Non-targeted Liquid Chromatography-mass Spectrometry Research Articles

Multi-omics analysis of biomarkers and molecular mechanism of rheumatoid arthritis with bone destruction.

Our study aimed to elucidate the role of metabolites, bacteria, and fungi in rheumatoid arthritis (RA) patients with bone destruction (BD(+)) and identify some biomarkers to predicate bone progression of RA. Plasma metabolites of the 127 RA patients and 69 healthy controls were conducted by using nontargeted liquid chromatography-mass spectrometry (LC-MS). The gut bacteria and fungi were assessed by 16S rRNA and internal transcribed spacer (ITS). Compared with RA patients without bone destruction (BD(-)), some metabolites, bacteria, and fungi were altered in BD(+). Seven metabolites were selected as key metabolites for classifying the BD(+) and BD(-) groups with moderate accuracy (AUC=0.71). Metabolites-groups, metabolites-metabolites, and metabolites-clinical factors had a certain correlation, and 7 metabolites were enriched in glycerophospholipid metabolism and L-arginine and proline metabolism pathways. The bacteria and fungi of the BD(+) group showed significant differences in composition and function compared with BD(-) group. The changed 4 bacteria and 12 fungi yielded accuracy (AUC=0.74 and AUC=0.87, respectively) for the two groups. Taking 7 metabolites, 4 bacteria, and 12 fungi as a panel for AUC analysis, an improved AUC of 0.99 significantly discriminated the two groups. The changed metabolites, gut bacteria, and fungi may affect the pathway related to L-arginine. Our nontargeted LC-MS, 16S rRNA, and ITS highlighted a novel link among the metabolites, bacteria, fungi, and pathology of BD(+), which could contribute to our understanding of the role of metabolites, bacteria, and fungi in BD(+) etiology and offered some novel biomarkers to predict the bone progression of RA.

Metabolomic and transcriptomic analyses reveal response mechanisms of juvenile flounder (Paralichthys olivaceus) to sublethal methylmercury

Although methylmercury (MeHg) has been recognized as a typical heavy metal posing huge damages to various life processes of fish, the response mechanisms of marine fish at early life stages (ELSs) to MeHg is still poorly understood. In this study, non-targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomic and transcriptomic approaches were used to explore response mechanisms of juvenile flounder (Paralichthys olivaceus) to long-term sublethal MeHg exposure (0 and 1.0 μg L-1; 30 d). After exposure, growth parameters of flounder were significantly decreased. Metabolomic and transcriptomic analyses of liver tissue showed obvious difference about biological pathways and identified biomarkers (around 2502 genes and 16 secondary metabolites). Those significantly differentially expressed genes (DEGs) and their enriched pathways were mainly related to immune response, oxidative stress, lipids metabolism, glycometabolism, amino acid and nucleotide metabolism and regulation of protein processes, while those identified secondary metabolites were mainly enriched in tryptophan metabolism, biosynthesis of unsaturated fatty acids, linoleic acid metabolism and glutathione metabolism. Additionally, multi-omic method was used to explore response mechanisms of key pathways under MeHg stress. In this regard, only 57 DEGs and 6 secondary metabolites were significantly enriched in 7 pathways to constitute an integrated regulatory network, including glutathione metabolism, thyroid hormone synthesis, linoleic acid metabolism, biosynthesis of unsaturated fatty acids, tryptophan metabolism pathway, serotonergic synapse and African trypanosomiasis. Above all, we could speculate that antioxidative function, lipids metabolism, nervous system and amino acid metabolism were the more sensitive targets in response to MeHg stress, which were conductive to deeply understand the response mechanisms of fish at ELSs under MeHg exposure. Those identified biomarkers could also be widely used for toxicological studies of pollutants and ecological risks monitoring.

