostatic and hedonic brain functions through improvements in microbiome-gut-brain communication. However, little is known if these results are applicable to humans. Therefore, we tested the effects of high-dosed prebiotic fibre on reward-related food decision-making in a randomised controlled within-subject cross-over study and assayed potential microbial and metabolic markers. DESIGN: 59 overweight young adults (19 females, 18-42 years, body mass index 25-30 kg/m2) underwent functional task MRI before and after 14 days of supplementary intake of 30 g/day of inulin (prebiotics) and equicaloric placebo, respectively. Short chain fatty acids (SCFA), gastrointestinal hormones, glucose/lipid and inflammatory markers were assayed in fasting blood. Gut microbiota and SCFA were measured in stool. RESULTS: Compared with placebo, participants showed decreased brain activation towards high-caloric wanted food stimuli in the ventral tegmental area and right orbitofrontal cortex after prebiotics (preregistered, family wise error-corrected p <0.05). While fasting blood levels remained largely unchanged, 16S-rRNA sequencing showed significant shifts in the microbiome towards increased occurrence of, among others, SCFA-producing Bifidobacteriaceae, and changes in >60 predicted functional signalling pathways after prebiotic intake. Changes in brain activation correlated with changes in Actinobacteria microbial abundance and associated activity previously linked with SCFA production, such as ABC transporter metabolism. CONCLUSIONS: In this proof-of-concept study, a prebiotic intervention attenuated reward-related brain activation during food decision-making, paralleled by shifts in gut microbiota. TRIAL REGISTRATION NUMBER: NCT03829189.

Metabolomics in human SGBS cells as new approach method for studying adipogenic effects: Analysis of the effects of DINCH and MINCH on central carbon metabolism.

Growing evidence suggests that exposure to certain metabolism-disrupting chemicals (MDCs), such as the phthalate plasticizer DEHP, might promote obesity in humans, contributing to the spread of this global health problem. Due to the restriction on the use of phthalates, there has been a shift to safer declared substitutes, including the plasticizer diisononyl-cyclohexane-1,2-dicarboxylate (DINCH). Notwithstanding, recent studies suggest that the primary metabolite monoisononyl-cyclohexane-1,2-dicarboxylic acid ester (MINCH), induces differentiation of human adipocytes and affects enzyme levels of key metabolic pathways. Given the lack of methods for assessing metabolism-disrupting effects of chemicals on adipose tissue, we used metabolomics to analyze human SGSB cells exposed to DINCH or MINCH. Concentration analysis of DINCH and MINCH revealed that uptake of MINCH in preadipocytes was associated with increased lipid accumulation during adipogenesis. Although we also observed intracellular uptake for DINCH, the solubility of DINCH in cell culture medium was limited, hampering the analysis of possible effects in the µM concentration range. Metabolomics revealed that MINCH induces lipid accumulation similar to peroxisome proliferator-activated receptor gamma (PPARG)-agonist rosiglitazone through upregulation of the pyruvate cycle, which was recently identified as a key driver of de novo lipogenesis. Analysis of the metabolome in the presence of the PPARG-inhibitor GW9662 indicated that the effect of MINCH on metabolism was mediated at least partly by a PPARG-independent mechanism. However, all effects of MINCH were only observed at high concentrations of 10 µM, which are three orders of magnitudes higher than the current concentrations of plasticizers in human serum. Overall, the assessment of the effects of DINCH and MINCH on SGBS cells by metabolomics revealed no adipogenic potential at physiologically relevant concentrations. This finding aligns with previous in vivo studies and supports the potential of our method as a New Approach Method (NAM) for the assessment of adipogenic effects of environmental chemicals.

Evaluating the performance of multi-omics integration: a thyroid toxicity case study.

