Rate Of Demethylation Research Articles

Assessment of mercury methylation and methylmercury demethylation potentials in water and sediments along the Wabigoon River system

Monomethylmercury (MMHg) plays a crucial role in the accumulation of mercury (Hg) within aquatic food chains. Since ambient levels of methylmercury are governed by the balance of simultaneous methylation and demethylation processes, determining in situ methylation and demethylation rates is critically important to understand the dynamics of methylmercury in the environment. This is especially important in the Wabigoon River system in Ontario, Canada, which is severely contaminated with Hg by a chlor-alkali facility operating in the 1960s, and still exhibits some of the highest recorded fish mercury concentrations in Canada. This work used a simultaneous addition of isotope enriched Hg and MMHg tracers to ascertain Hg methylation and MMHg demethylation potentials. At the locations investigated for this study, the most favourable conditions for Hg methylation were found at the Hydroelectric dam, being able to transform 4.2 % and 4.4 % of added Hg in water and sediments per day, respectively, to MMHg. This could correspond to 1.9 ng/L and 29 ng/g of new MMHg being produced from current ambient Hg. Clay Lake, which is considered a sink for mercury and exhibiting a seasonal anoxic environment at its bottom waters, also demonstrated significant MMHg generation, being able to produce 2.7 ng/L and 13 ng/g of MMHg per day, respectively. Demethylation rates in sediments of riverbed and wetland locations showed an average half-life for methylmercury of 2.1 days, indicating a rapid turnover of MMHg in the Wabigoon River. However, significantly lower demethylation rates were also measured near the inflow of Clay Lake, where it took up to 144 days for MMHg to decrease by 50 %. Generally, most of the investigated locations downstream of the pollution source displayed the potential to generate methylmercury, which could be distributed throughout the Wabigoon River system and therefore require attention with respect to future remediation activities.

Dimethylmercury as a Source of Monomethylmercury in a Highly Productive Upwelling System.

Monomethylmercury (MMHg) is a neurotoxicant that biomagnifies in marine food webs, reaching high concentrations in apex predators. To predict changes in oceanic MMHg concentrations, it is important to quantify the sources and sinks of MMHg. Here, we study mercury speciation in the California Current System through cruise sampling and modeling. Previous work in the California Current System has found that upwelling transports mercury-enriched deep waters to productive surface waters. These upwelled waters originate within the California Undercurrent water mass and are subsequently advected as a surface water parcel to the California Current. Between the two major water masses, we find that compared to the California Current, the California Undercurrent contains elevated dissolved total mercury (THg) and dimethylmercury (DMHg) concentrations by 59 and 69%, respectively. We explain that these differences result from losses during advection, specifically scavenging of THg and DMHg demethylation. We calculate a net DMHg demethylation rate of 2.0 ± 1.1% d-1 and build an empirically constrained mass budget model to demonstrate that net DMHg demethylation accounts for 61% of surface MMHg sources. These findings illustrate that DMHg is a significant source of MMHg in this region, challenging the current understanding of the major sources of marine MMHg.

Anticancer Drugs of Lysine Specific Histone Demethylase-1 (LSD1) Display Variable Inhibition on Nucleosome Substrates.

Lysine specific demethylase-1 (LSD1) serves as a regulator of transcription and represents a promising epigenetic target for anticancer treatment. LSD1 inhibitors are in clinical trials for the treatment of Ewing's sarcoma (EWS), acute myeloid leukemia, and small cell lung cancer, and the development of robust inhibitors requires accurate methods for probing demethylation, potency, and selectivity. Here, the inhibition kinetics on the H3K4me2 peptide and nucleosome substrates was examined, comparing the rates of demethylation in the presence of reversible [CC-90011 (PD) and SP-2577 (SD)] and irreversible [ORY-1001 (ID) and tranylcypromine (TCP)] inhibitors. Inhibitors were also subject to viability studies in three human cell lines and Western blot assays to monitor H3K4me2 nucleosome levels in EWS (TC-32) cells, enabling a correlation of drug potency, inhibition in vitro, and cell-based studies. For example, SP-2577, a drug in clinical trials for EWS, inhibits activity on small peptide substrates (Ki = 60 ± 20 nM) using an indirect coupled assay but does not inhibit demethylation on H3K4me2 peptides or nucleosomes using direct Western blot approaches. In addition, the drug has no effect on H3K4me2 levels in TC-32 cells. These data show that SP-2577 is not an LSD1 enzyme inhibitor, although the drug may function independent of demethylation due to its cytotoxic selectivity in TC-32 cells. Taken together, this work highlights the pitfalls of using coupled assays to ascribe a drug's mode of action, emphasizes the use of physiologically relevant substrates in epigenetic drug targeting strategies, and provides insight into the development of substrate-selective inhibitors of LSD1.

