bims-rimeca Biomed News
on RNA methylation in cancer
Issue of 2020‒12‒20
fifteen papers selected by
Sk Ramiz Islam
Saha Institute of Nuclear Physics
  1. The m6A methyltransferase METTL14 inhibits the proliferation, migration, and invasion of gastric cancer by regulating the PI3K/AKT/mTOR signaling pathway.
  2. Reversible N6-methyladenosine of RNA: The regulatory mechanisms on gene expression and implications in physiology and pathology.
  3. Integrated Analysis of m6A Methylome in Cisplatin-Induced Acute Kidney Injury and Berberine Alleviation in Mouse.
  4. Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6.
  5. Epitranscriptomic(N6-methyladenosine) Modification of Viral RNA and Virus-Host Interactions.
  6. The functions of N6-methyladenosine modification in lncRNAs.
  7. N6-Methyladenosine Regulates Host Responses to Viral Infection.
  8. The N6-methyladenosine RNA modification in acute myeloid leukemia.
  9. The N6-Methyladenosine Features of mRNA and Aberrant Expression of m6A Modified Genes in Gastric Cancer and Their Potential Impact on the Risk and Prognosis.
  10. Vascular Smooth Muscle FTO Promotes Aortic Dissecting Aneurysms via m6A Modification of Klf5.
  11. TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.
  12. Nucleotide resolution profiling of m3C RNA modification by HAC-seq.
  13. METTL14 Overexpression Promotes Osteosarcoma Cell Apoptosis and Slows Tumor Progression via Caspase 3 Activation.
  14. Oxidative demethylase ALKBH5 repairs DNA alkylation damage and protects against alkylation-induced toxicity.
  15. Base Pairing and Functional Insights into N3-Methylcytidine (m3C) in RNA.
  1. J Clin Lab Anal. 2020 Dec 12. e23655
    The m6A methyltransferase METTL14 inhibits the proliferation, migration, and invasion of gastric cancer by regulating the PI3K/AKT/mTOR signaling pathway.
    Xin Liu, Mingyang Xiao, Liang Zhang, Liuli Li, Guolian Zhu, Erdong Shen, Mingyue Lv, Xiaobo Lu, Zhe Sun.
      BACKGROUND: N6-methyladenosine (m6A) modification may participate in the regulation of occurrence and development of tumors. However, the m6A level and the potential regulatory mechanism of m6A in gastric cancer (GC) remain uncertain.METHODS: RNA m6A quantification assay was conducted to detect the m6A level in GC tissues and cell lines. Methyltransferase-like 14 (METTL14) expression in GC tissues was explored by bioinformatics and immunohistochemistry. Then, the function of METTL14 in GC cells was examined by CCK-8, colony formation assay, wound healing assay, and Transwell assay. Besides, Western blotting was conducted to probe the PI3K/AKT/mTOR pathway and the epithelial-mesenchymal transformation (EMT) pathway-related gene expression.
    RESULTS: The m6A modification level was decreased in GC and METTL14 was a key regulator resulting in m6A disorder in GC. METTL14 was downregulated in GC by analyzing both clinical samples and bioinformatics. METTL14 overexpression suppressed GC cell proliferation and aggression by deactivating the PI3K/AKT/mTOR pathway and the EMT pathway, respectively.
    CONCLUSIONS: Our findings indicate that METTL14 partakes in the biological process of GC as a tumor suppressor and may be an emerging biomarker in GC.
    Keywords:  RNA m6A modification; gastric cancer; methyltransferase-like 14; prognosis; tumor suppressor
    DOI:  https://doi.org/10.1002/jcla.23655
  2. Genes Dis. 2020 Dec;7(4): 585-597
    Reversible N6-methyladenosine of RNA: The regulatory mechanisms on gene expression and implications in physiology and pathology.
    Xinyu Fang, Mengyang Li, Tao Yu, Gaoli Liu, Jianxun Wang.
