Untargeted Metabolomics Strategy Research Articles

Heat exposure promotes sarcopenia via gut microbiota-derived metabolites.

The unprecedented rise in global ambient temperatures in the last decade has significantly impacted human health, yet how heat exposure affects the development of sarcopenia remains enigmatic. Here, we demonstrate that chronic heat exposure induces skeletal muscle volume loss, leading to muscle strength and functional decline in mice. The microbiota composition of heat-exposed mice was analyzed using 16S ribosomal DNA analysis. Liquid chromatography-mass spectrometry (LC-MS) was used to explore the effects of heat exposure on the blood metabolome and to further analyze the correlation between blood metabolism and gut microbiota. Transplantation of microbiota from heat-exposed mice to germ-free mice was sufficient to increase adverse effects on skeletal muscle function in the host. Mechanistically, using an untargeted metabolomics strategy, we reveal that altered gut microbiota due to high temperatures is associated with elevated serum levels of homocitrulline. Homocitrulline causes mitochondrial dysfunction in myocytes by exacerbating ferroptosis levels. And Nrf2 activator (Oltipraz) supplementation alleviates muscle atrophy and dysfunction induced by heat exposure. Our findings reveal the detrimental effects of heat exposure on muscle function and provide new strategies for treating sarcopenia.

Metabolomics study of Shenling Baizhu Powder in the treatment of multiple organ dysfunction syndrome in the elderly (MODSE) with malnutrition

Malnutrition is an important risk factor for multiple organ dysfunction syndrome in the elderly (MODSE) and seriously affects the occurrence, progression and prognosis of MODSE. Shenling Baizhu Power (SBP), a classic formula from traditional Chinese medicine (TCM), when integrated with enteral nutrition, has been proven to be an effective clinical strategy for treating the patients of MODSE with malnutrition. This study aimed to investigate the metabolic changes during disease occurrence and SBP treatment, and to discover potential metabolic biomarkers for the diagnosis and efficacy evaluation. An untargeted metabolomics strategy based on UHPLC-Q-Orbitrap-HRMS was performed to reveal the differential serum metabolites between MODSE patients with malnutrition (n=59) and healthy controls (n=33), and those between patients treated with enteral nutrition (n=31) and SBP combined with enteral nutrition (n=28). Significantly different metabolites were identified and mapped onto the network of metabolic pathways to explore the metabolic disorders caused by the disease and the metabolic regulatory mechanism of SBP. Additionally, the area under the curve (AUC) of the potential biomarkers was investigated for predicting the disease and the efficacy of SBP. Sixty differential metabolites were identified between the disease and control groups, which were mainly related to amino acid metabolism, energy metabolism and carbohydrate metabolism. In the same way, 50 differential metabolites associated with SBP treatment were identified, which improved metabolic abnormalities in vivo mainly by regulating the above-mentioned metabolic pathways. Finally, 13 differential metabolites in common were selected as the potential biomarkers and the AUC value of each biomarker was within the range of 0.8–1.0, indicating that these biomarkers had high prediction accuracy for the diagnosis and efficacy evaluation of MODSE with malnutrition. This study demonstrates that serum metabolomics approaches based on the UHPLC-Q-Orbitrap-HRMS platform can be applied as a tool to reveal the metabolic changes induced by MODSE with malnutrition and SBP can play an important role in the clinical application.

Metabolic Profiling Analysis of Congenital Adrenal Hyperplasia via an Untargeted Metabolomics Strategy.

Congenital adrenal hyperplasia (CAH) manifests as an autosomal recessive disorder characterized by defects in the enzymes responsible for steroid synthesis. This work aims to perform metabolic profiling of patients with CAH, screen key differential metabolites compared to the control group, and discover the associated metabolic pathways implicated in CAH. Serum samples obtained from 32 pediatric male patients with CAH and 31 healthy control group candidates were subjected to analysis using non-targeted metabolomics strategy using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). A total of 278 differential metabolites were identified and annotated in KEGG. Operating characteristic curves (ROC) measurement exhibited 9 metabolites exhibiting high efficacy in differential diagnosis, as evidenced by an area under ROC curve (AUC) exceeding 0.85. Pathway analysis uncovered notable disruptions in steroid hormone biosynthesis (p <0.0001), purine metabolism and irregularities in lipid metabolism and amino acid metabolism, including tyrosine and alanine, in CAH patients. These findings demonstrate that metabolic pathways of purine, amino acid and lipid metabolism, apart from steroid hormone biosynthesis, may be disrupted and associated with CAH. This study helps provide insight into the metabolic profile of CAH patients and offers a new perspective for monitoring and administering follow-up care to CAH patients.