Abstract 2463: Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients

Abstract Purpose: In Renal Cell Carcinoma (RCC) a crucial role is played by tumor microenvironment (TME). The high presence of T regulatory cells (Tregs) in TME cause a suppressive environment that promotes the tumor growth and its treatments failure. Tregs trafficking is controlled by CXCR4. In RCC, the new CXCR4 antagonist R54 was explored in peripheral blood Tregs isolated from primary RCC patients. Experimental Design: To investigate the mechanism by which the new CXCR4 antagonist R54 impairs Tregs activity, peripheral blood (PB) Tregs were explored on primary RCC patients. PB-Tregs were isolated from 77 RCC patients and 38 healthy donors-(HD) and CFSE-Tregs suppression assay, IL-35 secretion and Nrp-1+Tregs frequency was conducted. PB-Tregs were analyzed for PTEN, CD25, TGF-β1, FOXP3, DNMT1 expression. PTEN-pAKT signaling was evaluated in the presence of R54 and/or triciribine (TCB), Akt inhibitor. TSDR (Treg-specific Demethylated Region) methylation analysis was conducted. Result: Ex vivo R54 impaired PB-RCC-Tregs function, reduced IL-35 release and Nrp-1+Tregs. As PTEN and CD25 expression significantly decreased in R54-PB-RCC-Tregs, PTEN signaling was evaluated. While CXCL12 recruited PTEN+CD25+Tregs cells, R54 significantly reduced it in PB-RCC-Tregs. Tregs activation through IL-2/PMA impaired pAKT+Tregs while R54 increased it. The Akt inhibitor, triciribine, prevented the pAKT+Tregs R54 induction. As active Tregs present demethylated Treg-specific region (TSDR), a significant decrease in demethylation rate (DMR) of Foxp3-TSDR was observed in R54 treated PB-RCC-Tregs. As consequence of TSDR modulation, the expression of Tregs regulating genes was evaluated with significant reduction in DNMT1 and FOXP3 expression. Taken together, our findings demonstrated that R54 causes an impairment of peripheral Tregs functionality in primary RCC patients through regulation of PTEN/PI3K/AKT pathway, resulting in impaired Tregs activity. Conclusion: The CXCR4 antagonist R54 affects Tregs function in primary RCC patients through regulation of PTEN/PI3K/AKT pathway, reduction in TSDR demethylation and Foxp3 and DNMT1 expression. CXCR4 targeting is thus a strategy to inhibit Tregs activity contributing to the CXCR4 function in RCC tumor microenvironment. Citation Format: Giuseppina Rea, Sara Santagata, Anna Maria Bello, Anna Capiluongo, Maria Napolitano, Sonia Desicato, Alessandra Fragale, Crescenzo D'Alterio, Anna Maria Trotta, Caterina Ieranò, Luigi Portella, Marilena Di Napoli, Salvatore Di Maro, Florinda Feroce, Rosa Azzaro, Lucia Gabriele, Nicola Longo, Sandro Pignata, Sisto Perdonà, Stefania Scala. Targeting CXCR4 impaired T regulatory function through PTEN in renal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2463.

Abstract PR009: Tick tock trees: Reconstructing the evolutionary dynamics of human tissues using fluctuating methylation clocks

