Endogenous benzoic acid interferes with the signatures of amino acids and thiol compounds through perturbing N-methyltransferase, glutamate-cysteine ligase, and glutathione S-transferase activity in dairy products.

Endogenous benzoic acid causes adverse effects on individual health, but the potential mechanisms often remain elusive. The positive rate of benzoic acid in seventy-two goat milk samples in triplicate was 93.6 %, verifying the presence of endogenous benzoic acid. In this study, we investigated the differences in protein expression and metabolites among goat milk with different final concentrations of benzoic acid via combined proteomics and metabolomics (LOQ 3.25 to 56.63 µg L-1) analysis based on UHPLC-Q-Orbitrap HRMS. Integrated analysis showed that benzoic acid reduced the content of l-histidine (from 1.27 to 0.49 mg/L) and 1-methylhistidine (from 1.40 to 0.68 mg/L), due to the increase of benzoic acid (0-30 mg/L) concentration significantly reduced the level and activity of N-methyltransferase. Protein-metabolite interactions suggested that benzoic acid enhanced glutamate-cysteine ligase and glutathione S-transferase expression and affected l-glutamate (from 1.22 to 0.49 mg/L) and glutathione contents, eventually leading to the formation of off-flavors and oxidation of goat milk. Meanwhile, the level of l-phenylalanine (from 4.17 to 1.94 mg/L) and l-tyrosine (from 1.05 to 0.26 mg/L) progressively decreased with the increase of benzoic acid concentration, which had a deleterious effect on the nutritional value and flavor formation of goat milk. These findings clarified the mechanism by which low-dose benzoic acid negatively affects the nutritional quality and flavor formation of goat milk.

Functions, mechanisms, and therapeutic implications of METTL14 in human cancer.

RNA modification plays a crucial role in many biological functions, and its abnormal regulation is associated with the progression of cancer. Among them, N6-methyladenine (m6A) is the most abundant RNA modification. Methyltransferase-like 14 (METTL14) is the central component of the m6A methylated transferase complex, which is involved in the dynamic reversible process of m6A modification. METTL14 acts as both an oncogene and tumor suppressor gene to regulate the occurrence and development of various cancers. The abnormal m6A level induced by METTL14 is related to tumorigenesis, proliferation, metastasis, and invasion. To date, the molecular mechanism of METTL14 in various malignant tumors has not been fully studied. In this paper, we systematically summarize the latest research progress on METTL14 as a new biomarker for cancer diagnosis and its biological function in human tumors and discuss its potential clinical application. This study aims to provide new ideas for targeted therapy and improved prognoses in cancer.

Human TRMT112-Methyltransferase Network Consists of Seven Partners Interacting with a Common Co-Factor.

Methylation is an essential epigenetic modification mainly catalysed by S-Adenosyl methionine-dependent methyltransferases (MTases). Several MTases require a cofactor for their metabolic stability and enzymatic activity. TRMT112 is a small evolutionary conserved protein that acts as a co-factor and activator for different MTases involved in rRNA, tRNA and protein methylation. Using a SILAC screen, we pulled down seven methyltransferases-N6AMT1, WBSCR22, METTL5, ALKBH8, THUMPD2, THUMPD3 and TRMT11-as interaction partners of TRMT112. We showed that TRMT112 stabilises all seven MTases in cells. TRMT112 and MTases exhibit a strong mutual feedback loop when expressed together in cells. TRMT112 interacts with its partners in a similar way; however, single amino acid mutations on the surface of TRMT112 reveal several differences as well. In summary, mammalian TRMT112 can be considered as a central "hub" protein that regulates the activity of at least seven methyltransferases.

Increased DNA-incorporated thiopurine metabolite as a possible mechanism for leukocytopenia through cell apoptosis in inflammatory bowel disease patients with NUDT15 mutation.

