Representative Metabolites Research Articles

Secondary Metabolites Produced by the Dominant Fungus Eurotium cristatum in Liupao Tea and Their Hypolipidemic Activities by Regulating Liver Lipid Metabolism and Remodeling Gut Microbiota.

Liupao tea is a postfermented dark tea with hypolipidemic activity. Research on the active substances in Liupao tea has primarily focused on those derived from the tea itself, overlooking the secondary metabolites produced by its predominant fungus, Euirotium cristatum. In this study, E. cristatum CPCC 401251, the predominant strain found in Liupao tea under investigation, was isolated and analyzed. A total of 19 representative metabolites, including prenylbenzaldehydes, diketopiperazines, and anthraquinones, were obtained from its culture. Subsequent analysis revealed the presence of multiple secondary metabolites of E. cristatum CPCC 401251 in Liupao tea. The 19 compounds significantly reduced the lipid content in free fatty acid (FFA)-stimulated hepatocyte AML-12 cells to varying degrees. Considering the content, chemical class, and biological activity of secondary metabolites from E. cristatum CPCC 401251, compounds 1, 7, and 13 were selected to detect their hypolipidemic activities and potential mechanisms in hyperlipidemia golden hamsters. Compound 1 exerted a hypolipidemic effect by activating the AMPK signaling pathway, decreasing Scd1, and improving intestinal flora. Compounds 7 and 13 played a role in the hypolipidemic activity by regulating the gene expression related to lipid synthesis and degradation, including upregulating the mRNA levels of Pparα, Hsl, and Atgl, and decreasing the mRNA level of Scd1. These findings help us understand the dominant fungus E. cristatum secondary metabolites presenting in Liupao tea and their potential hypolipidemic contributions. This work improves the understanding of the active substances in Liupao tea and highlights the health-promoting effects of microorganisms in the fermented tea.

Research note: Changes in chicken egg yolk metabolome during its spray drying and storage.

The differences in metabolites between fresh egg yolk (FEY), spray-dried egg yolk powder (SEY), and stored egg yolk powder (S-SEY) were quantitatively compared through metabolomic analysis. Total of 1004 metabolites were identified in the three groups of egg yolk samples. In pairwise group analysis, 242 differential metabolites were identified in FEY and SEY, 311 differential metabolites were identified in FEY and S-SEY, and 157 differential metabolites were identified in SEY and S-SEY. The analysis of differential metabolites with the highest abundance showed that amino acids, carbohydrates and lipids in FEY would undergo oxidation reactions after spray drying and storage and thus led to significant changes in the type and abundance of metabolites. The representative differential metabolites were then screened out for judging the freshness of egg yolk powder. Therefore, the results are highly important for evaluating the quality of egg yolk powder and provide important information for understanding the nutritional changes of egg yolk after spray drying and storage.

Ecophysiological Trade-Off Strategies of Three Gramineous Crops in Response to Root Extracts of Phytolacca americana.

The invasive Phytolacca americana L. poses a significant threat to local agroforestry ecosystems due to its allelopathic toxicity. However, the ecophysiological response mechanisms of crops to allelochemicals remain unclear. This study investigated the seedling growth, physiological, and biochemical responses of three gramineous crops to the root extracts of P. americana and identified potential allelochemicals of the invader. The germination and seedling growth of three crops were inhibited by extracts differently, with high-concentration extracts causing more severe inhibition on seedling roots in hydroponic (>57%) than soil culture experiments (>18%). This inhibition may be related to representative secondary metabolites such as fatty acyls, alkaloids, and phenols. Despite the significant inhibition of high-concentration extracts on seedling growth, the levels of soluble sugar, soluble protein, and antioxidant enzymes increased synergistically. Under allelopathic stress, three species enhanced antioxidant enzyme activities and metabolite contents at the cost of reducing their shoot, root biomass, and root/shoot ratio. This may be an ecophysiological growth-defense strategy to bolster their resistance to allelopathy. Interestingly, transgenic rice exhibited greater sensitivity to allelochemicals than wild-type rice, resulting in more pronounced growth inhibition and increased levels of most metabolites and antioxidant enzymes. This study highlights the adaptive strategies of three gramineous crops to the allelopathy of invasive P. americana.