Abstract The development of cancer from healthy somatic cells is fundamentally an evolutionary process. Understanding this process is vital to improving its clinical management and developing new treatment strategies. Tracing individual human cells’ fates would simultaneously advance somatic evolutionary theory and our understanding of normal tissue function and its change through development, aging, and cancer progression. We cannot use human cell-labeling techniques in vivo. Still, population genetic and phylogenetic models can leverage the information recorded in heritable alterations to reconstruct the unobserved somatic cells’ history. Most such methods are limited by the few parameters they can estimate and restricted to neoplastic samples due to the lack of adequate evolutionary signal in healthy somatic cells. Gabbutt et al. recently ameliorated this limitation by introducing fluctuating methylation clocks (FMCs)—tissue-specific CpG sites that randomly change their methylation state—and a model that uses them to reconstruct the evolution of a clonal stem-cell population. In this application, all FMCs in a bulk sample are interrogated using a single standard methylation array to yield estimates of rates of methylation, demethylation and cell replacement, and the size of the stem-cell niche. This technique is especially suited to studying stem-cell niche dynamics in healthy or pre-malignant tissues organized in clonal cell populations where the whole population can be sampled together, like crypts (e.g., intestine, Barrett’s esophagus) and glands (e.g., endometrium). We have developed a multi-sample extension of this model that simultaneously reconstructs the evolutionary dynamics of stem cells within a niche and the niches within the tissue. This phylodynamic model produces additional estimates like ancestral relationships between sampled clonal populations, their calendar divergence times, the number of effective evolutionary niches, and how some of these parameters change over time. Here, we will present the method, demonstrate its validity, characterize its accuracy under a broad simulation parameter space, and showcase its application in analyzing human data from the following tissues: colon, small intestine, endometrium, and Barrett’s esophagus. We will also discuss the current model's limitations and future improvements that will discern between mechanisms of stem-cell-niche reproduction and incorporate geographical information. The stochastic nature of neoplastic progression makes it unlikely for any particular somatic alteration to be highly predictive of its outcome. This can explain why developing reliable traditional biomarkers has been so difficult. Alternatively, evolutionary biomarkers measure the characteristics of the process itself and thus should apply to all neoplasms. Parametric phylogenetic reconstruction models like the one introduced here will enable us to develop such universal biomarkers. Our ultimate goal is for evolutionary biomarkers to join evolutionary therapies in the revolution of the clinical management of cancer. Citation Format: Diego Mallo, Pablo Bousquets-Muñoz, Heather E. Grant, Darryl Shibata, Trevor A. Graham, Carlo C. Maley, Calum Gabbutt. Tick tock trees: Reconstructing the evolutionary dynamics of human tissues using fluctuating methylation clocks [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR009.

Methylation and Demethylation of Emerging Contaminants in Plants.

Many contaminants of emerging concern (CECs) have reactive functional groups and may readily undergo biotransformations, such as methylation and demethylation. These transformations have been reported to occur during human metabolism and wastewater treatment, leading to the propagation of CECs. When treated wastewater and biosolids are used in agriculture, CECs and their transformation products (TPs) are introduced into soil-plant systems. However, little is known about whether transformation cycles, such as methylation and demethylation, take place in higher plants and hence affect the fate of CECs in terrestrial ecosystems. In this study, we explored the interconversion between four common CECs (acetaminophen, diazepam, methylparaben, and naproxen) and their methylated or demethylated TPs in Arabidopsis thaliana cells and whole wheat seedlings. The methylation-demethylation cycle occurred in both plant models with demethylation generally taking place at a greater degree than methylation. The transformation rate of demethylation or methylation was dependent on the bond strength of R-CH3, with demethylation of methylparaben or methylation of acetaminophen being more pronounced. Although not explored in this study, these interconversions may exert influences on the behavior and biological activity of CECs, particularly in terrestrial ecosystems. The study findings demonstrated the prevalence of transformation cycles between CECs and their methylated or demethylated TPs in higher plants, contributing to a more complete understanding of risks of CECs in the human-wastewater-soil-plant continuum.

Differential response of Hg-methylating and MeHg-demethylating microbiomes to dissolved organic matter components in eutrophic lake water

Methylmercury (MeHg) production in aquatic ecosystems is a global concern because of its neurotoxic effect. Dissolved organic matter (DOM) plays a crucial role in biogeochemical cycling of Hg. However, owing to its complex composition, the effects of DOM on net MeHg production have not been fully understood. Here, the Hg isotope tracer technique combined with different DOM treatments was employed to explore the influences of DOM with divergent compositions on Hg methylation/demethylation and its microbial mechanisms in eutrophic lake waters. Our results showed that algae-derived DOM treatments enhanced MeHg concentrations by 1.42−1.53 times compared with terrestrial-derived DOM. Algae-derived DOM had largely increased the methylation rate constants by approximately 1−2 orders of magnitude compared to terrestrial-derived DOM, but its effects on demethylation rate constants were less pronounced, resulting in the enhancement of net MeHg formation. The abundance of hgcA and merB genes suggested that Hg-methylating and MeHg-demethylating microbiomes responded differently to DOM treatments. Specific DOM components (e.g., aromatic proteins and soluble microbial byproducts) were positively correlated with both methylation rate constants and the abundance of Hg-methylating microbiomes. Our results highlight that the DOM composition influences the Hg methylation and MeHg demethylation differently and should be incorporated into future Hg risk assessments in aquatic ecosystems.