      N6-methyladenosine (m6A) is the most abundant inner RNA modification in eukaryotes. Due to the development of RNA sequencing technology, the distribution pattern of m6A in the transcriptome has been uncovered. Dynamically, the reversible N6-methylation is mediated by two types of proteins, which are classified as "writers" and "erasers". Under the association of specific co-factors, writers show spatiotemporal N6-methyltransferase activity. Mechanically, m6A can be recognized by "reader" proteins or can directly modify RNA conformation, and it widely affects gene expression by mediating RNA stability, translation, splicing and export. m6A is involved in a series of physiology processes. Dysregulation of m6A is gradually defined as the pathogenesis of some diseases, e.g., cancer and cardiovascular disease. Therefore, a good understanding of m6A is essential for molecular biology and pathology research. In this article we systemically present an overview of the functions and mechanisms of identified m6A regulators. The discovered biological and pathological processes affected by m6A are also summarized. We hope that readers with related research interests benefit from our review.
    Keywords:  Diseases; Epigenetics; Gene expression; RNA; m6A; m6A regulator
    DOI:  https://doi.org/10.1016/j.gendis.2020.06.011
  3. Front Genet. 2020 ;11 584460
    Integrated Analysis of m6A Methylome in Cisplatin-Induced Acute Kidney Injury and Berberine Alleviation in Mouse.
    Jianxiao Shen, Wanpeng Wang, Xinghua Shao, Jingkui Wu, Shu Li, Xiajing Che, Zhaohui Ni.
      Background: N6-methyladenosine (m6A) is the most abundant modification known in mRNAs. It participates in a variety of physiological and pathological processes, such as metabolism, inflammation, and apoptosis.Aims: To explore the mechanism of m6A in cisplatin-induced acute kidney injury (AKI) and berberine alleviation in mouse.
    Methods: This study investigated the N6-methyladenosine (m6A) methylome of kidneys from three mouse groups: C57 mice (controls), those with CI-AKI (injury group, IG), and those pretreated with berberine (treatment group, TG). Methylated RNA Immunoprecipitation Next Generation Sequencing (MeRIP-seq) and RNA-seq were performed to identify the differences between the injury group and the control group (IvC) and between the treatment group and the injury group (TvI). Western blotting was performed to identify the protein levels of candidate genes.
    Results: In IvC, differentially methylated genes (DMGs) were enriched in metabolic processes and cell death. In TvI, DMGs were enriched in tissue development. Several genes involved in important pathways related to CI-AKI showed opposite methylation and expression trends in the IvC and TvI comparisons.
    Conclusion: m6A plays an important role in cisplatin induced AKI and berberine may alleviate this process.
    Keywords:  FGA; Havcr1; M6A; SLC12A1; berberine; cisplatin induced nephrotoxicity
    DOI:  https://doi.org/10.3389/fgene.2020.584460
  4. Nucleic Acids Res. 2020 Dec 16. pii: gkaa1193. [Epub ahead of print]
    Sequence- and structure-specific cytosine-5 mRNA methylation by NSUN6.
    Tommaso Selmi, Shobbir Hussain, Sabine Dietmann, Matthias Heiß, Kayla Borland, Sophia Flad, Jean-Michel Carter, Rebecca Dennison, Ya-Lin Huang, Stefanie Kellner, Susanne Bornelöv, Michaela Frye.
      The highly abundant N6-methyladenosine (m6A) RNA modification affects most aspects of mRNA function, yet the precise function of the rarer 5-methylcytidine (m5C) remains largely unknown. Here, we map m5C in the human transcriptome using methylation-dependent individual-nucleotide resolution cross-linking and immunoprecipitation (miCLIP) combined with RNA bisulfite sequencing. We identify NSUN6 as a methyltransferase with strong substrate specificity towards mRNA. NSUN6 primarily targeted three prime untranslated regions (3'UTR) at the consensus sequence motif CTCCA, located in loops of hairpin structures. Knockout and rescue experiments revealed enhanced mRNA and translation levels when NSUN6-targeted mRNAs were methylated. Ribosome profiling further demonstrated that NSUN6-specific methylation correlated with translation termination. While NSUN6 was dispensable for mouse embryonic development, it was down-regulated in human tumours and high expression of NSUN6 indicated better patient outcome of certain cancer types. In summary, our study identifies NSUN6 as a methyltransferase targeting mRNA, potentially as part of a quality control mechanism involved in translation termination fidelity.
    DOI:  https://doi.org/10.1093/nar/gkaa1193
  5. Front Cell Infect Microbiol. 2020 ;10 584283
    Epitranscriptomic(N6-methyladenosine) Modification of Viral RNA and Virus-Host Interactions.
