N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m6A as well as m6A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m6A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m6A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m6A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m6A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m6A pattern that is distinct from those in eukaryotes. Most m6A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m6A peaks indicates that the majority of m6A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m6A in these pathways.
Recent data indicates that nucleoside/nucleotide analogue (NUC) is effective in preventing and controlling hepatitis B virus (HBV) reactivation in HBV-carrying cancer patients who undergo chemotherapy, but the ideal antiviral agent and optimal application protocol still needs to be determined. Meanwhile, it is uncertain whether those with past HBV infection require antiviral prophylaxis during chemotherapy. This report retrospectively analyzed non-Hodgkin’s lymphoma (NHL) patients seen from January, 2004 to June, 2009 in West China Hospital. We found that the prevalence of chronic HBV infection in our NHL patients was 20.7 % while that of past HBV infection was 21.05 %. Compared with the high rate (25.6 %) of HBV reactivation in patients with chronic HBV infection, none of those with past HBV infection in fact had occult HBV infection thus none experienced reactivation. Of the 82 patients with chronic HBV infection who received chemotherapy, antiviral prophylaxis could significantly reduce the incidence of HBV reactivation (5.0 vs. 45.2 % in the control group) and the incidence of liver function damage (32.5 vs. 73.8 % in the control group). The results of the current study confirmed previous reports that prophylactic NUCs administration can effectively prevent HBV reactivation and significantly reduce the incidence of HBV reactivation especially for patients receiving rituximab-containing regimens. Due to the fact that none of individuals who had past HBV infection developed HBV reactivation reported in our study, antiviral prophylaxis may not be required for patients with past HBV infection. Close observation of alanine aminotransferase and HBV–DNA contributes to early diagnosis and timely treatment of HBV reactivation.
HBV reactivation; Nucleoside/nucleotide analogue; Rituximab; Non-Hodgkin’s lymphoma
N6-methyladenosine (m6A) is a prevalent modification of eukaryotic mRNAs. It regulates yeast cell fate and is essential to the development and fertility of metazoans. Although its presence in mRNA has been known since the early 1970s, the function of m6A remained a mystery until the spate of discoveries in the past three years. Here, we focus on the discovery of m6A “readers” (proteins that specifically recognize m6A), and their functions in tuning mRNA stability, as well as the broader significance of such m6A-dependent regulation of gene expression.
N6-methyladenosine; RNA methylation; RNA stability; RNA-binding protein; YTH domain; YTHDF2; gene expression regulation; m6A; reversible RNA modification
Recent discoveries of reversible N6-methyladenosine (m6A) methylation on messenger RNA (mRNA) and mapping of m6A methylomes in mammals and yeast have revealed potential regulatory functions of this RNA modification. In plants, defects in m6A methyltransferase cause an embryo-lethal phenotype, suggesting a critical role of m6A in plant development. Here, we profile m6A transcriptome-wide in two accessions of Arabidopsis thaliana and reveal that m6A is a highly conserved modification of mRNA in plants. Distinct from mammals, m6A in A. thaliana is enriched not only around the stop codon and within 3′ untranslated regions (3′ UTRs), but also around the start codon. Gene ontology analysis indicates that the unique distribution pattern of m6A in A. thaliana is associated with plant-specific pathways involving the chloroplast. We also discover a positive correlation between m6A deposition and the mRNA abundance, suggesting a regulatory role of m6A in plant gene expression.
N6-methyladenosine (m6A); RNA methylation; plant mRNA methylome; chloroplast; gene expression
An electrochemical membrane bioreactor (EMBR) has recently been developed for energy recovery and wastewater treatment. The hydrodynamics of the EMBR would significantly affect the mass transfers and reaction kinetics, exerting a pronounced effect on reactor performance. However, only scarce information is available to date. In this study, the hydrodynamic characteristics of the EMBR were investigated through various approaches. Tracer tests were adopted to generate residence time distribution curves at various hydraulic residence times, and three hydraulic models were developed to simulate the results of tracer studies. In addition, the detailed flow patterns of the EMBR were acquired from a computational fluid dynamics (CFD) simulation. Compared to the tank-in-series and axial dispersion ones, the Martin model could describe hydraulic performance of the EBMR better. CFD simulation results clearly indicated the existence of a preferential or circuitous flow in the EMBR. Moreover, the possible locations of dead zones in the EMBR were visualized through the CFD simulation. Based on these results, the relationship between the reactor performance and the hydrodynamics of EMBR was further elucidated relative to the current generation. The results of this study would benefit the design, operation and optimization of the EMBR for simultaneous energy recovery and wastewater treatment.