Integrated transcriptomics and metabolomics analysis of the intestine of cobia (Rachycentron canadum) under hypoxia stress

In intensive aquaculture systems, dissolved oxygen (DO) is the most critical and limiting factor for the development and health of aquatic organisms. Hypoxia is a common phenomenon in aquaculture. Its effects on fish’s growth and immune system have been widely reported, but the molecular mechanisms of hypoxic stress on damage to the fish gut are not fully understood. Additionally, studies on the effects of hypoxic stress on the intestinal metabolism of cobia (Rachycentron canadum) are still limited. Therefore, this study used cobia to explore the effects of hypoxic stress on intestinal metabolism. In this study, the combination of high-throughput RNA sequencing (RNA-Seq) and non-targeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics analyses were used to investigate changes in key genes and metabolites in the intestinal tract of juvenile cobia after 28 days of hypoxic stress (DO: 3.15 ± 0.21 mg/L). Transcriptomics analysis revealed the significant enrichment of 62 metabolic pathways, including glutathione metabolism, fat digestion and absorption, bile secretion, glycerolipid metabolism, tricarboxylic acid cycle, glycolysis/gluconeogenesis, and other pathways. Metabolomics analysis revealed that most of the significantly differential metabolites were related to the metabolism of amino acids and lipids. The combined transcriptomics and metabolomics analyses showed that the sustained hypoxic stress could cause some degree of oxidative damage to the gut of cobia, thereby interfering with its digestive absorption and physiological and metabolic processes, such as abnormalities in antioxidant and detoxification functions, disturbances in the metabolisms of amino acids, lipids, and carbohydrates, and reduction of ion transport capacity. These findings might help understand the molecular mechanisms of the intestinal tract under hypoxic stress in cobia, thereby reducing the damage caused by hypoxic stress to farmed fish.

Effects of exercise on NAFLD using non-targeted metabolomics in adipose tissue, plasma, urine, and stool

The mechanisms by which exercise benefits patients with non-alcoholic fatty liver disease (NAFLD), the most common liver disease worldwide, remain poorly understood. A non-targeted liquid chromatography-mass spectrometry (LC–MS)-based metabolomics analysis was used to identify metabolic changes associated with NAFLD in humans upon exercise intervention (without diet change) across four different sample types—adipose tissue (AT), plasma, urine, and stool. Altogether, 46 subjects with NAFLD participated in this randomized controlled intervention study. The intervention group (n = 21) performed high-intensity interval training (HIIT) for 12 weeks while the control group (n = 25) kept their sedentary lifestyle. The participants' clinical parameters and metabolic profiles were compared between baseline and endpoint. HIIT significantly decreased fasting plasma glucose concentration (p = 0.027) and waist circumference (p = 0.028); and increased maximum oxygen consumption rate and maximum achieved workload (p < 0.001). HIIT resulted in sample-type-specific metabolite changes, including accumulation of amino acids and their derivatives in AT and plasma, while decreasing in urine and stool. Moreover, many of the metabolite level changes especially in the AT were correlated with the clinical parameters monitored during the intervention. In addition, certain lipids increased in plasma and decreased in the stool. Glyco-conjugated bile acids decreased in AT and urine. The 12-week HIIT exercise intervention has beneficial ameliorating effects in NAFLD subjects on a whole-body level, even without dietary changes and weight loss. The metabolomics analysis applied to the four different sample matrices provided an overall view on several metabolic pathways that had tissue-type specific changes after HIIT intervention in subjects with NAFLD. The results highlight especially the role of AT in responding to the HIIT challenge, and suggest that altered amino acid metabolism in AT might play a critical role in e.g. improving fasting plasma glucose concentration.Trial registration ClinicalTrials.gov (NCT03995056).

Non-targeted metabolomics of saliva to explore potential biomarkers for gastric ulceration in pigs fed hemp

