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In living cells, some reactions can be conducted by more than one enzyme and sometimes it is difficult to establish which enzyme is responsible. Such is the case with proteins from the TET family, capable of converting 5-methyl-2’-deoxycytidine (5-mdC) in DNA to 5-(hydroxymethyl)-2’-deoxycytidine (5-hmdC) and further to 5-formyl-2’-deoxycytidine (5-fdC) and 5-carboxy-2’-deoxycytidine (5-cadC). The estimation of the efficiency of particular TETs in particular oxidative reactions and different cell types is important but experimentally difficult. Here, we propose an approach with mathematical modeling in which methylation and known deoxycytidine modification pathways are presented by 343 possible model versions with assumed different combinations of TET1, 2, and 3 activities in different pathways. Model parameters were calculated on the basis of 5-mdC, 5-hmdC, 5-fdC, 5-cadC, and 5-hmdU levels experimentally assessed in five human cultured cell lines and previously published. Selection of the model versions that give in simulations the best average fit to experimental data suggested that not all TET proteins participate in all modification reactions and that TET3 activity may be especially important in the reaction of 5-fdC removal.

[Objective] This work aims to construct the Tet-On inducible expression plasmid of methyl-binding protein 1 (MBD1) and 293T cells, to explore the effect of doxycycline (Dox) on the expression of MBD1, and to reflect the regulatory mechanism of MBD1 by analyzing the CpG methylation level of the plasmid promoter in the cells. [Method] The MBD1 expression sequence of 293T was amplified using MBD1 primers and ligated into the polyclonal site of Tet-On inducible expression plasmid. The successfully constructed plasmid was transfected into MBD1 KO 293T by electro-transfection, and the obtained MBD1-restored 293T was verified using fluorescence microscope and Western blot (MBD1 RE). MBD1 expression was induced using different concentrations of Dox, and the expression of MBD1 in monoclonal cells was detected by Western blot. MBD1 expression was induced using the same concentration of Dox, and the cells were collected at 12, 24, 48, 96, 144 and 192 h. The expression of MBD1 in the cells was detected by Western blot; and MBD1 expression in the cells was detected by bisulfite sequencing. The expression of MBD1 in the cells was detected by Western blot, and the CpG methylation of inducible plasmid promoter was detected by bisulfite sequencing after Dox induction. [Result] Tet-On inducible MBD1 plasmid was successfully constructed; MBD1 protein was re-expressed in the transfected cells after Dox induction compared with uninduced cells. The expression of MBD1 in the cells increased gradually with the increase of Dox concentration; the expression of MBD1 in the cells increased gradually from 12 to 96 h after induction with the same concentration of Dox, but the expression decreased after 144 h. The expression of CpG methylation of plasmid promoter was detected by bisulfite sequencing method; the CpG methylation of plasmid promoter was detected by bisulfite sequencing method. CpG methylation of the plasmid promoter showed that the methylation level of the plasmid promoter gradually increased at 144 and 192 h compared with that at 96 h. The methylation level of the plasmid promoter decreased and the expression of MBD1 increased after the addition of the methyltransferase inhibitor, decitabine, at 144 h. The expression of MBD1 in the cells also decreased by the addition of the methyltransferase inhibitor, decitabine, at 144 h after the induction. [Conclusion] Tet-On induced the re-expression of MBD1 protein in the cells and the expression was positively correlated with the Dox concentration, but the elevated level of intracellular plasmid CpG methylation after prolonged induction affected the expression of MBD1 in the cells. [ABSTRACT FROM AUTHOR]

Tigecycline is a last-resort antimicrobial in humans. Tetracyclines are the most widely used antimicrobials in livestock. Mobile tigecycline resistance genes [tet(X)] are disseminated worldwide, and tetracycline use may have promoted the selection of tet(X) genes. Thus, the selective pressure on tet(X) genes and their plasmids in livestock must be elucidated. We performed a retrospective study to clarify the prevalence of tigecycline-resistant Escherichia coli from pigs in Thailand. Screening for tigecycline resistance was performed on 107 E. coli strains from 25 samples, and tet(X)-carrying plasmids were characterized. tet(X) genes were cloned and expressed in E. coli. Bacterial growth rate in the presence of tetracycline as a result of the presence of tet(X) genes was also evaluated. Thirty-two tet(X4)-harboring tigecycline-resistant E. coli strains were detected in 10/25 samples (40%). The tet(X4) genes were carried on various Inc-type plasmids and flanked by ISCR2. The tet(X)-carrying plasmids were transferred to E. coli and Klebsiella pneumoniae. Acquisition of tet(X) genes and their plasmids improved bacterial growth in the presence of tetracycline. In summary, tetracycline use exerts selective pressure on tet(X) genes and their various backbone plasmids; therefore, a reduced amount of tetracycline use is important to limit the spreading of tet(X) genes. [ABSTRACT FROM AUTHOR]