Oral Decitabine/Cedazuridine Vs Intravenous Decitabine for Acute Myeloid Leukemia: Final Results of a Randomized, Crossover, Registration-Enabling, Pharmacokinetics Study

Background: Parenterally administered hypomethylating agents (HMAs), decitabine (DEC) and azacitidine (AZA), are approved in Europe for adult patients with acute myeloid leukemia (AML) who are not candidates for standard induction chemotherapy as single agent or in combination with venetoclax. Cedazuridine(C) is a novel, potent, and safe inhibitor of cytidine deaminase (CDA) and when given in combination with decitabine, cedazuridine enables efficient oral availability of decitabine.ASTX727 (DEC-C) is a fixed-dose combination (FDC) tablet of 35 mg DEC and 100 mg cedazuridine, DEC-C has been approved for MDS in the US, Canada, and Australia. Here we present final results using DEC-C in an AML population appropriate for single-agent decitabine treatment. Aims: Describe the final clinical outcomes of patients with AML with additional analysis of genetic profiles. Methods: The ASCERTAIN study used a randomized two-period, two-sequence, two-treatment, crossover study design. Patients were randomized 1:1 to either Sequence A: DEC-C (35 mg DEC/100 mg cedazuridine) in Cycle 1 followed by IV-DEC at 20 mg/m 2 in Cycle 2, or Sequence B: receiving IV-DEC in Cycle 1 followed by DEC-C on Cycle 2 to compare pharmacokinetics (PK) [primary endpoint Area Under the Curve (AUC) equivalence over 5 days of dosing]. All patients received DEC-C from Cycle 3 until treatment discontinuation to assess safety and clinical efficacy. Patients were eligible as per the EMA-approved decitabine label (newly diagnosed AML who are not candidates for standard induction chemotherapy). Clinical responses were assessed according to modified International Working Group (IWG) 2003 response criteria. Pre-treatment peripheral blood was used for DNA isolation from leukocytes, and molecular abnormalities identified using a NGS hematologic malignancy panel. Cox-regression analysis on the various factors (eg binary mutational status, CR, etc.) was used for analyses of risk-factors for overall survival (OS). Results: 89 patients were randomized, of whom 87 were treated. The median age of patients was 78.0 years (range, 61 to 92) with 31 (35.6%) males and 56 (64.4%) females. Cytogenetic risk classification was poor-risk in 33 (37.9%), intermediate-risk in 45 (51.7%), and 9 (10.4%) not evaluated patients. For the primary endpoint, preliminary PK data was available from 69 patients who successfully completed PK assessments for both IV-DEC and DEC-C cycles, and the DEC AUC 0-24 (h*ng/mL) 5-Day geometric mean estimate was 904 for DEC-C and 907 for IV-DEC resulting in an oral/IV geometric LSM AUC ratio of 99.64% (90% CI of 91.23-108.8%). Safety findings were consistent with those anticipated for IV-DEC (related Grade ≥ 3 AEs in more than 10% were thrombocytopenia, neutropenia, anaemia, and febrile neutropenia). As of the database lock (25May2023), median follow up was 24.5 (20.2, 25.0) months (25% of 19.7 and 75% of 25.2 months). The best response was complete response (CR) in 19/87 (21.8%, 95% CI: 13.7, 32.0%); CR with incomplete blood cell count recovery (CRi) in 5 patients (5.7%); and CR with incomplete platelet recovery (CRp) in 2 (2.3%); resulting in composite response rate [CR + CRi] of 27.6%. The median overall survival was approximately 8.9 months (95% CI: 6.0, 13.1).These results obtained with DEC-C are consistent with those observed for IV-DEC. Multiple genes, including ASXL1, TP53, RUNX1, and SRSF2 genes were present in more than 20% of the AML patients. TP53 mutation was associated with worse survival. Summary/Conclusion: This randomized phase 3 study in AML patients not candidates for standard induction chemotherapy demonstrates that Daily X 5 dosing of the oral FDC of DEC-C resulted in an equivalent DEC AUC exposure to IV-DEC at 20 mg/m 2 over 5 days. Pharmacodynamic data showed similar demethylation rates (≤1.1% difference). In addition, safety findings and observed clinical activity (response rates and OS) are consistent with published data from IV-DEC, suggesting that DEC-C has the potential to be an oral alternative to the standard IV-DEC Daily×5 regimen. These findings support the use of DEC-C in patients with AML who are not candidates for standard induction chemotherapy.

