Simultaneous mapping of active DNA demethylation and sister chromatid exchange in single cells.

To understand mammalian active DNA demethylation, various methods have been developed to map the genomic distribution of the demethylation intermediates 5-formylcysotine (5fC) and 5-carboxylcytosine (5caC). However, the majority of these methods requires a large number of cells to begin with. In this study, we describe low-input methylase-assisted bisulfite sequencing (liMAB-seq ) and single-cell MAB-seq (scMAB-seq), capable of profiling 5fC and 5caC at genome scale using ∼100 cells and single cells, respectively. liMAB-seq analysis of preimplantation embryos reveals the oxidation of 5mC to 5fC/5caC and the positive correlation between chromatin accessibility and processivity of ten-eleven translocation (TET) enzymes. scMAB-seq captures the cell-to-cell heterogeneity of 5fC and 5caC and reveals the strand-biased distribution of 5fC and 5caC. scMAB-seq also allows the simultaneous high-resolution mapping of sister chromatid exchange (SCE), facilitating the study of this type of genomic rearrangement. Therefore, our study not only establishes new methods for the genomic mapping of active DNA demethylation using limited numbers of cells or single cells but also demonstrates the utilities of the methods in different biological contexts.

Functional Diversity and Engineering of the Adenylation Domains in Nonribosomal Peptide Synthetases.

Nonribosomal peptides (NRPs) are biosynthesized by nonribosomal peptide synthetases (NRPSs) and are widely distributed in both terrestrial and marine organisms. Many NRPs and their analogs are biologically active and serve as therapeutic agents. The adenylation (A) domain is a key catalytic domain that primarily controls the sequence of a product during the assembling of NRPs and thus plays a predominant role in the structural diversity of NRPs. Engineering of the A domain to alter substrate specificity is a potential strategy for obtaining novel NRPs for pharmaceutical studies. On the basis of introducing the catalytic mechanism and multiple functions of the A domains, this article systematically describes several representative NRPS engineering strategies targeting the A domain, including mutagenesis of substrate-specificity codes, substitution of condensation-adenylation bidomains, the entire A domain or its subdomains, domain insertion, and whole-module rearrangements.

Genome-wide identification and comparative analysis of DNA methyltransferase and demethylase gene families in two ploidy Cyclocarya paliurus and their potential function in heterodichogamy.

BACKGROUND: DNA methylation is one of the most abundant epigenetic modifications, which plays important roles in flower development, sex differentiation, and regulation of flowering time. Its pattern is affected by cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase). At present, there are no reports on C5-MTase and dMTase genes in heterodichogamous Cyclocarya paliurus. RESULTS: In this study, 6 CpC5-MTase and 3 CpdMTase genes were identified in diploid (2n = 2 × = 32) C. paliurus, while 20 CpC5-MTase and 13 CpdMTase genes were identified in autotetraploid (2n = 4 × = 64). 80% of identified genes maintained relatively fixed positions on chromosomes during polyploidization. In addition, we found that some DRM subfamily members didn't contain the UBA domain. The transcript abundance of CpC5-MTase and CpdMTase in male and female flowers of two morphs (protandry and protogyny) from diploidy was analyzed. Results showed that all genes were significantly up-regulated at the stage of floral bud break (S2), but significantly down-regulated at the stage of flower maturation (S4). At S2, some CpC5-MTase genes showed higher expression levels in PG-M than in PG-F, whereas some CpdMTase genes showed higher expression levels in PA-M than in PA-F. In addition, these genes were significantly associated with gibberellin synthesis-related genes (e.g. DELLA and GID1), suggesting that DNA methylation may play a role in the asynchronous floral development process through gibberellin signal. CONCLUSIONS: These results broaden our understanding of the CpC5-MTase and CpdMTase genes in diploid and autotetraploid C. paliurus, and provide a novel insight into regulatory mechanisms of DNA methylation in heterodichogamy.

Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors.