BACKGROUND AND AIMS: Polymorphisms in the nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) gene are associated with thiopurine-induced leukopenia in patients with inflammatory bowel disease (IBD). NUDT15-associated subcellular thiopurine metabolism has not been investigated in primary lymphocytes. We hypothesized that NUDT15 mutation increases DNA-incorporated deoxythioguanosine (dTG) and induces apoptosis in lymphocytes. METHODS: DNA-incorporated dTG in peripheral blood mononuclear cells (PBMCs) and 6-thioguanine nucleotides (6-TGN) in red blood cells were measured in patients with IBD undergoing thiopurine treatment. The association of a single nucleotide polymorphism for NUDT15 (rs116855232) with dTGPBMC was examined. The pro-apoptotic effect of DNA-incorporated dTG was examined ex vivo in association with NUDT15 genotypes by co-culturing patient-derived peripheral CD4+ T lymphocytes with 6-thioguanine (6-TG). RESULTS: dTGPBMC was significantly higher in NUDT15 variants than in non-variants. dTGPBMC, but not 6-TGNRBC, negatively correlated with peripheral lymphocyte counts (r = - 0.31 and - 0.12, p = 0.012 and 0.173, respectively). DNA-incorporated dTG significantly accumulated to a greater extent in lymphocytes from NUDT15 variants when co-cultured with 6-TG ex vivo than in those from non-variants and was associated with decreased proliferation and increased apoptosis. CONCLUSION: Increased DNA-incorporated dTG may be responsible for thiopurine-induced leukocytopenia through cell apoptosis in IBD patients with NUDT15 mutation.

Localization of SARS-CoV-2 Capping Enzymes Revealed by an Antibody against the nsp10 Subunit.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease-19 pandemic. One of the key components of the coronavirus replication complex are the RNA methyltransferases (MTases), RNA-modifying enzymes crucial for RNA cap formation. Recently, the structure of the 2'-O MTase has become available; however, its biological characterization within the infected cells remains largely elusive. Here, we report a novel monoclonal antibody directed against the SARS-CoV-2 non-structural protein nsp10, a subunit of both the 2'-O RNA and N7 MTase protein complexes. Using this antibody, we investigated the subcellular localization of the SARS-CoV-2 MTases in cells infected with the SARS-CoV-2.

Exploring antimicrobial resistance to beta-lactams, aminoglycosides and fluoroquinolones in E. coli and K. pneumoniae using proteogenomics.

Antimicrobial resistance is mostly studied by means of phenotypic growth inhibition determinations, in combination with PCR confirmations or further characterization by means of whole genome sequencing (WGS). However, the actual proteins that cause resistance such as enzymes and a lack of porins cannot be detected by these methods. Improvements in liquid chromatography (LC) and mass spectrometry (MS) enabled easier and more comprehensive proteome analysis. In the current study, susceptibility testing, WGS and MS are combined into a multi-omics approach to analyze resistance against frequently used antibiotics within the beta-lactam, aminoglycoside and fluoroquinolone group in E. coli and K. pneumoniae. Our aim was to study which currently known mechanisms of resistance can be detected at the protein level using liquid chromatography-mass spectrometry (LC-MS/MS) and to assess whether these could explain beta-lactam, aminoglycoside, and fluoroquinolone resistance in the studied isolates. Furthermore, we aimed to identify significant protein to resistance correlations which have not yet been described before and to correlate the abundance of different porins in relation to resistance to different classes of antibiotics. Whole genome sequencing, high-resolution LC-MS/MS and antimicrobial susceptibility testing by broth microdilution were performed for 187 clinical E. coli and K. pneumoniae isolates. Resistance genes and proteins were identified using the Comprehensive Antibiotic Resistance Database (CARD). All proteins were annotated using the NCBI RefSeq database and Prokka. Proteins of small spectrum beta-lactamases, extended spectrum beta-lactamases, AmpC beta-lactamases, carbapenemases, and proteins of 16S ribosomal RNA methyltransferases and aminoglycoside acetyltransferases can be detected in E. coli and K. pneumoniae by LC-MS/MS. The detected mechanisms matched with the phenotype in the majority of isolates. Differences in the abundance and the primary structure of other proteins such as porins also correlated with resistance. LC-MS/MS is a different and complementary method which can be used to characterize antimicrobial resistance in detail as not only the primary resistance causing mechanisms are detected, but also secondary enhancing resistance mechanisms.

Exploration of the potential roles of m6A regulators in the uterus in pregnancy and infertility.