Propyl gallate (PG), an approved food additive, can be added to different foods and drugs to provide health benefits with minimal danger. However, no clinical application of PG as an antibacterial agent for the treatment of antimicrobial resistance (AMR) has been documented. The aim of this study was to elucidate the effects and mechanisms by which PG inhibits the activity of Tet(X4). Enzyme activity inhibition assay, antimicrobial tests, scanning electron microscopy (SEM) assay, molecular docking and dynamics simulation assays, and animal infection models were used to confirm the synergistic efficacy and mechanism. Here, we found that PG efficiently inhibited Tet(X4) enzyme activity (IC 50  = 34.83 μg/mL) while affecting the expression of tet(X4). PG has a synergistic effect with tigecycline (fractional inhibitory concentration index (FICI) < 0.5) against tet(X4)-positive Escherichia coli (E. coli) isolates of animal origin. The survival rates of G. mellonella larvae and the mouse systemic infection model increased by 60 % and 39 %, respectively. The combination of PG and tigecycline showed remarkable treatment benefits in terms of the bacterial load and inflammatory factors in mice. Our results indicate that PG is a valuable adjuvant with tetracyclines and can be considered to address the inevitable infection caused by tet(X4)-positive bacteria, which is a feasible way to extend the lifespan of existing antibiotics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Here, we report the genetic features and evolutionary mechanisms of two tet (M)-bearing plasmids (pTA2 and pTA7) recovered from swine Escherichia coli isolates. The genetic profiles of pTA2 and pTA7 and corresponding transconjugants were accessed by S1 nuclease pulsed-field gel electrophoresis and Southern hybridization, followed by whole genome sequencing and bioinformatics analysis. The biological influences of pTA2 and pTA7 were determined by stability and direct competition assays. Both pTA7 and pTA2 had the IncR backbone sequences but differed in the multidrug resistance region (MDR). The MDR of pTA2 consisted of sul3 , tet (M), qnr S1, bleO , oqxAB , floR , aadA1 , cmlA1 , aadA2 , and tet (A)- tetR (A) in addition to 22 insertion sequences. Notably, pTA2 carried the novel complex Tn 7124 (IS 26 -ctp-lp- tet (M)-hp-IS 406 tnp-IntI4-IS 26 ) harboring tet (M). The fragment carrying tet (M) (IS 26-ctp- lp- tet (M)-IS 406 tnp-ctp-aadA1 - cmlA1 - aadA2 - dfrA12- Int I1 ), named Tn 6942-like , and the two resistance modules IS Vsa3 - VirD2 - floR - lysR and tet (A)- tetR (A) were located in the MDR of pTA7. Both pTA2 and pTA7 were highly stable in E. coli DH5α cells with no fitness cost to the host or disadvantage in growth competition. These results indicate that transposons carrying tet (M) continuously integrate via mediation with an insertion sequence, which accelerates the transmission of tet (M) in E. coli isolates through integration of other drug-resistant genes, thereby posing a potential serious threat to the efficacy of clinical treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Liu, Qiao, Ma, Wang, Hu and Li.)

The plasmid-mediated tet (X4) gene has exhibited a high-level resistance to tigecycline (TGC), which has raised concerns globally regarding antibiotic resistance. Although the widespread tet (X4) has been found widely in Escherichia coli , it is scarcely found in other Enterobacteriaceae. This study aimed to characterize a ST469 Salmonella enterica serovar Rissen ( S. Rissen) isolate harboring tet (X4) from pork, which was identified and characterized via antimicrobial susceptibility testing, conjugation assays, plasmid curing testing, whole-genome sequencing, and bioinformatic analysis. Ten ST469 S. Rissen isolates of 223 Salmonella spp. isolates were isolated from food samples in China during 2021-2023. One of 10 S . Rissen isolates, SM2301, carrying tet (X4) conferred high-level resistance to TGC (minimum inhibitory concentration > 8 µg/mL). The tet (X4) could be conjugated into different recipients, including E. coli , S. enteritidis , and K. pneumoniae isolates. Plasmid curing confirmed that tet (X4) was plasmid-mediated. Genetic analysis revealed that the tet (X4) in the SM2301 isolate was located in the IncFIA(HI1)-IncHI1A-IncHI1B(R27) hybrid plasmid, and the structure of tet (X4) was abh - tet (X4)-IS CR2 . To the best of our knowledge, this is the first report of a tet (X4)-positive food-derived S. Rissen isolate. The extending bacterial species of tet (X4)-bearing plasmids suggested the increasing transmission risk of the mobile TGC resistance gene tet (X4) beyond E. coli . This study highlights the emerging and evolution risk of novel resistance genes across various bacterial species. Therefore, further surveillance is warranted to monitor the prevalence of tet (X4) in Salmonella spp. and other bacterial species.