Bacteria utilize type VI secretion system (T6SS) to deliver antibacterial toxins to target co-habiting bacteria. Here, we report that Burkholderia gladioli strain NGJ1 deploys certain T6SS effectors (TseTBg), having both DNase and RNase activities to kill target bacteria. RNase activity is prominent on NGJ1 as well as other bacterial RNA while DNase activity is pertinent to only other bacteria. The associated immunity (TsiTBg) proteins harbor non-canonical helix-turn-helix motifs and demonstrate transcriptional repression activity, similar to the antitoxins of type II toxin-antitoxin (TA) systems. Genome analysis reveals that homologs of TseTBg are either encoded as TA or T6SS effectors in diverse bacteria. Our results indicate that a new ORF (encoding a hypothetical protein) has evolved as a result of operonic fusion of TA type TseTBg homolog with certain T6SS-related genes by the action of IS3 transposable elements. This has potentially led to the conversion of a TA into T6SS effector in Burkholderia. Our study exemplifies that bacteria can recruit toxins of TA systems as T6SS weapons to diversify its arsenal to dominate during inter-bacterial competitions.

Epigenetic regulation of LINC01270 in breast cancer progression by mediating LAMA2 promoter methylation and MAPK signaling pathway.

Application of long non-coding RNAs (lncRNAs) for modulation of breast cancer (BC) has attracted much attention. Here, we probed into the role and underlying mechanism of long intergenic non-coding RNA 01270 (LINC01270) in BC. With the help of bioinformatics tools, we identified laminin subunit alpha 2 (LAMA2) as a BC-related differentially expressed gene to discern the effect of LAMA2 in BC cells. LAMA2 was initially poorly expressed while LINC01270 was highly expressed in BC. BC cells were subsequently treated with sh-LINC01270 or/and sh-LAMA2 for exploration of their regulatory mechanism in BC, which unfolded that LINC01270 inhibition up-regulated LAMA2 and inactivated the MAPK signaling pathway to suppress malignant characteristics of BC cells. Functional assays demonstrated that LINC01270 bound to DNMT1, DNMT3a, and DNMT3b promoted the methylation of CpG islands in LAMA2 promoter and inhibited the LAMA2 expression. Moreover, our data suggested that LAMA2 suppressed MAPK signaling pathway to inhibit BC cell malignant characteristics. The in vitro results were re-produced with the help of the in vivo experimentations. In conclusion, LINC01270 silencing inhibited the methylation of LAMA2 promoter to suppress the activation of MAPK signaling pathway, which subsequently restrained the BC progression. 1, Overexpression of LAMA2 inhibits malignant features of BC cells. 2, LINC01270 promotes LAMA2 promoter methylation by recruiting DNMTs to the LAMA2 promoter region. 3, 5-aza-dc reverses the promotion of LAMA2 promoter methylation by LINC01270. 4, LAMA2 inhibits malignant features of BC cells by suppressing the activation of MAPK signaling pathway.

Enhanced Fusobacterium nucleatum Genetics Using Host DNA Methyltransferases To Bypass Restriction-Modification Systems.