Gastric ulceration is a common disease in pig production worldwide and is associated with economic losses as well as animal health and welfare issues. The aim of this study was to explore potential salivary biomarkers for gastric ulceration in pigs. In addition, the aim was to study the effect of hemp on the incidence of gastric ulcers. Approximately 440 growing-finishing pigs in the period from 30 to 110 kg BW were allocated to four different diets: meal feed (Meal); pelleted feed (Pellets); pelleted feed added 4% hempseed cake (Hemp Cake); pelleted feed added 4% hempseed hulls (Hemp Hulls). The day before slaughter, saliva samples from each pig were collected. After slaughter, the stomachs were emptied to assess the consistency of the stomach content and examined for gastric ulceration using an index scale (0–10). Noticeable changes of the gastric mucosa (total index score ≥ 6) were observed in 291 pigs. The odds of having index scores 0–5 relative to index scores 6–8 and 9–10, respectively, were higher (P < 0.001) for pigs fed Meal compared to pigs fed Pellets. The odds of suffering from severe gastric ulcers tended (P = 0.08) to be lower in pigs fed Hemp Hulls compared to pigs fed Pellets. A non-targeted liquid chromatography mass spectrometry based metabolomics analysis was performed on saliva samples to determine any separation between pigs with healthy stomachs and those with gastric ulcers and to examine a possible correlation between gastric ulcer index and potential biomarkers. Partial least-squares discriminant analysis showed a separation between pigs with ulcers and those with healthy stomachs/hyperkeratosis (HK). Metabolites contributing to the separation between groups were identified. Levels of oxylipins deriving from linoleic acid were lower (P < 0.001) in pigs with ulcers compared to healthy/HK pigs. This may indicate a shift in the metabolic pathways towards more pro-inflammatory arachidonic acid-derived eicosanoids, which might reflect an increased inflammatory response. Thus, reduced levels of oxylipins derived from linoleic acid seemed to be associated with active gastric ulcers, and thereby they might function as biomarkers for gastric ulceration in pigs. In addition, supplementation of hempseed hulls had a beneficial effect on severe gastric ulcers, as hempseed hulls changed the consistency of the gastric content by conferring more solidness. However, it was not possible to observe any reliable separation between pigs fed pellets supplemented with hemp products and pigs fed non-supplemented pellets according to the identified salivary metabolites.

A plasma metabolite score of three eicosanoids predicts incident type 2 diabetes: a prospective study in three independent cohorts

IntroductionPeptide markers of inflammation have been associated with the development of type 2 diabetes. The role of upstream, lipid-derived mediators of inflammation such as eicosanoids, remains less clear. The aim...

Metabolite Profiling and Classification of Highbush Blueberry Leaves under Different Shade Treatments

Blueberry belongs to the genus Vaccinium L. in the Ericaceae and is an economically important shrub that produces small berries that are rich in nutrients. There were differences in the appearance of blueberry leaves under different shade treatments. To explore the differences in metabolites in blueberry leaves under different shading treatments, nontargeted liquid chromatography–mass spectrometry (LC–MS) metabonomic analysis was performed. Different shade intensities resulted in significant differences in the contents of metabolites. A total of 6879 known metabolites were detected, including 750 significantly differentially expressed metabolites, including mainly lipids and lipid-like molecules and phenylpropanoid and polyketide superclass members. Based on a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the flavone and flavonol biosynthesis pathways were the most significantly enriched. The results of this study provide a reference and scientific basis for the establishment of a high-quality and high-yield shaded blueberry cultivation system.

Electromagnetic field exposure-induced depression features could be alleviated by heat acclimation based on remodeling the gut microbiota

BackgroundElectromagnetic pollution cannot be ignored. Long-term low-dose electromagnetic field (EMF) exposure can cause central nervous system dysfunction without effective prevention. Materials/MethodsMale C57BL/6J mice (6–8 weeks, 17–20 g) were used in this study. Depression-like and anxiety-like behaviors detected by behavioral experiments were compared among different treatments. 16S rRNA gene sequencing and non-targeted liquid chromatography-mass spectrometry (LC-MS) metabolomics were used to explore the relationship between EMF exposure and heat acclimation (HA) effects on gut microbes and serum metabolites. ResultsBoth EMF and HA regulated the proportions of p_Firmicutes and p_Bacteroidota. EMF exposure caused the proportions of 6 kinds of bacteria, such as g_Butyricicoccus and g_Anaerotruncus, to change significantly (p < 0.05). HA restored the balance of gut microbes that was affected by EMF exposure and the proportion of probiotics (g_Lactobacillus) increased significantly (p < 0.01). Serum metabolite analysis suggested that HA alleviated the disturbance of serum metabolites (such as cholesterol and D-mannose) induced by EMF exposure. Both the metabolic KEGG pathways and PICRUSt functional analysis demonstrated that tryptophan metabolism, pyrimidine metabolism and amino acid biosynthesis were involved. ConclusionsEMF exposure not only led to depression-like neurobehavioral disorders, but also to gut microbiota imbalance. HA alleviated the depression features caused by EMF exposure. Based on the analysis of gut microbiota associated with serum metabolites, we speculated that gut microbiota might play a vital role in the cross-tolerance provided by HA.

