Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~ 3.8 Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.     

Differential expression of microRNAs associated with neurodegenerative diseases and diabetic nephropathy in protein l-isoaspartyl methyltransferase-deficient mice

Protein l -isoaspartyl methyltransferase (PIMT/PCMT1), an enzyme repairing isoaspartate residues in peptides and proteins that result from the spontaneous decomposition of normal l -aspartyl and l -asparaginyl residues during aging, has been revealed to be involved in neurodegenerative diseases (NDDs) and diabetes. However, the molecular mechanisms for a putative association of PIMT dysfunction with these diseases have not been clarified. Our study aimed to identify differentially expressed microRNAs (miRNAs) in the brain and kidneys of PIMT-deficient mice and uncover the epigenetic mechanism of PIMT-involved NDDs and diabetic nephropathy (DN). Differentially expressed miRNAs by sequencing underwent target prediction and enrichment analysis in the brain and kidney of PIMT knockout (KO) mice and age-matched wild-type (WT) littermates. Sequence analysis revealed 40 differentially expressed miRNAs in the PIMT KO mouse brain including 25 upregulated miRNAs and 15 downregulated miRNAs. In the PIMT KO mouse kidney, there were 80 differentially expressed miRNAs including 40 upregulated miRNAs and 40 downregulated miRNAs. Enrichment analysis and a systematic literature review of differentially expressed miRNAs indicated the involvement of PIMT deficiency in the pathogenesis in NDDs and DN. Some overlapped differentially expressed miRNAs between the brain and kidney were quantitatively assessed in the brain, kidney, and serum-derived exosomes, respectively. Despite being preliminary, these results may aid in investigating the pathological hallmarks and identify the potential therapeutic targets and biomarkers for PIMT dysfunction-related NDDs and DN.     

Synthetic construction of sugar-amino acid hybrid polymers involving globotriaose or lactose and evaluation of their biological activities against Shiga toxins produced by Escherichia coli O157:H7

Synthetic assembly of sugar moieties and amino acids in order to create “sugar-amino acid hybrid polymers” was accomplished by means of simple radical polymerization of carbohydrate monomers having an amino acid-modified polymerizable aglycon. Amines derived from globotriaoside and lactoside as glycoepitopes were condensed with known carbobenzyloxy derivatives, including Z-Gly, Z-l-Ala and Z-β-Ala, which had appropriate spacer ability and a chiral center to afford fully protected sugar-amino acid hybrid compounds in good yields. After deprotection followed by acryloylation, the water-soluble glycomonomers were polymerized with or without acrylamide in the presence of a radical initiator in water to give corresponding copolymers and homopolymers, which were shown by SEC analysis to have high molecular weights. Evaluation of the biological activities of the glycopolymers against Shiga toxins (Stxs) was carried out, and the results suggested that glycopolymers having highly clustered globotriaosyl residues had high affinity against Stx2 (K_D?=?2.7～4.0?µM) even though other glycopolymers did not show any affinity or showed very weak binding affinity. When Stx1 was used for the same assay, all of the glycopolymers having globotriaosyl residues showed high affinity (K_D?=?0.30～1.74?µM). Interestingly, couple of glycopolymers having lactosyl moieties had weaker binding affinity against Stx1. In addition, when cytotoxicity assays were carried out for both Stxs, glycopolymers having highly clustered globotriaosyl residues showed higher affinity than that of the copolymers, and only highly clustered-type glycopolymers displayed neutralization potency against Stx2.     

COMT Val158Met moderates the link between rank and aggression in a non‐human primate

The COMT Val¹⁵⁸Met polymorphism is one of the most widely studied genetic polymorphisms in humans implicated in aggression and the moderation of stressful life event effects. We screened a wild primate population for polymorphisms at the COMT Val¹⁵⁸Met site and phenotyped them for aggression to test whether the human polymorphism exists and is associated with variation in aggressive behavior. Subjects were all adults from 4 study groups (37 males, 40 females) of Assamese macaques (Macaca assamensis) in their natural habitat (Phu Khieo Wildlife Sanctuary, Thailand). We collected focal animal behavioral data (27 males, 36 females, 5964 focal hours) and fecal samples for non‐invasive DNA analysis. We identified the human COMT Val¹⁵⁸Met polymorphism (14 Met/Met, 41 Val/Met and 22 Val/Val). Preliminary results suggest that COMT genotype and dominance rank interact to influence aggression rates. Aggression rates increased with rank in Val/Val, but decreased in Met/Met and Val/Met individuals, with no significant main effect of COMT genotype on aggression. Further support for the interaction effect comes from time series analyses revealing that when changing from lower to higher rank position Val/Val individuals decreased, whereas Met/Met individuals increased their aggression rate. Contradicting the interpretation of earlier studies, we show that the widely studied Val¹⁵⁸Met polymorphism in COMT is not unique to humans and yields similar behavioral phenotypes in a non‐human primate. This study represents an important step towards understanding individual variation in aggression in a wild primate population and may inform human behavioral geneticists about the evolutionary roots of inter‐individual variation in aggression.     

