PubMed

ACS Omega. 2023 Jun 1;8(23):20755-20766. doi: 10.1021/acsomega.3c01370. eCollection 2023 Jun 13.ABSTRACTBiofluid metabolomics is a very appealing tool to increase the knowledge associated with pathophysiological mechanisms leading to better and new therapies and biomarkers for disease diagnosis and prognosis. However, due to the complex process of metabolome analysis, including the metabolome isolation method and the platform used to analyze it, there are diverse factors that affect metabolomics output. In the present work, the impact of two protocols to extract the serum metabolome, one using methanol and another using a mixture of methanol, acetonitrile, and water, was evaluated. The metabolome was analyzed by ultraperformance liquid chromatography associated with tandem mass spectrometry (UPLC-MS/MS), based on reverse-phase and hydrophobic chromatographic separations, and Fourier transform infrared (FTIR) spectroscopy. The two extraction protocols of the metabolome were compared over the analytical platforms (UPLC-MS/MS and FTIR spectroscopy) concerning the number of features, the type of features, common features, and the reproducibility of extraction replicas and analytical replicas. The ability of the extraction protocols to predict the survivability of critically ill patients hospitalized at an intensive care unit was also evaluated. The FTIR spectroscopy platform was compared to the UPLC-MS/MS platform and, despite not identifying metabolites and consequently not contributing as much as UPLC-MS/MS in terms of information concerning metabolic information, it enabled the comparison of the two extraction protocols as well as the development of very good predictive models of patient's survivability, such as the UPLC-MS/MS platform. Furthermore, FTIR spectroscopy is based on much simpler procedures and is rapid, economic, and applicable in the high-throughput mode, i.e., enabling the simultaneous analysis of hundreds of samples in the microliter range in a couple of hours. Therefore, FTIR spectroscopy represents a very interesting complementary technique not only to optimize processes as the metabolome isolation but also for obtaining biomarkers such as those for disease prognosis.PMID:37323376 | PMC:PMC10237515 | DOI:10.1021/acsomega.3c01370

Ther Adv Med Oncol. 2023 Jun 5;15:17588359231177021. doi: 10.1177/17588359231177021. eCollection 2023.ABSTRACTBACKGROUND: The exosome-focused translational research for afatinib (EXTRA) study is the first trial to identify novel predictive biomarkers for longer treatment efficacy of afatinib in patients with epidermal growth factor receptor (EGFR) mutation-positive nonsmall cell lung cancer (NSCLC) via a comprehensive association study using genomic, proteomic, epigenomic, and metabolomic analyses.OBJECTIVES: We report details of the clinical portion prior to omics analyses.DESIGN: A prospective, single-arm, observational study was conducted using afatinib 40 mg/day as an initial dose in untreated patients with EGFR mutation-positive NSCLC. Dose reduction to 20 mg every other day was allowed.METHODS: Progression-free survival (PFS), overall survival (OS), and adverse events (AEs) were evaluated.RESULTS: A total of 103 patients (median age 70 years, range 42-88 years) were enrolled from 21 institutions in Japan between February 2017 and March 2018. After a median follow-up of 35.0 months, 21% remained on afatinib treatment, whereas 9% had discontinued treatment because of AEs. The median PFS was 18.4 months, with a 3-year PFS rate of 23.3%. The median afatinib treatment duration in patients with final doses of 40 (n = 27), 30 (n = 23), and 20 mg/day (n = 35), and 20 mg every other day (n = 18) were 13.4, 15.4, 18.8, and 18.3 months, respectively. The median OS was not reached, with a 3-year OS rate of 58.5%. The median OS in patients who did (n = 25) and did not (n = 78) receive osimertinib during the entire course of treatment were 42.4 months and not reached, respectively (p = 0.654).CONCLUSIONS: As the largest prospective study in Japan, this study confirmed favorable OS following first-line afatinib in patients with EGFR mutation-positive NSCLC in a real-world setting. Further analysis of the EXTRA study is expected to identify novel predictive biomarkers for afatinib.TRIAL REGISTRATION: UMIN-CTR identifier (UMIN000024935, https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_his_list.cgi?recptno=R000028688.PMID:37323187 | PMC:PMC10262622 | DOI:10.1177/17588359231177021