Caco-2 cells, a human colorectal adenocarcinoma cell line, are widely used to model small intestinal epithelial cells in the drug development process because they can predict drug absorption with high accuracy. However, Caco-2 cells have several issues. First, Caco-2 cells have little expression of cytochrome P450 3A4 (CYP3A4), which is a major drug-metabolizing enzyme in the human intestine. We previously developed Caco-2 cells whose expression of CYP3A4 can be controlled using doxycycline (Dox) (CYP3A4-Caco-2 cells) (Ichikawa et al., Sci. Rep, 2021). However, since the Tet-On system was used to regulate CYP3A4 expression in these cells, there was concern about drug-drug interactions. The second issue is that in the human small intestine, carboxylesterase 2 (CES2) is more highly expressed than carboxylesterase 1 (CES1), while in Caco-2 cells CES1 is more highly expressed. The third issue is the low level expression of uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1), a phase II drug-metabolizing enzyme. In this study, we used genome-editing technology to establish CES1 -knockout Caco-2 cells whose CYP3A4 and UGT1A1 expression can be regulated by the Tet-Off system. These cell lines would be useful in pharmaceutical researches, including intestinal toxicological studies, as an in vitro model for orally administered drugs. [Display omitted] • CES1-KO/CYP3A4 Caco-2 cells and CES1-KO/CYP3A4-UGT1A1 Caco-2 cells are established. • CES1-KO/CYP3A4 Caco-2 cells can be used to predict absorption of prodrugs metabolized by CES. • CES1-KO/CYP3A4 Caco-2 cells have the potential to predict the metabolism of orally administered drugs involving in CYP3A4 in the small intestine. • CES1-KO/CYP3A4-UGT1A1 Caco-2 cells have the potential to predict the metabolism of orally administered drugs involving in both CYP3A4 and UGT1A1 in the small intestine. [ABSTRACT FROM AUTHOR]

Abstract Despite epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) have shown remarkable efficacy in patients with EGFR-mutant non-small cell lung cancer (NSCLC), acquired resistance inevitably develops, limiting clinical efficacy. We found that TET2 was poly-ubiquitinated by E3 ligase CUL7FBXW11 and degraded in EGFR-TKI resistant NSCLC cells. Genetic perturbation of TET2 rendered parental cells more tolerant to TKI treatment. TET2 was stabilized by MEK1 phosphorylation at Ser 1107, while MEK1 inactivation promoted its proteasome degradation by enhancing the recruitment of CUL7FBXW11. Loss of TET2 resulted in the upregulation of TNF/NF-κB signaling that confers the EGFR-TKI resistance. Genetic or pharmacological inhibition of NF-κB attenuate the TKI resistance both in vitro and in vivo. Our findings exemplified how a cell growth controlling kinase MEK1 leveraged the epigenetic homeostasis by regulating TET2, and demonstrated an alternative path of non-mutational acquired EGFR-TKI resistance modulated by TET2 deficiency. Therefore, combined strategy exploiting EGFR-TKI and inhibitors of TET2/NF-κB axis holds therapeutic potential for treating NSCLC patients who suffered from this resistance.

DNA methylation is an epigenetic mechanism involving the transfer of a methyl group onto the C5 position of the cytosine to form 5-methylcytosine (5mC). In general, DNA methylation in cancer is associated with the repression of the expression of tumor suppressor genes (TSG) and the demethylation with the overexpression of oncogenes. DNA methylation was considered a stable modification for a long time, but in 2009, it was reported that DNA methylation is a dynamic modification. The Ten-Eleven-Translocations (TET) enzymes include TET1, TET2, and TET3 and participate in DNA demethylation through the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC). The 5hmC oxidates to 5-formylcytosine (5fC) and 5-carboxylcitosine (5caC), which are replaced by unmodified cytosines via Thymine-DNA Glycosylase (TDG). Several studies have shown that the expression of TET proteins and 5hmC levels are deregulated in gynecological cancers, such as cervical (CC), endometrial (EC), and ovarian (OC) cancers. In addition, the molecular mechanisms involved in this deregulation have been reported, as well as their potential role as biomarkers in these types of cancers. This review shows the state-of-art TET enzymes and the 5hmC epigenetic mark in CC, EC, and OC. [ABSTRACT FROM AUTHOR]