Sex-dependent changes in the microbiota profile, serum metabolism, and hormone levels of growing pigs after dietary supplementation with Lactobacillus.

Dietary supplementation with Lactobacillus can improve the intestinal microbial balance and exerts beneficial effects on pig health. However, whether these effects in pigs show differences between the sexes remains poorly understood. Therefore, we analyzed the sex-dependent patterns in the fecal microbiota after dietary supplementation with Lactobacillus acidophilus ZLA012 through high-throughput sequencing, determined the metabolomic profile of serum in barrows (immature castrated males) and gilts (sexually immature females) through nontargeted liquid chromatography-mass spectrometry, and measured the levels of various hormones, such as insulin, growth hormone, serotonin, triiodothyronine, and thyroxine. Lactobacillus supplementation resulted in more obvious effects on the microbial diversity and composition in barrows than in gilts. Specifically, supplementation with L. acidophilus ZLA012 significantly increased the abundance of the phylum Bacteroidetes and decreased that of Firmicutes in barrows. In contrast, more notable effects on metabolites, particularly those involved in lipid metabolism and glycerophospholipid metabolism, were observed in gilts than in barrows after Lactobacillus supplementation. Megasphaera, Dialister, Gemmiger, Faecalibacterium, Bulleidia, and Prevotella were the core functional genera associated with the significantly affected metabolites, which are involved in the biosynthesis, degradation, and elongation of fatty acids. L. acidophilus ZLA012 treatment increased the serum insulin, total bile acid, lipoprotein lipase, and nicotinamide adenine dinucleotide (NAD)+ levels in gilts, whereas higher serotonin levels were found in barrows than in gilts. The results indicated that the separate housing of barrows and gilts might be beneficial for targeted dietary supplementation and application of Lactobacillus in pig production. KEY POINTS: • L. acidophilus exerted obvious effects on microbiota profiles for barrows than gilts. • Gilts treated by L. acidophilus had a greater variety of lipid metabolism compared with barrows. • Lactobacillus regulated the dynamic balance among the microbiota, metabolism, and hormone in pigs.

141 Diet supplementation alters oocyte lipid content and developmental competence in mares

The equine oocyte is dense in lipids, which may serve as an energy source for oocyte maturation and later embryonic development. However, the association between lipid content and fertility remains to be determined, as does the extent that diet can modify oocyte lipids. We hypothesised that diet supplementation can alter the oocyte lipid profile and subsequent developmental potential. In Study 1, we examined if oocyte triglyceride (TG) relative abundance was affected by dietary supplementation. Mares (16–22 years, n=9) were fed grass/alfalfa hay and supplemented daily with a combination of commercially available feed additives designed to promote equine wellness and fertility [Equine GI™ (147g daily), Potency® (28g daily), Motility Plus® (23g daily), Healthy Weight Oil (60mL daily), Platinum Performance Inc.]. Oocytes were collected from the mares before (PRE) and after ≥8 weeks (POST) of supplementation during the natural breeding season. In Study 2, we compared oocyte developmental potential after injection of sperm into oocytes obtained from mares supplemented for ≥8 weeks with the same additives (ADD, 18–24 yr, n=5) or from a similar group of mares supplemented with a grain control diet (450g of grain mix and 60ml of corn oil daily, GRN, 19–23 years, n=5). Oocytes were collected from dominant follicles (≥35mm) during oestrus and at 20±2 h after induction of follicular maturation. In Study 1, oocytes were denuded of cumulus cells after collection, snap frozen, and assessed for TG composition by nontargeted liquid chromatography-mass spectrometry using an Acquity UPLC system (Waters). In Study 2, recovered oocytes were placed in maturation medium for 22±2h before being injected with sperm from one stallion, and blastocyst formation was assessed in 7 or 8 days. A total of 100 annotated TG species were identified. Normalized peak areas for PRE and POST oocyte TG were compared using two-tailed, paired t-tests. Blastocyst development rates were compared by Fisher’s exact test. Relative abundance of 71 TG species differed (P ≤ 0.05) between PRE and POST; all TG species as well as total relative abundance of TG were higher in oocytes from PRE compared with POST. Blastocyst rates per sperm-injected oocyte were greater (P=0.03) for ADD (40%, 6/15) than for GRN (5%, 1/19). Dietary supplementation of the complex mix of nutrients to middle-aged and older mares resulted in reduced relative abundance of TG in oocytes and improved developmental potential. We determined that oocyte lipid content can be modified through diet. The extent that diet supplementation improved oocyte competence by altering the lipid profile is still to be determined.