Pharm Biol. 2023 Dec;61(1):927-937. doi: 10.1080/13880209.2023.2222755.ABSTRACTCONTEXT: Qingyi granules can be used to effectively treat patients with severe acute pancreatitis (SAP).OBJECTIVE: To elucidate the role of gut microbiota-mediated metabolism in the therapeutic effects of Qingyi granules.MATERIALS AND METHODS: Sprague-Dawley rats were grouped into the sham operation, SAP model, Qingyi granule intervention (Q, 1.8 g/kg) and emodin intervention (E, 50 mg/kg) groups and observed for 24 h. H&E staining and ELISA were used for histopathological analysis and serum enzyme and cytokine assays. 16S rDNA sequencing and UHPLC-HRMS were used for gut microbiota analysis and untargeted metabolomics.RESULTS: In SAP rats, Qingyi granules decreased the pancreatic pathological score (Q, 7.4 ± 1.14; SAP, 11.6 ± 1.14, p < 0.01); serum amylase (Q, 121.2 ± 6.7; SAP, 144.3 ± 8.86, p < 0.05), lipase (Q, 566 ± 20.34; SAP, 656.7 ± 29.32, p < 0.01), and diamineoxidase (Q, 492.8 ± 26.08; SAP, 566.1 ± 26.83, p < 0.05) activities; and IL-1β (Q, 29.48 ± 0.88; SAP, 36.17 ± 1.88, p < 0.01), IL-6 (Q, 112.2 ± 3.57; SAP, 128.9 ± 9.09, p < 0.05) and TNF-α (Q, 215.3 ± 8.67; SAP, 266.4 ± 28.03, p < 0.05) levels. SAP induced Helicobacter and Lactobacillus overgrowth and suppressed Romboutsia and Allobaculum growth and caused aberrations in bacterial metabolites, which were partly reversed by Qingyi granules.DISCUSSION AND CONCLUSIONS: Qingyi granules can modulate the gut microbiota and metabolic abnormalities to ameliorate SAP. Multi-omics approaches allow systematic study of the pharmacological mechanisms of compound prescriptions for critical illnesses.PMID:37323024 | DOI:10.1080/13880209.2023.2222755

Carbohydr Polym. 2023 Sep 15;316:121039. doi: 10.1016/j.carbpol.2023.121039. Epub 2023 May 25.ABSTRACTDietary fibers are known to modulate microbiome composition, but it is unclear to what extent minor fiber structural differences impact community assembly, microbial division of labor, and organismal metabolic responses. To test the hypothesis that fine linkage variations afford different ecological niches for distinct communities and metabolism, we employed a 7-day in vitro sequential batch fecal fermentation with four fecal inocula and measured responses using an integrated multi-omics approach. Two sorghum arabinoxylans (SAXs) were fermented, with one (RSAX) having slightly more complex branch linkages than the other (WSAX). Although there were minor glycoysl linkage differences, consortia on RSAX retained much higher species diversity (42 members) than on WSAX (18-23 members) with distinct species-level genomes and metabolic outcomes (e.g., higher short chain fatty acid production from RSAX and more lactic acid produced from WSAX). The major SAX-selected members were from genera of Bacteroides and Bifidobacterium and family Lachnospiraceae. Carbohydrate active enzyme (CAZyme) genes in metagenomes revealed broad AX-related hydrolytic potentials among key members; however, CAZyme genes enriched in different consortia displayed various catabolic domain fusions with diverse accessory motifs that differ among the two SAX types. These results suggest that fine polysaccharide structure exerts deterministic selection effect for distinct fermenting consortia.PMID:37321733 | DOI:10.1016/j.carbpol.2023.121039