UHPLC-MS-based untargeted metabolomic strategy to reveal the metabolic differences between cisplatin first- and second-generation apoptotic bodies from HK-2 cells

During the process of programmed cell death (apoptosis), the disassembly of apoptotic cells gives rise to apoptotic bodies (ABs) which play an important role in the pathogenesis of different diseases. ABs from renal human proximal tubular cells HK-2 on naïve cells was not always the same: ABs directly generated after exposure of human proximal tubular epithelial HK-2 cells to chemotherapeutic agent cisplatin (1st generation ABs) triggered apoptosis and inhibited cell proliferation, whereas ABs induced by 1st generation ABs (2nd generation ABs) enhanced HK-2 cell proliferation. To shed light on the mechanisms involved in these disparate ABs’ effects, a study of the changes in the metabolome of 1st and 2nd generation ABs produced by HK-2 cells in the cisplatin setting was performed by a reversed-phase ultra-high-performance liquid chromatography-mass spectrometry (RP-UHPLC-MS) untargeted metabolomic approach. Thus, ABs fluid and extracellular fluid from 1st and 2nd generation ABs were analyzed to obtain as much information as possible of the metabolites of these extracellular vesicles. Principal components analysis and partial least square discriminant analysis were conducted to find the statistically significant differences between both groups. A good clustering of each group was observed and variable importance in the projection values were selected to choose the molecular features which were identified according to the Metabolomics Standard Initiative (MSI) guidelines. Finally, the biological relevance of the achieved results was discussed.

An Untargeted Metabolomics Strategy to Identify Substrates of Known and Orphan E. coli Transporters.

Transport systems play a pivotal role in bacterial physiology and represent potential targets for medical and biotechnological applications. However, even in well-studied organisms like Escherichia coli, a notable proportion of transporters, exceeding as many as 30%, remain classified as orphans due to their lack of known substrates. This study leveraged high-resolution LC-MS-based untargeted metabolomics to identify candidate substrates for these orphan transporters. Human serum, including a diverse array of biologically relevant molecules, served as an unbiased source for substrate exposure. The analysis encompassed 26 paired transporter mutant contrasts (i.e., knockout vs. overexpression), compared with the wild type, revealing distinct patterns of substrate uptake and excretion across various mutants. The convergence of candidate substrates across mutant scenarios provided robust validation, shedding light on novel transporter-substrate relationships, including those involving yeaV, hsrA, ydjE, and yddA. Furthermore, several substrates were contingent upon the specific mutants employed. This investigation underscores the utility of untargeted metabolomics for substrate identification in the absence of prior knowledge and lays the groundwork for subsequent validation experiments, holding significant implications for both medical and biotechnological advancements.

LC-MS-based metabolomics reveals metabolite dynamic changes of beef after superchilling early post-mortem

To explore the underlying mechanisms by which superchilling (SC, −3 °C within 5 h of slaughter) improves beef tenderness, an untargeted metabolomics strategy was employed. M. Longissimus lumborum (LL) muscles from twelve beef carcasses were assigned to either SC or very fast chilling (VFC, 0 °C within 5 h of slaughter) treatments, with conventional chilling (CC, 0 ∼ 4 °C until 24 h post-mortem) serving as the control (6 per group). Biochemical properties and metabolites were investigated during the early post-mortem period. The results showed that the degradation of μ-calpain and caspase 3 occurred earlier in SC treated sample, which might be attributed to the accelerated accumulation of free Ca2+. The metabolomic profiles of samples from the SC and CC treatments were clearly distinguished based on partial least squares-discriminant analysis (PLS-DA) at each time point. It is noteworthy that more IMP and 4-hydroxyproline were found in the comparison between SC and CC treatments. According to the results of metabolic pathways analysis and the correlation analysis between traits related to tenderness and metabolites with significant differences (SC vs. CC), it can be suggested that the tenderization effect of the SC treatment may be related to the alteration of arginine and proline metabolism, and purine metabolism in the early post-mortem phase.