Viral reprogramming by nervous necrosis virus alters key metabolites and its pathways in sevenband grouper (Hyporthodus septemfasciatus) gills

Nervous necrosis virus (NNV) which mainly infects sevenband grouper (Hyporthodus Septemfasciatus) is considered a potential threat to the grouper aquaculture industry. The gills being one of the portal of entry and an active site of replication of fish viruses emphasises its role as a key region to study the metabolomic changes caused by viral reprograming and hijacking of metabolic pathways associated with immunity of the host. In the present study, liquid chromatography mass spectrometry (LC-MS) was used to detect changes of endogenous compounds of the grouper after NNV infection. A total of 75 metabolites of ten different pathways were identified. The metabolites were mainly associated with fatty acids, lipids, amino acids and nucleotides. The virus reprogramming lead to the downregulation of majority of the metabolites in their pathways. Arachidonic acid (AA), tryptophan, kynurenine and methandriol were selected as representative metabolites and challenge studies with NNV confirmed the fact that, metabolites controlled the replication of virus in a dose dependent manner. Immune gene expression studies also confirmed the effect of metabolites by upregulated expression of interleukins, cytokines and TLRs which are part of cellular immune response. This study shows the viral reprogramming of NNV in grouper gill cells resulting in alterations in basic metabolic pathways associated with normal functioning of the organism.

Untargeted metabolomics and mendelian randomization analysis identify alpha-linolenic acid and linoleic acid as novel biomarkers of perioperative neurocognitive dysfunction

Perioperative neurocognitive dysfunction (PND) occurs in elderly individuals undergoing anesthesia and surgery. To explore the potential molecular mechanisms, we performed right-sided cervical exploratory surgery under sevoflurane anesthesia in 18-month-old male Sprague-Dawley rats. Anxiety-depression-like behaviors and learning memory abilities were assessed using the Open Field Test (OFT) and Novel Object Recognition (NOR). Additionally, the hippocampus was collected one day after surgery for inflammatory factor detection, TUNEL staining, and metabolomics analysis. Mendelian randomization (MR) analyses were subsequently conducted to validate the causal relationships by using a series of GWAS datasets related to representative differential metabolites as exposures and cognitive impairment as endpoints. The results indicated that rats exposed to anesthesia and surgery exhibited poorer cognitive performance, significant elevations in hippocampal inflammatory factors such as IL-1β and TNF-α, and extensive neuronal apoptosis. LC-MS/MS-based untargeted metabolomics identified 19 up-regulated and 32 down-regulated metabolites in the test group, with 6 differential metabolites involved in metabolic pathways enriched according to the KEGG database. ROC analysis revealed a correlation between α-linolenic acid (ALA) and linoleic acid (LA) and the development of PND. Further MR analysis confirmed that ALA was significantly associated with cognitive performance and the risk of depression, while LA was significantly associated with the risk of memory loss. Taken together, our results identified ALA and LA as potentially powerful biomarkers for PND.

Possible immune mechanisms of gut microbiota and its metabolites in the occurrence and development of immune thrombocytopenia.

Immune thrombocytopenia (ITP) is an autoimmune disease characterized by increased platelet destruction and impaired production, leading to an elevated bleeding tendency. Recent studies have demonstrated an important link between the gut microbiota and the onset and progression of several immune diseases in humans, emphasizing that gut microbiota-derived metabolites play a non-negligible role in autoimmune diseases. The gut microbiota and its metabolites, such as short-chain fatty acids, oxidized trimethylamine, tryptophan metabolites, secondary bile acids and lipopolysaccharides, can alter intestinal barrier permeability by modulating immune cell differentiation and cytokine secretion, which in turn affects the systemic immune function of the host. It is therefore reasonable to hypothesize that ecological dysregulation of the gut microbiota may be an entirely new factor in the triggering of ITP. This article reviews the potential immune-related mechanisms of the gut microbiota and representative metabolites in ITP, as well as the important influence of leaky gut on the development of ITP, with a view to enriching the theoretical system of ITP-related gut microecology and providing new ideas for the study of ITP.