    Hasan Imam, Geon-Woo Kim, Aleem Siddiqui.
      N6-methyladenosine (m6A) is the most prevalent and internal modification of eukaryotic mRNA. Multiple m6A methylation sites have been identified in the viral RNA genome and transcripts of DNA viruses in recent years. m6A modification is involved in all the phases of RNA metabolism, including RNA stability, splicing, nuclear exporting, RNA folding, translational modulation, and RNA degradation. Three protein groups, methyltransferases (m6A-writers), demethylases (m6A-erasers), and m6A-binding proteins (m6A-readers) regulate this dynamic reversible process. Here, we have reviewed the role of m6A modification dictating viral replication, morphogenesis, life cycle, and its contribution to disease progression. A better understanding of the m6A methylation process during viral pathogenesis is required to reveal novel approaches to combat the virus-associated diseases.
    Keywords:  m6A modification; m6A-binding protein; m6A-eraser; m6A-writer; viral epitranscriptomics
    DOI:  https://doi.org/10.3389/fcimb.2020.584283
  6. Genes Dis. 2020 Dec;7(4): 598-605
    The functions of N6-methyladenosine modification in lncRNAs.
    Rong-Zhang He, Jing Jiang, Di-Xian Luo.
      Increasing evidence indicates that mRNAs are often subject to posttranscriptional modifications. Among them, N6-methyladenosine (m6A), which has been shown to play key roles in RNA splicing, stability, nuclear export, and translation, is the most abundant modification of RNA. Extensive studies of m6A modification of mRNAs have been carried out, while little is known about m6A modification of long non-coding RNAs (lncRNAs). Recently, several studies reported m6A modification of lncRNAs. In this review, we focus on these m6A-modified lncRNAs and discuss possible functions of m6A modification.
    Keywords:  Erasers; LncRNA; N6-methyladenosine; Readers; Writers
    DOI:  https://doi.org/10.1016/j.gendis.2020.03.005
  7. Trends Biochem Sci. 2020 Dec 09. pii: S0968-0004(20)30279-6. [Epub ahead of print]
    N6-Methyladenosine Regulates Host Responses to Viral Infection.
    Michael J McFadden, Stacy M Horner.
      Recent discoveries have revealed that, during viral infection, the presence of the RNA modification N6-methyladenosine (m6A) on viral and cellular RNAs has profound impacts on infection outcome. Although m6A directly regulates many viral RNA processes, its effects on cellular RNAs and pathways during infection have only recently begun to be elucidated. Disentangling the effects of m6A on viral and host RNAs remains a challenge for the field. m6A has been found to regulate host responses such as viral RNA sensing, cytokine responses, and immune cell functions. We highlight recent findings describing how m6A modulates host responses to viral infection and discuss future directions that will lead to a synergistic understanding of the processes by which m6A regulates viral infection.
    Keywords:  N(6)-methyladenosine; RNA sensing; cytokines; interferon (IFN); m(6)A
    DOI:  https://doi.org/10.1016/j.tibs.2020.11.008
  8. Curr Opin Hematol. 2020 Dec 16. Publish Ahead of Print
    The N6-methyladenosine RNA modification in acute myeloid leukemia.
    Eliza Yankova, Demetrios Aspris, Konstantinos Tzelepis.
      PURPOSE OF REVIEW: In recent years, the N6-methyladenosine (m6A) modification of RNA has been shown to play an important role in the development of acute myeloid leukemia (AML) and the maintenance of leukemic stem cells (LSCs). In this review we summarise the recent findings in the field of epitranscriptomics related to m6A and its relevance in AML.RECENT FINDINGS: Recent studies have focused on the role of m6A regulators in the development of AML and their potential as translational targets. The writer Methyltransferase Like 3 and its binding partner Methyltransferase Like 14, as well as the reader YTH domain-containing family protein 2, were shown to be vital for LSC survival, and their loss has detrimental effects on AML cells. Similar observations were made with the demethylases fat mass and obesity-associated protein and AlkB homologue 5 RNA demethylase. Of importance, loss of any of these genes has little to no effect on normal hemopoietic stem cells, suggesting therapeutic potential.
    SUMMARY: The field of epitranscriptomics is still in its infancy and the importance of m6A and other RNA-modifications in AML will only come into sharper focus. The development of therapeutics targeting RNA-modifying enzymes may open up new avenues for treatment of such malignancies.