Intestinal microbiota protects against methylmercury-induced neurotoxicity.

Methylmercury (MeHg) remains a global public health issue because of its frequent presence in human food sources obtained from the water. The excretion of MeHg in humans occurs slowly with a biological half-time of 32-47 days. Short-term MeHg exposure may cause long-lasting neurotoxicity. The excretion through feces is a major route in the demethylation of MeHg. Accumulating evidence suggests that the intestinal microbiota plays an important role in the demethylation of MeHg, thereby protecting the host from neurotoxic effects. Here, we discuss recent developments on the role of intestinal microbiota in MeHg metabolism, based on in vitro cell culture experiments, experimental animal studies and human investigations. Demethylation by intestinal bacteria is the rate-limiting step in MeHg metabolism and elimination. The identity of bacteria strains responsible for this biotransformation is currently unknown; however, the non-homogenous distribution of intestinal microbiota may lead to different demethylation rates in the intestinal tract. The maintenance of intestinal barrier function by intestinal microbiota may afford protection against MeHg-induced neurotoxicity, which warrant future investigations. We also discuss studies investigating the effects of MeHg exposure on the population structural stability of intestinal microbiota in several host species. Although this is an emerging area in metal toxicity, current research suggests that a change in certain phyla in the intestinal microbiota may indicate MeHg overexposure.

In Vivo Mercury (De)Methylation Metabolism in Cephalopods under Different pCO2 Scenarios.

This work quantified the accumulation efficiencies of Hg in cuttlefish, depending on both organic (MeHg) and inorganic (Hg(II)) forms, under increased pCO2 (1600 μatm). Cuttlefish were fed with live shrimps injected with two Hg stable isotopic tracers (Me202Hg and 199Hg(II)), which allowed for the simultaneous quantification of internal Hg accumulation, Hg(II) methylation, and MeHg demethylation rates in different organs. Results showed that pCO2 had no impact on Hg bioaccumulation and organotropism, and both Hg and pCO2 did not influence the microbiota diversity of gut and digestive gland. However, the results also demonstrated that the digestive gland is a key organ for in vivo MeHg demethylation. Consequently, cuttlefish exposed to environmental levels of MeHg could exhibit in vivo MeHg demethylation. We hypothesize that in vivo MeHg demethylation could be due to biologically induced reactions or to abiotic reactions. This has important implications as to how some marine organisms may respond to future ocean change and global mercury contamination.

Evaluating the influence of seasonal stratification on mercury methylation rates in the water column and sediment in a contaminated section of a western U.S.A. reservoir

Mercury methylation frequently occurs at the active oxic/anoxic boundary between the sediment bed and water column of lakes and reservoirs. Previous studies suggest that the predominant mercury methylation zone moves to the water column during periods of stratification and that high potential methylation rates (Km) in sediment require oxygenated overlying water. However, simultaneous measurements of methylmercury (MeHg) production in both the sediment and water column remain limited. Understanding the relative importance of sediment versus water column methylation and the impact of seasonal stratification on these processes has important implications for managing MeHg production. This study measured Km and potential demethylation rates (Kdm) using stable isotope tracers of unfiltered inorganic mercury and MeHg in sediments and water of the littoral and profundal zones of a shallow branch of the Nacimiento Reservoir in California's central coastal range. Field sampling was conducted once during winter (well-mixed/oxygenated conditions) and once during late summer (thermally stratified/anoxic conditions). The results showed very high ambient MeHg concentrations in hypolimnetic waters (up to 7.5 ng L−1; 79% MeHg/total Hg). During late summer, littoral sediments had higher Km (0.024 day−1) compared to profundal sediments (0.013 day−1). Anoxic water column Km were of similar magnitude to Km in the sediment (0.03 day−1). Following turnover, profundal sediment Km did not change significantly, but water column Km became insignificant. Summer and winter sediment Kdm were higher in profundal (2.35, 3.54 day−1, respectively) compared to the littoral sediments (0.52, 2.56 day−1, respectively). When modelled, Km in the water column could account for approximately 40% of the hypolimnetic MeHg. Our modelling results show that the remaining MeHg in the hypolimnion could originate from the profundal sediment. While further study is needed, these results suggest that addressing methylation in the water column and profundal sediment are of equal importance to any remediation strategy.

Pronounced sequence specificity of the TET enzyme catalytic domain guides its cellular function.