Multi-omics data integration has been repeatedly discussed as the way forward to more comprehensively cover the molecular responses of cells or organisms to chemical exposure in systems toxicology and regulatory risk assessment. In Canzler et al. (Arch Toxicol 94(2):371-388. https://doi.org/10.1007/s00204-020-02656-y ), we reviewed the state of the art in applying multi-omics approaches in toxicological research and chemical risk assessment. We developed best practices for the experimental design of multi-omics studies, omics data acquisition, and subsequent omics data integration. We found that multi-omics data sets for toxicological research questions were generally rare, with no data sets comprising more than two omics layers adhering to these best practices. Due to these limitations, we could not fully assess the benefits of different data integration approaches or quantitatively evaluate the contribution of various omics layers for toxicological research questions. Here, we report on a multi-omics study on thyroid toxicity that we conducted in compliance with these best practices. We induced direct and indirect thyroid toxicity through Propylthiouracil (PTU) and Phenytoin, respectively, in a 28-day plus 14-day recovery oral rat toxicity study. We collected clinical and histopathological data and six omics layers, including the long and short transcriptome, proteome, phosphoproteome, and metabolome from plasma, thyroid, and liver. We demonstrate that the multi-omics approach is superior to single-omics in detecting responses at the regulatory pathway level. We also show how combining omics data with clinical and histopathological parameters facilitates the interpretation of the data. Furthermore, we illustrate how multi-omics integration can hint at the involvement of non-coding RNAs in post-transcriptional regulation. Also, we show that multi-omics facilitates grouping, and we assess how much information individual and combinations of omics layers contribute to this approach.

The effect of polyphenols on DNA methylation-assessed biological age attenuation: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Epigenetic age is an estimator of biological age based on DNA methylation; its discrepancy from chronologic age warrants further investigation. We recently reported that greater polyphenol intake benefitted ectopic fats, brain function, and gut microbiota profile, corresponding with elevated urine polyphenols. The effect of polyphenol-rich dietary interventions on biological aging is yet to be determined. METHODS: We calculated different biological aging epigenetic clocks of different generations (Horvath2013, Hannum2013, Li2018, Horvath skin and blood2018, PhenoAge2018, PCGrimAge2022), their corresponding age and intrinsic age accelerations, and DunedinPACE, all based on DNA methylation (Illumina EPIC array; pre-specified secondary outcome) for 256 participants with abdominal obesity or dyslipidemia, before and after the 18-month DIRECT PLUS randomized controlled trial. Three interventions were assigned: healthy dietary guidelines, a Mediterranean (MED) diet, and a polyphenol-rich, low-red/processed meat Green-MED diet. Both MED groups consumed 28 g walnuts/day (+ 440 mg/day polyphenols). The Green-MED group consumed green tea (3-4 cups/day) and Mankai (Wolffia globosa strain) 500-ml green shake (+ 800 mg/day polyphenols). Adherence to the Green-MED diet was assessed by questionnaire and urine polyphenols metabolomics (high-performance liquid chromatography quadrupole time of flight). RESULTS: Baseline chronological age (51.3 ± 10.6 years) was significantly correlated with all methylation age (mAge) clocks with correlations ranging from 0.83 to 0.95; p < 2.2e - 16 for all. While all interventions did not differ in terms of changes between mAge clocks, greater Green-Med diet adherence was associated with a lower 18-month relative change (i.e., greater mAge attenuation) in Li and Hannum mAge (beta = - 0.41, p = 0.004 and beta = - 0.38, p = 0.03, respectively; multivariate models). Greater Li mAge attenuation (multivariate models adjusted for age, sex, baseline mAge, and weight loss) was mostly affected by higher intake of Mankai (beta = - 1.8; p = 0.061) and green tea (beta = - 1.57; p = 0.0016) and corresponded with elevated urine polyphenols: hydroxytyrosol, tyrosol, and urolithin C (p < 0.05 for all) and urolithin A (p = 0.08), highly common in green plants. Overall, participants undergoing either MED-style diet had ~ 8.9 months favorable difference between the observed and expected Li mAge at the end of the intervention (p = 0.02). CONCLUSIONS: This study showed that MED and green-MED diets with increased polyphenols intake, such as green tea and Mankai, are inversely associated with biological aging. To the best of our knowledge, this is the first clinical trial to indicate a potential link between polyphenol intake, urine polyphenols, and biological aging. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03020186.

Molecular Responses of Daphnids to Chronic Exposures to Pharmaceuticals.