Sci Total Environ. 2023 Jun 13:164857. doi: 10.1016/j.scitotenv.2023.164857. Online ahead of print.ABSTRACTSulfonamide antibiotics (SAs) are ubiquitous in surface water and soil environments, raising considerable concerns about their risk and removal. However, the impacts of various bromide ion (Br-) concentrations on the phytotoxicity, uptake and fate of SAs in plant growth and physiological metabolism of plants have not been well understood. Our research demonstrated that low concentrations of Br- (0.1, 0.5 mM) promoted the uptake and degradation of sulfadiazine (SDZ) in wheat and attenuated the phytotoxicity of SDZ. Additionally, we proposed a degradation pathway and identified the brominated product of SDZ (SDZBr), which attenuated the dihydrofolate synthesis inhibition by SDZ. The primary mechanism was that Br- reduced the level of reactive oxygen radicals (ROS) and alleviated oxidative damage. The production of SDZBr and the high consumption of H2O2 suggest the potential generation of reactive bromine species, contributing to the degradation of the electron-rich SDZ and thus reducing its toxicity. Moreover, metabolome analysis of wheat roots indicated that low concentrations of Br- stimulated the production of indoleacetic acid under SDZ stress, promoting growth and enhancing the uptake and degradation of SDZ. Conversely, high Br- (1 mM) concentration produced a deleterious effect. These findings provide valuable insights into the mechanisms of antibiotic removal, suggesting a potentially novel approach to plant-based antibiotic remediation.PMID:37321499 | DOI:10.1016/j.scitotenv.2023.164857

J Ethnopharmacol. 2023 Jun 13:116782. doi: 10.1016/j.jep.2023.116782. Online ahead of print.ABSTRACTETHNOPHARMACOLOGICAL RELEVANCE: Tripterygium wilfordii polyglycosides (TWP), extracted from the traditional Chinese herb Tripterygium wilfordii, has been widely used in the treatment of rheumatoid arthritis (RA). However, the toxicity of TWP to a variety of organs such as liver, kidney and testis greatly limits its clinical application. Salvia miltiorrhiza Bunge is often used in the treatment of RA due to its blood circulation promoting, stasis resolving, and anti-inflammatory effects. Salvia miltiorrhiza Bunge has also been reported to possess multiple organ protective effects.AIM OF THE STUDY: To investigate the influences of two main components of Salviorrhiza miltiorrhiza Bunge, hydrophilic salvianolic acids (SA) and lipophilic tanshinones (Tan), on the efficacy and toxicity of TWP in treating RA and to explore the underlying mechanisms.MATERIALS AND METHODS: SA and Tan were extracted from Salvia miltiorrhiza Bunge and the extracts were quantitated by HPLC and identified by UPLC-Q/TOF-MS. Then, a collagen-induced arthritis (CIA) rat model was established using bovine type II collagen (CII) and incomplete Freund's adjuvant (IFA). CIA rats were treated with TWP and/or SA/Tan. After 21 days of continuous treatment, arthritis symptoms and organs toxicity were evaluated. Meanwhile, serum metabolomics were investigated by the UPLC-Q/TOF-MS to understand the underlying mechanism.RESULTS: SA and Tan extracts could significantly alleviate arthritis symptoms in CIA rats and decrease the serum levels of inflammatory factors TNF-α, IL-1β and IL-6 when combined with TWP. Meanwhile, both extracts alleviated injury of liver, kidney and testis caused by TWP, and the hydrophilic extract SA was superior. Moreover, a total of 38 endogenous differential metabolites were identified between the CIA model group and the TWP group, among which 33 metabolites were significantly recovered after the combination of SA or Tan. Metabolic pathway analysis showed that SA and Tan can affect metabolic pathways including linoleic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism and steroid biosynthesis metabolism pathway.CONCLUSIONS: Our findings indicated for the first time that two Salviorrhiza miltiorrhiza Bunge extracts could improve the efficacy and reduce the toxicity of TWP in the treatment of RA by adjusting metabolic pathways, and the hydrophilic extract SA was superior.PMID:37321427 | DOI:10.1016/j.jep.2023.116782