Bacterial restriction-modification (R-M) systems are a first-line immune defense against foreign DNA from viruses and other bacteria. While R-M systems are critical in maintaining genome integrity, R-M nucleases unfortunately present significant barriers to targeted genetic modification. Bacteria of the genus Fusobacterium are oral, Gram-negative, anaerobic, opportunistic pathogens that are implicated in the progression and severity of multiple cancers and tissue infections, yet our understanding of their direct roles in disease have been severely hindered by their genetic recalcitrance. Here, we demonstrate a path to overcome these barriers in Fusobacterium by using native DNA methylation as a host mimicry strategy to bypass R-M system cleavage of transformed plasmid DNA. We report the identification, characterization, and successful use of Fusobacterium nucleatum type II and III DNA methyltransferase (MTase) enzymes to produce a multifold increase in gene knockout efficiency in the strain Fusobacterium nucleatum subsp. nucleatum 23726, as well as the first system for efficient gene knockouts and complementations in F. nucleatum subsp. nucleatum 25586. We show plasmid protection can be accomplished in vitro with purified enzymes, as well as in vivo in an Escherichia coli host that constitutively expresses F. nucleatum subsp. nucleatum MTase enzymes. In summary, this proof-of-concept study characterizes specific MTases that are critical for bypassing R-M systems and has enhanced our understanding of enzyme combinations that could be used to genetically modify clinical isolates of Fusobacterium that have thus far been inaccessible to molecular characterization. IMPORTANCE Fusobacterium nucleatum is an oral opportunistic pathogen associated with diseases that include cancer and preterm birth. Our understanding of how this bacterium modulates human disease has been hindered by a lack of genetic systems. Here, we show that F. nucleatum DNA methyltransferase-modified plasmid DNA overcomes the transformation barrier and has allowed the development of a genetic system in a previously inaccessible strain. We present a strategy that could potentially be expanded to enable the genetic modification of highly recalcitrant strains, thereby fostering investigational studies to uncover novel host-pathogen interactions in Fusobacterium.

Fibrillarin RNA methylase is an interacting protein of Cryptosporidium parvum calmodulin-like protein (CpCML).

Cryptosporidium parvum is an obligate protozoan parasite invading epithelial cells of small intestine of human and animals, and causing diarrheal disease. In apicomplexan parasites, calcium signaling can regulate many essential biological processes such as invasion and migration. As the main intracellular receptor for calcium ions, calmodulins control the activities of hundreds of enzymes and proteins. Calmodulin-like protein (CML) is an important member of the calmodulin family and may play a key role in C. parvum, however, the actual situation is still not clear. The present study aimed to identify the parasite interaction partner proteins of C. parvum calmodulin-like protein (CpCML). By constructing the cpcml bait plasmid, 5 potential CpCML - interacting proteins in C. parvum oocyst were screened by yeast-two-hybrid system (Y2H). Bimolecular fluorescence complementation (BiFC) and Co-immunoprecipitation (Co-IP) were performed as subsequent validations. Fibrillarin RNA methylase (FBL) was identified via this screening method as CpCML interacting protein in C. parvum. The identification of this interaction made it possible to get a further understanding of the function of CpCML and its contribution to the pathogenicity of C. parvum.

Structural and molecular perspectives of SARS-CoV-2.

Recent emergence of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transpired into pandemic coronavirus disease 2019 (COVID-19). SARS-CoV-2 has been rapidly transmitted across the globe within a short period of time, with more than 106 million cases and 2.3 million deaths. The continuous rise in worldwide cases of COVID-19, transmission dynamics of SARS-CoV-2 including re-infections and enormous case-fatality rates emphasizes the urgent need of potential preventive and therapeutic measures. The development of effective therapeutic and preventive measures relies on understanding the molecular and cellular mechanism of replication exhibited by SARS-CoV-2. The structure of SARS-CoV-2 is ranging from 90-120 nm that comprises surface viral proteins including spike, envelope, membrane which are attached in host lipid bilayer containing the helical nucleocapsid comprising viral RNA. Spike (S) glycoprotein initiates the attachment of SARS-CoV-2 with a widely expressed cellular receptor angiotensin-converting enzyme 2 (ACE2), and subsequent S glycoprotein priming via serine protease TMPRSS2. Prominently, comprehensive analysis of structural insights into the crucial SARS-CoV-2 proteins may lead us to design effective therapeutics molecules. The present article, emphasizes the molecular and structural perspective of SARS-CoV-2 including mechanistic insights in its replication.

A bacterial biosynthetic pathway for methylated furan fatty acids.