Integrated Metabolomics Approach Reveals the Dynamic Variations of Metabolites and Bioactivities in Paeonia ostii 'Feng Dan' Leaves during Development.

Paeonia ostii 'Feng Dan' is widely cultivated in China for its ornamental, medicinal, and edible properties. The whole plant of tree peony is rich in bioactive substances, while the comprehensive understanding of metabolites in the leaves is limited. In this study, an untargeted metabolomics strategy based on UPLC-ESI-TOF-MS was conducted to analyze the dynamic variations of bioactive metabolites in P. ostii 'Feng Dan' leaves during development. A total of 321 metabolites were rapidly annotated based on the GNPS platform, in-house database, and publications. To accurately quantify the selected metabolites, a targeted method of HPLC-ESI-QQQ-MS was used. Albiflorin, paeoniflorin, pentagalloylglucose, luteolin 7-glucoside, and benzoylpaeoniflorin were recognized as the dominant bioactive compounds with significant content variations during leaf development. Metabolite variations during the development of P. ostii 'Feng Dan' leaves are greatly attributed to the variations in antioxidant activities. Among all tested bacteria, the leaf extract exhibited exceptional inhibitory effects against Streptococcus hemolytis-β. This research firstly provides new insights into tree peony leaves during development. The stages of S1-S2 may be the most promising harvesting time for potential use in food or pharmaceutical purposes.

An untargeted metabolomics approach applied to the study of the bioavailability and metabolism of three different bioactive plant extracts in human blood samples.

Advances in the understanding of bioavailability and metabolism of bioactive compounds have been achieved primarily through targeted or semi-targeted metabolomics approaches using the hypothesis of potential metabolized compounds. The recent development of untargeted metabolomics approaches can present great advantages in this field, such as in the discovery of new metabolized compounds or to study the metabolism of compounds from multiple matrices simultaneously. Thus, this study proposes the use of an untargeted metabolomics strategy based on HPLC-ESI-QTOF-MS for the study of bioavailability and metabolism of bioactive compounds from different vegetal sources. Specifically, this study has been applied to plasma samples collected in an acute human intervention study using three matrices (Hibiscus sabdariffa, Silybum marianum and Theobroma cacao). This approach allowed the selection of those significant variables associated with exogenous metabolites derived from the consumption of bioactive compounds for their subsequent identification. As a result, 14, 25 and 3 potential metabolites associated with supplement intake were significantly detected in the plasma samples from volunteers who ingested the H. sabdariffa (HS), S. marianum (SM) and T. cacao (TC) extracts. Furthermore, Tmax values have been computed for each detected compound. The results highlight the potential of untargeted metabolomics for rapid and comprehensive analysis when working with a wide range of exogenous metabolites from different plant sources in biological samples.

Arginine Biosynthesis Pathway Found to Play a Key Role in the Neuroprotective Effect of Liu-Wei-Luo-Bi (LWLB) Granules in Diabetic db/db Mice with Peripheral Neuropathy Using an Untargeted Metabolomics Strategy.