Toxicol Appl Pharmacol. 2023 Jun 13:116597. doi: 10.1016/j.taap.2023.116597. Online ahead of print.ABSTRACTTacrolimus (TAC)-based treatment is associated with nephrotoxicity and hepatotoxicity; however, the underlying molecular mechanisms responsible for this toxicity have not been fully explored. This study elucidated the molecular processes underlying the toxic effects of TAC using an integrative omics approach. Rats were sacrificed after 4 weeks of daily oral TAC administration at a dose of 5 mg/kg. The liver and kidney underwent genome-wide gene expression profiling and untargeted metabolomics assays. Molecular alterations were identified using individual data profiling modalities and further characterized by pathway-level transcriptomics-metabolomics integration analysis. Metabolic disturbances were mainly related to an imbalance in oxidant-antioxidant status, as well as in lipid and amino acid metabolism in the liver and kidney. Gene expression profiles also indicated profound molecular alterations, including in genes associated with a dysregulated immune response, proinflammatory signals, and programmed cell death in the liver and kidney. Joint-pathway analysis indicated that the toxicity of TAC was associated with DNA synthesis disruption, oxidative stress, and cell membrane permeabilization, as well as lipid and glucose metabolism. In conclusion, our pathway-level integration of transcriptome and metabolome and conventional analyses of individual omics profiles, provided a more comprehensive picture of the molecular changes resulting from TAC toxicity. This study also serves as a valuable resource for subsequent investigations aiming to understand the mechanism underlying the molecular toxicology of TAC.PMID:37321324 | DOI:10.1016/j.taap.2023.116597

J Proteomics. 2023 Jun 13:104951. doi: 10.1016/j.jprot.2023.104951. Online ahead of print.ABSTRACTSpontaneous milk lipolysis refers to the breakdown of triacylglycerols in milk. Lipolysis impacts the organoleptic value of milk by causing off-flavours and reduces the technological properties of milk. Lipolysis is caused by lipoprotein lipase (LPL), a tightly regulated enzyme in milk. Our objective was to identify robust biomarkers of lipolysis and putative regulators of LPL enzyme in bovine milk. To achieve this goal, we used feed restriction as a lever to generate highly contrasted samples with regard to milk lipolysis. We combined statistical methods on proteomics data, milk lipolysis and LPL activity values. Following this strategy, we identified CD5L and GP2 as robust biomarkers of high lipolysis in cow milk. We also identified HID1, SURF4 and CUL9 as putative inhibitors of the lipolytic process in the milk. We thus proposed 5 putative biomarkers to be considered in future tools to manage milk lipolysis. SIGNIFICANCE: This manuscript is notable in three aspects. First, this is the first evaluation of the milk proteome relative to milk lipolysis or LPL activity. Second, the relationship between the abundance of proteins and milk traits was evaluated by a combination of univariate and multivariate analyses. Third, we provide a short list of five proteins to be tested in a larger population to feed the pipeline of biomarker discovery.PMID:37321301 | DOI:10.1016/j.jprot.2023.104951

Cell. 2023 Jun 8:S0092-8674(23)00543-3. doi: 10.1016/j.cell.2023.05.024. Online ahead of print.ABSTRACTThe human body contains thousands of metabolites derived from mammalian cells, the microbiota, food, and medical drugs. Many bioactive metabolites act through the engagement of G-protein-coupled receptors (GPCRs); however, technological limitations constrain current explorations of metabolite-GPCR interactions. Here, we developed a highly multiplexed screening technology called PRESTO-Salsa that enables simultaneous assessment of nearly all conventional GPCRs (>300 receptors) in a single well of a 96-well plate. Using PRESTO-Salsa, we screened 1,041 human-associated metabolites against the GPCRome and uncovered previously unreported endogenous, exogenous, and microbial GPCR agonists. Next, we leveraged PRESTO-Salsa to generate an atlas of microbiome-GPCR interactions across 435 human microbiome strains from multiple body sites, revealing conserved patterns of cross-tissue GPCR engagement and activation of CD97/ADGRE5 by the Porphyromonas gingivalis protease gingipain K. These studies thus establish a highly multiplexed bioactivity screening technology and expose a diverse landscape of human, diet, drug, and microbiota metabolome-GPCRome interactions.PMID:37321219 | DOI:10.1016/j.cell.2023.05.024