Infertility is a prevalent female reproductive disease worldwide. Currently, there are many unknown etiologies of infertility. N6-methyladenosine (m6A) is the most prevalent modification of eukaryotic mRNA. This study intended to investigate the implications of m6A regulators in the uterus for pregnancy and infertility. Pregnant ICR mice on days (D) 0, 4, 6, 10, and 15 were used to monitor m6A methylation in the uterus by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and then m6A methylation regulators were detected by real-time quantitative PCR (qPCR), western blot and immunohistochemistry (IHC). We found that m6A levels increased and that m6A regulators were expressed differently in the uterus during pregnancy. Then, we acquired expression data from endometrial tissue from women with infertility and recurrent pregnancy loss from the Gene Expression Omnibus (GEO) database. The expression of m6A regulators in infertility was significantly dysregulated according to the data mining technique. Specifically, the mRNA levels of METTL16 (p = 0.0147) and WTAP (p = 0.028) were lower and those of ALKBH5 (p = 0.0432) and IGF2BP2 (p = 0.0016) were higher in the endometrium of infertile patients. Meanwhile, many immunity-related pathways are abnormal in infertility, such as cytokine-cytokine receptor interactions, natural killer cell-mediated cytotoxicity and leukocyte transendothelial migration. In conclusion, we found that the m6A levels in the uterus increased as pregnancy progressed, and these regulators were dysregulated in the endometrium of infertility patients. These results suggest that m6A methylation may be very important in the establishment of implantation and maintenance of pregnancy and may become a new direction for research on infertility.

m6A regulators are associated with osteosarcoma metastasis and have prognostic significance: A study based on public databases.

BACKGROUND: Osteosarcoma represents the most common malignant bone tumor with high metastatic potential and inferior prognosis. RNA methylation (N6-methyladenosine [m6A]) is a prevalent RNA modification that epigenetically influences numerous biological processes including tumorigenesis. This study aims to determine that m6A regulators are significant biomarkers for osteosarcoma, and establish a prognostic model to predict the survival of patients. METHODS: In this study, we comprehensively analyzed the underlying associations between m6A regulators' mRNA expressions and metastasis as well as prognosis of osteosarcoma patients in the Cancer Genome Atlas. Multivariate Cox-regression analysis was used to screen regulators that were significantly associated with overall survival of osteosarcoma patients. Least absolute shrinkage and selection operator (LASSO) Cox-regression analysis was used for constructing m6A regulator-based osteosarcoma prognostic signature. RESULTS: Some of the regulators exhibited aberrant mRNA levels between osteosarcoma samples with and without metastasis. Multivariate Cox-regression analysis identified several regulators with potential prognostic significance. A risk score formula consisted of methyltransferase-like 3, YTH domains of Homo sapiens, and fat mass and obesity-associated protein was obtained through which patients could be prognostically stratified independently of potential confounding factors. The signature was also significantly associated with the metastatic potential of osteosarcoma. All the analyses could be well reproduced in another independent osteosarcoma cohort from the Gene Expression Omnibus. CONCLUSIONS: In conclusion, this study first revealed potential roles of m6A regulators in osteosarcoma metastasis and prognosis, which should be helpful for its clinical decision-making.

Towards personalised medicine in autoimmune hepatitis: Measurement of thiopurine metabolites results in higher biochemical response rates.