A Divalent Metal Cation-Metabolite Interaction Model Reveals Cation Buffering and Speciation.

I present the perspective that the divalent metalome and the metabolome can be modeled as a network of chelating interactions instead of separate entities. I review progress in understanding the complex cellular environment, in particular recent contributions to modeling metabolite-Mg2+ interactions. I then demonstrate a simple extension of these strategies based approximately on intracellular Escherichia coli concentrations. This model is composed of four divalent metal cations with a range of cellular concentrations and physical properties (Mg2+, Ca2+, Mn2+, and Zn2+), eight representative metabolites, and interaction constants. I applied this model to predict the speciation of divalent metal cations between free and metabolite-chelated species. This approach reveals potentially beneficial properties, including maintenance of free divalent metal cations at biologically relevant concentrations, buffering of free divalent metal cations, and enrichment of functional metabolite-chelated species. While currently limited by available interaction coefficients, this modeling strategy can be generalized to more complex systems. In summary, biochemists should consider the potential of cellular metabolites to form chelating interactions with divalent metal cations.

Untargeted Metabolomics of the Sea Cucumber, Bohadschia marmorata (Jaeger, 1833)

The marine invertebrates such as sea cucumber are regarded as sources of diverse bioactive compounds with promising health and nutritional benefits. Many commercially traded sea cucumber species have been explored, yet none of the local low-value types in the province of La Union, Philippines. This study aimed to establish a baseline metabolomic profile of Bohadschia marmorata (Jaeger, 1833). The identification of compounds in its body wall utilized an Orbitrap MS-based approach. Metabolites were annotated using Compound Discover 3.2, and similarity searches were performed from online databases. Eleven putatively identified compounds, which included saponin (3beta,5xi,9xi)-28-Hydroxy-28-oxoolean-12-en-3-yl-beta-Dgalac topyranosyl-(1-3)-[beta-D-glucopyranosyl-(1-2)]-beta-D-glucopyranosiduronic acid, fatty acids and their byproducts such as eicosanoid (11,12-Epoxy-(5Z,8Z,11Z)-icosatrienoic acid) and prostanoids (13,14-dihydro Prostaglandin F1?; 13,14-Dihydro-15-keto Prostaglandin A2), dicarboxylic acids (tetradecanedioic acid; dodecanedioic acid; suberic acid; NP-001596), and amino acid (4-Oxoproline). The metabolic profile of the B. marmorata body wall showed a diverse group of representative metabolites common among holothurians associated with innate defense mechanisms, structural composition, and products of metabolic pathways. There is, however, a need to further isolate these compounds using other methods and test their biological activities.

Metabolomic insights in advanced cardiomyopathy of chronic chagasic and idiopathic patients that underwent heart transplant

Heart failure (HF) studies typically focus on ischemic and idiopathic heart diseases. Chronic chagasic cardiomyopathy (CCC) is a progressive degenerative inflammatory condition highly prevalent in Latin America that leads to a disturbance of cardiac conduction system. Despite its clinical and epidemiological importance, CCC molecular pathogenesis is poorly understood. Here we characterize and discriminate the plasma metabolomic profile of 15 patients with advanced HF referred for heart transplantation – 8 patients with CCC and 7 with idiopathic dilated cardiomyopathy (IDC) – using gas chromatography/quadrupole time-of-flight mass spectrometry. Compared to the 12 heart donor individuals, also included to represent the control (CTRL) scenario, patients with advanced HF exhibited a metabolic imbalance with 21 discriminating metabolites, mostly indicative of accumulation of fatty acids, amino acids and important components of the tricarboxylic acid (TCA) cycle. CCC vs. IDC analyses revealed a metabolic disparity between conditions, with 12 CCC distinctive metabolites vs. 11 IDC representative metabolites. Disturbances were mainly related to amino acid metabolism profile. Although mitochondrial dysfunction and loss of metabolic flexibility may be a central mechanistic event in advanced HF, metabolic imbalance differs between CCC and IDC populations, possibly explaining the dissimilar clinical course of Chagas’ patients.