The reprogramming of parental methylomes is essential for embryonic development. In mammals, paternal 5-methylcytosines (5mCs) have been proposed to be actively converted to oxidized bases. These paternal oxidized bases and maternal 5mCs are believed to be passively diluted by cell divisions. By generating single-base resolution, allele-specific DNA methylomes from mouse gametes, early embryos and primordial germ cell (PGC), as well as single-base resolution maps of oxidized cytosine bases for early embryos, we report the existence of 5hmC and 5fC in both maternal and paternal genomes and find that 5mC or its oxidized derivatives, at the majority of demethylated CpGs, are converted to unmodified cytosines independent of passive dilution from gametes to 4-cell embryos. Therefore, we conclude that paternal methylome and at least a significant proportion of maternal methylome go through active demethylation during embryonic development. Additionally, all the known imprinting control regions (ICRs) were classified into germ-line or somatic ICRs.
The title compound crystallizes in the keto form and the carbonyl O atom forms an intramolecular N—H⋯O hydrogen bond with the neighbouring NH group. In the crystal, molecules are linked by pairs of N—H⋯O hydrogen bonds to form inversion dimers, which are linked via pairs of C—H⋯O hydrogen bonds, forming chains propagating along .
The title compound, C19H20N4O3S, was synthesized by refluxing equimolar amounts of 1-phenyl-3-methyl-4-propionylpyrazol-5-one and benzenesulfonyl hydrazide in ethanol. The compound crystallizes in the keto form and the carbonyl O atom forms an intramolecular N—H⋯O hydrogen bond with the neighbouring NH group. There is also C—H⋯O short contact involving the neighbouring phenyl ring. Probably as a result of this, the phenyl ring is inclined to the pyrazolone ring by only 7.58 (12)°. The dihedral angle between the phenyl ring and the benzenesulfonyl ring is 22.78 (11)°. In the crystal, molecules are linked by pairs of N—H⋯O hydrogen bonds, forming inversion dimers with an R
2(14) ring motif. The dimers are linked via pairs of C—H⋯O hydrogen bonds, forming chains propagating along .
crystal structure; pyrazolone derivative; hydrogen bonding; polymer chain
Background and objectives: Numerous studies have focused on the role of survivin in non-Hodgkin’s lymphomas (NHLs), but evidence regarding the prognostic value of survivin with respect to overall survival (OS) in NHL remains controversial. The aim of this study is to gain a better insight about the direct relationship between survivin expression and patients’ survival statuses. Materials and methods: Relevant publications addressing the association between survivin expression and OS in NHL patients were selected from PubMed, Embase, Chinese Biomedical Literature Database (CBM), China National Knowledge Infrastructure Database (CNKI), China Science and Technology Journal Database (VIP), Wanfang Database and the Cochrane library. Studies were pooled and summary hazard ratios (HR) were calculated. Sensitivity analyses and publication bias were also conducted. Statistical analysis was performed by STATA 12.0 software. Results: 12 studies met the inclusion criteria. Combined HRs suggested that survivin overexpression had an unfavorable impact on NHL patients’ survival (HR=1.55, 95% CI=1.12-2.13, P=0.008). Subgroup analyses according to the studies categorized by histological type, ethnicity, cutoff scores and follow-up period were also conducted, and all the above analyses supported the stability of the prognostic role of survivin. Conclusion: Our findings suggest that survivin high expression might be a poor prognostic factor for patients with NHL. However, further large scale studies are needed to confirm these findings.
Non-Hodgkin’s lymphoma; survivin; prognosis; meta-analysis
N6-methyladenosine (m6A) is an abundant internal
modification in eukaryotic mRNA and plays regulatory roles in mRNA metabolism.
