Search

Paraquat is one of the most widely used nonselective herbicides in agriculture. Due to its wide use, paraquat resistant weeds have emerged and is becoming a potential threat to agriculture. The molecular mechanisms of paraquat resistance in weeds remain largely unknown. Physiological studies indicated that the impaired translocation of paraquat and enhanced antioxidation could improve paraquat resistance in plants. However, the detoxification of paraquat via active metabolism by plants has not been reported to date. Here we report that an activated expression of At1g01600 encoding the P450 protein CYP86A4 confers paraquat resistance as revealed by the gain-of-function mutant paraquat tolerance 11D (pqt11D), in which a T-DNA with four 35S enhancers was inserted at 1646 bp upstream the ATG of At1g01600. The paraquat resistance can be recapitulated in Arabidopsis wild type by overexpressing AtPQT11 (At1g01600), while its knockout mutant is hypersensitive to paraquat. Moreover, AtPQT11 also confers paraquat resistance in E. coli when overexpressed. We further demonstrate that AtPQT11 has P450 enzyme activity that converts paraquat to N-demethyl paraquat nontoxic to Arabidopsis, therefore detoxifying paraquat in plants. Taken together, our results unequivocally demonstrate that AtPQT11/ CYP86A4 detoxifies paraquat via active metabolism, thus revealing a novel molecular mechanism of paraquat resistance in plants and providing a means potentially enabling crops to resist paraquat.

Activation of lignin by demethylation for improving the reactivity has attracted extensive attentions. However, it still faces many challenges, such as the unsatisfied increase of hydroxyl content and the undesired cracking of linear linkages. Here, the efficient demethylations for significantly increasing the hydroxyl content and protecting the structure of industrial lignin were explored using lewis acid as modification reagent. As BBr

Breast carcinoma is one of the common causes of cancer-related mortality in women. Maternally expressed gene 3 (MEG3), a lncRNA located at 14q32, can be involved in carcinogenesis. In this study, we discovered that MEG3 was downregulated by CpG hypermethylation within its gene promoter. Functionally, treatment of breast cancer cells with the DNA methylation inhibitor 5-AzadC as well as silencing of DNA methyltransferase-1 (DNMT1) could decrease the abnormal hypermethylation of the MEG3 promoter, reverse MEG3 expression, inhibit cell proliferation and promote cell apoptosis. In addition, we found that MEG3 expression was negatively correlated with DNMT1. Mechanistically, MEG3 knockdown combined with 5-AzadC or sh-DNMT1 treatment restored the expression of Notch1 receptor, leading to the Notch1 pathway activation, and promoted the progression of epithelial mesenchymal transformation (EMT). Finally, the mice tumor model experiments showed that DNMT1 knockdown can increase MEG3 expression and inhibit tumor growth. Collectively, our findings uncovered that DNMT1-mediated MEG3 demethylation leads to MEG3 upregulation, which in turn inhibits the Notch1 pathway and EMT process in breast cancer.

Oral squamous cell carcinoma (OSCC) is a frequently-diagnosed malignancy with high potential for proliferation and invasion. Histone methylation is known as a crucial mechanism that regulates pathological processes in various cancers, including OSCC.This study sought to delve into the molecular mechanism of lysine demethylase 3 A (KDM3A) in OSCC cell proliferation and invasion.Expression levels of KDM3A, lysin-9 of di-methylated histone H3 (H3K9me2), and doublecortin-like kinase 1 (DCLK1) in cells were determined by reverse-transcription quantitative polymerase chain reaction or Western blot analysis. Cell proliferation and invasion were evaluated by cell counting kit-8, colony formation, and Transwell assays. The enrichment of KDM3A and H3K9me2 on the DCLK1 promoter was determined by chromatin immunoprecipitation assay. The functional rescue experiment was performed with DCLK1 overexpression vector and si-KDM3A in CAL-27 and SCC-9 cells.KDM3A was elevated in OSCC cells. KDM3A knockdown suppressed OSCC proliferation and invasion, along with increased H3K9me2 level in OSCC cells. KDM3A and H3K9me2 were enriched on the DCLK1 promoter and inhibiting H3K9me2 improved DCLK1 expression levels. DCLK1 overexpression neutralized the inhibition of KDM3A knockdown on OSCC proliferation and invasion.KDM3A facilitated OSCC proliferation and invasion by eliminating H3K9me2 to upregulate DCLK1 expression levels.

Resistance to HER2-targeted therapy represents a significant challenge for the successful treatment of patients with breast cancer with HER2-positive tumors. Through a global mass spectrometry–based proteomics approach, we discovered that the expression of the N6-methyladenosine (m6A) demethylase ALKBH5 was significantly upregulated in HER2-targeted therapy-resistant breast cancer cells. Elevated expression of ALKBH5 was sufficient to confer resistance to HER2-targeted therapy, and specific knockdown of ALKBH5 rescued the efficacy of trastuzumab and lapatinib in resistant breast cancer cells. Mechanistically, ALKBH5 promoted m6A demethylation of GLUT4 mRNA and increased GLUT4 mRNA stability in a YTHDF2-dependent manner, resulting in enhanced glycolysis in resistant breast cancer cells. In breast cancer tissues obtained from patients with poor response to HER2-targeted therapy, increased expression of ALKBH5 or GLUT4 was observed and was significantly associated with poor prognosis in the patients. Moreover, suppression of GLUT4 via genetic knockdown or pharmacologic targeting with a specific inhibitor profoundly restored the response of resistant breast cancer cells to trastuzumab and lapatinib, both in vitro and in vivo. In conclusion, ALKBH5-mediated m6A demethylation of GLUT4 mRNA promotes resistance to HER2-targeted therapy, and targeting the ALKBH5/GLUT4 axis has therapeutic potential for treating patients with breast cancer refractory to HER2-targeted therapies. Significance: GLUT4 upregulation by ALKBH5-mediated m6A demethylation induces glycolysis and resistance to HER2-targeted therapy and represents a potential therapeutic target for treating HER2-positive breast cancer.

This study aims to investigate methylmercury (MeHg) demethylation processes in human gut. Here, we determined the compositions and MeHg demethylation rates of gut microbiota in residents from different Hg exposure levels (Wanshan (WS) town and Yangtou (YT) town) and different Hg exposure sources (Zhuchang (ZC) town and YT town) regions. MeHg and inorganic Hg exposure levels in residents of WS town were significantly higher than those of YT and ZC town. Desulfovibrio and Methanogens, which related to Hg methylation/demethylation, showed significantly higher abundance in WS and ZC, comparing with YT. In vitro experiments demonstrated that human intestinal microbiota could degrade MeHg directly. Besides, gut microbiota in WS and ZC exhibited significantly higher demethylation rates than YT, suggesting Desulfovibrio and Methanogens may play important roles in intestinal MeHg demethylation. This study highlights Hg exposure levels and sources may affect demethylation efficiency of gut microbiota, which provides new insights for MeHg demethylation processes in human body.