Plasmids carrying antibiotic resistance genes occur frequently in Aeromonas salmonicida subsp. salmonicida, an aquatic pathogen. In this study, we describe three new plasmids found in strains of A. salmonicida subsp. salmonicida from Québec, Canada: pAsa-2358, pAsa-2900, and pAsa-2900b. The last two plasmids have a tet(D) resistance gene against tetracycline, which has not been previously found in A. salmonicida. The only difference between these two plasmids is the presence of a florfenicol resistance gene (floR) surrounded by two copies of the same insertion sequence, indicating that pAsa-2900 and pAsa-2900b are plasmid variants. All three plasmids represent an issue for aquaculture because they bear resistance genes against antibiotics used to treat infected fish: floR and tet(A) for pAsa-2358; floR, sul2, and tet(D) for pAsa-2900; and sul2 and tet(D) for pAsa-2900b. Conjugation assays confirmed that the three plasmids are capable of conjugation with one A. salmonicida mesophilic strain, one A. hydrophila strain, and Escherichia coli DH5α; except for pAsa-2358 that is not able to conjugate into E. coli DH5α. Based on the results of antibiotic resistance assays, the tet(D) gene provides a greater resistance to tetracycline than the tet(A) gene found in many A. salmonicida subsp. salmonicida plasmids. This is the case whether the plasmids are in their parental A. salmonicida subsp. salmonicida strains or in Aeromonas sp. transconjugates. The presence of these three plasmids in A. salmonicida subsp. salmonicida strengthens the role of this bacterium as a reservoir for antibiotic resistance genes.

Summary: Mutations in ten-eleven translocation (TET) proteins are associated with human neurodevelopmental disorders. We find a function of Tet in regulating Drosophila early brain development. The Tet DNA-binding domain (TetAXXC) is required for axon guidance in the mushroom body (MB). Glutamine synthetase 2 (Gs2), a key enzyme in glutamatergic signaling, is significantly down-regulated in the TetAXXC brains. Loss of Gs2 recapitulates the TetAXXC phenotype. Surprisingly, Tet and Gs2 act in the insulin-producing cells (IPCs) to control MB axon guidance, and overexpression of Gs2 in IPCs rescues the defects of TetAXXC. Feeding TetAXXC with metabotropic glutamate receptor antagonist MPEP rescues the phenotype while glutamate enhances it. Mutants in Tet and Drosophila Fmr1, the homolog of human FMR1, have similar defects, and overexpression of Gs2 in IPCs also rescues the Fmr1 phenotype. We provide the first evidence that Tet controls the guidance of developing brain axons by modulating glutamatergic signaling.

ABSTRACTRecently discovered tet(X) gene variants have provided new insights into microbial antibiotic resistance mechanisms and their potential consequences for public health. This study focused on detection, analysis, and characterization of Tet(X4)-positive Enterobacterales from the gut microbiota of a healthy cohort of individuals in Singapore using cultivation-dependent and cultivation-independent approaches. Twelve Tet(X4)-positive Enterobacterales strains that were previously obtained from the cohort were fully genome-sequenced and comparatively analyzed. A metagenomic sequencing (MS) data set of the same samples was mined for contigs that harbored the tet(X4) resistance gene. The sequences of tet(X4)-containing contigs and plasmids sequences were compared. The presence of the resistance genes floR and estT (previously annotated as catD) was detected in the same cassette in 10 and 12 out of the 12 tet(X4)-carrying plasmids, respectively. MS detected tet(X4)-containing contigs in 2 out of the 109 subjects, while cultivation-dependent analysis previously reported a prevalence of 10.1%. The tet(X4)-containing sequences assembled from MS data are relatively short (~14 to 33 kb) but show high similarity to the respective plasmid sequences of the isolates. Our findings show that MS can complement efforts in the surveillance of antibiotic resistance genes for clinical samples, while it has a lower sensitivity than a cultivation-based method when the target organism has a low abundance. Further optimization is required if MS is to be utilized in antibiotic resistance surveillance.IMPORTANCEThe global rise in antibiotic resistance makes it necessary to develop and apply new approaches to detect and monitor the prevalence of antibiotic resistance genes in human populations. In this regard, of particular interest are resistances against last-resort antibiotics, such as tigecycline. In this study, we show that metagenomic sequencing can help to detect high abundance of the tigecycline resistance gene tet(X4) in fecal samples from a cohort of healthy human subjects. However, cultivation-based approaches currently remain the most reliable and cost-effective method for detection of antibiotic-resistant bacteria.

Introduction: The dissemination of strains producing tetracyclines monooxygenase Tet(X) from breeding farms to the natural environment poses a potential threat to public health., Methods: Antimicrobial susceptibility testing and WGS were performed to identify resistance phenotypes and genotypes. Cloning experiments, sequence alignment, and homology modeling were used to characterize the function and formation mechanisms of the recombinant variant. The mobilization potential of Tet(X) was assessed by collinearity analysis, conjugation experiments, and phylogenetic analysis., Results: Three tet (X)-producing Elizabethkingia meningoseptica strains were isolated from bullfrog breeding ponds, the sewage outlet, and downstream river in Zhejiang Province, China. These strains carry a novel Tet(X) variant, differing from Tet(X6) by seven residues, and possess the ability to degrade tetracyclines. Interestingly, the novel Tet(X) is a recombinant variant formed by homologous recombination of Tet(X6) and the C-terminal of Tet(X2). Further analysis revealed that Tet(X6) formed several Tet(X) variants, including Tet(X5), through homologous recombination. The novel tet (X) gene is located on a circularizable integrative and conjugative element (ICE EmeChn3 ), with IS wz1 participating in the recombination of its multi-drug resistance region, potentially facilitating the mobilization and recombination of tet (X) in early hosts. These three strains were clonally transmitted and shared a close genetic relationship (SNP < 62) with a clinically-sourced strain isolated from the same province., Discussion: To our knowledge, this is the first report of homologous recombination between Tet(X) variants with differing activities. These clonal strains provide evidence of the transmission of tet (X)-positive strains from aquaculture sewage to the natural environment, highlighting the need to strengthen the monitoring and management of this emerging farming model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jin, Jia, Jin, Zhu, Wang, Sun and Cui.)

