Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.     

Aptamer selection for the detection of Escherichia coli K88

In this study, the first group of single-stranded DNA aptamers that are highly specific to enterotoxigenic Escherichia coli (ETEC) K88 was obtained from an enriched oligonucleotide pool by the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure, during which the K88 fimbriae protein was used as the target and bovine serum albumin as counter targets. These aptamers were applied successfully in the detection of ETEC K88. They were then grouped under different families based on the similarity of their secondary structure and the homology of their primary sequence. Four sequences from different families were deliberately chosen for further characterization by fluorescence analysis. Having the advantage of high sensitivity, fluorescence photometry was selected as single-stranded DNA quantification method during the SELEX process. Aptamers with the highest specificity and affinity were analyzed to evaluate binding ability with E. coli. Since ETEC K88 is the only type of bacterium that expressed abundant K88 fimbriae, the selected aptamers against the K88 fimbriae protein were able to specifically identify ETEC K88 among other bacteria. This method of detecting ETEC K88 by aptamers can also be applied to bacteria other than ETEC K88.     

藤黄生孢链霉菌NRRL 2401遗传操作系统和基因文库的构建北大核心CSCD

【目的】建立藤黄生孢链霉菌NRRL 2401的遗传操作系统和基因文库,以便筛选次级代谢产物生物合成基因。【方法】利用大肠杆菌和链霉菌的属间接合转移的方法,以整合型载体pPM927、pSET152和复制型载体pJTU1278构建链霉菌遗传操作系统。以pCClFOS^(TM)载体,大肠杆菌EP1300^(TM)-T1~R为宿主菌构建Fosmid文库。随后,设计引物,利用"板池-行池-单克隆"的三级PCR方法对文库进行快速筛选。【结果】pPM927、pSET152和pJTU1278均成功转入藤黄生孢链霉菌NRRL 2401,其中pSET152载体的转化效率最高。构建了稳定高效的藤黄生孢链霉菌NRRL 2401的基因文库,含2880个克隆,平均插入片段大小约为35 kb,空载率小于1%,文库覆盖率为99.99%,覆盖基因组16.5倍。同时,初步筛选出可能含有吲哚霉素生物合成基因簇的9个阳性克隆。【结论】成功构建了稳定高效的藤黄生孢链霉菌NRRL 2401遗传操作系统和高质量的基因文库,为克隆该菌中次级代谢产物生物合成基因簇以及进一步遗传改造奠定了基础。     

定量蛋白质组分析蚯蚓血红蛋白样蛋白MSMEG_3312介导的分枝杆菌耐红霉素机制

[目的]细菌耐药机制是个复杂的机制,系统生物学是系统性揭示耐药机制的有力研究手段。我们课题组前期研究结果显示,蚯蚓血红蛋白样蛋白msmeg_3312基因敲除后能够增加耻垢分枝杆菌对红霉素的耐药性,本文系统研究MSMEG_3312参与红霉素耐药性形成的机制。[方法]首先纯化MSMEG_3312蛋白,利用光谱及圆二色谱描述MSMEG-3312蛋白。利用定量蛋白质组学的方法比较分析敲除菌株Δmsmeg_3312与野生型菌株mc² 155蛋白表达的差异,并通过qRT-PCR进行验证。利用红霉素ELASA试剂盒测定Δmsmeg_3312与mc² 155的胞内药物浓度。[结果]光谱及圆二色谱分析确定MSMEG_3312是蚯蚓血红蛋白样蛋白。定量蛋白质组学分析发现,红霉素未处理的条件下,相比于野生型菌株mc² 155,敲除菌株Δmsmeg_3312有包括3种转运蛋白在内的8种蛋白表达水平上调,14种蛋白表达下调;而红霉素处理后,Δmsmeg_3312中有448种蛋白差异表达,其中有11种转运蛋白表达上调,26种蛋白与氨基酸合成通路相关。胞内药物浓度检测显示敲除菌株Δmsmeg_3312的胞内红霉素浓度显著低于野生型菌株。[结论]蚯蚓血红蛋白样蛋白MSMEG_3312调控改变了细菌对红霉素药物处理的反应网络,其介导的红霉素耐药是一种集合抗生素耐受机制。     

Role of the sonchus yellow net virus N protein in formation of nuclear viroplasms

Sonchus yellow net virus is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (C. R. F. Martins, J. A. Johnson, D. M. Lawrence, T. J. Choi, A. Pisi, S. L. Tobin, D. Lapidus, J. D. O. Wagner, S. Ruzin, K. McDonald, and A. O. Jackson, J. Virol. 72:5669-5679, 1998). When expressed alone, the N protein localizes to the nuclei of plant and yeast (Saccharomyces cerevisiae) cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (M. M. Goodin, J. Austin, R. Tobias, M. Fujita, C. Morales, and A. O. Jackson, J. Virol. 75:9393-9406, 2001; M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, and A. O. Jackson, Plant J. 31:375-383, 2002). We now show that the N protein and various fluorescent derivatives form similar subnuclear foci in plant cells and that homologous interactions mediated by a helix-loop-helix region near the amino terminus are required for formation of the foci. Mutations within the helix-loop-helix region also interfere with N- and P-protein interactions that are required for N and P colocalization in the subnuclear foci. Affinity purification of N proteins harboring single mutations within the motif revealed that Tyr40 is critical for N-N and N-P interactions. Additional in vitro binding assays also indicated that the N protein binds to yeast and plant importin alpha homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin alpha binding. The P protein did not bind to the importin alpha homologues, suggesting that the N and P proteins use different pathways for nuclear entry. Our results in toto support a model suggesting that during infection, the N and P proteins enter the nucleus independently, that viroplasm formation requires homologous N-protein interactions, and that P protein targeting to the viroplasm requires N-P protein interactions that occur after N and P protein import into the nucleus.     

