Post-transcriptional modifications at the RNA level can regulate gene expression through cis- or trans-mechanisms, which are crucial for cell function and cell fate determination. To date, more than ...150 chemical modifications have been found on RNA, including m6A, m1A, pseudo-uridine and m5C.1,2 These modifications regulate gene expression at a critical level of the ‘epitranscriptome’. m6A is the most abundant internal mRNA and the most extensively studied modification to date. Previous research work showed that m6A tends to occur in the (G/A) (m6A) C canonical motif. m6A affects the metabolic process of mRNA such as splicing, nuclear export, degradation and translation to regulate gene expression and plays important roles in developmental, physiological and pathological processes.1 The earliest whole-transcriptome maps of m6A modifications were published in 2012 (m6A-Seq or MeRIP-Seq), using m6A antibodies to enrich m6A-containing RNA fragments, resulting in profiling maps with the resolution of 100–200 nt.3,4 The current mainstream sequencing method for RNA m6A is antibody-based MeRIP.
Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we ...introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.m6A-SAC-seq uses chemical labeling to quantify m6A at single-base resolution in the mammalian transcriptome.
Wastewater treatment plants (WWTPs) are significant contributors to energy consumption and anthropogenic greenhouse gas (GHG) emissions. For achieving carbon reduction in the wastewater treatment ...industry, the direct and indirect GHG emissions generated by WWTPs need to be understood from a holistic perspective. This study estimated GHG emissions from WWTPs at the country scale by integrating process-based life cycle assessment and statistical data. On-site data were collected from 17 WWTPs of various regions in China. Uncertainty analysis based on Monte Carlo was also performed, so as to provide more reliable results. The results show that life cycle GHG emissions generated from the wastewater treatment process vary from 0.29 kg CO2 eq/m3 to 1.18 kg CO2 eq/m3 based on 17 sample WWTPs. The key factors contributing to overall GHG emissions are also identified as carbon dioxide (fossil) and methane (fossil) to air mainly generated from electricity generation, and methane (biogenic) and nitrous oxide (biogenic) to air mainly generated from wastewater treatment. National average GHG emissions was evaluated with the value of 0.88 kg CO2 eq/m3, with on-site GHG emissions and off-site electricity-based GHG emissions accounting for 32% and 34%, respectively. The total GHG emissions generated from wastewater treatment are 56.46 billion kg CO2 eq in 2020, with Guangdong province having the dominant contribution. Policy suggestions (e.g., further adjusting the electricity grid toward a low carbon structure, improving technology to promote treatment efficiency and energy recovery) were highly recommended so that national GHG emissions of WWTPs can be reduced. In order to achieve the synergy of pollutant removal and GHG emission reduction, policy-making on wastewater treatment should be tailored to specific local conditions.
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•National GHG emissions of WWTPs were evaluated based on a bottom-up method.•GHG emissions varied from 0.29 to 1.18 kg CO2 eq/m3 based on LCA case study.•Average GHG emissions intensity for WWTPs in China was 0.88 kg CO2 eq/m3.•Geographic distribution of GHG emissions from WWTPs in China was explored.•Direct and electricity-based emissions occupied 32% and 34% of total GHG emissions.
Introduction Transcriptional modification of RNA in cells plays a crucial role in its stability, transportation, processing and thus regulation of gene expression. By modulating protein expression of ...m6A writers, erasers and readers in HIV-1 producer and target cells, several studies demonstrated the importance of the m6A pathway in regulating HIV-1 replication and viral protein expression in CD4+ T cell lines and primary CD4+ T cells 18–22. ...HIV-1 infection of CD4+ T cells upregulates m6A levels of cellular RNA 23 and HIV-1 protease cleaves the antiviral m6A reader protein YTHDF3 in the viral particle 22. Detection of viral RNA by these sensors triggers activation of several cellular kinases, which phosphorylate interferon regulatory factors 3 and 7 (IRF3 and IRF7) to induce IFN-I expression 26, 27. Un-paired t-test was used for statistical analysis. * P < 0.05, ** P < 0.005, compared with mock or Ctrl samples as indicated. https://doi.org/10.1371/journal.ppat.1009421.g001 To examine whether the sequence of RNA oligos is important for m6A-mediated suppression of IFN-I expression in differentiated U937 cells, we designed a different pair of RNA oligo with or without a single m6A modification based on the scrambled sequence of HIV-1 RNA oligo 2.