Background Studies have shown that histone H3 methylation is involved in regulating the differentiation of Bone Marrow Mesenchymal Stem Cells (BMSCs). KDM5B can specifically reduce the level of histone 3 lysine 4 trimethylation (H3K4me3), thereby activating the expression of related genes and participating in biological processes such as cell differentiation, embryonic development and tumor formation. Whether KDM5B is involved in the regulation of BMSCs differentiation into cardiomyocytes through the above manner has not been reported. Objective To investigate the effect of KDM5B on the induction and differentiation of swine BMSCs into myocardial cells in vitro. Methods Swine bone marrow BMSCs were isolated and cultured, and the overexpression, interference expression and blank vector of KMD5B were constructed and transfected by lentivirus. BMSCs was induced to differentiate into cardiomyocytes by 5-azacytidine (5-AZA) in vitro, and the differentiation efficiency was compared by immunofluorescence, RT-PCR, Western Blot and whole-cell patch clamp detection. Result Compared with the control group, the expression levels of histone H3K4me3 and pluripotency gene Nanog in KDM5B overexpression group were significantly decreased, while the expression level of key myocardial gene HCN4 and myocardial marker gene α-Actin and cTNT were significantly increased, and the Na+ current density on the surface of differentiated myocardial cell membrane was significantly increased. Meanwhile, the corresponding results of the KDM5B silent expression group were just opposite. Conclusions It indicated that enhanced KDM5B expression could promote the differentiation of BMSCs into cardiomyocytes and improve the differentiation efficiency by controlling H3K4 methylation levels.

The outcomes of older patients with acute myeloid leukaemia are inferior to their younger counterparts, because, in part, of a more aggressive disease biology and poorer tolerance of cytotoxic chemotherapy. Although intensive chemotherapy was historically considered the only effective treatment for these patients, many older patients are not suitable for intensive chemotherapy owing to comorbidities or general frailty. Determination of patient fitness for intensive chemotherapy is imperfect, and even older patients who appear to be suitable (also known as fit) for intensive chemotherapy can have high rates of morbidity and early and late mortality with this approach. Fortunately, the outcomes of older or unfit patients with acute myeloid leukaemia have substantially improved with the use of a hypomethylating agent plus venetoclax in the front-line setting. Although the formal approval of this combination is limited to patients aged 75 years or older, or those with a clinically significant comorbidity, the high response rates and survival improvement in these patients have led many practitioners to consider this low-intensity regimen in older patients without significant comorbidities and even in younger patients with high-risk disease features for whom the expected outcomes with intensive chemotherapy are poor. Modifications to the hypomethylating agent plus venetoclax backbone might further improve the outlook for these patients, particularly in some acute myeloid leukaemia subsets with a targetable mutation. In this Viewpoint, we review the retrospective and prospective data supporting both intensive chemotherapy and low-intensity venetoclax-based approaches in older patients with acute myeloid leukaemia. We also discuss our own approach to the management of older or unfit patients with acute myeloid leukaemia, including how cytomolecular features have a role in establishing the optimal front-line therapy.

The aim of this study was to investigate the expression and the promoter methylation level of PLAGL1 gene and the mechanism of epigallocatechin gallate (EGCG) that induces PLAGL1 gene demethylation and promotes the apoptosis of pheochromocytoma (PCC) in PC12 cell line.The PC12 cells were treated with 25, 50, 75, 100, and 150 μg/mL EGCG for 48 hours. MSP was used to examine PLAGL1 gene methylation and an MTT assay was performed to detect the cell proliferation. The cell apoptosis was detected using flow cytometry. The mRNA and protein expressions of DNMT1, PLAGL1, Wnt, and β-catenin were detected using RT-quantitative PCR and Western blot.EGCG dose-dependently reduced the cell viability and reversed PLAGL1 gene hypermethylation in PC12 cells (P0.05). The cell apoptosis was significantly increased in PC12 cells treated with EGCG. The EGCG treatment restored the expressions of PLAGL1 and downregulated the expression of DNMT1, Wnt, and β-catenin in PC12 cells (P0.05).The EGCG induces the demethylation process of PLAGL1 gene through down-regulating DNMT1 and restores the PLAGL1 mRNA and protein expression. The Wnt/β-catenin signaling pathway is involved in the regulation of PCC cell apoptosis promoted by EGCG inducing PLAGL1 gene demethylation.

AlkB homologue 5 (ALKBH5) is a ferrous iron and 2-oxoglutarate dependent oxygenase that demethylates RNA N6-methyladenosine (m6A), a post-transcriptional RNA modification with an emerging set of regulatory roles. Along with the fat mass and obesity-associated protein (FTO), ALKBH5 is one of only two identified human m6A RNA oxidizing enzymes and is a potential target for cancer treatment. Unlike FTO, ALKBH5 efficiently catalyzes fragmentation of its proposed nascent hemiaminal intermediate to give formaldehyde and a demethylated nucleoside. A detailed analysis of the molecular mechanisms used by ALKBH5 for substrate recognition and m6A demethylation is lacking. We report three crystal structures of ALKBH5 in complex with an m6A-ssRNA 8-mer substrate and supporting biochemical analyses. Strikingly, the single-stranded RNA substrate binds to the active site of ALKBH5 in a 5′-3′ orientation that is opposite to single-stranded or double-stranded DNA substrates observed for other AlkB subfamily members, including single-stranded DNA bound to FTO. The combined structural and biochemical results provide insight into the preference of ALKBH5 for substrates containing a (A/G)m6AC consensus sequence motif. The results support a mechanism involving formation of an m6A hemiaminal intermediate, followed by efficient ALKBH5 catalyzed demethylation, enabled by a proton shuttle network involving Lys132 and Tyr139.

5-Demethylated polymethoxyflavones (5-OH PMFs) are the most unique monodemethylated PMFs with relatively low polarities and are proved to possess better anticancer and anti-inflammatory effects than their respective permethoxylated ones. However, their detailed

Chemotherapy is used for recurrent and metastatic colorectal cancer, but the response rate of 5-fluorouracil (5-FU), the standard treatment for colorectal cancer, is low. We hypothesized that thymidine phosphorylase (TYMP) expression, a rate-limiting activating enzyme of 5-FU, is regulated by methylation of the gene promoter region, and demethylation of TYMP would increase sensitivity to 5-FU.HCT116 colon cancer cells were treated with 5-aza-2'-deoxycytidine, a demethylating agent, and changes in TYMP transcription and sensitivity to 5-FU were evaluated.TYMP expression increased over 54-fold in HCT116 transfected with TYMP. The cytotoxicity of 5-FU increased up to 5.5-fold. In comparison, in HCT116 treated with 5-aza-2'-deoxycytidine, TYMP expression increased 5.8-fold. However, the cytotoxicity of 5-FU remained unchanged.Demethylating agent alone did not promote the cytotoxicity of 5-FU against colorectal cancer. To further increase the sensitivity to 5-FU, combination with adjuvant therapy focusing on metabolic pathways other than the TYMP pathway appear necessary.

The implications of the CD40-CD40 ligand (CD40L) signaling pathway in systemic lupus erythematosus (SLE) were well documented, due to its important role among immune cells. Previous research found that 3-hydroxy butyrate dehydrogenase 2 (BDH2), a modulator of intracellular iron homeostasis and iron transportation promoted the pathogenic process of SLE by regulating the demethylation of cd70, cd11a, and cd40l genes among CD4 + T cells. The purpose of this study was to explore the role of BDH2 in oxidative damage-induced SLE. First, CD4 + T cells treated with H2O2 were injected into the tail vein of mice to establish a lupus model. CD40L knockdown significantly decreased CD40L expression on CD4 + T cells in the spleen of SLE mice. Compared with SLE model mice, the levels of serum anti-dsDNA antibody and urinary protein in the CD40L interference group were significantly decreased. CD40L knockdown alleviated the immune complex glomerulonephritis in syngeneic SLE mice. Moreover, the levels of IFN-γ and IL-2 were decreased. However, IL-4 and IL-10 levels were significantly upregulated in the serum of CD40L knockdown SLE mice, compared with SLE model mice. Accordingly, CD40L knockdown reduced Th1/Th2 percentage in SLE mice. Inhibiting the expression of BDH2 of CD4 + T cells promoted the demethylation of CD40L, while it inhibited cell proliferation, elevated oxidative stress through increased expression of CD40L, and thus, promoted the progress of SLE. Our results demonstrate that BDH2 aggravates the pathologic progression of SLE in mice, by increasing the demethylation level of CD40L among CD4 + T cells.