Liu-Wei-Luo-Bi (LWLB) granules was a Chinese compound prescription for treating diabetic peripheral neuropathy (DPN). The aim of this study was to investigate the effect of LWLB granules on diabetic mice with peripheral neuropathy and to elucidate the potential mechanism based on an untargeted metabolomics approach. One hundred forty db/db mice were randomly divided into seven groups: the Control group, DPN group, Mudan (MD) granules group, Epalrestat (Epa) group, and the LWLB low, medium, or high dose (LW-l, LW-m, or LW-h) group. After 12 weeks of treatment, body weight, blood glucose, mechanical pain threshold, motor conduction velocity (MCV), sensory conduction velocity (SCV), and Pathological Organization of the Sciatic and Caudal Nerves in mice were measured. Serum samples were collected for untargeted metabolomics analysis using liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and multivariate statistics. Disease-related pathways were screened out with function enrichment analyses of candidate biomarkers. LWLB granules can improve the peripheral neuropathy of type 2 diabetic mice with peripheral nerve conduction disorders, mainly through significantly improving the nerve conduction velocity (P < 0.05) and lowering the mechanical pain threshold (P < 0.05). A total of 43 metabolites were identified as potential biomarkers related to the therapeutic effect of LWLB granules. Fifty, 4, and 26; 23, 4, and 22; and 24, 1, and 16 biomarkers were discovered in the LW-l, LW-m, and LW-h groups at the 4th, 6th, and 12th weeks, respectively. Five, three, seven, five, and four metabolic pathways were found in MD, Epa, LW-l, LW-m, and LW-h groups, respectively. The arginine biosynthesis pathway is the overlapping pathway in LW-l, LW-m, and LW-h groups. LWLB granules have an obvious neuroprotective effect on diabetic peripheral neuropathy, and the metabolism mechanism of LWLB is mainly related to the arginine biosynthesis pathway on diabetic db/db mice with peripheral neuropathy.

Amniotic fluid metabolomics identifies impairment of glycerophospholipid and amino acid metabolism during congenital Zika syndrome development.

Our main goal is to identify the alterations in the amniotic fluid (AF) metabolome in Zika virus (ZIKV)-infected patients and their relation to congenital Zika syndrome (CZS) progression. We applied an untargeted metabolomics strategy to analyze seven AF of pregnant women: healthy women and ZIKV-infected women bearing non-microcephalic and microcephalic fetuses. Infected patients were characterized by glycerophospholipid metabolism impairment, which is accentuated in microcephalic phenotypes. Glycerophospholipid decreased concentration in AF can be a consequence of intracellular transport of lipids to the placental or fetal tissues under development. The increased intracellular concentration of lipids can lead to mitochondrial dysfunction and neurodegeneration caused by lipid droplet accumulation. Furthermore, the dysregulation of amino acid metabolism was a molecular fingerprint of microcephalic phenotypes, specifically serine, and proline metabolisms. Both amino acid deficiencies were related to neurodegenerative disorders, intrauterine growth retardation, and placental abnormalities. This study enhances our understanding of the development of CZS pathology and sheds light on dysregulated pathways that could be relevant for future studies.

Application of untargeted volatile profiling in inflammatory bowel disease research

Inflammatory bowel disease (IBD) diagnosis depends on criteria based on histological, endoscopic, radiological, and clinical results. These studies show drawbacks as being expensive, invasive, and time-consuming. In this work, an untargeted metabolomic strategy based on the monitoring of volatile compounds in serum by headspace gas chromatography–mass spectrometry is proposed as a complementary, fast, and efficient test for IBD patient diagnosis. To develop the method and build a chemometric model that allows the IBD diagnosis, serum samples including IBD patients and healthy volunteers were collected. Analyses were performed by incubating 400 µL of serum for 10 min at 90 °C. For data processing, an untargeted metabolomic strategy was used. A total of 96 features were detected, of which a total of 10 volatile compounds could be identified and confirmed by means of the analysis of real standards. The chemometric treatment consisted of a discriminant analysis of orthogonal partial least squares (OPLS-DA) obtaining a 100% of classification rate, since all the analyzed samples were correctly classified.Graphical abstract

Widely untargeted metabolomic profiling unearths metabolites and pathways involved in leaf senescence and N remobilization in spring-cultivated wheat under different N regimes.