A GC×GC-MS method based on solid-state modulator for non-targeted metabolomics: Comparison with traditional GC-MS method

The formidable challenge posed by the presence of extremely high amounts of compounds and large differences in concentrations in plasma significantly complicates non-targeted metabolomics analyses. In this study, a comprehensive two-dimensional gas chromatography-quadrupole mass spectrometry (GC×GC-qMS) method with a solid-state modulator (SSM) for non-targeted metabolomics in beagle plasma was first established based on a GC-MS method, and the qualitative and quantitative performance of the two platforms were compared. Identification of detected compounds was accomplished utilizing NIST database match scores, retention indices (RIs) and standards. Semi-quantification involved the calculation of peak area ratios to internal standards. Metabolite identification sheets were generated for plasma samples on both analytical platforms, featuring 22 representative metabolites chosen for validating qualitative accuracy, and for conducting comparisons of linearity, accuracy, precision, and sensitivity. The outcomes revealed a threefold increase in the number of identifiable metabolites on the GC×GC-MS platform, with lower limits of quantitation (LLOQs) reduced to 0.5–0.05 times those achieved on the GC-MS platform. Accuracy in quantification for both GC×GC-MS and GC-MS fell within the range of 85–115%, and the vast majority of intra- and inter-day precisions were within the range of 20%. These findings underscore that relative to the conventional GC-MS method, the GC×GC-MS method developed in this study, combined with SSM, exhibits enhanced qualitative capabilities, heightened sensitivity, and comparable accuracy and precision, rendering it more suitable for non-targeted metabolomics analyses.

Targeting gut microbiota to explore the Shanghuo effect of Qi-invigorating herbs

ScopeQi-invigorating herbs have been shown to have a tonic-like effect and to improve the body's immune system function. This study explores how high-dose Ginseng and Astragalus, two representative Qi-invigorating herbs, affect the immunes system of SD rats by regulating their gut microbiota. Method and resultsWe divided thirteen SD rats randomly into two groups, namely the control group and the Qi-invigorating group (Shenqi). The Shenqi rats received a mixture of Ginseng and Astragalus (Ginseng: 5 mg/kg, Astragalus: 15 mg/kg) for 28 days, once a day. After the treatment, we recorded the blood pressure and the heart rate of the rats. Metabolites of each group were detected via LC-MS, and we conducted gut microbiota profiling via 16 s rRNA sequencing. The blood pressure of the rats in the Shenqi group significantly increased compared with the control group (p<0.01), and the RGB value of the lingual picture also increased. At the genus level, we observed a higher relative abundance of Parabacteroides, Oscillibacter, and Romboutsia in the Shenqi group, and a higher relative abundance of Blautia, Subdoligranulum, and Anaerostipes in the control group. Furthermore, we also observed that the composition of the metabonomics significantly changed after the treatment of Qi-invigorating herbs. In total there were 2,862 differential metabolites (p<0.05), and we singled out 3alpha-Hydroxy-5beta-chola-7,9(11)-dien-24-oic Acid, (+)-22(29)-Hopen-6alpha-21beta-diol, 4,4′-Diapophytofluene, Cholic acid, and Wogonin as five of the most representative metabolites. ConclusionsOverall, we find that the composition and function of gut microbiota of rats in Shenqi group differed from that in the control group. Meanwhile, it can be summarized by our results that Shanghuo is related to the changes of intestinal flora and related metabolic pathways. What's more, modulating the gut microbiota and some metabolic pathways may be the mechanism by which Yiqi TCM treatment exerts its effects. Significance and impact of studyThis study confirms that Shanghuo is related to disorders of energy metabolism and changes in composition and function of gut microbiota. Overdose of Qi-invigorating herbs may change the composition and function of gut microbiota and metabolism and then generate adverse effect. However, modulating the composition and function of gut microbiota and even metabolism may also be the way Qi-invigorating herbs taking effects, which needs further investigation.