Ozone Exposure Induces Metabolic Disorders and NAD+ Depletion Through PARP1 Activation in Spinal Cord Neurons.

Background and Objective: Ozone therapy has shown therapeutic efficacy in different disorders particularly low back pain (LBP). However, ozone therapy has been associated with toxic effects on the respiratory, endocrine, cardiovascular systems as well as nervous system because of its strong oxidizing capacity. Recent studies have reported possible associations between ozone exposure and metabolic disorders, but the findings are controversial and little is known on the mechanisms of action. This study aims to investigate the cytotoxic effects of ozone exposure and possible mechanism of action in the animal model.Methods: Wistar neonate rats with the age of 24 h after birth were sacrificed by cervical dislocation under general anesthesia, then immersed in 75% alcohol and iodophor for 5 min, respectively. The spinal cord was isolated and cut to samples of ~1 mm3 and prepared for further experiments. The spinal cord neurons (SCNs) were exposed to ozone at different concentrations and then cultured in 96-well plates with glass bottom for 7 days. The cell viability, ATP levels and the NAD+ concentration were determined and compared between the different experimental groups and the control group.Results: Analyses of the data by non-targeted liquid chromatography-mass spectrometry (LC-MS) analysis determined the metabolic disorder in SCNs following the ozone exposure. Moreover, our assessments showed that ozone exposure resulted in DNA damage, poly (ADP)-ribose polymerase-1 (PARP1) excessive activation, nicotinamide adenine dinucleotide (NAD+) depletion and decrease of ATP level in SCNs. The PARP1 inhibitor can inhibit the cytotoxic effect of ozone to SCNs without inhibiting the activation of AMP-activated protein kinase (AMPK). Our findings revealed that the cytotoxic effects of ozone to SCNs might be mediated by excessive PARP1 activation and subsequent NAD+ depletion. Moreover, using PARP1 inhibitor can protect SCNs from cytotoxic effects of ozone by preventing NAD+ depletion during ozone exposure.Conclusion: Ozone exposure seems to induce metabolic disorders and NAD+ depletion through excessive PARP1 activation in SCNs.

Non-targeted metabolomic analysis predicts the therapeutic effects of exenatide on endothelial injury in patients with type 2 diabetes.

We aimed to investigate whether treatment with exenatide could ameliorate endothelial injury in patients with type 2 diabetes mellitus (T2DM), and to identify biomarkers for predicting amelioration of the endothelial injury induced by the treatment. Ninety-three patients with T2DM were recruited and treated with exenatide for 16 weeks. Enzyme-linked immunosorbent assays were performed at baseline and after the treatment to measure serum levels of endothelial injury markers, including soluble thrombomodulin (sTM). Patients were categorized as responders (n = 47) or non-responders (n = 46) based on median changes in their sTM levels. Serum levels of metabolites at baseline were measured with non-targeted liquid chromatography-mass spectrometry. The results obtained were evaluated with multivariate analysis. Treatment with exenatide for 16 weeks resulted in reduced body weight and improved levels of fasting plasma glucose, 2-hour postprandial plasma glucose, and HbA1c in patients with T2DM (all P < 0.05). Compared with baseline, serum levels of endothelial injury markers including sTM were significantly lowered after the treatment. Metabolites presented at significantly different levels in responders versus non-responders were considered as biomarkers for a therapeutic response of sTM to the exenatide treatment. Among those identified, 4-hydroxyproline and 12-oxo-9(Z)-dodecenoic acid were found to correlate most closely with the exenatide-induced endothelial protection response. The specificity and sensitivity of the multi-metabolite signature model contained higher 4-hydroxyproline and lower 12-oxo-9(Z)-dodecenoic acid were 53.3% and 92.3%, respectively, and the area under receiver operating characteristic curve was 69.2% (P < 0.001). Treatment with exenatide for 16 weeks ameliorates endothelial injury in patients with T2DM. Endothelial protection benefit from exenatide treatment was effectively predicted by the specific metabolomic combination of higher 4-hydroxyproline and lower 12-oxo-9(Z)-dodecenoic acid.