Progression of leaf senescence consists of both degenerative and nutrient recycling processes in crops including wheat. However, the levels of metabolites in flag leaves in spring-cultivated wheat, as well as biosynthetic pathways involved under different nitrogen fertilization regimes, are largely unknown. Therefore, the present study employed a widely untargeted metabolomic profiling strategy to identify metabolites and biosynthetic pathways that could be used in a wheat improvement program aimed at manipulating the rate and onset of senescence by handling spring wheat (Dingxi 38) flag leaves sampled from no-, low-, and high-nitrogen (N) conditions (designated Groups 1, 2, and 3, respectively) across three sampling times: anthesis, grain filling, and end grain filling stages. Through ultrahigh-performance liquid chromatography-tandem mass spectrometry, a total of 826 metabolites comprising 107 flavonoids, 51 phenol lipids, 37 fatty acyls, 37 organooxygen compounds, 31 steroids and steroid derivatives, 18 phenols, and several unknown compounds were detected. Upon the application of the stringent screening criteria for differentially accumulated metabolites (DAMs), 28 and 23 metabolites were differentially accumulated in Group 1_vs_Group 2 and Group 1_vs_Group 3, respectively. From these, 1-O-Caffeoylglucose, Rhoifolin, Eurycomalactone;Ingenol, 4-Methoxyphenyl beta-D-glucopyranoside, and Baldrinal were detected as core conserved DAMs among the three groups with all accumulated higher in Group 1 than in the other two groups. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that tropane, piperidine, and pyridine alkaloid biosynthesis; acarbose and validamycin biosynthesis; lysine degradation; and biosynthesis of alkaloids derived from ornithine, lysine, and nicotinic acid pathways were the most significantly (p < 0.05) enriched in Group 1_vs_Group 2, while flavone and flavonol as well as anthocyanins biosynthetic pathways were the most significantly (p < 0.05) enriched in Group 1_vs_Group 3. The results from this study provide a foundation for the manipulation of the onset and rate of leaf senescence and N remobilization in wheat.

Identifying Hair Biomarker Candidates for Alzheimer's Disease Using Three High Resolution Mass Spectrometry-Based Untargeted Metabolomics Strategies.

High-resolution mass spectrometry (HRMS)-based untargeted metabolomics strategies have emerged as an effective tool for discovering biomarkers of Alzheimer's disease (AD). There are various HRMS-based untargeted metabolomics strategies for biomarker discovery, including the data-dependent acquisition (DDA) method, the combination of full scan and target MS/MS, and the all ion fragmentation (AIF) method. Hair has emerged as a potential biospecimen for biomarker discovery in clinical research since it might reflect the circulating metabolic profiles over several months, while the analytical performances of the different data acquisition methods for hair biomarker discovery have been rarely investigated. Here, the analytical performances of three data acquisition methods in HRMS-based untargeted metabolomics for hair biomarker discovery were evaluated. The human hair samples from AD patients (N = 23) and cognitively normal individuals (N = 23) were used as an example. The most significant number of discriminatory features was acquired using the full scan (407), which is approximately 10-fold higher than that using the DDA strategy (41) and 11% higher than that using the AIF strategy (366). Only 66% of discriminatory chemicals discovered in the DDA strategy were discriminatory features in the full scan dataset. Moreover, compared to the deconvoluted MS/MS spectra with coeluted and background ions from the AIF method, the MS/MS spectrum obtained from the targeted MS/MS approach is cleaner and purer. Therefore, an untargeted metabolomics strategy combining the full scan with the targeted MS/MS method could obtain most discriminatory features along with a high quality MS/MS spectrum for discovering the AD biomarkers.

A Strategy for Differentiating Oak Barrel Aged and Non-Oak Barrel Aged Wines by Using UHPLC–HRMS Combined with Chemometrics

The time involved and the high economic cost of using oak barrels to age wines have led to a significant price difference compared to non-oak barrel aged wines and may lead to some fraudulent sales in the market. In this study, an untargeted metabolomic strategy was developed to detect the metabolite composition of oak barrel aged and non-oak barrel aged wines in both positive and negative ion modes by using UHPLC–HRMS combined with the recently developed chemometric method AntDAS. The results of partial least squares discrimination analysis (PLS-DA) showed that the samples were characterized into two categories. Finally, 51 compounds were identified in positive ion mode, while 26 compounds were identified in negative ion mode. The results indicate that combining UHPLC–HRMS with AntDAS can reveal the material basis of wines and has excellent potential to differentiate between oak barrel aged and non-oak barrel aged wines.

Development of an Untargeted Metabolomics Strategy to Study the Metabolic Rewiring of Dendritic Cells upon Lipopolysaccharide Activation.