The goal this review is to clarify the effects of the fat mass and obesity-associated protein (FTO) in lipid metabolism regulation and related underlying mechanisms through the FTO-mediated demethylation of m6A modification. FTO catalyzes the demethylation of m6A to alter the processing, maturation and translation of the mRNAs of lipid-related genes. FTO overexpression in the liver promotes lipogenesis and lipid droplet (LD) enlargement and suppresses CPT-1–mediated fatty acid oxidation via the SREBP1c pathway, promoting excessive lipid storage and nonalcoholic fatty liver diseases (NAFLD). FTO enhances preadipocyte differentiation through the C/EBPβ pathway, and facilitates adipogenesis and fat deposition by altering the alternative splicing of RUNX1T1, the expression of PPARγ and ANGPTL4, and the phosphorylation of PLIN1, whereas it inhibits lipolysis by inhibiting IRX3 expression and the leptin pathway, causing the occurrence and development of obesity. Suppression of the PPARβ/δ and AMPK pathways by FTO-mediated m6A demethylation damages lipid utilization in skeletal muscles, leading to the occurrence of diabetic hyperlipidemia. m6A demethylation by FTO inhibits macrophage lipid influx by downregulating PPARγ protein expression and accelerates cholesterol efflux by phosphorylating AMPK, thereby impeding foam cell formation and atherosclerosis development. In summary, FTO-mediated m6A demethylation modulates the expression of lipid-related genes to regulate lipid metabolism and lipid disorder diseases.

Neuronal pentraxin 2 (NPTX2), a secretory protein of neuronal pentraxins, was first identified in the nervous system. Several studies have shown that expression levels of NPTX2 are associated with the development of various cancers. However, whether NPTX2 is involved in prostate cancer progression is unclear. Herein, we found that NPTX2 is significantly reduced in prostate cancer tissues and cancer cell lines compared to control prostate tissues and control prostatic epithelial cell lines. Furthermore, the NPTX2 promoter is highly methylated in prostate cancer cells. Consistently, NPTX2 could be restored by treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (decitabine, 5-AZA-dC). Overexpression of NPTX2 inhibited prostate cancer cell proliferation both in vitro and in vivo. In conclusion, our study demonstrated that NPTX2 acts as a tumor suppressor gene in prostate cancer.

In anoxic environments, anaerobic microorganisms carrying the hgcAB gene cluster can mediate the transformation of inorganic mercury (Hg(II)) to monomethylmercury (MMHg). The kinetics of Hg(II) transformation to MMHg in periphyton from East Fork Poplar Creek (EFPC) in Oak Ridge, TN have previously been modeled using a transient availability model (TAM). The TAM for Hg(II) methylation combines methylation/demethylation kinetics with kinetic expressions for processes that decrease Hg(II) and MMHg availability for methylation and demethylation (multisite sorption of Hg(II) and MMHg, Hg(II) reduction/Hg(0) oxidation). In this study, the TAM is used for the first time to describe MMHg production in sediment. We assessed MMHg production in sediment microcosms using two different sediment types from EFPC: a relatively anoxic, carbon-rich sediment with higher microbial activity (higher CO2 production from sediment) and a relatively oxic, sandy, carbon-poor sediment with lower microbial activity (lower CO2 production from sediment). Based on 16s rRNA sequencing, the overall microbial community structure in the two sediments was retained during the incubations. However, the hgcA containing methanogenic Euryarchaeota communities differed between sediment types and their growth followed different trajectories over the course of incubations, potentially contributing to the distinct patterns of MMHg production observed. The general TAM paradigm performed well in describing MMHg production in the sediments. However, the MMHg production and ancillary data suggested the need to revise the model structure to incorporate terms for concentration-dependent microbial activity over the course of the incubations. We modified the TAM to include Monod-type kinetics for methylation and demethylation and observed an improved fit for the carbon-rich, microbially active sediment. Overall our work shows that the TAM can be applied to describe Hg(II) methylation in sediments and that including expressions accounting for concentration-dependent microbial activity can improve the accuracy of the model description of the data in some cases.

Background Identification of novel biomarker is important for development of molecular-targeted therapy agents for patients with hepatocellular carcinoma (HCC). This study aims to identify potential prognostic biomarkers and investigate epigenetic mechanism of HCC development. Methods Public bulk-RNA seq datasets and proteomic dataset were screened for identification of potential prognostic biomarkers for HCC patients. Public methylomic datasets were analyzed for deciphering the epigenetic mechanism regulating HCC-associated gene expression. Immunoblotting, immunohistochemistry, real-time PCR, and pyrosequencing were used to validate the findings from bioinformatic analyses. Results Minichromosome maintenance complex component 2 (MCM2) and nucleoporin 37 (NUP37) were overexpressed in human HCC tissues and hepatoma cell lines. MCM2 significantly positively correlated with NUP37 expression. Higher expression of MCM2 or NUP37 was significantly associated with advanced tumor stage and worse overall survival in 3 large independent HCC cohorts (n = 820). MCM2 and NUP37 overexpression are independent prognostic risk factors for HCC patients. Demethylation at an enhancer of MCM2 gene was a common event in patients with HCC, which significantly negatively correlated with MCM2 and NUP37 mRNA expression. Conclusions Demethylation at enhancer regulates MCM2 and NUP37 expression in HCC. MCM2 and NUP37 are promising prognostic biomarkers and potential targets for epigenetic therapy in HCC patients.

Folic acid (FA) supplementation in early pregnancy is recommended to protect against birth defects. But excess FA has exhibited neurodevelopmental toxicity. We previously reported that the mice treated with 2.5-fold the dietary requirement of FA one week before mating and throughout pregnancy and lactation displayed abnormal behaviors in the offspring. Here we found the levels of non-phosphorylated β-catenin (active) were increased in the brains of weaning and adult FA-exposed offspring. Meanwhile, demethylation of protein phosphatase 2 A catalytic subunit (PP2Ac), which suppresses its enzyme activity in regulatory subunit dependent manner, was significantly inhibited. Among the upstream regulators of β-catenin, PI3K/Akt/GSK-3β but not Wnt signaling was stimulated in FA-exposed brains only at weaning. In mouse neuroblastoma N2a cells, knockdown of PP2Ac or leucine carboxyl methyltransferase-1 (LCMT-1), or overexpression of PP2Ac methylation-deficient mutant decreased β-catenin dephosphorylation. These results suggest that excess FA may activate β-catenin via suppressing PP2Ac demethylation, providing a novel mechanism for the influence of FA on neurodevelopment.

Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.