Eicosanoid Inflammatory Mediators Are Robustly Associated With Blood Pressure in the General Population.

BackgroundEpidemiological and animal studies have associated systemic inflammation with blood pressure (BP). However, the mechanistic factors linking inflammation and BP remain unknown. Fatty acid–derived eicosanoids serve as mediators of inflammation and have been suggested to regulate renal vascular tone, peripheral resistance, renin‐angiotensin system, and endothelial function. We hypothesize that specific proinflammatory and anti‐inflammatory eicosanoids are linked with BP.Methods and ResultsWe studied a population sample of 8099 FINRISK 2002 participants randomly drawn from the Finnish population register (53% women; mean age, 48±13 years) and, for external validation, a sample of 2859 FHS (Framingham Heart Study) Offspring study participants (55% women; mean age, 66±9 years). Using nontargeted liquid chromatography–mass spectrometry, we profiled 545 distinct high‐quality eicosanoids and related oxylipin mediators in plasma. Adjusting for conventional hypertension risk factors, we observed 187 (34%) metabolites that were significantly associated with systolic BP (P<Bonferroni‐corrected threshold of 0.05/545). We used forward selection linear regression modeling in FINRISK to define a general formula for individual eicosanoid risk score. Individuals of the top risk score quartile in FINRISK had a 9.0 (95% CI, 8.0–10.1) mm Hg higher systolic BP compared with individuals in the lowest quartile in fully adjusted models. Observed metabolite associations were consistent across FINRISK and FHS.ConclusionsPlasma eicosanoids demonstrate strong associations with BP in the general population. As eicosanoid compounds affect numerous physiological processes that are central to BP regulation, they may offer new insights about the pathogenesis of hypertension, as well as serve as potential targets for therapeutic intervention.

Impact of substituting compound feed with increasing levels of fresh grass-clover on nitrogen metabolism and plasma metabolites of sows

The purpose of this study was to identify biomarkers for fresh grass-clover intake and determine the digestibility and energy value of fresh grass to estimate voluntary fresh grass-clover intake in sows kept on pasture according to organic production practice. A total of sixteen multiparous dry sows (Danish Landrace x Danish Yorkshire) were housed in metabolism cages for 2 periods of 5 d. To study dietary effects, sows were fed one of four mixed treatments where increasing proportions of a basal commercial organic sow compound feed was partly replaced with 0, 2, 4, or 6 kg fresh grass-clover collected 3 wk after the previous cut. Sows were fed similar amounts of metabolizable energy (ME). Total collection of urine and feces was performed on a daily basis for 5d, and blood samples were collected from the jugular vein on the last day of feeding. Plasma metabolites were analyzed using a non-targeted liquid chromatography-mass spectrometry (LCMS) approach. Analyzed plasma metabolites (area under the curve in arbitrary units from the LCMS) were screened for correlation with grass intake. Data on nutrient digestibility and plasma metabolites were analyzed using a MIXED procedure while accounting for repeated measurements. Apparent total tract digestibilities of dry matter, organic matter, nitrogen, and energy of fresh grass-clover were 72, 64, 71, and 68%, respectively, using the regression method. The N intake increased linearly with increasing fresh grass-clover intake (P < 0.001). There was no evidence of altered N deposition and N utilization in response to increasing grass intake (P > 0.05). There was a linear increase in plasma urea content (2.64 to 4.39 mM) when grass intake increased from 0 to 6 kg/d (P = 0.02). Plasma glucose, lactate, creatinine and NEFA, and triglycerides were not affected by increased grass-clover intake. The daily ME contribution from fresh grass-clover (MJ/d) was found to be highly positively correlated with plasma pipecolic acid (Y = 0.0289 X; r2 = 0.91; P < 0.001) and a metabolite tentatively identified as plasma bisnorbiotin (Y = 0.482 X; r2 = 0.92; P < 0.001). In conclusion, fresh grass-clover intake of sows was highly correlated with plasma pipecolic acid and plasma bisnorbiotin concentration and apparent total tract digestibility of DM, OM, N, and energy estimated for 100% fresh grass-clover intake in dry sows was 64 to 72% using the regression method.