Dendritic cells (DCs) are essential immune cells for defense against external pathogens. Upon activation, DCs undergo profound metabolic alterations whose precise nature remains poorly studied at a large scale and is thus far from being fully understood. The goal of the present work was to develop a reliable and accurate untargeted metabolomics workflow to get a deeper insight into the metabolism of DCs when exposed to an infectious agent (lipopolysaccharide, LPS, was used to mimic bacterial infection). As DCs transition rapidly from a non-adherent to an adherent state upon LPS exposure, one of the leading analytical challenges was to implement a single protocol suitable for getting comparable metabolomic snapshots of those two cellular states. Thus, a thoroughly optimized and robust sample preparation method consisting of a one-pot solvent-assisted method for the simultaneous cell lysis/metabolism quenching and metabolite extraction was first implemented to measure intracellular DC metabolites in an unbiased manner. We also placed special emphasis on metabolome coverage and annotation by using a combination of hydrophilic interaction liquid chromatography and reverse phase columns coupled to high-resolution mass spectrometry in conjunction with an in-house developed spectral database to identify metabolites at a high confidence level. Overall, we were able to characterize up to 171 unique meaningful metabolites in DCs. We then preliminarily compared the metabolic profiles of DCs derived from monocytes of 12 healthy donors upon in vitro LPS activation in a time-course experiment. Interestingly, the resulting data revealed differential and time-dependent activation of some particular metabolic pathways, the most impacted being nucleotides, nucleotide sugars, polyamines pathways, the TCA cycle, and to a lesser extent, the arginine pathway.

Untargeted metabolomic analysis to explore the impact of soil amendments in a non-conventional wastewater treatment

As non-conventional wastewater treatment, vegetation filters make the most of the natural attenuation processes that occur in soil to remove contaminants, while providing several environmental benefits. However, this practice may introduce contaminants of emerging concern (CECs) and their transformation products (TPs) into the environment. A potential improvement to the system was tested using column experiments containing soil (S) and soil amended with woodchips (SW) or biochar (SB) irrigated with synthetic wastewater that included 11 selected CECs. This study evaluated: i) known CECs attenuation and ii) unknown metabolites formation. Known CECs attenuation was assessed by total mass balance by considering both water and soil media. An untargeted metabolomic strategy was developed to assess the formation of unknown metabolites and to identify them in water samples. The results indicated that SB enhanced CECs attenuation and led to the formation of fewer metabolites. Sorption and biodegradation processes were favored by the bigger surface area of particles in SB column, especially for compounds with negative charges. Incorporating woodchips into soil shortened retention times in the column, which reduced attenuation phenomena and resulted in the formation of significantly more metabolites. Incomplete biodegradation reactions, fostered by shorter retention times in SW column could mainly explain these results.

The complete change in bile acids and steroids in systematic metabolomics applied to the intrahepatic cholestasis of pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific hepatobiliary disease, leading to an abnormal increase in total bile acid in the blood of pregnant women. To systematically explore the similarities and differences in metabolites and metabolic pathways among three types of biological samples from ICP women, a study of 18 ICP and 6 healthy (as a normal control) pregnant women was performed to investigate their clinical information and biochemical features. Based on validated LC-MS/MS methods 1-5 for hydrophilic and hydrophobic metabolites (molecular weight <2000 Dalton), an untargeted-metabolomic strategy was applied to 24 pregnant women to determine the metabolites from 22 serum, 15 placental and 22 urine samples. Then 1137 metabolites from serum, 876 metabolites from placental tissue and 311 metabolites from urine with a coefficient of variation <30% in the pooled quality control samples were found. Furthermore, orthogonal partial least squares-discriminate analysis (OPLS-DA), correlation analysis, chemical enrichment analysis and metabolic pathway analysis were carried out by a bioinformatics process. On the OPLS-DA model analysis, the metabolites in urine were better than those in serum or placental tissue to reflect the metabolic changes of ICP disease. Some metabolites were significantly changed in serum (n = 71), placental tissue (n = 46) and urine (n = 36), such as bile acids, triacylglycerols, lysoPCs, and steroids. Primary bile acid biosynthesis was the main metabolic pathway in ICP disease, and taurine and hypotaurine metabolism and sphingolipid metabolism were also found. More specifically, bile acids increased and steroids decreased in the serum, placental and urine samples. For complex metabolic diseases such as ICP disease, untargeted-metabolomic analysis of multiple biological samples could provide a systematic understanding of the changes in metabolic types and pathways.