Pharmaceutical compounds are among several classes of contaminants of emerging concern, such as pesticides, heavy metals and personal care products, all of which are a major concern for aquatic ecosystems. The hazards posed by the presence of pharmaceutical is one which affects both freshwater organisms and human health-via non-target effects and by the contamination of drinking water sources. The molecular and phenotypic alterations of five pharmaceuticals which are commonly present in the aquatic environment were explored in daphnids under chronic exposures. Markers of physiology such as enzyme activities were combined with metabolic perturbations to assess the impact of metformin, diclofenac, gabapentin, carbamazepine and gemfibrozil on daphnids. Enzyme activity of markers of physiology included phosphatases, lipase, peptidase, ß-galactosidase, lactate dehydrogenase, glutathione-S-transferase and glutathione reductase activities. Furthermore, targeted LC-MS/MS analysis focusing on glycolysis, the pentose phosphate pathway and the TCA cycle intermediates was performed to assess metabolic alterations. Exposure to pharmaceuticals resulted in the changes in activity for several enzymes of metabolism and the detoxification enzyme glutathione-S-transferase. Metabolic perturbations on key pathways revealed distinct groups and metabolic fingerprints for the different exposures and their mixtures. Chronic exposure to pharmaceuticals at low concentrations revealed significant alterations of metabolic and physiological endpoints.

The effect of high-polyphenol Mediterranean diet on visceral adiposity: the DIRECT PLUS randomized controlled trial.

BACKGROUND: Mediterranean (MED) diet is a rich source of polyphenols, which benefit adiposity by several mechanisms. We explored the effect of the green-MED diet, twice fortified in dietary polyphenols and lower in red/processed meat, on visceral adipose tissue (VAT). METHODS: In the 18-month Dietary Intervention Randomized Controlled Trial PoLyphenols UnproceSsed (DIRECT-PLUS) weight-loss trial, 294 participants were randomized to (A) healthy dietary guidelines (HDG), (B) MED, or (C) green-MED diets, all combined with physical activity. Both isocaloric MED groups consumed 28 g/day of walnuts (+ 440 mg/day polyphenols). The green-MED group further consumed green tea (3-4 cups/day) and Wolffia globosa (duckweed strain) plant green shake (100 g frozen cubes/day) (+ 800mg/day polyphenols) and reduced red meat intake. We used magnetic resonance imaging (MRI) to quantify the abdominal adipose tissues. RESULTS: Participants (age = 51 years; 88% men; body mass index = 31.2 kg/m2; 29% VAT) had an 89.8% retention rate and 79.3% completed eligible MRIs. While both MED diets reached similar moderate weight (MED: - 2.7%, green-MED: - 3.9%) and waist circumference (MED: - 4.7%, green-MED: - 5.7%) loss, the green-MED dieters doubled the VAT loss (HDG: - 4.2%, MED: - 6.0%, green-MED: - 14.1%; p < 0.05, independent of age, sex, waist circumference, or weight loss). Higher dietary consumption of green tea, walnuts, and Wolffia globosa; lower red meat intake; higher total plasma polyphenols (mainly hippuric acid), and elevated urine urolithin A polyphenol were significantly related to greater VAT loss (p < 0.05, multivariate models). CONCLUSIONS: A green-MED diet, enriched with plant-based polyphenols and lower in red/processed meat, may be a potent intervention to promote visceral adiposity regression. TRIAL REGISTRATION: ClinicalTrials.gov , NCT03020186.

Bidirectional Role of NLRP3 During Acute and Chronic Cholestatic Liver Injury.

BACKGROUND AND AIMS: Cholestatic liver injury leads to cell death and subsequent inflammation and fibrosis. As shown for primary biliary cholangitis (PBC), the mechanisms and circuits between different cell death pathways leading to disease progression are incompletely defined. Common bile duct ligation (BDL) is a well-established murine model to mimic cholestatic liver injury. Here, we hypothesized that pyroptotic cell death by the Nucleotide-Binding Domain, Leucine-Rich-Containing Family, Pyrin Domain-Containing-3 (Nlrp3) inflammasome plays an essential role during human and murine cholestasis. APPROACH AND RESULTS: NLRP3 activation was analyzed in humans with cholestatic liver injury. Wild-type (WT) and Nlrp3-/- mice were subjected to BDL for 2 or 28 days. Chronic cholestasis in humans and mice is associated with NLRP3 activation and correlates with disease activity. Acute BDL in Nlrp3-deficient mice triggered increased inflammation as well as liver injury, associated with stronger apoptotic and necroptotic cell death. In contrast, NLRP3 deletion led to decreased liver injury and inflammation in chronic cholestasis. Moreover, bridging fibrosis was observed in WT, but not in NLRP3 knockout, mice 28 days after BDL. In contrast, lack of NLRP3 expression attenuated kidney injury and fibrosis after acute and chronic BDL. Importantly, administration of MCC950, an NLRP3 small molecule inhibitor, reduced BDL-induced disease progression in WT mice. CONCLUSIONS: NLRP3 activation correlates with disease activity in patients with PBC. NLRP3 has a differential role during acute and chronic cholestatic liver injury in contrast to kidney injury. Disease progression during chronic cholestasis can be targeted through small molecules and thus suggests a potential clinical benefit for humans, attenuating liver and kidney injury.