Metabolomic analysis revealed the edible and extended-application potential of specific Polygonum multiflorum tissues

The diverse applications of various tissues of Polygonum Multiflorum (PM) encompass the use of its leaf and bud as tea and vegetables, as well as the utilization of its expanded root tubers and caulis as medicinal substances. However, previous studies in the field of metabolomics have primarily focused on the medicinal properties of PM. In order to investigate the potential for broader applications of other tissues within PM, a metabolomic analysis was conducted for the first time using UPLC-Q-TOF-MS/MS on 15 fresh PM tissues. A total of 231 compounds, including newly discovered compounds such as torosachrysone and dihydro-trihydroxystilbene acid derivatives, were identified within PM. Through clustering analysis, the PM tissues were categorized into edible and medicinal parts, with edible tissues exhibiting higher levels of phenolic acids, organic acids, and flavonoids, while the accumulation of quinones, dianthrones, stilbenes, and xanthones was observed in medicinal tissues. Comparative analysis demonstrated the potential application of discarded tissues, such as unexpanded root tuber (an industrial alternative to expanded root tuber) and young caulis (with edible potential). Moreover, the quantification of representative metabolites indicated that flowers and buds contained significant amounts of flavonoids or phenolic acids, suggesting their potential as functional food. Additionally, the edible portion of PM exhibited a high content of quercitrin, ranging from 0.59 to 10.37 mg/g. These findings serve as a valuable point of reference for the expanded utilization of PM tissues, thereby mitigating resource waste in this plant.

Predictive metabolomic signatures for safety assessment of three plastic nanoparticles using intestinal organoids

Nanoplastic particles are pervasive environmental contaminants with potential health risks, while mouse intestinal organoids provide accurate in vitro models for studying these interactions. Metabolomics, especially through LC-MS, enables detailed cellular response studies, and there's a novel interest in comparing metabolic changes across nanoparticle species using gut organoids. This study used a mouse intestinal organoid combined with cell model to explore the differences in metabolites and toxicity mechanisms induced by exposure to three nanoplastics (PS, PTFE, and PMMA). The results showed that PS, PTFE, and PMMA exposure reduced mitochondrial membrane potential, intracellular ROS accumulation and oxidative stress, and inhibited the AKT/mTOR signaling pathway. Non-targeted metabolomics results confirmed that three types of nanoplastic particles regulate cellular status by regulating fatty acid metabolism, nucleotide metabolism, necroptosis and autophagy pathways. More importantly, these representative metabolites were further validated in model groups after mouse intestinal organoids and HCT116 cells were exposed to the respective NPs, indicating that organoid metabolomics results can be used to effectively predict toxicity. Untargeted metabolomics is sensitive enough to detect subtle metabolomic changes when functional cellular analysis shows no significant differences. Overall, our study reveals the underlying metabolic mechanism of NPs-induced intestinal organoid toxicity and provides new insights into the possible adverse consequences of NPs.

An Automated Centrifugal Microfluidic Platform for Efficient Multistep Blood Sample Preparation and Clean-Up towards Small Ion-Molecule Analysis.

Sample preparation for mass spectroscopy typically involves several liquid and solid phase clean-ups, extractions, and other unit operations, which are labour-intensive and error-prone. We demonstrate a centrifugal microfluidic platform that automates the whole blood sample's preparation and clean-up by combining traditional liquid-phase and multiple solid-phase extractions for applications in mass spectroscopy (MS)-based small molecule detection. Liquid phase extraction was performed using methanol to precipitate proteins in plasma separated from a blood sample under centrifugal force. The preloaded solid phase composed of C18 beads then removed lipids with a combination of silica particles, which further cleaned up any remaining proteins. We further integrated the application of this sample prep disc with matrix-assisted laser desorption/ionization (MALDI) MS by using glancing angle deposition films, which further cleaned up the processed sample by segregating the electrolyte background from the sample salts. Additionally, hydrophilic interaction liquid chromatography (HILIC) MS was employed for detecting targeted free amino acids. Therefore, several representative ionic metabolites, including several amino acids and organic acids from blood samples, were analysed by both MALDI-MS and HILIC-MS to demonstrate the performance of this sample preparation disc. The fully automated blood sample preparation procedure only took 35 mins, with a throughput of three parallel units.