    DOI:  https://doi.org/10.1097/MOH.0000000000000636
  9. Front Genet. 2020 ;11 561566
    The N6-Methyladenosine Features of mRNA and Aberrant Expression of m6A Modified Genes in Gastric Cancer and Their Potential Impact on the Risk and Prognosis.
    Liang Sang, Liping Sun, Ang Wang, Han Zhang, Yuan Yuan.
      Although N6-methyladenosine (m6A) mRNA methylation is known to be closely related to tumor events, its role in carcinogenesis and the development of gastric cancer (GC) is not yet clear. The aim of this study was to identify common m6A features and novel aberrant expression of m6A modified genes in GC and to further explore their potential impact on risk and prognosis. Three paired GC and paracancerous (PCa) tissues were collected to perform an m6A sequencing by MeRIP-seq and microarray assays. The expression profile of m6A and mRNA were determined. Gene function note and enrichment analysis were performed, and protein-protein interaction networks of differentially m6A methylated genes (DMGs) were generated using the DAVID and STRING databases, respectively. Validation of the m6A related differentially expressed genes by matching TCGA and GTEx data and human tissues. Clinical and pathological correlation and survival analysis were performed by TCGA data. The m6A motif sequence GGACAR (R = U or A) C was the consensus in both GC and PCa tissues. m6A peaks were significantly related to different coordinates, however, for most samples, the end of the coding sequence (CDS) was more prominent than the start of CDS. The genes with higher levels of m6A in their mRNAs were mainly enriched in transcriptional misregulation in carcinogenesis pathways, whereas the genes with decreased methylation mainly regulated digestion and absorption of protein. There are genes with differential m6A modifications in GC and paired PCa tissues, and these genes are mainly enriched in transcriptional misregulation and digestion/absorption pathways. m6A-GC with the down- and up-regulated genes may play an important role in gastric carcinogenesis, which can affect the risk and prognosis in GC.
    Keywords:  M6A; expression; gastric cancer; methylation; prognosis; risk
    DOI:  https://doi.org/10.3389/fgene.2020.561566
  10. Front Cardiovasc Med. 2020 ;7 592550
    Vascular Smooth Muscle FTO Promotes Aortic Dissecting Aneurysms via m6A Modification of Klf5.
    Dong Ma, Xiao Liu, Jin-Jin Zhang, Jun-Jian Zhao, Yan-Jie Xiong, Quan Chang, Hong-Yan Wang, Peng Su, Jia Meng, Yong-Bo Zhao.
      Background: Aortic dissecting aneurysm (ADA) represents an aortic remodeling disease with a high mortality rate. Fat mass and obesity-associated protein (FTO) exerts RNA demethylation function to regulate gene expression related to stem cell differentiation, DNA damage repair, and tumorigenesis, but the role of FTO in ADA is still unclear. Methods: The expression and location of FTO in 43 ADA tissues and 11 normal tissues were determined by RT-qPCR, WB, immunohistochemistry, and immunofluorescence staining. Detecting proliferation and migration of VSMCs. M6A methylated RNA immuno-precipitation qRT-PCR and dual luciferase reporter assay were performed for determining m6A level and interaction between m6A modulation and Klf5 mRNA, respectively. Results: FTO are highly expressed in VSMCs. FTO was positively correlated with BMI, triglyceride, and D-dimer (all P < 0.05). Functionally, both AngII-induced FTO expression and over expression of FTO promote cell proliferation and migration, whereas knockdown of FTO inhibits these functions. Mechanically, we identified Krüppel-like factor 5 (Klf5) as a target of FTO mediating m6A modification. Overexpression of FTO reduced m6A modification on Klf5 mRNA and promoted Klf5 mRNA expression. Furthermore, the p-GSK3β and Klf5 levels increased after FTO overexpression. Finally, knockdown of FTO suppresses the p-GSK3β levels and Klf5 expression regardless of AngII treatment. Conclusions: Our study revealed that FTO expression significantly contributes to the phenotype conversion of VSMCs and the ADA by the demethylation function (m6A), thereby providing a novel therapeutic target.