Phytomedicine. 2023 Jun 2;117:154908. doi: 10.1016/j.phymed.2023.154908. Online ahead of print.ABSTRACTBACKGROUND: Abnormal endocrine metabolism caused by polycystic ovary syndrome combined with insulin resistance (PCOS-IR) poses a serious risk to reproductive health in females. Quercitrin is a flavonoid that can efficiently improve both endocrine and metabolic abnormalities. However, it remains unclear if this agent can exert therapeutic effect on PCOS-IR.METHODS: The present study used a combination of metabolomic and bioinformatic methods to screen key molecules and pathways involved in PCOS-IR. A rat model of PCOS-IR and an adipocyte IR model were generated to investigate the role of quercitrin in regulating reproductive endocrine and lipid metabolism processes in PCOS-IR.RESULTS: Peptidase M20 domain containing 1 (PM20D1) was screened using bioinformatics to evaluate its participation in PCOS-IR. PCOS-IR regulation via the PI3K/Akt signaling pathway was also investigated. Experimental analysis showed that PM20D1 levels were reduced in insulin-resistant 3T3-L1 cells and a letrozole PCOS-IR rat model. Reproductive function was inhibited, and endocrine metabolism was abnormal. The loss of adipocyte PM20D1 aggravated IR. In addition, PM20D1 and PI3K interacted with each other in the PCOS-IR model. Furthermore, the PI3K/Akt signaling pathway was shown to participate in lipid metabolism disorders and PCOS-IR regulation. Quercitrin reversed these reproductive and metabolic disorders.CONCLUSION: PM20D1 and PI3K/Akt were required for lipolysis and endocrine regulation in PCOS-IR to restore ovarian function and maintain normal endocrine metabolism. By upregulating the expression of PM20D1, quercitrin activated the PI3K/Akt signaling pathway, improved adipocyte catabolism, corrected reproductive and metabolic abnormalities, and had a therapeutic effect on PCOS-IR.PMID:37321077 | DOI:10.1016/j.phymed.2023.154908

BMC Bioinformatics. 2023 Jun 15;24(1):250. doi: 10.1186/s12859-023-05383-0.ABSTRACTMetabolomics is a dynamic tool for elucidating biochemical changes in human health and disease. Metabolic profiles provide a close insight into physiological states and are highly volatile to genetic and environmental perturbations. Variation in metabolic profiles can inform mechanisms of pathology, providing potential biomarkers for diagnosis and assessment of the risk of contracting a disease. With the advancement of high-throughput technologies, large-scale metabolomics data sources have become abundant. As such, careful statistical analysis of intricate metabolomics data is essential for deriving relevant and robust results that can be deployed in real-life clinical settings. Multiple tools have been developed for both data analysis and interpretations. In this review, we survey statistical approaches and corresponding statistical tools that are available for discovery of biomarkers using metabolomics.PMID:37322419 | DOI:10.1186/s12859-023-05383-0

Nat Immunol. 2023 Jun 15. doi: 10.1038/s41590-023-01513-1. Online ahead of print.ABSTRACTDespite the success of COVID-19 vaccines, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have emerged that can cause breakthrough infections. Although protection against severe disease has been largely preserved, the immunological mediators of protection in humans remain undefined. We performed a substudy on the ChAdOx1 nCoV-19 (AZD1222) vaccinees enrolled in a South African clinical trial. At peak immunogenicity, before infection, no differences were observed in immunoglobulin (Ig)G1-binding antibody titers; however, the vaccine induced different Fc-receptor-binding antibodies across groups. Vaccinees who resisted COVID-19 exclusively mounted FcγR3B-binding antibodies. In contrast, enhanced IgA and IgG3, linked to enriched FcγR2B binding, was observed in individuals who experienced breakthrough. Antibodies unable to bind to FcγR3B led to immune complex clearance and resulted in inflammatory cascades. Differential antibody binding to FcγR3B was linked to Fc-glycosylation differences in SARS-CoV-2-specific antibodies. These data potentially point to specific FcγR3B-mediated antibody functional profiles as critical markers of immunity against COVID-19.PMID:37322179 | DOI:10.1038/s41590-023-01513-1

Nat Commun. 2023 Jun 15;14(1):3566. doi: 10.1038/s41467-023-39372-x.NO ABSTRACTPMID:37322043 | DOI:10.1038/s41467-023-39372-x