This review deals with the functional characteristics and biological roles of enzymes participating in DNA methylation and demethylation as key factors in epigenetic regulation of gene expression. The set of enzymes that carry out such processes in human cells is limited to representatives of two families, namely DNMT (DNA methyltransferases) and TET (DNA dioxygenases). The review presents detailed information known today about each functionally important member of these families and describes the catalytic activity and roles in the mammalian body while also providing examples of dysregulation of the expression and/or activity of these enzymes in conjunction with the development of some human disorders, including cancers, neurodegenerative diseases, and developmental pathologies. By combining the up-to-date information on the dysfunction of various enzymes that control the DNA “methylome” in the human body, we hope not only to draw attention to the importance of the maintenance of a required DNA methylation level (ensuring epigenetic regulation of gene expression and normal functioning of the entire body) but also to help identify new targets for directed control over the activity of the enzymes that implement the balance between processes of DNA methylation and demethylation.

Modifications of mRNA, especially methylation of adenosine, have recently drawn much attention. The much rarer modification, 5-hydroxymethylation of cytosine (5hmC), is not well understood and is the subject of this study. Vertebrate Tet proteins are 5-methylcytosine (5mC) hydroxylases and catalyze the transition of 5mC to 5hmC in DNA. These enzymes have recently been shown to have the same function in messenger RNAs in both vertebrates and in Drosophila. The Tet gene is essential in Drosophila as Tet knock-out animals do not reach adulthood. We describe the identification of Tet-target genes in the embryo and larval brain by mapping one, Tet DNA-binding sites throughout the genome and two, the Tet-dependent 5hmrC modifications transcriptome-wide. 5hmrC modifications are distributed along the entire transcript, while Tet DNA-binding sites are preferentially located at the promoter where they overlap with histone H3K4me3 peaks. The identified mRNAs are preferentially involved in neuron and axon development and Tet knock-out led to a reduction of 5hmrC marks on specific mRNAs. Among the Tet-target genes were the robo2 receptor and its slit ligand that function in axon guidance in Drosophila and in vertebrates. Tet knock-out embryos show overlapping phenotypes with robo2 and both Robo2 and Slit protein levels were markedly reduced in Tet KO larval brains. Our results establish a role for Tet-dependent 5hmrC in facilitating the translation of modified mRNAs primarily in cells of the nervous system.

DNA demethylases TET2 and TET3 play a fundamental role in thymic invariant natural killer T (iNKT) cell differentiation by mediating DNA demethylation of genes encoding for lineage specifying factors. Paradoxically, differential gene expression analysis revealed that significant number of genes were upregulated upon TET2 and TET3 loss in iNKT cells. This unexpected finding could be potentially explained if loss of TET proteins was reducing the expression of proteins that suppress gene expression. In this study, we discover that TET2 and TET3 synergistically regulate Drosha expression, by generating 5hmC across the gene body and by impacting chromatin accessibility. As DROSHA is involved in microRNA biogenesis, we proceed to investigate the impact of TET2/3 loss on microRNAs in iNKT cells. We report that among the downregulated microRNAs are members of the Let-7 family that downregulate in vivo the expression of the iNKT cell lineage specifying factor PLZF. Our data link TET proteins with microRNA expression and reveal an additional layer of TET mediated regulation of gene expression., Competing Interests: Dr. TÄ is a Director of Data Science at Covera Health. No funding from Covera Health was received for this study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gioulbasani, Äijö, Valenzuela, Bettes and Tsagaratou.)

DNA cytosine methylation is an epigenetic marker which regulates many cellular processes. Mammalian genomes typically maintain consistent methylation patterns over time, except in specific regulatory regions like promoters and certain types of enhancers. The dynamics of DNA methylation is controlled by a complex cellular machinery, in which the enzymes DNMT3 and TET play a major role. This study explores the identification of differentially methylated cytosines (DMCs) in TET and DNMT3 knockout mutants in mice and human embryonic stem cells. We investigate (i) whether a large language model can be trained to recognize DMCs in human and mouse from the sequence surrounding the cytosine of interest, (ii) whether a classifier trained on human knockout data can predict DMCs in the mouse genome (and vice versa), (iii) whether a classifier trained on DNMT3 knockout can predict DMCs for TET knockout (and vice versa). Our study identifies statistically significant motifs associated with the prediction of DMCs each mutant, casting a new light on the understanding of DNA methylation dynamics in stem cells. Our software tool is available at https://github.com/ucrbioinfo/dmc&#95;prediction., Competing Interests: Competing interests The authors declare that they have no competing interests.