Evaluation of Antioxidant Activity and Biotransformation of Opuntia Ficus Fruit: The Effect of In Vitro and Ex Vivo Gut Microbiota Metabolism.

Opuntia ficus-indica biological effects are attributed to several bioactive metabolites. However, these actions could be altered in vivo by biotransformation reactions mainly via gut microbiota. This study assessed gut microbiota effect on the biotransformation of O. ficus-indica metabolites both in vitro and ex vivo. Two-time aliquots (0.5 and 24 h) from the in vitro assay were harvested post incubation of O. ficus-indica methanol extract with microbial consortium, while untreated and treated samples with fecal bacterial culture from the ex vivo assay were prepared. Metabolites were analyzed using UHPLC-QTOF-MS, with flavonoid glycosides completely hydrolyzed in vitro at 24 h being converted to two major metabolites, 3-(4-hydroxyphenyl)propanoic acid and phloroglucinol, concurrent with an increase in the gallic acid level. In case of the ex vivo assay, detected flavonoid glycosides in untreated sample were completely absent from treated counterpart with few flavonoid aglycones and 3-(4-hydroxyphenyl)propanoic acid in parallel to an increase in piscidic acid. In both assays, fatty and organic acids were completely hydrolyzed being used as energy units for bacterial growth. Chemometric tools were employed revealing malic and (iso)citric acids as the main discriminating metabolites in vitro showing an increased abundance at 0.5 h, whereas in ex vivo assay, (iso)citric, aconitic and mesaconic acids showed an increase at untreated sample. Piscidic acid was a significant marker for the ex vivo treated sample. DPPH, ORAC and FRAP assays were further employed to determine whether these changes could be associated with changes in antioxidant activity, and all assays showed a decline in antioxidant potential post biotransformation.

Differences in the sperm metabolomes of smoking and nonsmoking men†.

Currently, spermiogram analysis is the most relevant method used to clarify the potential infertility of a couple. However, in some cases, the reasons for infertility remain obscure. Smoking is among the factors that have been described to adversely affect male fertility. Smoking increases oxidative stress and thus promotes various pathological processes. Comparative studies, particularly those on metabolomic changes in sperm and seminal plasma caused by smoking, have not yet been published. Thus, the present pilot study aimed at the mass spectrometric characterization of the metabolomes of specimens from both smoking and nonsmoking subjects and the comparison of the evaluated data in terms of sperm apoptosis and spermiogram parameters. The results provided evidence that the conventional spermiogram is not altered in smokers compared to nonsmokers. However, a more careful investigation of sperm cells by metabolomic profiling reveals profound effects of smoking on sperm: first, nitrogen oxide synthase, a marker of oxidative stress, is activated. Second, the uptake of fatty acids into sperm mitochondria is reduced, leading to an impaired energy supply. Third, phenylalanine hydroxylation and tryptophan degradation, which are both indications of altered tetrahydrobiopterin biosynthesis, are reduced. Moreover, flow cytometry approaches indicated increased sperm caspase-3 activity, a sign of apoptosis. The present study clearly shows the negative effects of smoking on semen quality. Especially for idiopathic cases, metabolomic profiling can help to shed light on male subfertility or infertility.

Reduced lipolysis in lipoma phenocopies lipid accumulation in obesity.