Biochem Biophys Res Commun. 2023 Jun 7;671:292-300. doi: 10.1016/j.bbrc.2023.06.027. Online ahead of print.ABSTRACTAging adipose tissue exhibits elevated inflammation and oxidative stress that are major sources of age-related metabolic dysfunction. However, the exact metabolic changes associated with inflammation and oxidative stress are unclear. To address this topic, we assessed variation in metabolic phenotypes of adipose tissue from 18 months adult sedentary (ASED), 26 months old sedentary (OSED), and 8 months young sedentary (YSED). The results of metabolomic analysis showed that ASED and OSED group had higher palmitic acid, elaidic acid, 1-heptadecanol, and α-tocopherol levels than YSED, but lower sarcosine levels. Furthermore, stearic acid was specifically elevated in ASED compared with YSED. Cholesterol was upregulated specifically in the OSED group compared with YSED, whereas linoleic acid was downregulated. In addition, ASED and OSED had more inflammatory cytokines, lower antioxidant capacity, and higher expression of ferroptosis-related genes than YSED. Moreover, mitochondrial dysfunction associated with abnormal cardiolipin synthesis was more pronounced in the OSED group. In conclusion, both ASED and OSED can affect the FA metabolism and increase oxidative stress in adipose tissue, leading to inflammation. In particular, linoleic acid content specifically decreases in OSED, which associated with abnormal cardiolipin synthesis and mitochondrial dysfunction in adipose tissue.PMID:37320861 | DOI:10.1016/j.bbrc.2023.06.027

PLoS One. 2023 Jun 15;18(6):e0287121. doi: 10.1371/journal.pone.0287121. eCollection 2023.ABSTRACTMedulloblastoma is one of the most frequent malignant brain tumors in infancy and childhood. Early diagnosis and treatment are quite crucial for the prognosis. However, the pathogenesis of medulloblastoma is still not completely clarified. High-resolution mass spectrometry has enabled a comprehensive investigation on the mechanism of disease from the perspective of metabolism. Herein, we compared the difference of metabolic profiles of serum between medulloblastoma (n = 33) and healthy control (HC, n = 16) by using UPLC-Q/E-MS/MS. Principal component analysis and orthogonal projections to latent structures discriminant analysis (OPLS-DA) intuitively revealed the significantly distinct metabolic profiles between medulloblastoma and HC (p < 0.01 for permutation test on OPLS-DA model). Total of 25 significantly changed metabolites were identified. ROC analysis reported that six of them (Phosphatidic acid (8:0/15:0), 3'-Sialyllactose, Isocoproporphyrin, Acetylspermidine, Fructoseglycine and 3-Hydroxydodecanedioate) showed high specificity and precision to be potential diagnosis biomarkers (AUC > 0.98). Functional analysis discovered that there are four pathways notably perturbed for medulloblastoma. These pathways are related with the dysfunction of arachidonic acid metabolism, steroid hormone biosynthesis, and folate-related metabolism. The target intervention on these pathways may reduce the mortality of medulloblastoma.PMID:37319142 | DOI:10.1371/journal.pone.0287121

Proc Natl Acad Sci U S A. 2023 Jun 20;120(25):e2219373120. doi: 10.1073/pnas.2219373120. Epub 2023 Jun 15.ABSTRACTFungus-growing ants depend on a fungal mutualist that can fall prey to fungal pathogens. This mutualist is cultivated by these ants in structures called fungus gardens. Ants exhibit weeding behaviors that keep their fungus gardens healthy by physically removing compromised pieces. However, how ants detect diseases of their fungus gardens is unknown. Here, we applied the logic of Koch's postulates using environmental fungal community gene sequencing, fungal isolation, and laboratory infection experiments to establish that Trichoderma spp. can act as previously unrecognized pathogens of Trachymyrmex septentrionalis fungus gardens. Our environmental data showed that Trichoderma are the most abundant noncultivar fungi in wild T. septentrionalis fungus gardens. We further determined that metabolites produced by Trichoderma induce an ant weeding response that mirrors their response to live Trichoderma. Combining ant behavioral experiments with bioactivity-guided fractionation and statistical prioritization of metabolites in Trichoderma extracts demonstrated that T. septentrionalis ants weed in response to peptaibols, a specific class of secondary metabolites known to be produced by Trichoderma fungi. Similar assays conducted using purified peptaibols, including the two previously undescribed peptaibols trichokindins VIII and IX, suggested that weeding is likely induced by peptaibols as a class rather than by a single peptaibol metabolite. In addition to their presence in laboratory experiments, we detected peptaibols in wild fungus gardens. Our combination of environmental data and laboratory infection experiments strongly support that peptaibols act as chemical cues of Trichoderma pathogenesis in T. septentrionalis fungus gardens.PMID:37319116 | DOI:10.1073/pnas.2219373120