保乳手术与改良根治术治疗Ⅰ-Ⅱ期乳腺癌患者的疗效及生存状况比较研究

目的:比较分析乳腺癌保乳手术和根治术的临床疗效及患者生存状况。方法:回顾性分析2012年6月至2015年6月在我院乳腺外科行手术治疗的乳腺癌患者92例的临床资料,其中行保乳手术患者24例(保乳组),行根治手术患者68例(根治组),两组患者术后均采用个性化综合治疗巩固疗效,对比观察两组疗效及预后状况;通过乳腺癌生命质量测定量表(FACT-B)检测对比两组患者术后1、2年的生存质量;并对比两组患者术后乳房美容效果。结果:保乳组患者手术时间、术中出血量、引流量以及引流时间均明显较根治组少,差异有统计学意义(P0.05),而淋巴结清扫数则无明显差异(P0.05);两组患者上肢水肿发生率无明显差异(P0.05),而保乳组切缘皮瓣缺血发生率低于根治组(P0.05);两组患者术后2年生存率、复发率以及远处转移率无统计学差异(P0.05)。保乳组患者术后1、2年生理状况、情感状况、社会状况、功能状况、其他因素及生活质量综合评分均显著高于根治组,差异有统计学意义(P0.05);保乳组术后乳房美容效果的优良率显著高于根治组,差异有统计学意义(P0.05)。结论:相比于根治术,保乳手术不仅具有创伤小、术后恢复快的优势,患者预后状况与根治术相当,同时可更好的改善患者生存质量,术后乳房美容效果较好,临床应用价值更高。     

肝癌乙型肝炎病毒感染患者术后恩替卡韦抗病毒治疗的临床疗效及安全性评价

目的:探讨肝癌乙型肝炎病毒(HBV)感染患者根治性切除术后采用恩替卡韦抗病毒治疗的临床疗效及安全性。方法:收集2015年1月-2017年8月在我院行根治性切除术的肝癌HBV感染患者279例为研究对象,以血清HBV-DNA载量10~5 copies/ml为界限,分为高病毒复制组128例,低病毒复制组151例,按照随机数字表法将高病毒复制组分为高-治疗组64例、高-对照组64例,将低病毒复制组分为低-治疗组76例、低-对照组75例。高-治疗组和低-治疗组术后给予恩替卡韦0.5 mg/d,高-对照组和低-对照组未行抗病毒治疗。比较手术前、术后7 d各组的血清HBV-DNA水平,血清白蛋白(ALB)、谷丙转氨酶(ALT)、前白蛋白(PA),以及术后并发症的发生情况。结果:高-治疗组、高-对照组、低-治疗组、低-对照组术后血清ALB、PA均较治疗前降低,血清ALT均较治疗前升高,且高-治疗组或低-治疗组术后血清ALB、PA均高于高-对照组或低-对照组,血清ALT水平均低于高-对照组或低-对照组,差异均有统计学意义(P0.05);高-治疗组或低-治疗组术后血清HBV-DNA水平均低于治疗前,且均低于同期高-对照组或低-对照组,差异均有统计学意义(P0.05)。高-治疗组与高-对照组、低-治疗组与低-对照组患者术后并发症发生率均无统计学差异(P0.05)。结论:恩替卡韦能显著改善肝癌HBV感染患者术后的血清HBV-DNA载量水平和肝功能,安全性高,值得临床推广。     

Specificity of MLL1 and TET3 CXXC domains towards naturally occurring cytosine modifications

CXXC domains have traditionally been considered as CpG specific DNA binding domains that are repelled by cytosine modifications. This view has recently been challenged by the demonstration that CXXC domain of TET3 has relaxed sequence specificity and binds with the highest affinity to symmetric DNA duplex containing 5caCpG. Here, we present a comparative analysis of the MLL1-CXXC and TET3-CXXC sequence specificity and tolerance to cytosine modifications (5-methyl, 5-hydroxymethyl, 5-formyl, 5-carboxyl) in CpG and non-CpG context. For the first time, we take into consideration possible interference from cytosine bases elsewhere in the sequence. We show that despite similar overall structure, MLL1-CXXC has greater sequence and modification specificity than TET3-CXXC. MLL1-CXXC is specific only for CpG and does not tolerate any cytosine modifications. In contrast, TET3-CXXC does not require the CpG context of cytosine bases. Methyl-, formyl- and carboxyl-modifications are tolerated by TET3-CXXC, but only preceding G. Based on our and other data we propose a parsimonious model of MLL1-CXXC and TET3-CXXC DNA binding. This model explains why the binding of modified DNA duplexes by TET3-CXXC requires in some cases a register shift and is therefore context-dependent.     

Old and new glycopeptide antibiotics: From product to gene and back in the post-genomic era

Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by multi-drug resistant Gram-positive pathogens. First-generation glycopeptides (vancomycin and teicoplanin) are produced by soil-dwelling actinomycetes. Second-generation glycopeptides (dalbavancin, oritavancin, and telavancin) are semi-synthetic derivatives of the progenitor natural products. Herein, we cover past and present biotechnological approaches for searching for and producing old and new glycopeptide antibiotics. We review the strategies adopted to increase microbial production (from classical strain improvement to rational genetic engineering), and the recent progress in genome mining, chemoenzymatic derivatization, and combinatorial biosynthesis for expanding glycopeptide chemical diversity and tackling the never-ceasing evolution of antibiotic resistance.     

Computational characterization of substrate and product specificities,and functionality of S‐adenosylmethionine binding pocket in histone lysine methyltransferases from Arabidopsis,rice and maize

Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax‐like protein (ATX), Su (var)3‐9 h omologs protein (SUVH), Su(var)3‐9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S‐adenosylmethionine (SAM) binding pocket and a post‐SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein‐protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre‐SET and post‐SET domains in HKMTs of the aforementioned plant species lead to variations in class‐specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.