Comparative secretome metabolic dysregulation by six engineered dietary nanoparticles (EDNs) on the simulated gut microbiota

The potential use of engineered dietary nanoparticles (EDNs) in diet has been increasing and poses a risk of exposure. The effect of EDNs on gut bacterial metabolism remains largely unknown. In this study, liquid chromatography–mass spectrometry (LC-MS) based metabolomics was used to reveal significantly altered metabolites and metabolic pathways in the secretome of simulated gut microbiome exposed to six different types of EDNs (Chitosan, cellulose nanocrystals (CNC), cellulose nanofibrils (CNF) and polylactic-co-glycolic acid (PLGA); two inorganic EDNs including TiO2 and SiO2) at two dietary doses. We demonstrated that all six EDNs can alter the composition in the secretome with distinct patterns. Chitosan, followed by PLGA and SiO2, has shown the highest potency in inducing the secretome change with major pathways in tryptophan and indole metabolism, bile acid metabolism, tyrosine and phenol metabolism. Metabolomic alterations with clear dose response were observed in most EDNs. Overall, phenylalanine has been shown as the most sensitive metabolites, followed by bile acids such as chenodeoxycholic acid and cholic acid. Those metabolites might be served as the representative metabolites for the EDNs-gut bacteria interaction. Collectively, our studies have demonstrated the sensitivity and feasibility of using metabolomic signatures to understand and predict EDNs-gut microbiome interaction.

Research Note: Comparison of water-soluble metabolites in egg yolk, yolk granules, and yolk plasma based on quantitative metabolomic analysis

Egg yolk (EY) can be divided into yolk granules (EYG) and yolk plasma (EYP) after water dilution and centrifugation. EYP had a significantly higher lipid content and is predominantly in the form of low-density lipoprotein (LDL) while EYG has a higher protein content. In order to explore the nutritional differences between EY, EYP and EYG, quantitative metabolomic analysis was carried out by ultra-high-performance liquid chromatography/tandem mass spectrometry (UHPLC-MS/MS). The results showed that a total of 588 small molecule metabolites were identified, mainly glycerol phospholipids (51.59-66.46%), coenzymes, and vitamins (1.21-1.84%). A quantitative comparison showed that amino acids and their metabolites were the most representative metabolites of differential abundance in the contrast of EY and EYG, EYP and EYG. Carbohydrates and their metabolites were the representative metabolites of differential abundance between EY and EYP. The study provided important information for understanding the metabolic composition of EY, EYG, and EYP, as well as providing a reference for utilizing the whole egg.

Chemical profiles, differentiation, and quality evaluation of Radix et Rhizoma Thalictri Foliolosi based on LC-MS

Radix et Rhizoma Thalictri Foliolosi (RRTF) belongs to one of the alkaloid-rich traditional Chinese medicines in Ranunculaceae, which possesses anti-inflammatory, anti-tumor, and several other pharmacological activities. However, due to lack of research on chemical composition, serious confusion in the origin, and ambiguity in pharmacological mechanisms, it is quite urgent to establish quality control standards based on modern research and to increase the widespread usage. Aiming to clarify the differential compounds among three species of RRTF (TFD, TFB, and TCW), targeted and untargeted acquisition strategies based on high resolution mass spectrometry were established. Plant metabolomics analysis and multivariate statistical analysis were accomplished to screen out differential markers which were answerable for categorizing different species of RRTF. A network pharmacology analysis was further performed to predict the bioactive constituents and pharmacological mechanisms. Moreover, multi-components quantitative analysis under multiple reaction monitoring mode and multiple logistic regression analysis were conducted to estimate the rationality of the quality markers (Q-markers). Ultimately, the targeted alkaloid detection list was built as premise relying on alkaloid cleavage pathway, and a total 87 compounds were identified. The 25 representative differential metabolites were screened out successfully and divided into three categories to differentiate TFD, TFB, and TCW. 14 active components and 25 presumptive targets of RRTF were found to play a central role according to network pharmacology analysis. The abundance of screened 12 Q-marker showed significant differences in the three varieties. In conclusion, the study systematically investigated the material basis of RRTF, distinguished and evaluated the quality of RRTF effectively, and predicted its pharmacodynamic material basis.