    Keywords:  FTO; GSK3β; Klf5; aortic dissecting aneurysm; vascular smooth muscle cells
    DOI:  https://doi.org/10.3389/fcvm.2020.592550
  11. Nucleic Acids Res. 2020 Dec 11. pii: gkaa1162. [Epub ahead of print]
    TonEBP recognizes R-loops and initiates m6A RNA methylation for R-loop resolution.
    Hyun Je Kang, Na Young Cheon, Hyun Park, Gyu Won Jeong, Byeong Jin Ye, Eun Jin Yoo, Jun Ho Lee, Jin-Hoe Hur, Eun-A Lee, Hongtae Kim, Kyoo-Young Lee, Soo Youn Choi, Whaseon Lee-Kwon, Kyungjae Myung, Ja Yil Lee, Hyug Moo Kwon.
      R-loops are three-stranded, RNA-DNA hybrid, nucleic acid structures produced due to inappropriate processing of newly transcribed RNA or transcription-replication collision (TRC). Although R-loops are important for many cellular processes, their accumulation causes genomic instability and malignant diseases, so these structures are tightly regulated. It was recently reported that R-loop accumulation is resolved by methyltransferase-like 3 (METTL3)-mediated m6A RNA methylation under physiological conditions. However, it remains unclear how R-loops in the genome are recognized and induce resolution signals. Here, we demonstrate that tonicity-responsive enhancer binding protein (TonEBP) recognizes R-loops generated by DNA damaging agents such as ultraviolet (UV) or camptothecin (CPT). Single-molecule imaging and biochemical assays reveal that TonEBP preferentially binds a R-loop via both 3D collision and 1D diffusion along DNA in vitro. In addition, we find that TonEBP recruits METTL3 to R-loops through the Rel homology domain (RHD) for m6A RNA methylation. We also show that TonEBP recruits RNaseH1 to R-loops through a METTL3 interaction. Consistent with this, TonEBP or METTL3 depletion increases R-loops and reduces cell survival in the presence of UV or CPT. Collectively, our results reveal an R-loop resolution pathway by TonEBP and m6A RNA methylation by METTL3 and provide new insights into R-loop resolution processes.
    DOI:  https://doi.org/10.1093/nar/gkaa1162
  12. Nucleic Acids Res. 2020 Dec 11. pii: gkaa1186. [Epub ahead of print]
    Nucleotide resolution profiling of m3C RNA modification by HAC-seq.
    Jia Cui, Qi Liu, Erdem Sendinc, Yang Shi, Richard I Gregory.
      Cellular RNAs are subject to a myriad of different chemical modifications that play important roles in controlling RNA expression and function. Dysregulation of certain RNA modifications, the so-called 'epitranscriptome', contributes to human disease. One limitation in studying the functional, physiological, and pathological roles of the epitranscriptome is the availability of methods for the precise mapping of individual RNA modifications throughout the transcriptome. 3-Methylcytidine (m3C) modification of certain tRNAs is well established and was also recently detected in mRNA. However, methods for the specific mapping of m3C throughout the transcriptome are lacking. Here, we developed a m3C-specific technique, Hydrazine-Aniline Cleavage sequencing (HAC-seq), to profile the m3C methylome at single-nucleotide resolution. We applied HAC-seq to analyze ribosomal RNA (rRNA)-depleted total RNAs in human cells. We found that tRNAs are the predominant m3C-modified RNA species, with 17 m3C modification sites on 11 cytoplasmic and 2 mitochondrial tRNA isoacceptors in MCF7 cells. We found no evidence for m3C-modification of mRNA or other non-coding RNAs at comparable levels to tRNAs in these cells. HAC-seq provides a novel method for the unbiased, transcriptome-wide identification of m3C RNA modification at single-nucleotide resolution, and could be widely applied to reveal the m3C methylome in different cells and tissues.
    DOI:  https://doi.org/10.1093/nar/gkaa1186
  13. Cancer Manag Res. 2020 ;12 12759-12767
    METTL14 Overexpression Promotes Osteosarcoma Cell Apoptosis and Slows Tumor Progression via Caspase 3 Activation.
    Ziwen Liu, Ning Liu, Zhipeng Huang, Wenbo Wang.