N6-methyladenosine (m6A) is a prevalent RNA modification that plays a key role in regulating eukaryotic cellular mRNA functions. RNA m6A modification is regulated by two groups of cellular proteins, ...writers and erasers that add or remove m6A, respectively. HIV-1 RNA contains m6A modifications that modulate viral infection and gene expression in CD4+ T cells. However, it remains unclear whether m6A modifications of HIV-1 RNA modulate innate immune responses in myeloid cells that are important for antiviral immunity. Here we show that m6A modification of HIV-1 RNA suppresses the expression of antiviral cytokine type-I interferon (IFN-I) in differentiated human monocytic cells and primary monocyte-derived macrophages. Transfection of differentiated monocytic U937 cells with HIV-1 RNA fragments containing a single m6A-modification significantly reduced IFN-I mRNA expression relative to their unmodified RNA counterparts. We generated HIV-1 with altered m6A levels of RNA by manipulating the expression of the m6A erasers (FTO and ALKBH5) or pharmacological inhibition of m6A addition in virus-producing cells, or by treating HIV-1 RNA with recombinant FTO in vitro. HIV-1 RNA transfection or viral infection of differentiated U937 cells and primary macrophages demonstrated that HIV-1 RNA with decreased m6A levels enhanced IFN-I expression, whereas HIV-1 RNA with increased m6A modifications had opposite effects. Our mechanistic studies indicated that m6A of HIV-1 RNA escaped retinoic acid-induced gene I (RIG-I)-mediated RNA sensing and activation of the transcription factors IRF3 and IRF7 that drive IFN-I gene expression. Together, these findings suggest that m6A modifications of HIV-1 RNA evade innate immune sensing in myeloid cells.
DNA methylation in the promoters of plant genes sometimes leads to transcriptional repression, and the loss of DNA methylation in methyltransferase mutants results in altered gene expression and ...severe developmental defects. However, many cases of naturally occurring DNA methylation variations have been reported, whereby altered expression of differentially methylated genes is responsible for agronomically important traits. The ability to manipulate plant methylomes to generate epigenetically distinct individuals could be invaluable for breeding and research purposes. Here, we describe "epimutagenesis," a method to rapidly generate DNA methylation variation through random demethylation of the Arabidopsis thaliana genome. This method involves the expression of a human ten-eleven translocation (TET) enzyme, and results in widespread hypomethylation that can be inherited to subsequent generations, mimicking mutants in the maintenance of DNA methyltransferase met1. Application of epimutagenesis to agriculturally significant plants may result in differential expression of alleles normally silenced by DNA methylation, uncovering previously hidden phenotypic variations.
Mps one binder 2 (MOB2) regulates the NDR kinase family, however, whether and how it is implicated in cancer remain unknown. Here we show that MOB2 functions as a tumor suppressor in glioblastoma ...(GBM). Analysis of MOB2 expression in glioma patient specimens and bioinformatic analyses of public datasets revealed that MOB2 was downregulated at both mRNA and protein levels in GBM. Ectopic MOB2 expression suppressed, while depletion of MOB2 enhanced, the malignant phenotypes of GBM cells, such as clonogenic growth, anoikis resistance, and formation of focal adhesions, migration, and invasion. Moreover, depletion of MOB2 increased, while overexpression of MOB2 decreased, GBM cell metastasis in a chick chorioallantoic membrane model. Overexpression of MOB2-mediated antitumor effects were further confirmed in mouse xenograft models. Mechanistically, MOB2 negatively regulated the FAK/Akt pathway involving integrin. Notably, MOB2 interacted with and promoted PKA signaling in a cAMP-dependent manner. Furthermore, the cAMP activator Forskolin increased, while the PKA inhibitor H89 decreased, MOB2 expression in GBM cells. Functionally, MOB2 contributed to the cAMP/PKA signaling-regulated inactivation of FAK/Akt pathway and inhibition of GBM cell migration and invasion. Collectively, these findings suggest a role of MOB2 as a tumor suppressor in GBM via regulation of FAK/Akt signaling. Additionally, we uncover MOB2 as a novel regulator in cAMP/PKA signaling. Given that small compounds targeting FAK and cAMP pathway have been tested in clinical trials, we suggest that interference with MOB2 expression and function may support a theoretical and therapeutic basis for applications of these compounds.