Omadacycline and eravacycline are gradually being used as new tetracycline antibiotics for the clinical treatment of Gram-negative pathogens. Affected by various tetracycline-inactivating enzymes, there have been reports of resistance to eravacycline and omadacycline in recent years. We isolated a strain carrying the mobile tigecycline resistance gene tet (X4) from the feces of a patient in Zhejiang Province, China. The strain belongs to the rare ST485 sequence type. The isolate was identified as Klebsiella pneumoniae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MICs of antimicrobial agents were determined using either the agar dilution method or the micro broth dilution method. The result showed that the isolate was resistant to eravacycline (MIC = 32 mg/L), omadacycline (MIC > 64 mg/L), and tigecycline (MIC > 32 mg/L). Whole-genome sequencing revealed that the tet (X4) resistance gene is located on the IncFII(pCRY) conjugative plasmid. tet (X4) is flanked by IS Vsa3 , and we hypothesize that this association contributes to the spread of the resistance gene. Plasmids were analyzed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, and electrotransformation experiment. We successfully transferred the plasmid carrying tet (X4) to the recipient bacteria by electrotransformation experiment. Compared with the DH-5α, the MICs of the transformant L3995-DH5α were increased by eight-fold for eravacycline and two-fold higher for omadacycline. Overall, the emergence of plasmid-borne tet (X4) resistance gene in a clinical isolate of K. pneumoniae ST485 underscores the essential requirement for the ongoing monitoring of tet (X4) to prevent and control its further dissemination in China.IMPORTANCEThere are still limited reports on Klebsiella pneumoniae strains harboring tetracycline-resistant genes in China, and K. pneumoniae L3995hy adds a new example to those positive for the tet (X4) gene. Importantly, our study raises concerns that plasmid-mediated resistance to omadacycline and eravacycline may spread further to a variety of ecological and clinical pathogens, limiting the choice of medication for extensively drug-resistant bacterial infections. Therefore, it is important to continue to monitor the prevalence and spread of tet (X4) and other tetracyclines resistance genes in K. pneumoniae and diverse bacterial populations., Competing Interests: The authors declare no conflict of interest.

Objective: The aim of the present study is to explore the impact of the tet(A) type I variant (tetA-v1) on its fitness effect in Klebsiella pneumoniae., Methods: Clinical K. pneumoniae strains were utilized as parental strains to generate strains carrying only the plasmid vector (pBBR1MCS-5) or the tetA-v1 recombinant plasmid (ptetA-v1). Antimicrobial susceptibility testing was conducted to estimate the contribution of tetA-v1 to drug resistance. Plasmid stability was evaluated by serial passage over 10 consecutive days in the absence of tigecycline. Biological fitness was examined through growth curve analysis, in vitro competition assays and a neutropenic mouse thigh infection model., Results: A 2-4-fold increase in tigecycline MIC was observed following the acquisition of tetA-v1. Without tigecycline treatment, the stability of ptetA-v1 plasmids has been decreasing since day 1. The ptetA-v1 plasmid in Kp89, Kp91, and Kp93 exhibited a decrease of about 20% compared to the pBBR1MCS-5 plasmid. The acquisition of the tetA-v1 gene could inhibit the growth ability of K. pneumoniae strains both in vitro and in vivo. tetA-v1 gene imposed a fitness cost in K. pneumoniae, particularly in the CRKP strain Kp51, with a W value of approximately 0.56., Conclusion: The presence of tetA-v1 is associated with a significant fitness cost in K. pneumoniae in the absence of tigecycline, both in vitro and in vivo., Competing Interests: Competing interests All authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: The aim of this study was to characterise the first complete genome of Porphyromonas pogonae strain PP01-1 of human origin in China., Methods: The Illumina NovaSeq 6000 (200X coverage) and Nanopore MinION platforms (100× coverage) were used for genome sequencing. A de novo hybrid assembly of short Illumina reads and long MinION reads was performed using Unicycler (v.0.5.0). Genome annotation of PP01-1 was performed using the prokaryotic gene-prediction tool Prokka1.14.6. The genome was further analysed using several bioinformatics tools, including ResFinder, VFDB, VirulenceFinder, Type Strain Genome Server, AntiSMASH, PathogenFinder, MobileElementfinder, CRISPRFinder, and IslandViewer., Results: The assembled circular genome of P. pogonae strain PP01-1 was 2 916 423 bp in length, with a GC content of 41.0%, and no plasmid sequence was detected. A total of 2399 coding sequences were predicted by Prokka. PP01-1 harbours antimicrobial resistance genes bla OXA-347 (β-lactamase resistance), tet(Q) (tetracycline resistance), and floR (chloramphenicol and florfenicol resistance)., Conclusions: Here, we are the first to report the complete genome of P. pogonae strain PP01-1 of human origin. In this strain, we first identified bla OXA-347 and tet(Q) in P. pogonae, which will pave the way for further analysis that could identify the potential mechanism of antibiotic resistance and virulence factors in P. pogonae., Competing Interests: Competing interests None declared., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP), a superbug that can be difficult or impossible to treat, has become a worldwide problem. This study presents the first report of a CRKP strain carrying a plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) and the subsequent analysis of its genomic features., Methods: Isolation and identification of bacteria, antimicrobial susceptibility test, whole genome sequencing, and conjugation experiments assay were conducted in clinical epidemiological investigations and plasmid genetic characterisation analysis., Results: A total of 116 strains of bacteria were isolated from patients with bloodstream infections (BSI) between 2018 and 2023. A total of 89.66% of the isolates were carbapenem-resistant Enterobacteriaceae (CRE), with the majority (75/116) being CRKP. Among these, a novel plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) simultaneously was found in CRKP46, and the three genes mediated conjugation by IS26, ISAba125, and IS26, respectively. This plasmid conferred carbapenem resistance to E. coli J53 after conjugative transfer, which was 2 times greater than that of CRKP46., Conclusion: The present study identified the occurrence of a rare plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A), and the spread of these genes was mediated by the corresponding mobile elements. The increased carbapenem resistance created by this novel plasmid challenges public health security and poses a potential threat to human health; therefore, it deserves attention., (Copyright © 2024 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.)