Cryo-EM structures of human organic anion transporting polypeptide OATP1B1

Members of the solute carrier organic anion transporting polypeptide (OATPs) family function as transporters for a large variety of amphipathic organic anions including endogenous metabolites and clinical drugs, such as bile salts, steroids, thyroid hormones, statins, antibiotics, antivirals, and anticancer drugs. OATP1B1 plays a vital role in transporting such substances into the liver for hepatic clearance. FDA and EMA recommend conducting in vitro testing of drug–drug interactions (DDIs) involving OATP1B1. However, the structure and working mechanism of OATPs still remains elusive. In this study, we determined cryo-EM structures of human OATP1B1 bound with representative endogenous metabolites (bilirubin and estrone-3-sulfate), a clinical drug (simeprevir), and a fluorescent indicator (2′,7′-dichlorofluorescein), in both outward- and inward-open states. These structures reveal major and minor substrate binding pockets and conformational changes during transport. In combination with mutagenesis studies and molecular dynamics simulations, our work comprehensively elucidates the transport mechanism of OATP1B1 and provides the structural basis for DDI predictions involving OATP1B1, which will greatly promote our understanding of OATPs.

Assessment of the stability of antimicrobials and resistance genes during short- and long-term storage condition: accounting for uncertainties in bioanalytical workflows

Unravelling complexities in antimicrobial agent–microbe interactions in the context of antimicrobial resistance (AMR) requires robust analytical workflows accounting for all uncertainties. Temporal storage of wastewater samples under refrigerated or frozen conditions prior to chemical and biological analysis is widely used to facilitate laboratory routine but may affect stability of analytes over time. Yet, little knowledge exists regarding stability of biological and chemical determinants in environmental samples, which hampers validity of research outputs. This study examines, for the first time, the stability of 32 antimicrobials (AAs) including commonly used classes of antibiotics and their representative metabolites and variation of 5 antibiotic resistance genes (ARGs) (ermB, sul1, tetW, blaCTX-M, qnrS), as well as intI1 and 16S rRNA genes in a reference wastewater sample stored under freezing condition for up to 1 year. Ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) and quantitative PCR (qPCR) techniques were adopted to measure concentration of AAs and ARGs, respectively. Results suggested that parent compounds are less affected by freezing storage compared to the metabolites. β-Lactams, clindamycin, and N-desmethyl clindamycin are the most-affected compounds which were poorly recovered (34–67%) from the starting concentration. By contrast, sulfonamides, macrolides, quinolones, and azoles are generally stable under freezing condition. No consistent differences were observed in gene copies between fresh and frozen samples, and ermB and tetW showed the highest variabilities at 30% under freezing condition. Overall, this study adds to the current knowledge on environmental AMR monitoring and emphasises the need for standardised protocols for AMR monitoring in the wastewater samples.Graphical

Optimization of the first extraction protocol for metabolomic studies of Brucella abortus.

Brucellosis is a zoonosis prevalent worldwide and very recurrent in less developed or developing regions. This zoonosis affects livestock, generating high financial losses to producers, in addition to transmitting diseases to humans through meat consumption or handling contaminated products and animals. In this study, five extraction methods for Brucella abortus intracellular metabolites, using different solvent compositions and cell membrane disruption procedures, were evaluated. Derivatized extracts were analyzed by GC-HRMS. Raw data were processed in XCMS Online and the results were evaluated through multivariate statistical analysis using the MetaboAnalyst platform. The identification of the extracted metabolites was performed by the Unknowns software using the NIST 17.L library. The extraction performance of each method was evaluated for thirteen representative metabolites, comprising four different chemical classes. Most of these compounds are reported in the cell membrane composition of Gram-negative bacteria. The method based on extraction with methanol/chloroform/water presented the best performance in the evaluation of the extracted compounds and in the statistical results. Therefore, this method was selected for extracting intracellular metabolites from cultures of Brucella abortus for untargeted metabolomics analysis.