Deep eutectic solvent (DES) is a kind of green solvent for biorefinery, which favors the progress of being more environmentally friendly and effective. A better understanding of structural changes of lignin is necessary to optimize pretreatment conditions and efficient utilization of the resultant lignin. The current study reported the structural features of lignin recovered from alkaline ChCl/imidazole and ChCl/urea DES pretreatment, and the mechanism of lignin modification was revealed. The profiling demonstrated that lignin samples possessed a high purity (>94.4%), low molecular weight ranging from 1544 to 2562 g/mol and an excellent uniformity (PDI < 1.6). Noteworthy, the content of β-O-4' linkages in lignin was over 75% (i.e. 72.2%-77.4% retention); S/G ratio was increased whereas the content of -OCH3 groups were decreased. It was revealed that slight cleavage of β-O-4' linkages, preferential breakdown of G units, and demethylation reaction were occurred during alkaline ChCl-based DES pretreatment. Specifically, cleavage of ester linkages between PB and lignin macromolecule was taking place during ChCl/imidazole pretreatment at a high temperature; whereas oxidation only appeared in ChCl/urea system. Despite the modification, well β-O-4' preserved and less condensed lignin samples were recovered after low-temperature pretreatment. Consequently, high contents of phenol derivatives (26.3-30.6%) were achieved in lignin oil. The present study provides critical information on alkaline ChCl-based DES pretreatment, which will contribute to the valorization of lignin by-products and will be beneficial to the development of biorefineries.

Objectives DNA hydroxymethylation may be induced by oxidative stress in lupus patients, so we investigated the association between DNA hydroxymethylation and demethylation with the antioxidant response. Methods A case–control study was performed including lupus patients and matched healthy controls. Serum concentration of glutathione (GSH), glutathione disulphide (GSSG), superoxide dismutase (SOD) and total antioxidant capacity (TAC), 5-mC and 5-hmC were determined. Results One hundred and forty-two patients and 34 controls were included. 5-hmC levels were lower in SLE patients than in controls. GSH and GSSG values were lower in patients, while SOD levels were higher in patients. TAC did not show significant differences, but higher demethylation and lower hydroxydemethylation were associated to increased TAC values. Lower demethylation was associated with cytopenia and lower hydroxymethylation with longer course of the disease. Lower levels of GSH and GSSG and higher SOD values were associated with accumulated damage assessed by SLICC-ACR. Conclusions Lower hydroxymethylation in patients than in controls was observed. Moreover, higher demethylation and lower hydroxymethylation leads to high TAC levels. DNA hydroxymethylation seems to be related to longer course of the disease.

Background Isocitrate Dehydrogenase 1/2 (IDH1/2) mutations are diagnostic for Astrocytoma or Oligodendroglioma, IDH-mutant. In these IDH-mutant gliomas, retinoic acid-related gene expression is commonly silenced by DNA hypermethylation. DNA demethylating agents can epigenetically reprogram IDH-mutant cells and reduce proliferation, likely by re-expression of silenced tumor suppressor pathways. We hypothesized that DNA demethylation might restore the retinoic acid pathway and slow tumor growth. This was the rationale for a preclinical evaluation combining the DNA demethylating agent, 5-Azacytidine (5-Aza), and retinoic acid pathway activation with all-trans retinoic acid (atRA) in IDH-mutant glioma. Methods In this study, we evaluated the effect of 5-Aza and atRA combination on cell proliferation, apoptosis, and gene expression in human glioma cells. In addition, the efficacy of this combination was tested in patient-derived xenograft (PDX) bearing the IDH1R132H mutation, utilizing subcutaneous and orthotopic models. Results 5-Aza reduced the DNA methylation profile and increased the gene expression of retinoic acid-related genes. Combination of 5-Aza and atRA reduced cell growth, increased differentiation marker expression, and apoptosis in IDH1R132H glioma cells. Mechanistically, 5-Aza sensitized IDHIR132H glioma cells to atRA via upregulation of the retinoic acid pathway. Importantly, the drug combination reduced significantly the growth rate of subcutaneous tumors, but in an orthotopic mouse model, the combination did not improve survival and 5-Aza alone provided the best survival benefit. Conclusion Use of DNA demethylating agent in combination with retinoids shows promise, but further optimization and preclinical studies are required for treatment of intracranial IDH-mutant gliomas.

The public and environmental health consequences of mercury (Hg) methylation have drawn much attention and considerable research to Hg methylation processes and their dynamics in diverse environmen...

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We investigated the role of epigenetic DNA modifications in childhood acute lymphoblastic leukemia (ALL) diagnostics and therapy monitoring. We analyzed the levels of 5-methyl-2′-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC–MS/MS). Moreover, the expression of Ten Eleven Translocation enzymes (TETs) at the mRNA and protein levels was determined. Additionally, the ascorbate level in the blood plasma was analyzed. Before treatment, the ALL patients had profoundly higher levels of the analyzed modified DNA in their urine than the controls. After chemotherapy, we observed a statistically significant decrease in active demethylation products in urine, with a final level similar to the level characteristic of healthy children. The level of 5-hmdC in the DNA of the leukocytes in blood of the patient group was significantly lower than that of the control group. Our data suggest that urinary excretion of epigenetic DNA modification may be a marker of pediatric ALL status and a reliable marker of chemotherapy response.

Methoxy-prism[5]arene PrS[5]Me is demethylated by a supramolecularly assisted reaction. In the presence of a tetramethylammonium cation, PrS[5]Me is demethylated by BBr3 in high yield, while in its absence a 55/40 mixture of PrS[5]OH/PrS[6]OH is formed. The dealkylation of prismarenes, such as PrS[6]R (R = Et, nPr) and c-PrS[5]Me, can be easily obtained in high yields in the presence of BBr3.

Berberine (BBR) is an isoquinoline alkaloid extracted from Coptis chinensis that improves diabetes, hyperlipidemia and inflammation. Due to the low oral bioavailability of BBR, its mechanism of action is closely related to the gut microbiota. This study focused on the CYP51 enzyme of intestinal bacteria to elucidate a new mechanism of BBR transformation by demethylation in the gut microbiota through multiple analytical techniques. First, the docking of BBR and CYP51 was performed; then, the pharmacokinetics of BBR was determined in ICR mice in vivo, and the metabolism of BBR in the liver, kidney, gut microbiota and single bacterial strains was examined in vitro. Moreover, 16S rRNA analysis of ICR mouse feces indicated the relationship between BBR and the gut microbiota. Finally, recombinant E. coli containing cyp51 gene was constructed and the CYP51 enzyme lysate was induced to express. The metabolic characteristics of BBR were analyzed in the CYP51 enzyme lysate system. The results showed that CYP51 in the gut microbiota could bind stably with BBR, and the addition of voriconazole (a specific inhibitor of CYP51) slowed down the metabolism of BBR, which prevented the production of the demethylated metabolites thalifendine and berberrubine. This study demonstrated that CYP51 promoted the demethylation of BBR and enhanced its intestinal absorption, providing a new method for studying the metabolic transformation mechanism of isoquinoline alkaloids in vivo., Graphical abstract Image 1, Highlights • The demethylation metabolism of natural drugs difficult to absorb through the gut microbiota was first reported. • Six different methods were presented to explain the metabolic mechanism of natural isoquinoline alkaloids. • The findings provided a new idea for studying the mechanism of drug metabolism of gut microbiota.