Tigecycline is a last-resort drug used to treat serious infections caused by multidrug-resistant bacteria. tet (X4) is a recently discovered plasmid-mediated tigecycline resistance gene that confers high-level resistance to tigecycline and other tetracyclines. Since the first discovery of tet (X4) in 2019, it has spread rapidly worldwide, and as a consequence, tigecycline has become increasingly ineffective in the clinical treatment of multidrug-resistant infections. In this study, we identified and analyzed tet (X4)-positive Escherichia coli isolates from duck farms in Hunan Province, China. In total, 976 samples were collected from nine duck farms. Antimicrobial susceptibility testing and whole-genome sequencing (WGS) were performed to establish the phenotypes and genotypes of tet (X4)-positive isolates. In addition, the genomic characteristics and transferability of tet (X4) were determined based on bioinformatics analysis and conjugation. We accordingly detected an E. coli strain harboring tet (X4) and seven other resistance genes in duck feces. Multi-locus sequence typing analysis revealed that this isolate belonged to a new clone, and subsequent genetic analysis indicated that tet (X4) was carried in a 4608-bp circular intermediate, flanked by IS Vsa3 -ORF2- abh elements. Moreover, it exhibited transferability to E. coli C600 with a frequency of 10 -5 . The detection of tet (X4)-harboring E, coli strains on duck farms enhances our understanding of tigecycline resistance dynamics. The transferable nature of the circular intermediate of tet (X4) contributing to the spread of tigecycline resistance genes poses a substantial threat to healthcare. Consequently, vigilant monitoring and proactive measures are necessary to prevent their spread., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Jiang, Yang, Xiao, Xiao, Hu, Yang, Sun and Li.)

Mammalian embryo development is initiated by the union of paternal and maternal gametes. Upon fertilization, their epigenome landscape is transformed through a series of finely orchestrated mechanisms that are crucial for survival and successful embryogenesis. Specifically, maternal or oocyte-specific reprogramming factors modulate germ cell specific epigenetic marks into their embryonic states. Rapid and dynamic changes in epigenetic marks such as DNA methylation and histone modifications are observed during early embryo development. These changes govern the structure of embryonic genome prior to zygotic genome activation. Differential changes in epigenetic marks are observed between paternal and maternal genomes because the structure of the parental genomes allows interaction with specific oocyte reprogramming factors. For instance, the paternal genome is targeted by the TET family of enzymes which oxidize the 5-methylcytosine (5mC) epigenetic mark into 5-hydroxymethylcytosine (5hmC) to lower the level of DNA methylation. The maternal genome is mainly protected from TET3-mediated oxidation by the maternal factor, STELLA. The TET3-mediated DNA demethylation occurs at the global level and is clearly observed in many mammalian species. Other epigenetic modulating enzymes, such as DNA methyltransferases, provide fine tuning of the DNA methylation level by initiating de novo methylation. The mechanisms which initiate the epigenetic reprogramming of gametes are critical for proper activation of embryonic genome and subsequent establishment of pluripotency and normal development. Clinical cases or diseases linked to mutations in reprogramming modulators exist, emphasizing the need to understand mechanistic actions of these modulators. In addition, embryos generated via in vitro embryo production system often present epigenetic abnormalities. Understanding mechanistic actions of the epigenetic modulators will potentially improve the well-being of individuals suffering from these epigenetic disorders and correct epigenetic abnormalities in embryos produced in vitro . This review will summarize the current understanding of epigenetic reprogramming by TET enzymes during early embryogenesis and highlight its clinical relevance and potential implication for assisted reproductive technologies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Montgomery, Uh and Lee.)