BACKGROUND & AIMS: Patients with autoimmune hepatitis (AIH) usually receive maintenance therapy with thiopurines, such as azathioprine (AZA) or mercaptopurine. Genetic polymorphisms in AZA metabolism can lead to variations in thioguanine nucleotide (TGN) and 6-methylmercaptopurine, both of which can cause adverse drug reactions (ADRs). In inflammatory bowel disease, a therapeutic TGN range (225-450 pmol/8x108 erythrocytes) has been identified to optimise effectiveness. We evaluated the benefits of a personalised medicine approach to thiopurine dosing, in comparison to standard weight-based dosing. METHODS: A retrospective matched cohort study of 214 patients with AIH who were seen at King's College between 1999-2019 was performed. Metabolite levels were measured in 109 patients. The control group included 105 patients on weight-based thiopurine dosing with no metabolite monitoring. RESULTS: Biochemical response (BR) occurred more frequently at 6-month follow-up in patients with metabolite monitoring compared to those on a weight-based regimen (77% vs. 60%, p = 0.008). This remained true with data analysis based on clinicians who measure metabolites and those who do not (BR at 6 months: 84% vs. 64%, p = 0.016). Patients with BR had TGN levels within the therapeutic range of 225-450 pmol/8x108 erythrocytes significantly more often than those who failed to achieve or lost BR (40% vs. 13%, p <0.0001). Moreover, TGN levels within the pre-defined therapeutic range predicted more stable disease within 6 months of testing compared to levels outside the range (p <0.0001). A high proportion of patients with sub-therapeutic TGN levels (75-225 pmol/8x108 erythrocytes) remained in BR (75% vs. 81%, p = 0.589) with fewer ADRs (44% vs. 86%, p = 0.0002) when compared to patients with therapeutic TGN levels. CONCLUSION: A strategy of personalised medicine using metabolite levels can optimise treatment regimens in AIH, resulting in fewer ADRs whilst maintaining BR. LAY SUMMARY: This study looked to see if measuring the breakdown products of a medication used in autoimmune hepatitis increases the chances of gaining good control of the disease, when compared to a group of patients who were on a dose of this medication based on their weight. A group of 214 patients with autoimmune hepatitis were split into 2 groups: roughly half had their medication dose adjusted according to measurements of breakdown products in the blood, whilst the other half received their weight-based dose as normal. The results confirmed that using a personalised approach and checking drug breakdown products resulted in fewer side effects and potentially improved control of disease.

In silico identification and structure function analysis of a putative coclaurine N-methyltransferase from Aristolochia fimbriata.

In this study we isolated and performed in silico analysis of a putative coclaurine N-methyltransferase (CNMT) from the basal angiosperm Aristolochia fimbriata. The Aristolochiaceae plant family produces alkaloids similar to the Papavaraceae family, and CNMTs are central enzymes in biosynthesis pathways producing compounds of ethnopharmacological interest. We used bioinformatics and computational tools to predict a three-dimensional homology model and to investigate the putative function of the protein and its mechanism for methylation. The putative CNMT is a unique (S)-adenosyl-L-methionine (SAM)-dependent N-methyltransferase, catalyzing transfer of a methyl group from SAM to the amino group of coclaurine. The model revealed a mixed α/ß structure comprising seven twisted ß-strands surrounded by twelve α-helices. Sequence comparisons and the model indicate an N-terminal catalytic Core domain and a C-terminal domain, of which the latter forms a pocket for coclaurine. An additional binding pocket for SAM is connected to the coclaurine binding pocket by a small opening. CNMT activity is proposed to follow an SN2-type mechanism as observed for a similarly conformed enzyme. Residues predicted for the methyl transfer reaction are Tyr79 and Glu96, which are conserved in the sequence from A. fimbriata and in homologous N-methyltransferases. The isolated CNMT is the first to be investigated from any basal angiosperm.

A single residue determines substrate preference in benzylisoquinoline alkaloid N-methyltransferases.

N-methylation is a recurring feature in the biosynthesis of many plant specialized metabolites, including alkaloids. A crucial step in the conserved central pathway that provides intermediates for the biosynthesis of benzylisoquinoline alkaloids (BIAs) involves conversion of the secondary amine (S)-coclaurine into the tertiary amine (S)-N-methylcoclaurine by coclaurine N-methyltransferase (CNMT). Subsequent enzymatic steps yield the core intermediate (S)-reticuline, from which various branch pathways for the biosynthesis of major BIAs such as morphine, noscapine and sanguinarine diverge. An additional N-methylation yielding quaternary BIAs is catalyzed by reticuline N-methyltransferase (RNMT), such as in the branch pathway leading to the taxonomically widespread and ecologically significant alkaloid magnoflorine. Despite their functional differences, analysis of primary sequence information has been unable to accurately distinguish between CNMT-like and RNMT-like enzymes, necessitating laborious in vitro screening. Furthermore, despite a recent emphasis on structural characterization of BIA NMTs, the features and mechanisms underlying the CNMT-RNMT functional dichotomy were unknown. We report the identification of structural variants tightly correlated with function in known BIA NMTs and show through reciprocal mutagenesis that a single residue acts as a switch between CNMT- and RNMT-like functions. We use yeast in vivo screening to show that this discovery allows for accurate prediction of activity strictly from primary sequence information and, on this basis, improve the annotation of previously reported putative BIA NMTs. Our results highlight the unusually short mutational distance separating ancestral CNMT-like enzymes from more evolutionarily advanced RNMT-like enzymes, and thus help explain the widespread yet sporadic occurrence of quaternary BIAs in plants. While this is the first report of structural variants controlling mono-versus di-methylation activity among plant NMT enzymes, comparison with bacterial MT enzymes also suggests possible convergent evolution.