Asian grapevine leaf rust, caused by Neophysopella meliosmae-myrianthae and N. tropicalis, is often controlled by quinone outside inhibitor (QoI) and demethylation inhibitor (DMI) fungicides in Brazil. Here, we evaluated the sensitivity of 55 Neophysopella spp. isolates to pyraclostrobin (QoI) and tebuconazole (DMI). To elucidate the resistance mechanisms, we analyzed the sequences of the cytochrome b (CYTB) and cytochrome P450 sterol 14α-demethylase (CYP51) target proteins of QoI and DMI fungicides, respectively. The CYP51 expression levels were also determined in a selection of isolates. In leaf disc assays, the mean 50% effective concentration (EC50) value for pyraclostrobin was about 0.040 µg/ml for both species. CYTB sequences were identical among all 55 isolates, which did not contain an intron immediately after codon 143. No amino acid substitution was identified at codons 129, 137, and 143. The mean EC50 value for tebuconazole was 0.62 µg/ml for N. tropicalis and 0.46 µg/ml for N. meliosmae-myrianthae, and no CYP51 sequence variation was identified among isolates of the same species. However, five N. meliosmae-myrianthae isolates grew on leaf discs treated at 10 µg/ml tebuconazole, and these were further exposed to tebuconazole selection pressure. Tebuconazole-adapted laboratory isolates of N. meliosmae-myrianthae showed an eight- to 25-fold increase in resistance after four rounds of selection that was not associated with CYP51 target alterations. In comparison with sensitive isolates, CYP51 expression was induced in the presence of tebuconazole in three out of four tebuconazole-adapted isolates tested. These results suggest a potential risk for QoI and DMI resistance development in Neophysopella spp.

Wei Shang, Xin Zhao, Fan Yang, Dongyi Wang, Le Lu, Zihan Xu, Zhiming zhao, Hui Cai, Junyi Shen Department of Integrated Traditional and Western Medicine, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, 210002, People’s Republic of ChinaCorrespondence: Junyi Shen, Email sauron84@163.comBackground: Compared with traditional drugs, nanomaterial drugs have the benefits of improving the solubility, bioavailability, and absorption rate of insoluble drugs. Nanoporous complexes can increase the efficiency with which drugs can penetrate the blood-brain barrier and reach target organs. Ginsenoside Rg1 is an effective drug that promotes angiogenesis. Ginsenoside Rg1 composite nanoparticles were employed to induce the expression of several key epigenetic enzymes and then activate the VEGF and Notch pathways after the onset of ischemic brain lesions.Methods: We constructed nanoparticles to fully encapsulate the therapeutic drug (ginsenoside Rg1), which can be transferred into brain tissue via the receptor-mediated transfer of drug-encapsulated nanoparticles. Evaluation of the therapeutic effect of ginsenoside Rg1 complex nanovesicles (CNV) was performed by in vitro and in vivo experiments. Real-time polymerase chain reaction (RT- PCR), Western blot, immunohistochemistry staining (IHC), and Co-immunoprecipitation (co-IP) were employed to screen for epigenetic enzymes with an up-regulated expression post ginsenoside Rg1-CNV intervention. RNA sequencing, shRNA knockdown, and chromatin Immunoprecipitation (ChIP) sequencing were performed to detect the target genes of ginsenoside Rg1-CNV that regulate angiogenesis. Then, bioinformatic analysis was performed to investigate the mechanism of action of epigenetic modifying enzymes in regulating target genes.Results: The average of the synthesized ginsenoside Rg1-CNV was 203.78± 6.83 nm, the polydispersion index was 0.135± 0.007, and the Zeta potential was 23.13± 1.65 mV. Through in vivo and in vitro experiments, we found that it promotes the proliferation, migration, and tubular formation of brain microvascular endothelial cells (BMECs). Meanwhile, the intervention of ginsenoside Rg1-CNV promoted the demethylation of H3K27me3 within the promoter region of VEGF-A and Jagged1 genes and reduced the H3K27me3 modification within this region.Conclusion: The ginsenoside Rg1 nanoparticles may be an available blood-brain barrier penetrating agent for ischemic stroke.Keywords: ginsenoside Rg1, nanoparticles, angiogenesis, blood-brain barrier, epigenetic, ischemic stroke

In this work, nine new bromophenol derivatives were designed and synthesized. The alkylation reactions of (2-bromo-4,5-dimethoxyphenyl)methanol (7) with substituted benzenes 8–12 produced new diaryl methanes 13–17. Targeted bromophenol derivatives 18–21 were synthesized via the O-Me demethylation of diaryl methanes with BBr3. Moreover, the synthesized bromophenol compounds were tested with some metabolic enzymes such as acetylcholinesterase (AChE), carbonic anhydrase I (CA I), and II (CA II) isoenzymes. The novel synthesized bromophenol compounds showed Ki values that ranged from 2.53 ± 0.25 to 25.67 ± 4.58 nM against hCA I, from 1.63 ± 0.11 to 15.05 ± 1.07 nM against hCA II, and from 6.54 ± 1.03 to 24.86 ± 5.30 nM against AChE. The studied compounds in this work exhibited effective hCA isoenzyme and AChE enzyme inhibition effects. The results show that they can be used for the treatment of glaucoma, epilepsy, Parkinson’s as well as Alzheimer’s disease (AD) after some imperative pharmacological studies that would reveal their drug potential.

Methylmercury (MeHg) persists today as a priority public health concern. Mechanisms influencing MeHg metabolism, kinetics, and toxicity outcomes are therefore essential knowledge for informing exposure risks. Evidence points to different toxic potencies of MeHg and inorganic mercury (Hg2+), highlighting the role for biotransformation (demethylation) in regulating MeHg toxicokinetics/dynamics. Whereas microbial MeHg demethylation in the gut is seen to influence elimination kinetics, the potential for systemic demethylation in tissues and target organs to influence MeHg toxicity remains uncertain. To investigate the consequences of systemic MeHg demethylation across development, we engineered transgenic Drosophila to express the bacterial organomercurial lyase enzyme (merB) in a targeted and tissue-specific manner. With all combinations of merB-induced demethylation, ubiquitously (via an actin promoter) or in a tissue-specific manner (ie, gut, muscle, neurons), we observe a rescue of MeHg-induced eclosion failure at the pupal to adult transition. In MeHg-fed larvae with ubiquitous or targeted (gut and muscle) merB expression, we see a significant decrease in MeHg body burden at the pupal stage relative to control flies. We also observe a significant increase in the MeHg elimination rate with merB demethylation induced in adults (control, t1/2 = 7.2 days; merB flies, t1/2 = 3.1 days). With neuronal-specific merB expression, we observe a rescue of MeHg-induced eclosion failure without a decrease in Hg body burden, but a redistribution of Hg away from the brain. These results demonstrate the previously unidentified potential for intracellular MeHg demethylation to promote transport and elimination of Hg, and reduce developmental MeHg toxicity. Impact Statement: These findings demonstrate the potential for MeHg demethylation in situ to contribute significantly to the MeHg elimination and distribution kinetics of whole animals and thereby affords a means of protection against the toxic insult of MeHg. Therefore, this study reveals important insight into processes that can determine an individual’s resistance or susceptibility to MeHg and provides rationale for therapies targeting a novel metabolism-based pathways to alleviate toxicity risk stemming from MeHg exposure.