The Ten-Eleven Translocation (TET) family genes are implicated in a wide array of biological functions across various human cancers. Nonetheless, there is a scarcity of studies that comprehensively analyze the correlation between TET family members and the molecular phenotypes and clinical characteristics of different cancers. Leveraging updated public databases and employing several bioinformatics analysis methods, we assessed the expression levels, somatic variations, methylation levels, and prognostic values of TET family genes. Additionally, we explored the association between the expression of TET family genes and pathway activity, tumor microenvironment (TME), stemness score, immune subtype, clinical staging, and drug sensitivity in pan-cancer. Molecular biology and cytology experiments were conducted to validate the potential role of TET3 in tumor progression. Each TET family gene displayed distinct expression patterns across at least ten detected tumors. The frequency of Single Nucleotide Variant (SNV) in TET genes was found to be 91.24%, primarily comprising missense mutation types, with the main types of copy number variant (CNV) being heterozygous amplifications and deletions. TET1 gene exhibited high methylation levels, whereas TET2 and TET3 genes displayed hypomethylation in most cancers, which correlated closely with patient prognosis. Pathway activity analysis revealed the involvement of TET family genes in multiple signaling pathways, including cell cycle, apoptosis, DNA damage response, hormone AR, PI3K/AKT, and RTK. Furthermore, the expression levels of TET family genes were shown to impact the clinical staging of tumor patients, modulate the sensitivity of chemotherapy drugs, and thereby influence patient prognosis by participating in the regulation of the tumor microenvironment, cellular stemness potential, and immune subtype. Notably, TET3 was identified to promote cancer progression across various tumors, and its silencing was found to inhibit tumor malignancy and enhance chemotherapy sensitivity. These findings shed light on the role of TET family genes in cancer progression and offer insights for further research on TET3 as a potential therapeutic target for pan-cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zhang, Zheng, Liu, Li, Xing, Zhang, Chen, Wang, Shao, Li, Jiang, Pan, Liu, Xu and Wu.)

The emergence and widespread of tigecycline resistance undoubtedly poses a serious threat to public health globally. The exploration of combination therapies has become preferred antibacterial strategies to alleviate this global burden. In this study, tigecycline-resistant tet (X4)-positive Escherichia coli were selected for adjuvant screening. Interestingly, 9-aminominocycline (9-AMC), one of the tigecycline metabolites, exhibits synergistic antibacterial activity with tigecycline using checkerboard assay. The efficacy in vitro and in vivo was evaluated, and the synergistic mechanism was further explored. The results suggested that 9-AMC combined with tigecycline could inhibit the growth of antibiotic resistant bacteria, efficiently retard the evolution of tet (X4) gene and narrow the drug mutant selection window. In addition, the combination of tigecycline and 9-AMC could destroy the normal membrane structure of bacteria, inhibit the formation of biofilm, remarkably reduce the level of intracellular ATP level, and accelerate the oxidative damage of bacteria. Furthermore, 9-AMC is more stable in the bind of Tet(X4) inactivating enzyme. The transcriptomics analysis revealed that the genes related to the 9-AMC and tigecycline were mainly enriched in ABC transporters. Collectively, the results reveal the potentiation effects on tigecycline and the probability of 9-AMC as a novel tigecycline adjuvant against tet (X4)-positive Escherichia coli, which provides new insights for adjuvant screening., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Sun, Zhang, Ma, Ali, Wu and Li.)

Carbapenem-resistant Acinetobacter baumannii (CRAb) is an important pathogen causing serious nosocomial infections. We describe an outbreak of CRAb in an intensive care unit in the Netherlands in 2021. During an outbreak of non-resistant A. baumannii , while infection control measures were in place, CRAb isolates carrying highly similar bla NDM-1 - and tet(x3)- encoding plasmids were isolated from three patients over a period of several months. The chromosomal and plasmid sequences of the CRAb and non-carbapenemase-carrying A. baumannii isolates cultured from patient materials were analysed using hybrid assemblies of short-read and long-read sequences. The CRAb isolates revealed that the CRAb outbreak consisted of two different strains, carrying similar plasmids. The plasmids contained multiple antibiotic resistance genes including the tetracycline resistance gene tet(x3) , and the bla NDM-1 and bla OXA-97 carbapenemase genes. We determined minimal inhibitory concentrations (MICs) for 13 antibiotics, including the newly registered tetracycline antibiotics eravacycline and omadacycline. The CRAb isolates showed high MICs for tetracycline antibiotics including eravacycline and omadacycline, except for minocycline which had a low MIC. In this study we show the value of sequencing multidrug-resistant A. baumannii for outbreak tracking and guiding outbreak mitigation measures.