      Background: As a key enzyme of m6A methylation modification, methyltransferase-like 14 (METTL14) is involved in many physiological and pathophysiological processes. This study aims to explore the effect of METTL14 on the viability of osteosarcoma cells and explain the underlying molecular mechanism.Methods: We detected the content of METTL14 in osteosarcoma tissue by qRT-PCR and Western blot. Experiments such as transwell, EdU, and CCK-8 have demonstrated the effect of METTL14 on osteosarcoma cell activity. In addition, the regulation of caspase-3 by METL14 was determined by Western blot. We used caspase-3 inhibitor to further reverse the effect of METTL14 on osteosarcoma cell apoptosis.
    Results: We found that the expression of METTL14 in osteosarcoma cells was reduced compared with normal tissues. METTL14 overexpression significantly reduced the proliferation, migration, invasion and apoptosis of osteosarcoma cells. Inhibition of METL14 showed the opposite result. We have demonstrated that METTL14 finally achieves apoptosis by activating caspase-3.
    Conclusion: We have demonstrated that METTL14 has effects on osteosarcoma cell proliferation, migration, and invasion and promotes cell apoptosis by activating caspase-3, which may become a potential therapeutic target for osteosarcoma.
    Keywords:  METTL14; m6A methylation; osteosarcoma
    DOI:  https://doi.org/10.2147/CMAR.S284273
  14. Biochem Biophys Res Commun. 2020 Dec 12. pii: S0006-291X(20)32194-X. [Epub ahead of print]534 114-120
    Oxidative demethylase ALKBH5 repairs DNA alkylation damage and protects against alkylation-induced toxicity.
    Deepa Akula, Timothy R O'Connor, Roy Anindya.
      DNA integrity is challenged by both exogenous and endogenous alkylating agents. DNA repair proteins such as Escherichia coli AlkB family of enzymes can repair 1-methyladenine and 3-methylcytosine adducts by oxidative demethylation. Human AlkB homologue 5 (ALKBH5) is RNA N6-methyladenine demethylase and not known to be involved in DNA repair. Herein we show that ALKBH5 also has weak DNA repair activity and it can demethylate DNA 3-methylcytosine. The mutation of the amino acid residues involved in demethylation also abolishes the DNA repair activity of ALKBH5. Overexpression of ALKBH5 decreases the 3-methylcytosine level in genomic DNA and reduces the cytotoxic effects of the DNA damaging alkylating agent methyl methanesulfonate. Thus, demethylation by ALKBH5 might play a supporting role in maintaining genome integrity.
    Keywords:  ALKBH2; ALKBH3; ALKBH5; AlkB; Alkyl adducts; DNA alkylation; DNA repair; Demethylation; Fe(II)/2OG-Dependent dioxygenase; N3-methylcytosine
    DOI:  https://doi.org/10.1016/j.bbrc.2020.12.017
  15. ACS Chem Biol. 2020 Dec 17.
    Base Pairing and Functional Insights into N3-Methylcytidine (m3C) in RNA.
    Song Mao, Phensinee Haruehanroengra, Srivathsan V Ranganathan, Fusheng Shen, Thomas J Begley, Jia Sheng.
      N3-methylcytidine (m3C) is present in both eukaryotic tRNA and mRNA and plays critical roles in many biological processes. We report the synthesis of the m3C phosphoramidite building block and its containing RNA oligonucleotides. The base-pairing stability and specificity studies show that the m3C modification significantly disrupts the stability of the Watson-Crick C:G pair. Further m3C decreases the base pairing discrimination between C:G and the other mismatched C:A, C:U, and C:C pairs. Our molecular dynamic simulation study further reveals the detailed structural insights into the m3C:G base pairing pattern in an RNA duplex. More importantly, the biochemical investigation of m3C using reverse transcription in vitro shows that N3-methylation specifies the C:A pair and induces a G to A change using HIV-1-RT, MMLV-RT, and MutiScribe-RT enzymes, all with relatively low replication fidelity. For other reverse transcriptases with higher fidelity like AMV-RT, the methylation could completely shut down DNA synthesis. Our work provides detailed insights into the thermostability of m3C in RNA and a foundation for developing new molecular tools for mapping m3C in different RNA contexts and exploring the biochemical and biomedical potentials of m3C in the design and development of RNA based therapeutics.
    DOI:  https://doi.org/10.1021/acschembio.0c00735
This page is being maintained by Thomas Krichel. It was last updated on 2021‒07‒23 at 10:24:08.