Fatty acids play many important roles in cells and also in industrial processes. Furan fatty acids (FuFAs) are present in the lipids of some plant, fish, and microbial species and appear to function as second messengers in pathways that protect cells from membrane-damaging agents. We report here the results of chemical, genetic, and synthetic biology experiments to decipher the biosynthesis of the monomethylated FuFA, methyl 9-(3-methyl-5-pentylfuran-2-yl) nonanoate (9M5-FuFA), and its dimethyl counterpart, methyl 9-(3,4-dimethyl-5-pentylfuran-2-yl) nonanoate (9D5-FuFA), in two α-proteobacteria. Each of the steps in FuFA biosynthesis occurs on pre-existing phospholipid fatty acid chains, and we identified pathway intermediates and the gene products that catalyze 9M5-FuFA and 9D5-FuFA synthesis in Rhodobacter sphaeroides 2.4.1 and Rhodopseudomonas palustris CGA009. One previously unknown pathway intermediate was a methylated diunsaturated fatty acid, (10E,12E)-11-methyloctadeca-10,12-dienoic acid (11Me-10t,12t-18:2), produced from (11E)-methyloctadeca-11-enoic acid (11Me-12t-18:1) by a newly identified fatty acid desaturase, UfaD. We also show that molecular oxygen (O2) is the source of the oxygen atom in the furan ring of 9M5-FuFA, and our findings predict that an O2-derived oxygen atom is incorporated into 9M5-FuFA via a protein, UfaO, that uses the 11Me-10t,12t-18:2 fatty acid phospholipid chain as a substrate. We discovered that R. palustris also contains a SAM-dependent methylase, FufM, that produces 9D5-FuFA from 9M5-FuFA. These results uncover the biochemical sequence of intermediates in a bacterial pathway for 9M5-FuFA and 9D5-FuFA biosynthesis and suggest the existence of homologs of the enzymes identified here that could function in FuFA biosynthesis in other organisms.

Nonsense-mediated mRNA decay modulates Arabidopsis flowering time via the SET DOMAIN GROUP 40-FLOWERING LOCUS C module.

The nonsense-mediated mRNA decay (NMD) surveillance system clears aberrant mRNAs from the cell, thus preventing the accumulation of truncated proteins. Although loss of the core NMD proteins UP-FRAMESHIFT1 (UPF1) and UPF3 leads to late flowering in Arabidopsis, the underlying mechanism remains elusive. Here, we showed that mutations in UPF1 and UPF3 cause temperature- and photoperiod-independent late flowering. Expression analyses revealed high FLOWERING LOCUS C (FLC) mRNA levels in upf mutants; in agreement with this, the flc mutation strongly suppressed the late flowering of upf mutants. Vernalization accelerated flowering of upf mutants in a temperature-independent manner. FLC transcript levels rose in wild-type plants upon NMD inhibition. In upf mutants, we observed increased enrichment of H3K4me3 and reduced enrichment of H3K27me3 in FLC chromatin. Transcriptome analyses showed that SET DOMAIN GROUP 40 (SDG40) mRNA levels increased in upf mutants, and the SDG40 transcript underwent NMD-coupled alternative splicing, suggesting that SDG40 affects flowering time in upf mutants. Furthermore, NMD directly regulated SDG40 transcript stability. The sdg40 mutants showed decreased H3K4me3 and increased H3K27me3 levels in FLC chromatin, flowered early, and rescued the late flowering of upf mutants. Taken together, these results suggest that NMD epigenetically regulates FLC through SDG40 to modulate flowering time in Arabidopsis.

RNA methylation and cancer treatment.

To this date, over 100 different types of RNA modification have been identified. Methylation of different RNA species has emerged as a critical regulator of transcript expression. RNA methylation and its related downstream signaling pathways are involved in plethora biological processes, including cell differentiation, sex determination and stress response, and others. It is catalyzed by the RNA methyltransferases, is demethylated by the demethylases (FTO and ALKBH5) and read by methylation binding protein (YTHDF1 and IGF2BP1). Increasing evidence indicates that this process closely connected to cancer cell proliferation, cellular stress, metastasis, immune response. And RNA methylation related protein has been becoming a promising targets of cancer therapy. This review outlines the relationship between different types of RNA methylation and cancer, and some FTO inhibitors in cancer treatment.