Fat mass and obesity-associated protein (FTO) regulating the N6-methyladenine (m

© 2022 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.Background: Lipopolysaccharide-responsive beige-like anchor protein (LRBA) deficiency and cytotoxic T-lymphocyte protein-4 (CTLA-4) insufficiency are recently described disorders that present with susceptibility to infections, autoimmunity, and lymphoproliferation. Clinical and immunological comparisons of the diseases with long-term follow-up have not been previously reported. We sought to compare the clinical and laboratory manifestations of both diseases and investigate the role of flow cytometry in predicting the genetic defect in patients with LRBA deficiency and CTLA-4 insufficiency. Methods: Patients were evaluated clinically with laboratory assessments for lymphocyte subsets, T follicular helper cells (TFH), LRBA expression, and expression of CD25, FOXP3, and CTLA4 in regulatory T cells (Tregs) at baseline and 16 h post-stimulation. Results: LRBA-deficient patients (n = 29) showed significantly early age of symptom onset, higher rates of pneumonia, autoimmunity, chronic diarrhea, and failure to thrive compared to CTLA-4 insufficiency (n = 12). In total, 29 patients received abatacept with favorable responses and the overall survival probability was not different between transplanted versus non-transplanted patients in LRBA deficiency. Meanwhile, higher probability of survival was observed in CTLA-4-insufficient patients (p = 0.04). The T-cell subsets showed more deviation to memory cells in CTLA-4-insufficiency, accompanied by low percentages of Treg and dysregulated cTFH cells response in both diseases. Cumulative numbers of autoimmunities positively correlated with cTFH frequencies. Baseline CTLA-4 expression was significantly diminished in LRBA deficiency and CTLA-4 insufficiency, but significant induction in CTLA-4 was observed after short-term T-cell stimulation in LRBA deficiency and controls, while this elevation was less in CTLA-4 insufficiency, allowing to differentiate this disease from LRBA deficiency with high sensitivity (87.5%) and specificity (90%). Conclusion: This cohort provided detailed clinical and laboratory comparisons for LRBA deficiency and CTLA-4 insufficiency. The flow cytometric approach is useful in predicting the defective gene; thus, targeted sequencing can be conducted to provide rapid diagnosis and treatment for these diseases impacting the CTLA-4 pathway.

Pancreatic cancer (PC) is an aggressive human cancer. Appropriate methods for the diagnosis and treatment of PC have not been found at the genetic level, thus making epigenetics a promising research path in studies of PC. Histone methylation is one of the most complicated types of epigenetic modifications and has proved crucial in the development of PC. Histone methylation is a reversible process regulated by readers, writers, and erasers. Some writers and erasers can be recognized as potential biomarkers and candidate therapeutic targets in PC because of their unusual expression in PC cells compared with normal pancreatic cells. Based on the impact that writers have on the development of PC, some inhibitors of writers have been developed. However, few inhibitors of erasers have been developed and put to clinical use. Meanwhile, there is not enough research on the reader domains. Therefore, the study of erasers and readers is still a promising area. This review focuses on the regulatory mechanism of histone methylation, and the diagnosis and chemotherapy of PC based on it. The future of epigenetic modification in PC research is also discussed.

The effects of low-dose radiation (LDR, ≤0.1 Gy) on living organisms have been the hot areas of radiation biology but do not reach a definitive conclusion yet. So far, few studies have adequately accounted for the male reproductive system responses to LDR, particularly the regulation of testosterone content. Hence, this study was designed to evaluate the effects of LDR on Leydig cells and testicular tissue, especially the ability to synthesize testosterone. We found that less than 0.2-Gy 60 Co gamma rays did not cause significant changes in the hemogram index and the body weight; also, pathological examination did not find obvious structural alterations in testis, epididymis, and other radiation-sensitive organs. Consistently, the results from in vitro showed that only more than 0.5-Gy gamma rays could induce remarkable DNA damage, cycle arrest, and apoptosis. Notably, LDR disturbed the contents of testosterone in mice serums and culture supernatants of TM3 cells and dose dependently increased the expression of 3β-HSD. After cotreatment with trilostane (Tril), the inhibitor of 3β-HSD, increased testosterone could be partially reversed. Besides, DNA damage repair-related enzymes, including DNMT1, DNMT3B, and Sirt1, were increased in irradiated TM3 cells, accompanying by evident demethylation in the gene body of 3β-HSD. In conclusion, our results strongly suggest that LDR could induce obvious perturbation in the synthesis of testosterone without causing organic damage, during which DNA demethylation modification of 3β-HSD might play a crucial role and would be a potential target to prevent LDR-induced male reproductive damage.

Background The long noncoding RNA, cardiac autophagy inhibitory factor (CAIF), and microRNA (miR)‐16 are reported to be involved in lipopolysaccharide (LPS)‐induced inflammatory responses and cell apoptosis in many diseases. Herein, we investigated the interaction between CAIF and miR‐16 in sepsis‐induced chronic heart failure (CHF). Methods The expression of CAIF and miR‐16 in plasma samples from sepsis‐induced CHF patients (n = 60) and healthy controls (n = 60) were measured using quantitative reverse‐transcription polymerase chain reaction (qRT‐PCR). The correlations between CAIF and miR‐16 across plasma samples from patients with sepsis‐induced CHF and healthy controls were analyzed using linear regression. The messenger RNA (mRNA) levels of inducible nitric oxide synthase, C‐C motif chemokine 2 (CCL2), growth‐regulated alpha protein (CXCL1), and interleukin‐6 (IL‐6) were evaluated using qRT‐PCR while nuclear factor κB activation was evaluated using luciferase assay. Results The expression levels of CAIF and miR‐16 were downregulated in the plasma of sepsis‐induced CHF patients and were positively correlated in these patients. In cardiomyocytes, LPS treatment dose‐dependently decreased CAIF and miR‐16 levels. CAIF overexpression increased miR‐16 expression by demethylating miR‐16. CAIF and/or miR‐16 overexpression suppressed LPS‐induced CCL2, CXCL1, and IL‐6 expression at both the mRNA and protein levels. Analysis of cell apoptosis and western blot analysis showed that CAIF and/or miR‐16 overexpression inhibited LPS‐induced cardiomyocyte apoptosis by reducing Bax and cleaved caspase 3 levels and enhancing Bcl‐2 levels. Conclusion Our study is the first to report the abnormal expression of CAIF and miR‐16 in heart disease. CAIF plays a protective role in sepsis‐induced CHF by inhibiting cardiomyocyte apoptosis and inflammation, possibly by regulating miR‐16 demethylation.

Plants have a close relationship with their root microbiota, which comprises a complex microbial network. Histone methylation is an important epigenetic modification influencing multiple plant traits; however, little is known about the role of plant histone methylation in the assembly and network structure of the root microbiota. In this study, we established that the rice (Oryza sativa) histone methylation regulates the structure and composition of the root microbiota, especially the hub species in the microbial network. DJ-jmj703 (defective in histone H3K4 demethylation) and ZH11-sdg714 (defective in H3K9 methylation) showed significant different root microbiota compared with the corresponding wild types at the phylum and family levels, with a consistent increase in the abundance of Betaproteobacteria and a decrease in the Firmicutes. In the root microbial network, 35 of 44 hub species in the top 10 modules in the tested field were regulated by at least one histone methylation-related gene. These observations establish that the rice histone methylation plays a pivotal role in regulating the assembly of the root microbiota, providing insights into the links between plant epigenetic regulation and root microbiota.