TET (ten-eleven translocation) enzymes catalyze the oxidation of 5-methylcytosine bases in DNA, thus driving active and passive DNA demethylation. Here, we report that the catalytic domain of mammalian TET enzymes favor CGs embedded within basic helix-loop-helix and basic leucine zipper domain transcription factor–binding sites, with up to 250-fold preference in vitro. Crystal structures and molecular dynamics calculations show that sequence preference is caused by intrasubstrate interactions and CG flanking sequence indirectly affecting enzyme conformation. TET sequence preferences are physiologically relevant as they explain the rates of DNA demethylation in TET-rescue experiments in culture and in vivo within the zygote and germ line. Most and least favorable TET motifs represent DNA sites that are bound by methylation-sensitive immediate-early transcription factors and octamer-binding transcription factor 4 (OCT4), respectively, illuminating TET function in transcriptional responses and pluripotency support.

Mercury Methylation Potentials in Sediments of an Ancient Cypress Wetland Using Species-Specific Isotope Dilution GC-ICP-MS.

Wetlands are of a considerable environmental value as they provide food and habitat for plants and animals. Several important chemical transformations take place in wetland media, including the conversion of inorganic mercury (Hg) to monomethylmercury (MeHg), a toxic compound with a strong tendency for bioconcentration. Considering the fact that wetlands are hotspots for Hg methylation, we investigated, for the first time, Hg methylation and demethylation rates in an old growth cypress wetland at Sky Lake in the Mississippi Delta. The Sky Lake ecosystem undergoes large-scale water level fluctuations causing alternating periods of oxic and anoxic conditions in the sediment. These oscillating redox conditions, in turn, can influence the transformation, speciation, and bioavailability of Hg. In the present study, sediment cores from the wetland and Sky Lake itself were spiked with enriched stable isotope tracers of inorganic Hg and MeHg and allowed to incubate (in-situ) before freezing, sectioning, and analysis. Methylation rates (day−1) ranged from 0.012 ± 0.003 to 0.054 ± 0.019, with the lowest rate in the winter and the highest in the summer. Demethylation rates were about two orders of magnitude higher, and also greater in the warmer seasons (e.g., 1.84 ± 0.78 and 4.63 ± 0.51 for wetland sediment in the winter and summer, respectively). Methylation rates were generally higher in the open water sediment compared to wetland sediment, with the latter shaded and cooler. Both methylation (r = 0.76, p = 0.034) and demethylation (0.97, p = 0.016) rates (day−1) were positively correlated with temperature, but not with most other water quality parameters. MeHg concentration in the water was correlated with pH (r = 0.80, p < 0.05), but methylation rates were only marginally correlated (r = 0.71). Environmental factors driving microbial production of MeHg in the system include warm temperatures, high levels of labile natural organic matter, and to a lesser extent the relatively low pH and the residence time of the water. This study also provides baseline data that can be used to quantify the impacts of modifying the natural flow of water to the system on Hg methylation and demethylation rates.

Impacts of Mercury Exposure Levels and Sources on the Demethylation of Methylmercury Through Human Gut Microbiota.

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.

Occurrence of methylmercury in aerobic environments: Evidence of mercury bacterial methylation based on simulation experiments

Methylmercury (MeHg) is mainly produced by anaerobic δ-proteobacteria such as sulfate-reducing bacteria (SRB). However, mercury bio-methylation has also been found to occur in the aerobic soil of the Three Gorges Reservoir (TGR). Using γ-proteobacterial TGR bacteria (TGRB) and δ-proteobacterial Desulfomicrobium escambiense strains, the efficiency of mercury methylation and demethylation was evaluated using an isotope tracer technique. Kinetics simulation showed that the bacterial Hg methylation rate (km) of TGRB3 was 4.36 × 10−9 pg·cell−1·h−1, which was significantly lower than that of D. escambiense (170.74 ×10−9 pg·cell−1·h−1) under anaerobic conditions. Under facultative and/or aerobic conditions, D. escambiense could not survive, while the km of TGRB3 were 0.35 × 10−9 and 0.29 × 10−9 pg·cell−1·h−1, respectively. Furthermore, the bacterial MeHg tolerance threshold of TGRB3 was 3.47 × 10−9 pg·cell−1, which was 98.6-fold lower than that of D. escambiense under anaerobic conditions. However, the MeHg tolerance threshold of TGRB3 remained at 0.50–0.52 × 10−9 pg·cell−1 under facultative and/or aerobic conditions. Notably, bacterial Hg methylation rates (km) were higher than the corresponding bacterial MeHg demethylation rates (kd1). These results establish the contribution of some aerobic and/or facultative anaerobic bacteria to net environmental MeHg production in terrestrial ecosystems and provide a novel understanding of the biogeochemical cycle of MeHg. SynopsisHg methylation of facultative and/or aerobic bacteria may contribute to the net production of environmental methylmercury in terrestrial ecosystems.