However, methods to precisely locate the m6A modification remain
limited. We present here a photo-crosslinking-assisted m6A sequencing
strategy (PA-m6A-seq) to more accurately define sites with
m6A modification. Using this strategy, we obtained a
high-resolution map of m6A in a human transcriptome. The map
resembles the general distribution pattern observed previously, and reveals new
m6A sites at base resolution. Our results provide insight into
the relationship between the methylation regions and the binding sites of
N6-methyladenosine; photo-crosslinking; RNA modification; transcriptome sequencing
DNA methylation (5-methylcytosine,
5mC) plays critical biological
functions in mammals and plants as a vital epigenetic marker. The
Ten-Eleven translocation dioxygenases (TET1, 2, and 3) have been found
to oxidize 5mC to 5-hydroxymethylcytosine (5hmC) and then to 5-formylcytosine
(5fC) and 5-carboxylcytosine (5caC) in mammalian cells. We report
herein three mushroom TET homologues from Coprinopsis cinerea that can mediate 5mC oxidation. Specifically, one homologue (CC1G_05589,
CcTET) shows similar activity to its mammalian TET homologues. Biochemically,
CcTET actively converts 5mC to 5hmC, 5fC, and 5caC under natural conditions
(pH 7.0). Interestingly, CcTET also converts the majority of 5mC to
5fC under slightly acidic (pH 5.8) and neutral conditions. Kinetics
analyses of the oxidation by CcTET under neutral conditions indicate
that conversion of 5mC to 5hmC and 5hmC to 5fC are faster than that
of 5fC to 5caC, respectively. Our results provide an example of a
TET homologue in a non-mammalian organism that exhibits full 5mC-to-5caC
oxidation activity and a slight preference to producing 5fC. The preferential
accumulation of 5fC in the in vitro oxidation reactions
under both neutral and acidic conditions may have biological implications
for 5mC oxidation in fungi species.
ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N6-methyladenosine (m6A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65 Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m6A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m6A demethylation.
ALKBH5; crystal structure; demethylation; N6-hydroxymethyladenosine
The xeroderma pigmentosum C (XPC) complex initiates nucleotide excision repair by recognizing DNA lesions before recruiting downstream factors. How XPC detects structurally diverse lesions embedded within normal DNA is unknown. Here we present a crystal structure that captures the yeast XPC orthologue (Rad4) on a single register of undamaged DNA. The structure shows that a disulphide-tethered Rad4 flips out normal nucleotides and adopts a conformation similar to that seen with damaged DNA. Contrary to many DNA repair enzymes that can directly reject non-target sites as structural misfits, our results suggest that Rad4/XPC uses a kinetic gating mechanism whereby lesion selectivity arises from the kinetic competition between DNA opening and the residence time of Rad4/XPC per site. This mechanism is further supported by measurements of Rad4-induced lesion-opening times using temperature-jump perturbation spectroscopy. Kinetic gating may be a general mechanism used by site-specific DNA-binding proteins to minimize time-consuming interrogations of non-target sites.
XPC nucleotide excision repair factor is key to starting the repair of diverse helix-distorting DNA lesions caused by environmental insults. Here, the authors propose a kinetic gating mechanism whereby XPC recognizes DNA lesions by preferentially opening damaged sites while readily diffusing away from undamaged sites.
5-hydroxymethylcytosine (5hmC), an oxidized derivative of 5-methylcytosine (5mC), has been implicated as an important epigenetic regulator of mammalian development. Current procedures use DNA sequencing methods to discriminate 5hmC from 5mC, limiting their accessibility to the scientific community. Here we report a method that combines TET-assisted bisulfite conversion with Illumina 450 K DNA methylation arrays for a low-cost high-throughput approach that distinguishes 5hmC and 5mC signals at base resolution. Implementing this approach, termed “TAB-array”, we assessed DNA methylation dynamics in the differentiation of human pluripotent stem cells into cardiovascular progenitors and neural precursor cells. With the ability to discriminate 5mC and 5hmC, we identified a large number of novel dynamically methylated genomic regions that are implicated in the development of these lineages. The increased resolution and accuracy afforded by this approach provides a powerful means to investigate the distinct contributions of 5mC and 5hmC in human development and disease.
5-hydroxymethylcytosine; TAB-array; DNA methylation; Human pluripotent stem cells; Epigenetics; Differentiation; Neuronal cells; Cardiovascular cells
The N6-methyladenosine (m6A) modification of mRNA has a crucial function in regulating pluripotency in murine stem cells: it facilitates resolution of naïve pluripotency towards differentiation.