Function of the MYND Domain and C-Terminal Region in Regulating the Subcellular Localization and Catalytic Activity of the SMYD Family Lysine Methyltransferase Set5.

SMYD lysine methyltransferases target histones and nonhistone proteins for methylation and are critical regulators of muscle development and implicated in neoplastic transformation. They are characterized by a split catalytic SET domain and an intervening MYND zinc finger domain, as well as an extended C-terminal domain. Saccharomyces cerevisiae contains two SMYD proteins, Set5 and Set6, which share structural elements with the mammalian SMYD enzymes. Set5 is a histone H4 lysine 5, 8, and 12 methyltransferase, implicated in the regulation of stress responses and genome stability. While the SMYD proteins have diverse roles in cells, there are many gaps in our understanding of how these enzymes are regulated. Here, we performed mutational analysis of Set5, combined with phosphoproteomics, to identify regulatory mechanisms for its enzymatic activity and subcellular localization. Our results indicate that the MYND domain promotes Set5 chromatin association in cells and is required for its role in repressing subtelomeric genes. Phosphoproteomics revealed extensive phosphorylation of Set5, and phosphomimetic mutations enhance Set5 catalytic activity but diminish its ability to interact with chromatin in cells. These studies uncover multiple regions within Set5 that regulate its localization and activity and highlight potential avenues for understanding mechanisms controlling the diverse roles of SMYD enzymes.

Liquid chromatography-electrospray ionization-tandem mass spectrometric assay for d-aspartate N-methyltransferase activity in ark shells.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the separation and quantification of the enantiomers of N-methylaspartate and N-methylglutamate, after derivatization with Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide was established. The time required for the LC-ESI-MS/MS analysis was within 20 min and the detection limit was approximately 10 fmol per injection, demonstrating that this method can be used for the rapid determination of D-aspartate N-methyltransferase activity in the ark shell clam Scapharca broughtonii.Abbreviations: NMDA: N-methyl-D-aspartate; NMLA: N-methyl-L-aspartate; NMDG: N-methyl-D-glutamate; NMLG: N-methyl-L-glutamate; NMA: N-methylaspartate; NMG: N-methylglutamate; HPLC: high-performance liquid chromatography; SAM: S-adenosyl-L-methionine; OPA: o-phthalaldehyde; LC-ESI-MS/MS: liquid chromatography-electrospray ionization-tandem mass spectrometry; FDLA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-leucinamide; FDAA: Nα-(5-fluoro-2,4-dinitrophenyl)-L-alaninamide; ESI: electrospray ionization; LC-ESI-MS: liquid chromatography-electrospray ionization-mass spectrometry; MS/MS: tandem mass spectrometry.

Correlation between Thiopurine S-Methyltransferase Genotype and Adverse Events in Inflammatory Bowel Disease Patients.

Background and Objectives: In patients with inflammatory bowel diseases (IBD), the use of azathioprine results in adverse events at a rate of 5% to 20%. The aim of the study was to assess a possible correlation between genetic variability of the enzyme thiopurine S-methyltransferase (TPMT) and the development of toxicity to azathioprine. Materials and Methods: A retrospective, single center, blind, case-control study was conducted on 200 IBD patients, of whom 60 cases suspended azathioprine due to toxicity (leukopenia, pancreatitis, hepatitis, and nausea or vomiting), and 140 controls continued treatment with the drug without adverse events. Results: In the entire cohort, only 8 cases of heterozygous mutations of TPMT were observed, corresponding to 4% mutated haplotype rate, much lower than that reported in literature (close to 10%). No homozygous mutation was found. Regarding the TPMT allelic variants, we did not find any statistically significant difference between patients who tolerated azathioprine and those who suffered from adverse events. (OR = 0.77, 95% CI = 0.08-7.72; p = 0.82). Conclusions: According to our study, in IBD patients, the search for TPMT gene mutations before starting treatment with azathioprine is not helpful in predicting the occurrence of adverse events. Importantly, patients with allelic variants should not be denied the therapeutic option of azathioprine, as they may tolerate this drug.