Anal Chem. 2023 Jun 15. doi: 10.1021/acs.analchem.3c00909. Online ahead of print.ABSTRACTAmino metabolites are essential for life activities and can be used clinically as biomarkers for disease diagnosis and treatment. Solid-phase-supported chemoselective probes can simplify sample handling and enhance detection sensitivity. However, the low efficiency and complicated preparation of traditional probes limit their further application. In this work, a novel solid-phase-supported probe Fe3O4-SiO2-polymers-phenyl isothiocyanate (FSP-PITC) was developed by immobilizing phenyl isothiocyanate on magnetic beads with disulfide as an orthogonal cleavage site, which can couple amino metabolites directly regardless of whether proteins and other matrixes were removed. After purification, the targeted metabolites were released by dithiothreitol and detected by high-resolution mass spectrometry. The simplified processing steps shorten the analysis time, and the introduction of polymers results in a 100-1000-fold increase in probe capacity. With high stability and specificity, FSP-PITC pretreatment allows accurate qualitative and quantitative (R2 > 0.99) analysis, facilitating the detection of metabolites in subfemtomole quantities. Using this strategy, 4158 metabolite signals were detected in negative ion mode. Among them, 352 amino metabolites including human cells (226), serum (227), and mouse samples (274) were searched from the Human Metabolome Database. These metabolites participate in metabolic pathways of amino acids, biogenic amine, and the urea cycle. All these results indicate that FSP-PITC is a promising probe for novel metabolite discovery and high-throughput screening.PMID:37318774 | DOI:10.1021/acs.analchem.3c00909

Biotechnol Lett. 2023 Jun 15. doi: 10.1007/s10529-023-03404-9. Online ahead of print.ABSTRACTPURPOSE: The accumulation of carbon dioxide during large-scale culture of animal cells brings adverse effects, appropriate aeration strategies alleviate CO2 accumulation while improper reactor operation may lead to the presence of low CO2 partial pressure (pCO2) condition as occurs in many industrial cases. Thus, this study aims to reveal the in-depth influence of low pCO2 on Chinese Hamster Ovary (CHO) cells for providing a reference for design space determination of CO2 control with regard to the Quality by Design (QbD) guidelines.METHODS AND RESULTS: The headspace air over purging caused the ultra-low pCO2 (ULC) where the monoclonal antibody production as well as the aerobic metabolic activity were reduced. Intracellular metabolomics analysis indicated a less efficient aerobic glucose metabolic state under ULC conditions. Based on the increase of intracellular pH and lactate dehydrogenase activity, the shortage of intracellular pyruvate could be the cause of the deficient aerobic metabolism, which could be partially mitigated by pyruvate addition under ULC conditions. Finally, a semi-empirical mathematical model was used to better understand, predict and control the occurrence of extreme pCO2 conditions during the cultures of CHO cells.CONCLUSION: Low pCO2 steers CHO cells into a defective metabolic state. A predictive relation among pCO2, lactate, and pH control was applied to get new insights into CHO cell culture for better and more robust metabolic behavior and process performance and the determination of QbD design space for CO2 control.PMID:37318718 | DOI:10.1007/s10529-023-03404-9