Metagenome of gut microbes has been implicated in metabolism, immunity, and health maintenance of its host. However, in most of previous studies, the microbiota was sampled from feces instead of gastrointestinal (GI) tract. In this study, we compared the microbial populations from feces at four different developmental stages and contents of four intestinal segments at maturity to examine the dynamic shift of microbiota in pigs and investigated whether adult porcine fecal samples could be used to represent samples of the GI tract. Analysis results revealed that the ratio of Firmicutes to Bacteroidetes from the feces of the older pigs (2-, 3-, 6- month) were 10 times higher compared to those from piglets (1-month). As the pigs matured, so did it seem that the composition of microbiome became more stable in feces. In adult pigs, there were significant differences in microbial profiles between the contents of the small intestine and large intestine. The dominant genera in the small intestine belonged to aerobe or facultative anaerobe categories, whereas the main genera in the large intestine were all anaerobes. Compared to the GI tract, the composition of microbiome was quite different in feces. The microbial profile in large intestine was more similar to feces than those in the small intestine, with the similarity of 0.75 and 0.38 on average, respectively. Microbial functions, predicted by metagenome profiles, showed the enrichment associated with metabolism pathway and metabolic disease in large intestine and feces while higher abundance of infectious disease, immune function disease, and cancer in small intestine. Fecal microbes also showed enriched function in metabolic pathways compared to microbes from pooled gut contents. Our study extended the understanding of dynamic shift of gut microbes during pig growth and also characterized the profiles of bacterial communities across GI tracts of mature pigs.
Hematopoietic stem cell differentiation involves the silencing of self-renewal genes and induction of a specific transcriptional program. Identification of multiple covalent cytosine modifications raises the question of how these derivatized bases influence stem cell commitment. Using a replicative primary human hematopoietic stem/progenitor cell differentiation system, we demonstrate dynamic changes of 5-hydroxymethylcytosine (5-hmC) during stem cell commitment and differentiation to the erythroid line-age. Genomic loci that maintain or gain 5-hmC density throughout erythroid differentiation contain binding sites for erythroid transcription factors and several factors not previously recognized as erythroid-specific factors. The functional importance of 5-hmC was demonstrated by impaired erythroid differentiation, with augmentation of myeloid potential, and disrupted 5-hmC patterning in leukemia patient-derived CD34+ stem/early progenitor cells with TET methylcytosine dioxygenase 2 (TET2) mutations. Thus, chemical conjugation and affinity purification of 5-hmC-enriched sequences followed by sequencing serve as resources for deciphering functional implications for gene expression during stem cell commitment and differentiation along a particular lineage.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from human pathogens Staphylococcus aureus and Pseudomonas aeruginosa can be readily inhibited by reactive oxygen species (ROS)-mediated direct oxidation of their catalytic active cysteines. Because of the rapid degradation of H2O2 by bacterial catalase, only steady-state but not one-dose treatment with H2O2 rapidly induces glycolysis and the pentose phosphate pathway (PPP). We conducted transcriptome sequencing (RNA-seq) analyses to globally profile the bacterial transcriptomes in response to a steady level of H2O2, which revealed profound transcriptional changes, including the induced expression of glycolytic genes in both bacteria. Our results revealed that the inactivation of GAPDH by H2O2 induces metabolic levels of glycolysis and the PPP; the elevated levels of fructose 1,6-biphosphate (FBP) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) lead to dissociation of their corresponding glycolytic repressors (GapR and HexR, respectively) from their cognate promoters, thus resulting in derepression of the glycolytic genes to overcome H2O2-stalled glycolysis in S. aureus and P. aeruginosa, respectively. Both GapR and HexR may directly sense oxidative stresses, such as menadione.
Endogenous and exogenous factors constantly challenge cellular DNA, generating cytotoxic and/or mutagenic DNA adducts. As a result, organisms have evolved different mechanisms to defend against the deleterious effects of DNA damage. Among these diverse repair pathways, direct DNA-repair systems provide cells with simple yet efficient solutions to reverse covalent DNA adducts. In this review, we focus on recent advances in the field of direct DNA repair, namely, photolyase-, alkyltransferase-, and dioxygenase-mediated repair processes. We present specific examples to describe new findings of known enzymes and appealing discoveries of new proteins. At the end of this article, we also briefly discuss the influence of direct DNA repair on other fields of biology and its implication on the discovery of new biology.