A Reader-Based Assay for m6A Writers and Erasers.

We have developed a homogeneous time-resolved fluorescence (HTRF)-based enzyme assay to measure the catalytic activity of N6-methyladenosine (m6A) methyltransferases and demethylases. The assay detects m6A modifications using the natural m6A-binding proteins (m6A readers). The reaction product or substrate m6A-containing RNA and the m6A reader protein are fluorescently labeled such that their proximity during binding initiates Förster resonance energy transfer (FRET). We show that our HTRF assay can be used for high-throughput screening, which will facilitate the discovery of small-molecule modulators of m6A (de)methylases.

Versatile Electrochemiluminescence and Electrochemical "On-Off" Assays of Methyltransferases and Aflatoxin B1 Based on a Novel Multifunctional DNA Nanotube.

Herein, a new multifunctional DNA nanotube (DNANT) was self-assembled and used to load Ru(phen)32+ and methylene blue (MB) as amplified signal probes for versatile electrochemiluminescence (ECL) and electrochemical (EC) "on-off" assays of Dam methylase (MTase) and aflatoxin B1 (AFB1). The DNA nanotube as a carrier could immobilize numerous MB or Ru(phen)32+ species in the double-stranded DNA (dsDNA) to significantly amplify signals, which enabled highly sensitive ECL and electrochemical detection of dual targets. The target Dam MTase first catalyzed the methylation of hairpin DNA (H1), and then the methylated DNA was cleaved by endonuclease DpnI to expose a single-strand DNA. After the Ru(phen)32+-DNANT or MB-DNANT signal probes were assembled to the electrode by hybridization, remarkable "signal on" states for amplified ECL or EC assays of MTase were obtained. Furthermore, in the presence of the target AFB1, the structure of DNANTs collapsed due to the specific binding of AFB1 to aptamer S2 in NTs, which led to the release of signal probes (Ru(phen)32+ or MB) from the electrode to achieve "signal off" state for dual detection of AFB1. Taking advantage of the multifunctional DNANT amplification signal probes, the versatile biosensors showed good analytical performance with very wide linear ranges (0.001-100 U mL-1 and 0.0001-100 ng mL-1 for MTase and AFB1 assay by DPV) and lower detection limits (2.1 × 10-4 U mL-1 and 0.018 pg mL-1 for MTase and AFB1 by DPV). This is the first time that ECL and EC "on-off" methods have been achieved separately for dual target assays, which opens a new avenue of DNANT-based signal amplification strategyies for the versatile design of biosensors in various biological detections.

The potential impact of naturally produced antibiotics, environmental factors, and anthropogenic pressure on the occurrence of erm genes in urban soils.

The occurrence of environmental antibiotic resistance genes (ARGs) are often attributed to selective pressure from antibiotics from point source pollution. However, the potential effects of natural production of antibiotics, environmental factors, and anthropogenic pressure on the development and spread of ARGs have not been fully investigated. This study evaluated the occurrence and distribution of erythromycin resistance methylase (erm) genes in urban soils. The ermA, ermB, ermC, ermD, ermF, ermG, ermT, and ermY genes were detected with detection frequencies ranging from 20% to 80% and abundances ranging between 5.95 × 101 and 6.94 × 106 copies g-1 dw soil. Both polyketide synthase (PKS) type I and type II biosynthesis genes-which are responsible for biosynthesis of polyketides, such as erythromycin-were detected in all soil samples with a range between 5.77 × 102 and 9.39 × 106 copies g-1 dw soil. The abundances of PKS genes were significantly correlated with 16S rRNA genes (r = 0.487 to 0.741, p < 0.001) and absolute abundances of ermB, ermC, ermD, ermG, and ermY (r = 0.302-0.490, p < 0.05), suggesting that the wide occurrence of ARGs in soils could be potentially driven by naturally produced antibiotics. Erythromycin was strongly correlated with ermB, ermC, ermF and ermY genes (r = 0.462 to 0.667, p < 0.05), but no significant correlation was observed between macrolides and PKS genes, suggesting other environmental factors may have contributed to detected macrolides. The fact that erm gene presented higher extent of variability than PKS genes in different land use types suggests that anthropogenic activity might also influence the occurrence of erm genes in urban soils.