Background Chemotherapy resistance remains a barrier to improving the prognosis of epithelial ovarian cancer (EOC). ALKBH5 has recently been shown to be one of the RNA N6-methyladenosine (m6A) demethyltransferases associated with various cancers, but its role in cancer therapeutic resistance remains unclear. This study aimed to investigate the role of AlkB homolog 5 (ALKBH5) in cisplatin-resistant EOC. Methods Functional assays were performed both in vitro and in vivo. RNA sequencing (RNA-seq), m6A-modified RNA immunoprecipitation sequencing (MeRIP-seq), chromatin immunoprecipitation, RNA immunoprecipitation, and luciferase reporter and actinomycin-D assays were performed to investigate RNA/RNA interaction and m6A modification of the ALKBH5-HOXA10 loop. Results ALKBH5 was upregulated in cisplatin-resistant EOC and promoted cancer cell cisplatin resistance both in vivo and in vitro. Notably, HOXA10 formed a loop with ALKBH5 and was found to be the upstream transcription factor of ALKBH5. HOXA10 overexpression also facilitated EOC cell chemoresistance both in vivo and in vitro. Collective results of MeRIP-seq and RNA-seq showed that JAK2 is the m6A-modified gene targeted by ALKBH5. The JAK2/STAT3 signaling pathway was activated by overexpression of the ALKBH5-HOXA10 loop, resulting in EOC chemoresistance. Cell sensitivity to cisplatin was rescued by ALKBH5 and HOXA10 knockdown or inhibition of the JAK2/STAT3 signaling pathway in EOC cells overexpressing ALKBH5-HOXA10. Conclusions The ALKBH5-HOXA10 loop jointly activates the JAK2/STAT3 signaling pathway by mediating JAK2 m6A demethylation, promoting EOC resistance to cisplatin. Thus, inhibition of the expression of the ALKBH5-HOXA10 loop may be a potential strategy to overcome cisplatin resistance in EOC.

In hard coal mines with methane, there is often a need to apply demethylation in order to keep the methane concentration not exceeding 2% in the ventilation air. The basic demethylation method in longwall areas is through drainage boreholes made in the roof rocks of the coal bed, from top gate, in front of the longwall. The drainage boreholes are usually made in bundles, in a fan-shaped arrangement, with several boreholes in each bundle. The paper presents the results of measurements and tests of the efficiency of a bundle of four drainage boreholes drilled approximately 100 m in front of the longwall face. The efficiency of individual boreholes was analyzed in time and depending on the distance of borehole outlets from the longwall face. It was found that there is a large variation in the extraction of air-methane mixture by individual drainage boreholes, as well as large differences in the efficiency of individual drainage boreholes during the longwall extraction process.

Improving the chemotherapy resistance of temozolomide (TMZ) is of great significance in the treatment of glioblastoma multiforme (GBM). Long non‐coding RNA just proximal to the X‐inactive specific transcript (JPX) has been proven to be involved in cancer progression. However, the intrinsic significance and molecular mechanism by which JPX orchestrates GBM progression and TMZ chemotherapy resistance remain poorly understood. Here, JPX was found to be significantly elevated in GBM tissues and cell lines, and patients with high expressions of JPX showed significantly worse prognoses. Functional experiments revealed its carcinogenic roles in GBM cell proliferation, TMZ chemoresistance, anti‐apoptosis, DNA damage repair, and aerobic glycolysis. Mechanistically, JPX formed a complex with phosphoinositide dependent kinase‐1 (PDK1) messenger RNA (mRNA) and promoted its stability and expression. Furthermore, an RNA immunoprecipitation (RIP) experiment showed that JPX interacted with N6‐methyladenosine (m6A) demethylase FTO alpha‐ketoglutarate dependent dioxygenase (FTO) and enhanced FTO‐mediated PDK1 mRNA demethylation. JPX exerted its GBM‐promotion effects through the FTO/PDK1 axis. Taken together, these findings reveal the key role of JPX in promoting GBM aerobic glycolysis and TMZ chemoresistance in an m6A‐dependent manner. Thus, it comprises a promising novel therapeutic target for GBM chemotherapy., Our findings uncover a key role of just proximal to the X‐inactive specific transcript in promoting glioblastoma multiforme (GBM) aerobic glycolysis and temozolomide chemoresistance in a N6‐methyladenosine (m6A)‐dependent manner, providing a promising novel therapeutic target for GBM chemotherapy.

The epigenetic marker 5‐methyl‐2′‐deoxycytidine (5mdC) is the most prevalent modification to DNA. It is removed inter alia via an active demethylation pathway: oxidation by Ten‐Eleven Translocation 5‐methyl cytosine dioxygenase (TET) and subsequent removal via base excision repair or direct demodification. Recently, we have shown that the synthetic iron(IV)‐oxo complex [FeIV(O)(Py5Me2H)]2+ (1) can serve as a biomimetic model for TET by oxidizing the nucleobase 5‐methyl cytosine (5mC) to its natural metabolites. In this work, we demonstrate that nucleosides and even short oligonucleotide strands can also serve as substrates, using a range of HPLC and MS techniques. We found that the 5‐position of 5mC is oxidized preferably by 1, with side reactions occurring only at the strand ends of the used oligonucleotides. A detailed study of the reactivity of 1 towards nucleosides confirms our results; that oxidation of the anomeric center (1′) is the most common side reaction., The synthetic iron(IV)‐oxo complex [FeIV(O)(Py5Me2H)]2+ serves as a biomimetic model for TET by oxidizing the nucleobase 5‐methyl cytosine (5mC) to its natural metabolites. In this work, we demonstrate that nucleosides and short oligonucleotide strands serve as substrates. The 5‐position of 5mC is oxidized preferably and highly selectively by the complex.

DMIs (sterol demethylation inhibitors) provide an important mode of action for control of Venturia inaequalis. They target the enzyme 14a-demethylase, encoded by the Cyp51 gene, which acts on an essential precursor for ergosterol biosynthesis. One mechanism, which leads to reduced DMI sensitivity of fungi, is based on mutations in Cyp51. In contrast to other phytopathogenic fungi, only little information is available on changes in the Cyp51 gene of V. inaequalis and their potential influence on DMI sensitivity. In this study, 126 monoconidial isolates were generated from field samples of V. inaequalis collected in apple orchards in different countries in 2018 and 2019. The Cyp51 gene of all isolates was sequenced. Furthermore, a new liquid culture method for V. inaequalis was established, which provides strongly sporulating cultures as a basis for the sensitivity tests. The sequence analysis revealed 15 different point mutations, and each of them resulted in an amino acid exchange in the 14α-demethylase. For some of them, corresponding mutations had been reported in other plant pathogens and shown to influence DMI sensitivity. We measured fungal growth in microtiter-plate tests to evaluate the influence of the amino acid changes in the Cyp51 gene on the sensitivity of the isolates towards the DMIs mefentrifluconazole, difenoconazole, and myclobutanil. While some mutations (e.g. those in the YGYG region 443–446) showed little impact on DMI sensitivity, others caused higher EC50 values, in particular Y133F and M141T. The sensitivity reducing effects differed for the three DMIs and were most pronounced for myclobutanil and less for mefentrifluconazole. The variability in the DMI sensitivity in each Cyp51 haplotype suggests the presence of additional resistance mechanisms caused by genetic background.

BACKGROUND Cannabis sativa L. (hemp) is a medicinal plant producing various cannabinoids. Its consumption is legalized for medical use due to the alleged positive health effects of these cannabinoids. To satisfy the demand, C. sativa plants are propagated in contained growth chambers. During indoor propagation, pesticides usually are used to ensure efficient production. However, pesticide registration and safe application in C. sativa has not been investigated in detail. RESULTS With this study the metabolic degradation of pesticides in recently established C. sativa callus cultures was examined. Tebuconazole, metalaxyl-M fenhexamid, flurtamone and spirodiclofen were applied at 10 μm for 21 days. Results were compared with metabolism data obtained from Brassica napus L., Glycine max (L.) Merr., Zea mays L. and Tritium aestivum L. callus cultures as well as in metabolism guideline studies. The successfully established C. sativa callus cultures were able to degrade pesticides by oxidation, demethylation, and cleavage of ester bonds in phase I, as well as glycosylation and conjugation with malonic acid in phase II and III. Initial metabolites were detected after Day (D)7 and were traced at D21. CONCLUSION The resulting pathways demonstrate the same main degradation strategies as crop plants. Because metabolites could be the main residue, the exposure of consumers to these residues will be of high importance. We present here an in vitro assay for a first estimation of pesticide metabolism in C. sativa. © 2021 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