Lignin demethylation for modifying halloysite nanotubes towards robust phenolic foams with excellent thermal insulation and flame retardancy

AbstractThe demethylation of lignin was conducted under HBr catalysis and microwave irradiation resulting in a demethylation rate of 21.4% as calculated from1H NMR. The demethylated lignin (DL) was applied to modify halloysite nanotubes (HNTs) forming a nano‐hybrid containing 16.5 wt% of DL according to TGA results, and showing rough surfaces dotted with DL particles as observed through TEM. HNTs‐DL was introduced into resol resin synthesis where DL molecules participate in phenol‐formaldehyde polycondensation, functioning as a binder to connect the inorganic HNTs framework and the organic resol resin molecules, giving an increase in flow activation energy as calculated from rheological results. The prepared phenolic foam containing 0.4 wt% of HNTs‐DL shows a significant increase by 37.7% in compressive strength and a decrease by 27.3% in pulverization ratio compared to the neat phenolic foam, and exhibits uniform cells with the highest cell density as observed in SEM, explaining its improved performance in thermal insulation and flame retardancy.

Correlation between promoter methylation of the LDH-C4 gene and DNMT expression in breast cancer and their prognostic significance.

DNA methylation plays an important role in tumorigenesis and development. The potential of aberrant DNA methylation to act as a biomarker for tumor diagnosis and prognostic evaluation is currently being explored. Lactate dehydrogenase C4 (LDH-C4) is a member of the cancer/testis antigen family that is expressed in a broad range of human tumor types, with particularly high expression patterns observed in lung cancer, melanoma and breast cancer. However, whether the methylation of the promoter region of LDH-C4 can be used as a tumor marker and its association with prognosis remain poorly understood. The present study aimed to determine the potential of the methylation status of LDH-C4 and DNA methyltransferase (DNMT) expression to be biological markers for the prognostic evaluation of breast cancer. Methylation-specific PCR was conducted to evaluate alterations in the methylation levels of LDH-C4. Immunohistochemical analysis was performed to assess the expression levels of DNMTs in breast cancer tissues. The association between the methylation status of LDH-C4 or the expression of DNMTs, and clinical pathological parameters was also evaluated. The results of the present study revealed that the demethylation rate of LDH-C4 in breast cancer tissues was significantly increased compared with that of adjacent tissues, and associations were identified between the demethylation of LDH-C4 and the histological grade, estrogen receptor, progesterone receptor and HER-2 status, and lymph node metastasis. The level of LDH-C4 demethylation was negatively correlated with the expression of DNMTs. Demethylation of the LDH-C4 promoter and DNMT expression predicted an unfavorable prognosis of patients with breast cancer. In addition, demethylation of the LDH-C4 promoter, high expression of DNMT3a and DNMT3b, histological grade and lymph node metastasis were all discovered to be independent prognostic factors in patients with breast cancer. In conclusion, results of the present study indicated that the demethylation of LDH-C4 and DNMT expression levels may be closely associated with the occurrence and development of breast cancer, in addition to lymph node metastasis. Thus, both may be used to assist the clinical evaluation of prognosis.

Inhibition of methanogenesis leads to accumulation of methylated arsenic species and enhances arsenic volatilization from rice paddy soil

Flooded soils are important environments for the biomethylation and subsequent volatilization of arsenic (As), a contaminant of global concern. Conversion of inorganic to methylated oxyarsenic species is thought to be the rate-limiting step in the production and emission of volatile (methyl)arsines. While methanogens and sulfate-reducing bacteria (SRB) have been identified as important regulators of methylated oxyarsenic concentrations in anaerobic soils, the effects of these microbial groups on biovolatilization remain unclear. Here, microcosm and batch incubation experiments with an Arkansas, USA, rice paddy soil were performed in conjunction with metabolic inhibition to test the effects of methanogenic activity on As speciation and biovolatilization. Inhibition of methanogenesis with 2-bromoethanesulfonate (BES) led to the accumulation of methylated oxyarsenic species, primarily dimethylarsinic acid (DMAs(V)), and a four-fold increase in As biovolatilization compared to a control soil. Our results support a conceptual model that methanogenic activity suppresses biovolatilization by enhancing As demethylation rates. This work refines understanding of biogeochemical processes regulating As biovolatilization in anaerobic soil environments, and extends recent insights into links between methanogenesis and As metabolism to soils from the mid-South United States rice production region.