This randomized controlled trial aimed to evaluate the effectiveness of an online application based on HAPA theory on oral hygiene promotion in young adults with fixed orthodontic appliances.
A ...WeChat mini-program (called "Clean Teeth") based on HAPA theory was designed beforehand to improve oral-health behaviors and oral hygiene, and 44 participants aged 17-29 with fixed orthodontic appliances were recruited. Participants of the experimental group (n = 22) received the "Clean Teeth" mini-program, in addition to care as usual, and the control group (n = 22) only received routine oral health education. Data were collected during three orthodontic check-ups: baseline (T0), 6 weeks of follow-up (T1), and 12 weeks of follow-up (T2). All participants completed questionnaires assessing oral health behaviors and the psychosocial factors of the HAPA model and accepted the clinical examinations involving the dental plaque index and the gingival bleeding index.
After a 12-week intervention, the plaque index and gingival bleeding index in the experimental group were significantly lower than that in the control group. The psycho-social parameters of social effects, expected outcomes, and action control were improved significantly after treatment, among which social effects increased significantly only in the experimental group but not in the control group.
The HAPA theory-based mini-program had positive effects on oral-health behavior promotion and oral hygiene among young adults with fixed orthodontic appliances. Trial registration This study was retrospectively registered in the Chinese Clinical Trial Registry, with the number CTR2200056731, dated 12/02/2022. http://www.chictr.org.cn/index.aspx .
Histone methylation occurs on both lysine and arginine residues, and its dynamic regulation plays a critical role in chromatin biology. Here we identify the UHRF1 PHD finger (PHDUHRF1), an important ...regulator of DNA CpG methylation, as a histone H3 unmodified arginine 2 (H3R2) recognition modality. This conclusion is based on binding studies and cocrystal structures of PHDUHRF1 bound to histone H3 peptides, where the guanidinium group of unmodified R2 forms an extensive intermolecular hydrogen bond network, with methylation of H3R2, but not H3K4 or H3K9, disrupting complex formation. We have identified direct target genes of UHRF1 from microarray and ChIP studies. Importantly, we show that UHRF1's ability to repress its direct target gene expression is dependent on PHDUHRF1 binding to unmodified H3R2, thereby demonstrating the functional importance of this recognition event and supporting the potential for crosstalk between histone arginine methylation and UHRF1 function.
► In vitro binding and ITC identify PHDUHRF1 domain as an unmodified histone H3R2 reader ► Crystal structure shows hydrogen bonding, electrostatics, and hydrophobic interactions ► Genome-wide analyses demonstrate a general transcriptional role for UHRF1 ► Binding unmodified H3R2 is critical for UHRF1 to regulate gene expression
Metallic multilayered nanofilms have been extensively studied owing to their unique physical properties and applications. However, studies on the thermal conductivity and electrical resistivity of ...metallic multilayered nanofilms, as their important physical properties, are seldom reported. In this work, Cu/W multilayered nanofilms with periodic thickness varying from 6 to 150 nm were deposited by magnetron sputtering. The resistivities of the Cu/W multilayered nanofilms increase with the decrease of periodic thickness, especially when the periodic thickness is smaller than 37 nm. The resistivities of the multilayered nanofilms fit well with the Fuchs–Sondheimer and Mayadas–Shatzkes (FS–MS) model, which considers both interface scattering and grain boundary scattering. The thermal conductivities of the Cu/W multilayered nanofilms were measured by the three-omega (3ω) method, which decrease with a decrease of periodic thickness initially and increase at the smallest periodic thickness of 6 nm. The Boltzmann transport equation (BTE)-based model was used, to explain the periodic thickness-dependent thermal conductivity of metallic multilayered nanofilms by considering the contributions from both phonon and electron heat transport processes, where the calculated thermal conductivities agree well with the measured ones. The electrical resistivity and thermal conductivity strongly depend on the microstructures of the multilayered nanofilms.
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