Simple direct repair mechanisms can fix DNA damage without breaking the DNA backbone. These essentially error-free processes include photolyase-, alkyltransferase-, and dioxygenase-mediated mechanisms.
To investigate whether a combination of demineralized bone matrix (DBM) and bone marrow mesenchymal stem cells (BMSCs) infected with adenovirus-mediated- bone morphogenetic protein (Ad-BMP-2) and transforming growth factor-β3 (Ad-TGF-β3) promotes the repair of the full-thickness cartilage lesions in pig model.
BMSCs isolated from pig were cultured and infected with Ad-BMP-2(B group), Ad-TGF-β3 (T group), Ad-BMP-2 + Ad-TGF-β3(BT group), cells infected with empty Ad served as a negative group(N group), the expression of the BMP-2 and TGF-β3 were confirmed by immunofluorescence, PCR, and ELISA, the expression of SOX-9, type II collagen(COL-2A), aggrecan (ACAN) in each group were evaluated by real-time PCR at 1w, 2w, 3w, respectively. The chondrogenic differentiation of BMSCs was evaluated by type II collagen at 21d with immunohistochemical staining. The third-passage BMSCs infected with Ad-BMP-2 and Ad-TGF-β3 were suspended and cultured with DBM for 6 days to construct a new type of tissue engineering scaffold to repair full-thickness cartilage lesions in the femur condyles of pig knee, the regenerated tissue was evaluated at 1,2 and 3 months after surgery by gross appearance, H&E, safranin O staining and O'driscoll score.
Ad-BMP-2 and Ad-TGF-β3 (BT group) infected cells acquired strong type II collagen staining compared with Ad-BMP-2 (B group) and Ad-TGF-β3 (T group) along. The Ad-BMP-2 and Ad-TGF-β3 infected BMSCs adhered and propagated well in DBM and the new type of tissue engineering scaffold produced hyaline cartilage morphology containing a stronger type II collagen and safranin O staining, the O'driscoll score was higher than other groups.
The DBM compound with Ad-BMP-2 and Ad-TGF-β3 infected BMSCs scaffold has a good biocompatibility and could well induce cartilage regeneration to repair the defects of joint cartilage. This technology may be efficiently employed for cartilage lesions repair in vivo.
Hypoxia induces the epithelial-mesenchymal transition, EMT, to promote cancer metastasis. In addition to transcriptional regulation mediated by hypoxia-inducible factors, HIFs, other epigenetic mechanisms of gene regulation, such as histone modifications and DNA methylation, are utilized under hypoxia. However, whether DNA demethylation mediated by TET1, a DNA dioxygenase converting 5-methylcytosine, 5mC, into 5-hydroxymethylcytosine, 5hmC, plays a role in hypoxia-induced EMT is largely unknown.
We show that TET1 regulates hypoxia-responsive gene expression. Hypoxia/HIF-2α regulates the expression of TET1. Knockdown of TET1 mitigates hypoxia-induced EMT. RNA sequencing and 5hmC sequencing identified the set of TET1-regulated genes. Cholesterol metabolic process genes are among the genes that showed high prevalence and statistical significance. We characterize one of the genes, INSIG1 (insulin induced gene 1), to confirm its expression and the 5hmC levels in its promoter. Knockdown of INSIG1 also mitigates hypoxia-induced EMT. Finally, TET1 is shown to be a transcriptional co-activator that interacts with HIF-1α and HIF-2α to enhance their transactivation activity independent of its enzymatic activity. TET1 acts as a co-activator to further enhance the expression of INSIG1 together with HIF-2α. We define the domain in HIF-1α that interacts with TET1 and map the domain in TET1 that confers transactivation to a 200 amino acid region that contains a CXXC domain. The TET1 catalytically inactive mutant is capable of rescuing hypoxia-induced EMT in TET1 knockdown cells.
These findings demonstrate that TET1 serves as a transcription co-activator to regulate hypoxia-responsive gene expression and EMT, in addition to its role in demethylating 5mC.
Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0513-0) contains supplementary material, which is available to authorized users.