Effects of 24-Epibrassinolide on DNA Methylation Variation in Soybean (Glycine max) Leaf and Root Under Saline-Alkali Stress

Saline-alkali stress is major stress that severely reduces plant growth and productivity, it is necessary to make clear whether exogenous 24-epibrassinolide (EBR) can improve the salt-alkali resistance of soybean (Glycine max) by affecting its DNA methylation. In this study, the effects of EBR on soybean adaptation to saline-alkali stress, genomic DNA methylation level and pattern changes in saline-alkali-stressed leaf and root with or without EBR treatment were compared using methylation-sensitive amplified polymorphism (MSAP). In the results, saline-alkali stress increased DNA methylation levels in leaf and root, with higher respective hemi-methylation and global methylation rates observed in leaf (6.22, 22.24%) than root (5.72, 21.76%). EBR application reduced leaf and root DNA methylation levels, with leaf hemi-methylation rate (6.15%) exceeding that of root (4.25%) and leaf global methylation rate (21.79%) below that of root (22.51%). There were distinct DNA remethylation and demethylation variations across different tissues and treatments, demethylation in leaves was dominant. Meanwhile, untreated saline-alkali-stressed roots exhibited major demethylation-based variations, while remethylation variations predominated post-treatment. Under saline-alkali stress, root remethylation and demethylation rates (6.17, 7.55%, respectively) both exceeded respective leaf rates (5.18 and 7.46%); however, post-EBR treatment, root methylation rate (6.45%) exceeded leaf rate (5.38%), while root demethylation rate (6.13%) fell below leaf rate (6.94%). In conclusion, exogenous EBR application to saline-alkali-stressed soybean can influence leaf and root genomic DNA methylation levels and patterns via distinct tissue-specific methylation mechanisms.

Microbial Diversity and Mercury Methylation Activity in Periphytic Biofilms at a Run-of-River Hydroelectric Dam and Constructed Wetlands.

ABSTRACTPeriphytic biofilms have the potential to greatly influence the microbial production of the neurotoxicant monomethylmercury in freshwaters although few studies have simultaneously assessed periphyton mercury methylation and demethylation rates and the microbial communities associated with these transformations. We performed a field study on periphyton from a river affected by run-of-river power plants and artificial wetlands in a boreal landscape (Québec, Canada). In situ incubations were performed on three sites using environmental concentrations of isotopically enriched monomethylmercury (MM198Hg) and inorganic mercury (200Hg) for demethylation and methylation rate measurements. Periphytic microbial communities were investigated through 16S rRNA gene analyses and metagenomic screenings for the hgcA gene, involved in mercury methylation. Positive mercury methylation rates ([5.9 ± 3.4] × 10−3 day−1) were observed only in the wetlands, and demethylation rates averaged 1.78 ± 0.21 day−1 for the three studied sites. The 16S rRNA gene analyses revealed Proteobacteria as the most abundant phylum across all sites (36.3% ± 1.4%), from which families associated with mercury methylation were mostly found in the wetland site. Metagenome screening for HgcA identified 24 different hgcA sequences in the constructed wetland site only, associated with 8 known families, where the iron-reducing Geobacteraceae were the most abundant. This work brings new information on mercury methylation in periphyton from habitats of impacted rivers, associating it mostly with putative iron-reducing bacteria.IMPORTANCE Monomethylmercury (MMHg) is a biomagnifiable neurotoxin of global concern with risks to human health mostly associated with fish consumption. Hydroelectric reservoirs are known to be sources of MMHg many years after their impoundment. Little is known, however, on run-of-river dams flooding smaller terrestrial areas, although their numbers are expected to increase considerably worldwide in decades to come. Production of MMHg is associated mostly with anaerobic processes, but Hg methylation has been shown to occur in periphytic biofilms located in oxic zones of the water column. Therefore, in this study, we investigated in situ production of MMHg by periphytic communities in habitats impacted by the construction of a run-of-river dam by combining transformation rate measurements with genomic approaches targeting hgcAB genes, responsible for mercury methylation. These results provide extended knowledge on mercury methylators in river ecosystems impacted by run-of-river dams in temperate habitats.