BACKGROUND: Elucidation of lipid metabolism and accumulation mechanisms is of paramount importance to understanding obesity and unveiling therapeutic targets. In vitro cell models have been extensively used for these purposes, yet, they do not entirely reflect the in vivo setup. Conventional lipomas, characterized by the presence of mature adipocytes and increased adipogenesis, could overcome the drawbacks of cell cultures. Also, they have the unique advantage of easily accessible matched controls in the form of subcutaneous adipose tissue (SAT) from the same individual. We aimed to determine whether lipomas are a good model to understand lipid accumulation. METHODS: We histologically compared lipomas and control SAT, followed by assessment of the lipidome using high-resolution 1H NMR spectroscopy and ESI-IT mass spectrometry. RNA-sequencing was used to obtain the transcriptome of lipomas and the matched SAT. RESULTS: We found a significant increase of small-size (maximal axis < 70 µm) and very big (maximal axis > 150 µm) adipocytes within lipomas. This suggests both enhanced adipocyte proliferation and increased lipid accumulation. We further show that there is no significant change in the lipid composition compared to matched SAT. To better delineate the pathophysiology of lipid accumulation, we considered two groups with different genetic backgrounds: (1) lipomas with HMGA2 fusions and (2) without gene fusions. To reduce the search space for genes that are relevant for lipid pathophysiology, we focused on the overlapping differentially expressed (DE) genes between the two groups. Gene Ontology analysis revealed that DE genes are enriched in pathways related to lipid accumulation. CONCLUSIONS: We show that the common shared lipid accumulation mechanism in lipoma is a reduction in lipolysis, with most gene dysregulations leading to a reduced cAMP in the adipocyte. Superficial lipomas could thus be used as a model for lipid accumulation through altered lipolysis as found in obese patients.

Prenatal paraben exposure and atopic dermatitis-related outcomes among children.

BACKGROUND: Parabens, widely used as preservatives in cosmetics, foods, and other consumer products, are suspected of contributing to allergy susceptibility. The detection of parabens in the placenta or amniotic fluid raised concerns about potential health consequences for the child. Recently, an increased asthma risk following prenatal exposure has been reported. Here, we investigated whether prenatal paraben exposure can influence the risk for atopic dermatitis (AD). METHODS: 261 mother-child pairs of the German mother-child study LINA were included in this analysis. Eight paraben species were quantified in maternal urine obtained at gestational week 34. According to the parental report of physician-diagnosed AD from age 1 to 8 years, disease onset, and persistence, childhood AD was classified into four different phenotypes. RESULTS: 4.6% (n = 12) and 12.3% (n = 32) of the children were classified as having very early-onset AD (until age two) either with or without remission, 11.9% (n = 31) as early-onset (after age two), and 3.1% (n = 8) as childhood-onset AD (after age six). Exposure to ethylparaben and n-butylparaben was associated with an increased risk to develop very early-onset AD without remission (EtP: adj.OR/95% CI:1.44/1.04-2.00,nBuP:adj.OR/95% CI:1.95/1.22-3.12). The effects of both parabens were predominant in children without a history of maternal AD and independent of children's sex. CONCLUSION: Prenatal EtP or nBuP exposure may increase children's susceptibility for persistent AD with disease onset at very early age. This association was particularly pronounced in children without a history of maternal AD, indicating that children without a genetic predisposition are more susceptible to paraben exposure.

Quantification of glyphosate and aminomethylphosphonic acid from microbiome reactor fluids.

RATIONALE: Glyphosate is one of the most widely used herbicides and it is suspected to affect the intestinal microbiota through inhibition of aromatic amino acid synthesis via the shikimate pathway. In vitro microbiome bioreactors are increasingly used as model systems to investigate effects on intestinal microbiota and consequently methods for the quantitation of glyphosate and its degradation product aminomethylphosphonic acid (AMPA) in microbiome model systems are required. METHODS: An optimized protocol enables the analysis of both glyphosate and AMPA by simple extraction with methanol:acetonitrile:water (2:3:1) without further enrichment steps. Glyphosate and AMPA are separated by liquid chromatography on an amide column and identified and quantified with a targeted tandem mass spectrometry method using a QTRAP 5500 system (AB Sciex). RESULTS: Our method has a limit of detection (LOD) in extracted water samples of <2 ng/mL for both glyphosate and AMPA. In complex intestinal medium, the LOD is 2 and 5 ng/mL for glyphosate and AMPA, respectively. These LODs allow for measurement at exposure-relevant concentrations. Glyphosate levels in a bioreactor model of porcine colon were determined and consequently it was verified whether AMPA was produced by porcine gut microbiota. CONCLUSIONS: The method presented here allows quantitation of glyphosate and AMPA in complex bioreactor fluids and thus enables studies of the impact of glyphosate and its metabolism on intestinal microbiota. In addition, the extraction protocol is compatible with an untargeted metabolomics analysis, thus allowing one to look for other perturbations caused by glyphosate in the same sample.

Prospects and challenges of multi-omics data integration in toxicology.