A conserved motif in human BTG1 and BTG2 proteins mediates interaction with the poly(A) binding protein PABPC1 to stimulate mRNA deadenylation.

Antiproliferative BTG/Tob proteins interact directly with the CAF1 deadenylase subunit of the CCR4-NOT complex. This binding requires the presence of two conserved motifs, boxA and boxB, characteristic of the BTG/Tob APRO domain. Consistently, these proteins were shown to stimulate mRNA deadenylation and decay in several instances. Two members of the family, BTG1 and BTG2, were reported further to associate with the protein arginine methyltransferase PRMT1 through a motif, boxC, conserved only in this subset of proteins. We recently demonstrated that BTG1 and BTG2 also contact the first RRM domain of the cytoplasmic poly(A) binding protein PABPC1. To decipher the mode of interaction of BTG1 and BTG2 with partners, we performed nuclear magnetic resonance experiments as well as mutational and biochemical analyses. Our data demonstrate that, in the context of an APRO domain, the boxC motif is necessary and sufficient to allow interaction with PABPC1 but, unexpectedly, that it is not required for BTG2 association with PRMT1. We show further that the presence of a boxC motif in an APRO domain endows it with the ability to stimulate deadenylation in cellulo and in vitro. Overall, our results identify the molecular interface allowing BTG1 and BTG2 to activate deadenylation, a process recently shown to be necessary for maintaining T-cell quiescence.

DNA adenine methylase, not the PstI restriction-modification system, regulates virulence gene expression in Shiga toxin-producing Escherichia coli.

We previously reported a distinct methylome between the two Shiga toxin-producing Escherichia coli (STEC) O145:H28 strains linked to the 2010 U.S. lettuce-associated outbreak (RM13514) and the 2007 Belgium ice cream-associated outbreak (RM13516), respectively. This difference was thought to be attributed to a prophage encoded type II restriction-modification system (PstI R-M) in RM13514. Here, we characterized this PstI R-M system in comparison to DNA adenine methylase (Dam), a highly conserved enzyme in γ proteobacteria, by functional genomics. Deficiency in Dam led to a differential expression of over 1000 genes in RM13514, whereas deficiency in PstI R-M only impacted a few genes transcriptionally. Dam regulated genes involved in diverse functions, whereas PstI R-M regulated genes mostly encoding transporters and adhesins. Dam regulated a large number of genes located on prophages, pathogenicity islands, and plasmids, including Shiga toxin genes, type III secretion system (TTSS) genes, and enterohemolysin genes. Production of Stx2 in dam mutant was significantly higher than in RM13514, supporting a role of Dam in maintaining lysogeny of Stx2-prophage. However, following mitomycin C treatment, Stx2 in RM13514 was significantly higher than that of dam or PstI R-M deletion mutant, implying that both Dam and PstI R-M contributed to maximum Stx2 production.

Essential role of Salmonella Enteritidis DNA adenine methylase in modulating inflammasome activation.

BACKGROUND: Salmonella Enteritidis (SE) is one of the major foodborne zoonotic pathogens of worldwide importance which can induce activation of NLRC4 and NLRP3 inflammasomes during infection. Given that the inflammasomes play an essential role in resisting bacterial infection, Salmonella has evolved various strategies to regulate activation of the inflammasome, most of which largely remain unclear. RESULTS: A transposon mutant library in SE strain C50336 was screened for the identification of the potential factors that regulate inflammasome activation. We found that T3SS-associated genes invC, prgH, and spaN were required for inflammasome activation in vitro. Interestingly, C50336 strains with deletion or overexpression of Dam were both defective in activation of caspase-1, secretion of IL-1ß and phosphorylation of c-Jun N-terminal kinase (Jnk). Transcriptome sequencing (RNA-seq) results showed that most of the differentially expressed genes and enriched KEGG pathways between the C50336-VS-C50336Δdam and C50336-VS-C50336::dam groups overlapped, which includes multiple signaling pathways related to the inflammasome. C50336Δdam and C50336::dam were both found to be defective in suppressing the expression of several anti-inflammasome factors. Moreover, overexpression of Dam in macrophages by lentiviral infection could specifically enhance the activation of NLRP3 inflammasome independently via promoting the Jnk pathway. CONCLUSIONS: These data indicated that Dam was essential for modulating inflammasome activation during SE infection, there were complex and dynamic interplays between Dam and the inflammasome under different conditions. New insights were provided about the battle between SE and host innate immunological mechanisms.