A combined proteomic and metabolomic analyses of the priming phase during rat liver regeneration

By comparing differentially abundant proteins and metabolites, the protein expression, metabolic changes and metabolic regulation mechanisms during the priming phase of liver regeneration (LR) were investigated. We combined proteomic analysis via isobaric tags for relative and absolute quantification (iTRAQ) with metabolomic analysis via nontargeted liquid chromatography-mass spectrometry (LC-MS). LC-MS was used to examine 29 energy metabolites expression alterations in targeted metabolomics. A total number of 441 differentially expressed proteins and 65 metabolites were identified. PSMB10, PSMB5, RCG_63409, PSME4 and PSMB7 were key node proteins, these proteins are involved in the proteasome pathway. The most strongly enriched transcription factor motif was TP63. These results point out a critical role of the proteasome pathway (defense mechanisms) and of TP63 (metabolic regulator) as the key transcription factor during the priming phase of LR. Metabolomic and metabolite analysis showed that profiling indicates upregulation of arginine biosynthesis and glycolysis as the main ATP-delivering pathway. Integrative proteomic and metabolomic analysis showed that biomolecular changes were primarily related to the neurological disease, cell death and survival and cell morphology. What's more, neurotransmitters may play an important role in the regulation of LR.

Food Phenotyping: Recording and Processing of Non-Targeted Liquid Chromatography Mass Spectrometry Data for Verifying Food Authenticity.

Experiments based on metabolomics represent powerful approaches to the experimental verification of the integrity of food. In particular, high-resolution non-targeted analyses, which are carried out by means of liquid chromatography-mass spectrometry systems (LC-MS), offer a variety of options. However, an enormous amount of data is recorded, which must be processed in a correspondingly complex manner. The evaluation of LC-MS based non-targeted data is not entirely trivial and a wide variety of strategies have been developed that can be used in this regard. In this paper, an overview of the mandatory steps regarding data acquisition is given first, followed by a presentation of the required preprocessing steps for data evaluation. Then some multivariate analysis methods are discussed, which have proven to be particularly suitable in this context in recent years. The publication closes with information on the identification of marker compounds.

Engineered butyrate-producing bacteria prevents high fat diet-induced obesity in mice

BackgroundObesity is a major problem worldwide and severely affects public safety. As a metabolite of gut microbiota, endogenous butyric acid participates in energy and material metabolism. Considering the serious side effects and weight regain associated with existing weight loss interventions, novel strategies are urgently needed for prevention and treatment of obesity.ResultsIn the present study, we engineered Bacillus subtilis SCK6 to exhibited enhanced butyric acid production. Compared to the original Bacillus subtilis SCK6 strain, the genetically modified BsS-RS06550 strain had higher butyric acid production. The mice were randomly divided into four groups: a normal diet (C) group, a high-fat diet (HFD) group, an HFD + Bacillus subtilis SCK6 (HS) group and an HFD + BsS-RS06550 (HE) group. The results showed BsS-RS06550 decreased the body weight, body weight gain, and food intake of HFD mice. BsS-RS06550 had beneficial effects on blood glucose, insulin resistance and hepatic biochemistry. After the 14-week of experiment, fecal samples were collected for nontargeted liquid chromatography-mass spectrometry analysis to identify and quantify significant changes in metabolites. Sixteen potentially significant metabolites were screened, and BsS-RS06550 was shown to potentially regulate disorders in glutathione, methionine, tyrosine, phenylalanine, and purine metabolism and secondary bile acid biosynthesis.ConclusionsIn this study, we successfully engineered Bacillus subtilis SCK6 to have enhanced butyric acid production. The results of this work revealed that the genetically modified live bacterium BsS-RS06550 showed potential anti-obesity effects, which may have been related to regulating the levels of metabolites associated with obesity. These results indicate that the use of BsS-RS06550 may be a promising strategy to attenuate obesity.