Exposure of cells or organisms to chemicals can trigger a series of effects at the regulatory pathway level, which involve changes of levels, interactions, and feedback loops of biomolecules of different types. A single-omics technique, e.g., transcriptomics, will detect biomolecules of one type and thus can only capture changes in a small subset of the biological cascade. Therefore, although applying single-omics analyses can lead to the identification of biomarkers for certain exposures, they cannot provide a systemic understanding of toxicity pathways or adverse outcome pathways. Integration of multiple omics data sets promises a substantial improvement in detecting this pathway response to a toxicant, by an increase of information as such and especially by a systemic understanding. Here, we report the findings of a thorough evaluation of the prospects and challenges of multi-omics data integration in toxicological research. We review the availability of such data, discuss options for experimental design, evaluate methods for integration and analysis of multi-omics data, discuss best practices, and identify knowledge gaps. Re-analyzing published data, we demonstrate that multi-omics data integration can considerably improve the confidence in detecting a pathway response. Finally, we argue that more data need to be generated from studies with a multi-omics-focused design, to define which omics layers contribute most to the identification of a pathway response to a toxicant.

Serum metabolites and risk of myocardial infarction and ischemic stroke: a targeted metabolomic approach in two German prospective cohorts.

Metabolomic approaches in prospective cohorts may offer a unique snapshot into early metabolic perturbations that are associated with a higher risk of cardiovascular diseases (CVD) in healthy people. We investigated the association of 105 serum metabolites, including acylcarnitines, amino acids, phospholipids and hexose, with risk of myocardial infarction (MI) and ischemic stroke in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam (27,548 adults) and Heidelberg (25,540 adults) cohorts. Using case-cohort designs, we measured metabolites among individuals who were free of CVD and diabetes at blood draw but developed MI (n = 204 and n = 228) or stroke (n = 147 and n = 121) during follow-up (mean, 7.8 and 7.3 years) and among randomly drawn subcohorts (n = 2214 and n = 770). We used Cox regression analysis and combined results using meta-analysis. Independent of classical CVD risk factors, ten metabolites were associated with risk of MI in both cohorts, including sphingomyelins, diacyl-phosphatidylcholines and acyl-alkyl-phosphatidylcholines with pooled relative risks in the range of 1.21-1.40 per one standard deviation increase in metabolite concentrations. The metabolites showed positive correlations with total- and LDL-cholesterol (r ranged from 0.13 to 0.57). When additionally adjusting for total-, LDL- and HDL-cholesterol, triglycerides and C-reactive protein, acyl-alkyl-phosphatidylcholine C36:3 and diacyl-phosphatidylcholines C38:3 and C40:4 remained associated with risk of MI. When added to classical CVD risk models these metabolites further improved CVD prediction (c-statistics increased from 0.8365 to 0.8384 in EPIC-Potsdam and from 0.8344 to 0.8378 in EPIC-Heidelberg). None of the metabolites was consistently associated with stroke risk. Alterations in sphingomyelin and phosphatidylcholine metabolism, and particularly metabolites of the arachidonic acid pathway are independently associated with risk of MI in healthy adults.

Biochemical Foundations of Health and Energy Conservation in Hibernating Free-ranging Subadult Brown Bear Ursus arctos.

Brown bears (Ursus arctos) hibernate for 5-7 months without eating, drinking, urinating, and defecating at a metabolic rate of only 25% of the summer activity rate. Nonetheless, they emerge healthy and alert in spring. We quantified the biochemical adaptations for hibernation by comparing the proteome, metabolome, and hematological features of blood from hibernating and active free-ranging subadult brown bears with a focus on conservation of health and energy. We found that total plasma protein concentration increased during hibernation, even though the concentrations of most individual plasma proteins decreased, as did the white blood cell types. Strikingly, antimicrobial defense proteins increased in concentration. Central functions in hibernation involving the coagulation response and protease inhibition, as well as lipid transport and metabolism, were upheld by increased levels of very few key or broad specificity proteins. The changes in coagulation factor levels matched the changes in activity measurements. A dramatic 45-fold increase in sex hormone-binding globulin levels during hibernation draws, for the first time, attention to its significant but unknown role in maintaining hibernation physiology. We propose that energy for the costly protein synthesis is reduced by three mechanisms as follows: (i) dehydration, which increases protein concentration without de novo synthesis; (ii) reduced protein degradation rates due to a 6 °C reduction in body temperature and decreased protease activity; and (iii) a marked redistribution of energy resources only increasing de novo synthesis of a few key proteins. The comprehensive global data identified novel biochemical strategies for bear adaptations to the extreme condition of hibernation and have implications for our understanding of physiology in general.

Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children.

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype-related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular "commuting" enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole-genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non-regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c-Jun N-terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

Higher plasma levels of lysophosphatidylcholine 18:0 are related to a lower risk of common cancers in a prospective metabolomics study.

BACKGROUND: First metabolomics studies have indicated that metabolic fingerprints from accessible tissues might be useful to better understand the etiological links between metabolism and cancer. However, there is still a lack of prospective metabolomics studies on pre-diagnostic metabolic alterations and cancer risk. METHODS: Associations between pre-diagnostic levels of 120 circulating metabolites (acylcarnitines, amino acids, biogenic amines, phosphatidylcholines, sphingolipids, and hexoses) and the risks of breast, prostate, and colorectal cancer were evaluated by Cox regression analyses using data of a prospective case-cohort study including 835 incident cancer cases. RESULTS: The median follow-up duration was 8.3 years among non-cases and 6.5 years among incident cases of cancer. Higher levels of lysophosphatidylcholines (lysoPCs), and especially lysoPC a C18:0, were consistently related to lower risks of breast, prostate, and colorectal cancer, independent of background factors. In contrast, higher levels of phosphatidylcholine PC ae C30:0 were associated with increased cancer risk. There was no heterogeneity in the observed associations by lag time between blood draw and cancer diagnosis. CONCLUSION: Changes in blood lipid composition precede the diagnosis of common malignancies by several years. Considering the consistency of the present results across three cancer types the observed alterations point to a global metabolic shift in phosphatidylcholine metabolism that may drive tumorigenesis.

Optimization of metabolomics of defined in vitro gut microbial ecosystems.

The metabolic functionality of a microbial community is a key to the understanding of its inherent ecological processes and the interaction with the host. However, the study of the human gut microbiota is hindered by the complexity of this ecosystem. One way to resolve this issue is to derive defined communities that may be cultured ex vivo in bioreactor systems and used to approximate the native ecosystem. Doing so has the advantage of experimental reproducibility and ease of sampling, and furthermore, in-depth analysis of metabolic processes becomes highly accessible. Here, we review the use of bioreactor systems for ex vivo modelling of the human gut microbiota with respect to analysis of the metabolic output of the microbial ecosystem, and discuss the possibility of mechanistic insights using these combined techniques. We summarize the different platforms currently used for metabolomics and suitable for analysis of gut microbiota samples from a bioreactor system. With the help of representative datasets obtained from a series of bioreactor runs, we compare the outputs of both NMR and mass spectrometry based approaches in terms of their coverage, sensitivity and quantification. We also discuss the use of untargeted and targeted analyses in mass spectroscopy and how these techniques can be combined for optimal biological interpretation. Potential solutions for linking metabolomic and phylogenetic datasets with regards to active, key species within the ecosystem will be presented.

Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers.

BACKGROUND: There is evidence that microRNAs (miRNAs) are sensitive to environmental stressors, including tobacco smoke. On the other hand, miRNAs are involved in immune regulation, such as regulatory T (Treg) cell differentiation. The aim of the present study was to investigate the association between prenatal tobacco smoke exposure, miRNAs, and Treg cell numbers. METHODS: Within a prospective mother-child study (Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk), we analyzed the expression of miR-155 and miR-223 together with Treg cell numbers in maternal blood during pregnancy, as well as in cord blood (n = 441). Tobacco smoke exposure was assessed based on questionnaire answers and maternal urine cotinine levels. Additionally, the concentration of smoking-related volatile organic compounds was measured in dwellings of study participants. RESULTS: Both maternal and cord blood miR-223 expressions were positively correlated with maternal urine cotinine levels. An association was also found between maternal miR-223 expression and indoor concentrations of benzene and toluene. High miR-223 expression was associated with lower Treg cell numbers in maternal and cord blood. Furthermore, children with lower Treg cell numbers at birth had a higher risk of atopic dermatitis during the first 3 years of life. The concentration of the toluene metabolite S-benzylmercapturic acid in maternal urine was associated with decreased cord blood, but not maternal blood, miR-155 expression. A relationship between miR-155 expression and Treg cell numbers was not found. CONCLUSIONS: For the first time, we show that maternal tobacco smoke exposure during pregnancy correlates with the level of miRNA-223 expression in blood, with an effect on children's cord blood Treg cell numbers and subsequent allergy risk.