Prevalence and molecular epidemiology of Staphylococcus aureus nasal colonization in four nursing home residents in Crete, Greece.

Nursing homes are considered as reservoirs for methicillin-resistant Staphylococcus aureus (MRSA). The present study investigated the point prevalence and molecular epidemiology of S. aureus colonization among nursing home residents. The study population comprised of 227 residents, living in four nursing homes of the Heraklion, Crete, Greece area, between January and December 2015. From each nursing home, swabs from the anterior nares of all eligible participants were obtained within a 2-week period. The isolated S. aureus strains were identified and screened by standard microbiological and molecular epidemiological methods. S. aureus carriage was found in 62 out of 227 participants (38.4%) with 33 out of 62 (53.2%) being MRSA. The median age was 83 years (range 52-103). Females were more frequently colonized [47 (75.8%)]. All 33 methicillin resistant Staphylococcus aureus (MRSA) isolates were mecA-positive carrying SCCmec type IV, 30 (91%) the fnbA, and 17 (51.5%) the PVL genes. Thirty-two (97%) belonged to a single pulsotype C; among them, the PVL-positives belonged to ST80 clone, whereas, the PVL-negatives to ST225. Among the 33 MRSA isolates, 32 (97%) were clindamycin-resistant, carrying the ermA gene. Methicillin-susceptible Staphylococcus aureus (MSSA) strains showed polyclonality and 76% were PVL-positive. In conclusion the present study has shown that nursing homes in our area can be regarded as important reservoirs for community-associated MRSA (CA-MRSA).

Systematic Analysis of the DNA Methylase and Demethylase Gene Families in Rapeseed (Brassica napus L.) and Their Expression Variations After Salt and Heat stresses.

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.

Spontaneous development of hepatocellular carcinoma with cancer stem cell properties in PR-SET7-deficient livers.

PR-SET7-mediated histone 4 lysine 20 methylation has been implicated in mitotic condensation, DNA damage response and replication licensing. Here, we show that PR-SET7 function in the liver is pivotal for maintaining genome integrity. Hepatocyte-specific deletion of PR-SET7 in mouse embryos resulted in G2 phase arrest followed by massive cell death and defect in liver organogenesis. Inactivation at postnatal stages caused cell duplication-dependent hepatocyte necrosis, accompanied by inflammation, fibrosis and compensatory growth induction of neighboring hepatocytes and resident ductal progenitor cells. Prolonged necrotic regenerative cycles coupled with oncogenic STAT3 activation led to the spontaneous development of hepatic tumors composed of cells with cancer stem cell characteristics. These include a capacity to self-renew in culture or in xenografts and the ability to differentiate to phenotypically distinct hepatic cells. Hepatocellular carcinoma in PR-SET7-deficient mice displays a cancer stem cell gene signature specified by the co-expression of ductal progenitor markers and oncofetal genes.

The Antitumoral Effect of the S-Adenosylhomocysteine Hydrolase Inhibitor, 3-Deazaneplanocin A, is Independent of EZH2 but is Correlated with EGFR Downregulation in Chondrosarcomas.