Plasma lipid profile associates with the improvement of psychological well-being in individuals with perceived stress symptoms

Psychological stress is a suggested risk factor of metabolic disorders, but molecular mediators are not well understood. We investigated the association between the metabolic profiles of fasting plasma and the improvement of psychological well-being using non-targeted liquid chromatography-mass spectrometry (LC-MS) platform. The metabolic profiles of volunteers participating in the face-to-face intervention group (n = 60) in a randomised lifestyle intervention were compared to ones of controls (n = 64) between baseline and 36-week follow-up. Despite modest differences in metabolic profile between groups, we found associations between phosphatidylcholines (PCs) and several parameters indicating stress, adiposity, relaxation, and recovery. The relief of heart-rate-variability-based stress had positive, while improved indices of recovery and relaxation in the intervention group had an inverse association with the reduction of e.g. lysophosphatidylcholines (LPC). Interleukin-1 receptor antagonist and adiposity correlated positively with the suppressed PCs and negatively with the elevated plasmalogens PC(P-18:0/22:6) and PC(P-18:0/20:4). Also, we found changes in an unknown class of lipids over time regardless of the intervention groups, which also correlated with physiological and psychological markers of stress. The associations between lipid changes with some markers of psychological wellbeing and body composition may suggest the involvement of these lipids in the shared mechanisms between psychological and metabolic health.

Which is the urine sample material of choice for metabolomics-driven biomarker studies?

Urine-based metabolomics-driven strategies for the discovery of biomarkers are increasingly developed and applied in analytical chemistry. But valid, data-based recommendations for a urine sample material of choice are lacking. We investigated first and second morning urine (MU), which are the most commonly used urine specimens. Potential major factors biasing metabolomics biomarker results in these sample materials were studied. First, 35 1st and 2nd MU samples were collected from healthy, young men after an overnight fast. Subsequently, two subgroups were built, one having fast food at lunch and dinner (n=17), the other vegetarian meals (n=18). Again 1st and 2nd MU were collected. Non-targeted liquid chromatography-mass spectrometry was applied for analyses. More than half of the >5400 urinary ion features showed a significant difference between 1st and 2nd MU. Just two fast food meals on previous day significantly affected around 30% of all metabolites in 1st and 2nd MU. In contrast, the effects of two vegetarian meals in 2nd MU were only minor. Additionally, we describe 47 metabolites in urine, possible hits in biomarker studies, which are susceptible to the diet the day before sample collection. They should be handled with caution until validation in diet-controlled studies. Based on our results we think the second MU, ideally collected after standardized vegetarian meals and drinking only water on the previous day, is most suitable for valid analysis of biomarkers in urine.

Integrated proteomics and metabolomics analysis reveals the antifungal mechanism of the C-coordinated O-carboxymethyl chitosan Cu(II) complex

With wide application in agriculture, copper fungicides have undergone three stages of development: inorganic copper, synthetic organic copper, and natural organic copper. Using chitin/chitosan (CS) as a substrate, the natural organic copper fungicide C-coordinated O-carboxymethyl chitosan Cu(II) complex (O-CSLn-Cu) was developed in the laboratory. Taking Phytophthora capsici Leonian as an example, we explored the antifungal mechanism of O-CSLn-Cu by combining tandem mass tag (TMT)-based proteomics with non-targeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. A total of 1172 differentially expressed proteins were identified by proteomics analysis. According to the metabolomics analysis, 93 differentially metabolites were identified. Acetyl-CoA-related and membrane localized proteins showed significant differences in the proteomics analysis. Most of the differential expressed metabolites were distributed in the cytoplasm, followed by mitochondria. The integrated analysis revealed that O-CSLn-Cu could induce the “Warburg effect”, with increased glycolysis in the cytoplasm and decreased metabolism in the mitochondria. Therefore, P. capsici Leonian had to compensate for ATP loss in the TCA cycle by increasing the glycolysis rate. However, this metabolic shift could not prevent the death of P. capsici Leonian. To verify this hypothesis, a series of biological experiments, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), and enzyme activity measurements were carried out. The results suggest that O-CSLn-Cu causes mitochondrial injury, which consequently leads to excessive ROS levels and insufficient ATP levels, thereby killing P. capsici Leonian.