Appl Environ Microbiol. 2023 Jun 15:e0023823. doi: 10.1128/aem.00238-23. Online ahead of print.ABSTRACTMetabolic degeneracy describes the phenomenon that cells can use one substrate through different metabolic routes, while metabolic plasticity, refers to the ability of an organism to dynamically rewire its metabolism in response to changing physiological needs. A prime example for both phenomena is the dynamic switch between two alternative and seemingly degenerate acetyl-CoA assimilation routes in the alphaproteobacterium Paracoccus denitrificans Pd1222: the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). The EMCP and the GC each tightly control the balance between catabolism and anabolism by shifting flux away from the oxidation of acetyl-CoA in the tricarboxylic acid (TCA) cycle toward biomass formation. However, the simultaneous presence of both the EMCP and GC in P. denitrificans Pd1222 raises the question of how this apparent functional degeneracy is globally coordinated during growth. Here, we show that RamB, a transcription factor of the ScfR family, controls expression of the GC in P. denitrificans Pd1222. Combining genetic, molecular biological and biochemical approaches, we identify the binding motif of RamB and demonstrate that CoA-thioester intermediates of the EMCP directly bind to the protein. Overall, our study shows that the EMCP and the GC are metabolically and genetically linked with each other, demonstrating a thus far undescribed bacterial strategy to achieve metabolic plasticity, in which one seemingly degenerate metabolic pathway directly drives expression of the other. IMPORTANCE Carbon metabolism provides organisms with energy and building blocks for cellular functions and growth. The tight regulation between degradation and assimilation of carbon substrates is central for optimal growth. Understanding the underlying mechanisms of metabolic control in bacteria is of importance for applications in health (e.g., targeting of metabolic pathways with new antibiotics, development of resistances) and biotechnology (e.g., metabolic engineering, introduction of new-to-nature pathways). In this study, we use the alphaproteobacterium P. denitrificans as model organism to study functional degeneracy, a well-known phenomenon of bacteria to use the same carbon source through two different (competing) metabolic routes. We demonstrate that two seemingly degenerate central carbon metabolic pathways are metabolically and genetically linked with each other, which allows the organism to control the switch between them in a coordinated manner during growth. Our study elucidates the molecular basis of metabolic plasticity in central carbon metabolism, which improves our understanding of how bacterial metabolism is able to partition fluxes between anabolism and catabolism.PMID:37318336 | DOI:10.1128/aem.00238-23

Microbiol Spectr. 2023 Jun 15:e0002223. doi: 10.1128/spectrum.00022-23. Online ahead of print.ABSTRACTAlicyclobacillus acidoterrestris, which has strong acidophilic and heat-resistant properties, can cause spoilage of pasteurized acidic juice. The current study determined the physiological performance of A. acidoterrestris under acidic stress (pH 3.0) for 1 h. Metabolomic analysis was carried out to investigate the metabolic responses of A. acidoterrestris to acid stress, and integrative analysis with transcriptome data was also performed. Acid stress inhibited the growth of A. acidoterrestris and altered its metabolic profiles. In total, 63 differential metabolites, mainly enriched in amino acid metabolism, nucleotide metabolism, and energy metabolism, were identified between acid-stressed cells and the control. Integrated transcriptomic and metabolomic analysis revealed that A. acidoterrestris maintains intracellular pH (pHi) homeostasis by enhancing amino acids decarboxylation, urea hydrolysis, and energy supply, which was verified using real-time quantitative PCR and pHi measurement. Additionally, two-component systems, ABC transporters, and unsaturated fatty acid synthesis also play crucial roles in resisting acid stress. Finally, a model of the responses of A. acidoterrestris to acid stress was proposed. IMPORTANCE Fruit juice spoilage caused by A. acidoterrestris contamination has become a major concern and challenge in the food industry, and this bacterium has been suggested as a target microbe in the design of the pasteurization process. However, the response mechanisms of A. acidoterrestris to acid stress still remain unknown. In this study, integrative transcriptomic, metabolomic, and physiological approaches were used to uncover the global responses of A. acidoterrestris to acid stress for the first time. The obtained results can provide new insights into the acid stress responses of A. acidoterrestris, which will point out future possible directions for the effective control and application of A. acidoterrestris.PMID:37318333 | DOI:10.1128/spectrum.00022-23