BACKGROUND/AIMS: 3-Deazaneplanocin, DZNep, has been reported to inhibit the EZH2 histone methylase and to induce cell apoptosis in chondrosarcomas (CS). The present study aims to confirm the therapeutic potential of EZH2 inhibitors and investigate the molecular mechanisms of DZNep in chondrosarcomas. METHODS: CS cell lines and primary cultures were used. Apoptosis was investigated using PARP cleavage, caspase 3/7 activity, or Apo2.7 expression. S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were quantified by UHPLC-MS/MS. Differentially expressed genes in treated-chondrosarcomas and chondrocytes were researched by microarray analysis. RESULTS: DZNep induced apoptosis in chondrosarcomas both in vivo and in vitro. However, this effect was not correlated to EZH2 expression nor activity, and EZH2 knock-down by siRNA did not reduce CS viability. Additionally, the reduction of H3K27me3 induced by GSK126 or tazemetostat (EPZ-6438) did not provoke chondrosarcoma death. However, as expected, DZNep induced SAH accumulation and reduced SAM:SAH ratio. Further, microarray analysis suggests a key role of EGFR in antitumoral effect of DZNep, and pharmacological inhibition of EGFR reduced chondrosarcoma survival. CONCLUSION: EZH2 is not an adequate target for chondrosarcoma treatment. However, DZNep induces apoptosis in chondrosarcomas in vitro and in vivo, by a mechanism likely mediated though EGFR expression. Consequently, it would be worth initiating clinical trials to evaluating efficiency to S-adenosylhomocysteine hydrolase or EGFR inhibitors in patients with chondrosarcomas.

High distribution of 16S rRNA methylase genes rmtB and armA among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran.

The emergence of 16S rRNA methylase genes encoded on plasmids confers high-level aminoglycoside resistance (HLAR). This study aimed to investigate the prevalence of 16S rRNA methylases among Enterobacter cloacae strains isolated from an Ahvaz teaching hospital, Iran. A total of 68 E. cloacae clinical strains were collected between November 2017 and September 2018. The MICs of aminoglycosides were assessed using the agar dilution method. The presence of 16S rRNA methylase genes, including armA, rmtA to rmtH, and nmpA was evaluated by PCR. The transferability of 16S rRNA methylase-harboring plasmids was evaluated by conjugation assay. The genetic diversity of all isolates was evaluated by ERIC-PCR. The armA and rmtB genes were the only 16S rRNA methylase genes detected in this study (29 out of 68 isolates; 42.64%). The transferability by conjugation was observed in 23 rmtB or/and armA positive donors. HLAR phenotype was in 33 of 68 strains. Ten clonal types were obtained by ERIC-PCR and significant associations (p < 0.05) were between the clone types and aminoglycoside susceptibility, as well as with profile of the 16S rRNA methylase genes. In conclusion, both horizontal transfer and clonal spread are responsible for dissemination of the rmtB and armA genes among E. cloacae strains.

Control of proline accumulation under drought via a novel pathway comprising the histone methylase CAU1 and the transcription factor ANAC055.

Proline plays a crucial role in the drought stress response in plants. However, there are still gaps in our knowledge about the molecular mechanisms that regulate proline metabolism under drought stress. Here, we report that the histone methylase encoded by CAU1, which is genetically upstream of P5CS1 (encoding the proline biosynthetic enzyme Δ1-pyrroline-5-carboxylate synthetase 1), plays a crucial role in proline-mediated drought tolerance. We determined that the transcript level of CAU1 decreased while that of ANAC055 (encoding a transcription factor) increased in wild-type Arabidopsis under drought stress. Further analyses showed that CAU1 bound to the promoter of ANAC055 and suppressed its expression via H4R3sme2-type histone methylation in the promoter region. Thus, under drought stress, a decreased level of CAU1 led to an increased transcript level of ANAC055, which induced the expression of P5CS1 and increased proline level independently of CAS. Drought tolerance and the level of proline were found to be decreased in the cau1 anac055 double-mutant, while proline supplementation restored drought sensitivity in the anac055 mutant. Our results reveal the details of a novel pathway leading to drought tolerance mediated by CAU1.