Sequential Fractionation Strategy Identifies Three Missing Proteins in the Mitochondrial Proteome of Commonly Used Cell Lines.

Mitochondria are undeniably the cell powerhouse, directly affecting cell survival and fate. Growing evidence suggest that mitochondrial protein repertoire affects metabolic activity and plays an important role in determining cell proliferation/differentiation or quiescence shift. Consequently, the bioenergetic status of a cell is associated with the quality and abundance of the mitochondrial populations and proteomes. Mitochondrial morphology changes in the development of different cellular functions associated with metabolic switches. It is therefore reasonable to speculate that different cell lines do contain different mitochondrial-associated proteins, and the investigation of these pools may well represent a source for mining missing proteins (MPs). A very effective approach to increase the number of IDs through mass spectrometry consists of reducing the complexity of the biological samples by fractionation. The present study aims at investigating the mitochondrial proteome of five phenotypically different cell lines, possibly expressing some of the MPs, through an enrichment-fractionation approach at the organelle and protein level. We demonstrate a substantial increase in the proteome coverage, which, in turn, increases the likelihood of detecting low abundant proteins, often falling in the category of MPs, and resulting, for the present study, in the identification of METTL12, FAM163A, and RGS13. All MS data have been deposited to the MassIVE data repository ( https://massive.ucsd.edu ) with the data set identifier MSV000082409 and PXD010446.

Precise Antibody-Independent m6A Identification via 4SedTTP-Involved and FTO-Assisted Strategy at Single-Nucleotide Resolution.

Innovative detection techniques to achieve precise m6A distribution within mammalian transcriptome can advance our understanding of its biological functions. We specifically introduced the atom-specific replacement of oxygen with progressively larger atoms (sulfur and selenium) at 4-position of deoxythymidine triphosphate to weaken its ability to base pair with m6A, while maintaining A-T* base pair virtually the same as the natural one. 4SedTTP turned out to be an outstanding candidate that endowed m6A with a specific signature of RT truncation, thereby making this "RT-silent" modification detectable with the assistance of m6A demethylase FTO through next-generation sequencing. This antibody-independent, 4SedTTP-involved and FTO-assisted strategy is applicable in m6A identification, even for two closely gathered m6A sites, within an unknown region at single-nucleotide resolution.

Analytical and clinical validation of an LC-MS/MS method to measure thiopurine S-methyltransferase activity by quantifying d3-6-MMP.

BACKGROUND: Identification of patients with thiopurine S-methyltransferase (TPMT) deficiency prior to thiopurine drug therapy has become routine clinical practice worldwide. To measure TPMT activity, traditional radiochemical assays have been replaced by chromatographic methods. METHOD: Inspired by the increasing number of isotope labelled sources that may be of benefit for the TPMT assay, a new LC-MS/MS method for TPMT activity was developed and validated. Isotope labelled d3-S-adenosyl-l-methionine (d3-SAM) was selected for the enzymatic methylation of mercaptopurine during sample incubation; d3-6-methylmercaptopurine (d3-6-MMP) with d2-2, 8-hypoxanthine as the internal standard was quantified to ascertain individual TPMT activity. RESULTS: The validation of the analytical part of this method showed good linearity (coefficient of determination 0.9999 in the range of 1-500 ng/mL) with the intra-and inter-day impression CV% between 7.6% and 9.1% and 3.7% and 9.2%, respectively. Recovery ranged from 94.9% to 112.3%. The specificity of the enzymatic reaction was validated by using 108 clinical check samples. After compared with traditional radiochemical assay and genotype results, all homozygous and heterozygous deficiency clinical checks fitted into the nominal groups, inter-batch and intra-batch impression CV% were between 2.3% and 9.7%. CONCLUSION: With the inclusion of isotope labelled substrate, interfering non-enzymatic methylation no longer results in potential false assignment of abnormal patients. Furthermore, the method can be applied to patients who have already been prescribed thiopurine drugs. This new LC-MS/MS is therefore a favourable clinical routine application to test TPMT activity, as it shows excellent performance in identifying patients with TPMT deficiency.