Characterization of melanin produced by a wild-type strain of Bacillus cereus

Bacillus cereus 58 (Bc58) is a UV-resistant wild type strain that has an ability to produce a sorrel pigment induced by L-tyrosine. The Fourier-transform infrared (FT-IR) spectra and chemical tests of its pigment are similar to that of the standard melanin (Sigma). A bioassay shows that the LC₅₀ of a Bacillus thuringiensis (Bt) formulation added with the melanin of Bc58 and exposed to UV for 5 h is 16.1 μg/ml, which is similar to that of the Bt formulation without UV treatment, however, it is almost double that of the Bt formulation exposed to UV without the melanin of Bc58. The result of SDS-PAGE indicates that the melanin of Bc58 can protect the insecticidal crystal proteins from degradation. This suggests that it is an excellent UV protective agent for the insecticidal crystal proteins of the Bt formulation. Translated from Microbiology, 2006, 33(1): 42–45 [译自: 微生物学通报]     

Etiological characteristics of chlamydia trachoma conjunctivitis of Primary Boarding School students in the Qinghai Tibetan area

The aim of this study was to investigate the etiological characteristics of Chlamydia trachomatis conjunctivitis among resident students at primary schools in the Qinghai Tibetan area in order to understand the distribution of C. trachomatis and other pathogenic microorganisms, to detect the isolation rate of infectious pathogens, and to provide an evidence for further targeted efforts in the prevent of sporadic trachoma efforts. From two primary schools in Qinghai Province, ocular samples from 35 students who were clinically diagnosed as trachoma cases and 60 normal controls were obtained by swabbing their upper eyelids and lower conjunctival sacs. Samples were preserved at 4°C and airlifted to Beijing Tongren Hospital within 24 h. Real-time polymerase chain reaction(RT-PCR) was used to screen for C. trachomatis, and nested PCR was used to amplify a fragment of the omp A gene for serotype confirmation. Bacterial cultivation and sensitivity tests were conducted based on the 2015 version of the Clinical and Laboratory Standards Institute. Adenovirus, herpes simplex virus, cytomegalovirus, and Epstein-Barr virus were screened by RT-PCR. Among the 35 students with trachoma, 8 came from the Jianshetang Primary School and 27 came from the Central Primary School. Two novel C. trachomatis B serotypes(Gen Bank accession numbers KU737520 and KU737521) were detected based on a sequence analysis of the omp A gene. Single C. trachomatis infections accounted for 42.86%(9/21) of the cases, and infections with multiple bacteria, particularly Haemophilus influenzae, Staphylococcus aureus, Moraxella catarrhalis, and Streptococcus pneumoniae, accounted for the remaining 57.14%(12/21). Of the 14 C. trachomatis-negative samples, one was positive for adenoviral infection(serotype D) and 13 were positive for bacterial infections(H. influenzae, M. catarrhalis, S. pneumoniae, S. aureus, streptococci other than S. pneumoniae, Staphylococcus epidermidis, Corynebacterium, and Arthrobacterium). In addition to C. trachomatis, the other bacteria and virus that were detected in the boarding students of primary schools in the Qinghai Tibetan area should be emphasized in trachoma prevention and control.     

两种肚倍型蚜虫之两种同功酶及可溶性蛋白的比较

应用电泳分析方法,对青麸杨上幼锤形信子与长枣形倍子内蚜虫进行了酯酶同功酶、过氧化物同功酶及可溶蛋白质的比较研究。结果表明：经形态学鉴定同为肚倍蚜可致瘿形成纺锤形与长枣形倍子。2种倍形内蚜虫在可溶性蛋白及酯酶同功酶上有较大差异。相异系数分别为02571,0067。说明肚倍蚜种内已出现分化,不同变形体倍子内蚜虫在分子水平上已具有向不同生物型分化的趋势。     

论提高广西桂林漓江上游水源径流量的可能性

论提高广西桂林漓江上游水源径流量的可能性邓世宗唐俊（广西农业大学林学院,南宁５３０００１）（广西林业勘测设计院,南宁５３００１１）ＰｏｏｉｂｉｌｉｔｙｏｆＩｎｃｒｅａｓｉｎｇＷａｔｅｒＤｉｓｃｈａｒｇｅｉｎＵｐｐｅｒＲｅａｃｈｅｓｏｆＬｉｊｉａｎｇＲ...     

Elevated auxin and reduced cytokinin contents in rootstocks improve their performance and grafting success

Plant grafting is an important technique for horticultural and silvicultural production. However, many rootstock plants suffer from undesirable lateral bud outgrowth, low grafting success rates or poor rooting. Here, we used a root‐predominant gene promoter (SbUGT) to drive the expression of a tryptophan‐2‐monooxygenase gene (iaaM) from Agrobacterium tumefaciens to increase auxin levels in tobacco. The transgenic plants, when used as a rootstock, displayed inhibited lateral bud outgrowth, enhanced grafting success rate and improved root initiation. However, root elongation and biomass of SbUGT::iaaM transgenic plants were reduced compared to those of wild‐type plants. In contrast, when we used this same promoter to drive CKX (a cytokinin degradation gene) expression, the transgenic tobacco plants displayed enhanced root elongation and biomass. We then made crosses between the SbUGT::CKX and SbUGT::iaaM transgenic plants. We observed that overexpression of the CKX gene neutralized the negative effects of auxin overproduction on root elongation. Also, the simultaneous expression of both the iaaM and CKX genes in rootstock did not disrupt normal growth and developmental patterns in wild‐type scions. Our results demonstrate that expression of both the iaaM and CKX genes predominantly in roots of rootstock inhibits lateral bud release from rootstock, improves grafting success rates and enhances root initiation and biomass.