N6 -methyladenosine (m6 A) is an abundant methylation mark in eukaryotic mRNAs. It is installed and removed by methyltransferases ("writers") and demethylases ("erasers"), respectively. A recent study has demonstrated that alpha-ketoglutarate-dependent dioxygenase homolog 10B (ALKBH10B) is an mRNA m6 A eraser affecting floral transition in Arabidopsis thaliana. However, the roles of m6 A eraser proteins, including ALKHB10B, in plant adaptation to abiotic stresses are largely unknown. In this study, we aimed to determine the role of ALKBH10B in the response of A. thaliana to abiotic stresses and abscisic acid (ABA). The m6 A level increased in response to salt stress, and m6 A levels in alkbh10b mutants were higher than those in the wild-type under both normal and salt stress conditions. Germination of alkbh10b mutant seeds was markedly delayed under salt stress but not under dehydration, cold, or ABA conditions. Seedling growth and survival rate of alkbh10b mutants were enhanced under salt stress. Notably, salt-tolerant phenotypes of alkbh10b mutants were correlated with decreased levels of several m6 A-modified genes, including ATAF1, BGLU22, and MYB73, which are negative effectors of salt stress tolerance. In response to ABA, both seedling and root growth of alkbh10b mutants were inhibited via upregulating ABA signaling-related genes, including ABI3 and ABI4. Collectively, these findings indicate that ALKBH10B-mediated m6 A demethylation affects the transcript levels of stress-responsive genes, which are important for seed germination, seedling growth, and survival of Arabidopsis thaliana in response to salt stress or ABA.

Fusarium graminearum is one of the most important causal agents of Fusarium head blight disease and is controlled mainly by chemicals such as demethylation inhibitor (DMI) fungicides. FgCYP51B is one of the DMI targets in F. graminearum, and Tyrosine123 (Y123) is an important amino acid in F. graminearum CYP51B, located in one of predicted substrate binding pockets based on the binding mode between DMIs and CYP51B. Previous studies suggest that resistance to DMI fungicides is attributed primarily to point mutations in the CYP51 gene and that the Y123H mutation in F. verticillioides CYP51 confers prochloraz resistance in the laboratory. To investigate the function of FgCYP51B Y123 residue in the growth and development, pathogenicity, and DMI resistance, we generated and analyzed the FgCYP51B Y123H mutant. Results revealed that the Y123H mutation led to reduced conidial sporulation and affected ascospore development; moreover, the mutation conferred reduced sensitivity to prochloraz. Quantitative PCR and molecular docking were performed to investigate the resistance mechanism. Results indicated that Y123H mutation changed the target gene expression and decreased the binding affinity of FgCYP51 to prochloraz. These results will attract more attention to the potential DMI-resistant mutation of F. graminearum and increase our understanding of the DMI resistance mechanism.

Background: Gelsemium elegans Benth（G. elegans） is a well-known toxic plant. Alkaloids are main active components of G. elegans. Currently, the metabolism of several alkaloids, such as gelsenicine, koumine, and gelsemine, has been widely studied. However, as one of the most important alkaloids in G. elegans, the metabolism of humantenine has not been studied yet. Methods: In order to elaborate on the in vitro metabolism of humantenine, a comparative analysis of its metabolic profile in human, pig, goat and rat liver microsomes was carried out using high-performance chromatography/quadrupole time-of-flight mass spectrometry (HPLC/QqTOF-MS) for the first time. Results: Totally, ten metabolites of humantenine were identified in liver microsomes from human (HLMs), pig (PLMs), goat (GLMs) and rat (RLMs) based on the accurate MS/MS spectra. Five metabolic pathways of humantenine, including demethylation, dehydrogenation, oxidation, dehydrogenation and oxidation, and demethylation and oxidation, were proposed in this study. There were qualitative and quantitative species differences in the metabolism of humantenine among the four species. Conclusions: The in vitro metabolism of humantenine in HLMs, PLMs, GLMs and RLMs was studied by a sensitive and specific detection method based on HPLC/QqTOF-MS. The results indicated that there were species-related differences in the metabolism of humantenine. This work might be of great significance for the further research and explanation of species differences in terms of toxicological effects of G. elegans.

RNA N6-methyladenosine (m6A) modifications are essential in plants. Here, we show that transgenic expression of the human RNA demethylase FTO in rice caused a more than threefold increase in grain yield under greenhouse conditions. In field trials, transgenic expression of FTO in rice and potato caused ~50% increases in yield and biomass. We demonstrate that the presence of FTO stimulates root meristem cell proliferation and tiller bud formation and promotes photosynthetic efficiency and drought tolerance but has no effect on mature cell size, shoot meristem cell proliferation, root diameter, plant height or ploidy. FTO mediates substantial m6A demethylation (around 7% of demethylation in poly(A) RNA and around 35% decrease of m6A in non-ribosomal nuclear RNA) in plant RNA, inducing chromatin openness and transcriptional activation. Therefore, modulation of plant RNA m6A methylation is a promising strategy to dramatically improve plant growth and crop yield.

Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant. In order to better understand the decomposition process of TBBPA and clarify the evolution process of the main pyrolysis products, the density functional theory (DFT) method PBE0/6-311G(d) has been used to investigate the pyrolysis mechanisms of TBBPA in this study. Seven possible pyrolysis reaction paths were proposed, and the kinetic parameters in all pyrolysis paths were calculated. The calculation results indicate that in initial degradation of TBBPA without the involvement of hydrogen radical, the demethylation reaction is the main pyrolysis reaction channel, and the keto-enol tautomerization reaction is the main competitive pyrolysis reaction channel. The brominated cyclohexadienone formed through the keto-enol tautomerization is prone to further debromination to generate Br radical. The involvement of hydrogen radical significantly lowers the energy barrier of TBBPA decomposition. When a hydrogen radical is involved in the pyrolysis process, the debromination reaction becomes the major pyrolysis reaction channel, and the homolytic cleavage of Caromatic-C bond becomes the major competitive pyrolysis reaction channel.

Methylmercury (MeHg) contamination in paddy fields is a significant environmental issue globally since over half of the population of our planet consumes rice. MeHg is a neurotoxin produced by microorganisms in oxygen-limited environments. Microbial effect on MeHg production is a hotspot of research; however, it has been largely ignored how the oxidation-reduction potential (Eh) shapes MeHg formation. Here, we elucidated Hg (de)-methylation in a contaminated soil by increasing Eh stepwise from -300 to +300 mV using a sophisticated biogeochemical microcosm. At the Eh range from -300 to -100 mV, high MeHg concentration and dissolved total Hg (THg) concentration were found due to a high relative abundance of Hg-methylation bacteria (e.g., Desulfitobacterium spp.), acidification, and reductive dissolution of Fe(oxyhydr)oxides. At the Eh range from 0 to +200 mV, the formation of colloids leads to adsorption of Hg and as a result colloidal Hg increased. MeHg reduction with Eh (-300 to +200 mV) increase was mainly attributed to a reduced Hg methylation, as dissolved THg and relative abundance of Desulfitobacterium spp. decreased by 50 and 96%, respectively, at Eh of +200 mV as compared to Eh of -300 mV. Mercury demethylation might be less important since the relative abundance of demethylation bacteria (Clostridium spp.) also decreased over 93% at Eh of +200 mV. These new results are crucial for predicting Hg risks in paddy fields.