UHPLC-QTOF-MS-based untargeted metabolomics and mineral element analysis insight into the geographical differences of Chrysanthemum morifolium Ramat cv. “Hangbaiju” from different origins

Chrysanthemum morifolium Ramat cv. “Hangbaiju” (HBJ), known as one of the “eight flavors of Zhejiang”, is commonly used as a classical tea material for both food and medicine over three thousand years in China. The quality of HBJ is closely related to its geographical origins. However, the mechanism underlying the geographical differences of HBJ remains to be elucidated. In this study, an untargeted metabolomic strategy based on UHPLC-QTOF-MS was established to discover the differential metabolites in HBJ samples from four different origins and explore the possible relationship with mineral elements in planting soils by chemometric analysis. Eight compounds were screened and identified as the key differential metabolites in HBJ samples from different origins. Among them, four important pharmacodynamic compounds including L-arginine, rutin, chlorogenic acid and apigenin-7-O-glucoside are the most abundant in HBJ samples from Tongxiang region, which suggests that HBJ planted in Tongxiang has higher medicinal values. Pearson correlation analysis revealed that the contents of soil mineral elements are positively correlated with those of chlorogenic acid, rutin, apigenin-7-O-glucoside in HBJ samples. Furthermore, an interrelationship model based on random forest algorithm was established to successfully predict the contents of differential metabolites in HBJ samples by soil mineral elements. All these results indicated that the contents of differential metabolites in HBJ samples seemed to be affected by soil mineral elements and therefore resulted in the geographical differences of HBJ.

Illuminating the dark metabolome of Pseudo-nitzschia-microbiome associations.

The exchange of metabolites mediates algal and bacterial interactions that maintain ecosystem function. Yet, while thousands of metabolites are produced, only a few molecules have been identified in these associations. Using the ubiquitous microalgae Pseudo-nitzschia sp., as a model, we employed an untargeted metabolomics strategy to assign structural characteristics to the metabolites that distinguished specific diatom-microbiome associations. We cultured five species of Pseudo-nitzschia, including two species that produced the toxin domoic acid, and examined their microbiomes and metabolomes. A total of 4826 molecular features were detected by tandem mass spectrometry. Only 229 of these could be annotated using available mass spectral libraries, but by applying new in silico annotation tools, characterization was expanded to 2710 features. The metabolomes of the Pseudo-nitzschia-microbiome associations were distinct and distinguished by structurally diverse nitrogen compounds, ranging from simple amines and amides to cyclic compounds such as imidazoles, pyrrolidines and lactams. By illuminating the dark metabolomes, this study expands our capacity to discover new chemical targets that facilitate microbial partnerships and uncovers the chemical diversity that underpins algae-bacteria interactions.

Differential metabolism between biofilm and suspended Pseudomonas aeruginosa cultures in bovine synovial fluid by 2D NMR-based metabolomics

Total joint arthroplasty is a common surgical procedure resulting in improved quality of life; however, a leading cause of surgery failure is infection. Periprosthetic joint infections often involve biofilms, making treatment challenging. The metabolic state of pathogens in the joint space and mechanism of their tolerance to antibiotics and host defenses are not well understood. Thus, there is a critical need for increased understanding of the physiological state of pathogens in the joint space for development of improved treatment strategies toward better patient outcomes. Here, we present a quantitative, untargeted NMR-based metabolomics strategy for Pseudomonas aeruginosa suspended culture and biofilm phenotypes grown in bovine synovial fluid as a model system. Significant differences in metabolic pathways were found between the suspended culture and biofilm phenotypes including creatine, glutathione, alanine, and choline metabolism and the tricarboxylic acid cycle. We also identified 21 unique metabolites with the presence of P. aeruginosa in synovial fluid and one uniquely present with the biofilm phenotype in synovial fluid. If translatable in vivo, these unique metabolite and pathway differences have the potential for further development to serve as targets for P. aeruginosa and biofilm control in synovial fluid.