Ability of Reptalus quinquecostatus (Hemiptera: Cixiidae) to inoculate stolbur phytoplasma to artificial feeding medium

Laboratory trials were carried out on wild individuals of Reptalus quinquecostatus (Cixiidae), a potential vector of stolbur phytoplasma to grapevine, to assess its ability to inoculate the phytoplasma in artificial feeding medium. Seventy‐seven specimens of the cixiid were tested on a sucrose–TE (Tris–ethylenediaminetetraacetic acid) diet and 62 of them survived less than 24 h. Polymerase chain reaction (PCR) assays performed on the insect bodies detected the presence of stolbur phytoplasma, with an infection rate of 32.5%. Restriction fragment length polymorphism analysis of the tuf gene, amplified by PCR, revealed Vergilbungskrankheit type I (VK‐I) in 20 specimens, VK‐II in 4 specimens and both types in 1 specimen. Ten of the 25 infected R. quinquecostatus specimens successfully inoculated VK‐I in the sucrose solution, that is, a 40% inoculation efficiency despite the brief survival. The results indicate that R. quinquecostatus is a competent species to transmit the stolbur phytoplasma in artificial conditions. The repeated observation of adults feeding on grapevine strengthens the hypothesis that the species is a vector of stolbur phytoplasma to this plant.     

柯萨奇病毒A组16型研究进展

柯萨奇病毒A组16型(CA16)是引起手足口病(HFMD)的主要病原体,与肠道病毒71型(EV71)交替或共同流行;特别是近年在西太平洋地区呈现流行强度高、重症和死亡人数多的特点,已成为该地区的重大公共卫生问题。研发安全有效的疫苗是控制HFMD流行的有效手段。由于EV71所致疾病在重症和死亡病例中所占比例高,对其疫苗研发得到了广泛关注,全病毒灭活疫苗已进入III期临床,有望即将应用于婴幼儿HFMD的防控。EV71疫苗的顺利研发随之也增加了对CA16疫苗研发的迫切性。近年来日本、新加坡以及中国台湾地区逐渐开始关注CA16相关的研究,我国也有多家企业开展CA16疫苗的研发。本文就CA16的病原学,流行病学,实验室诊断,治疗和预防等方面进行了综述。     

Multilocus genotyping of a ‘Candidatus Phytoplasma aurantifolia’‐related strain associated with cauliflower phyllody disease in China

A new cauliflower disease characterised by the formation of leaf‐like inflorescences and malformed flowers occurred in a seed production field located in Yunnan, a southwest province of China. Detection of phytoplasma‐characteristic 16S rRNA gene sequences in DNA samples from diseased plants linked the cauliflower disease to phytoplasmal infection. Results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence indicated that the cauliflower‐infecting agent is a ‘Candidatus Phytoplasma aurantifolia’‐related strain and is a new member of the peanut witches'‐broom phytoplasma group, subgroup A (16SrII‐A). Multilocus genotyping showed close genetic relationship between this cauliflower phytoplasma and a broad host range phytoplasma lineage found only in East Asia thus far. Molecular markers present in the secY and rp loci distinguished this phytoplasma from other members of the subgroup 16SrII‐A.     

Two new miR‐16 targets: caprin‐1 and HMGA1, proteins implicated in cell proliferation

Background information. miRNAs (microRNAs) are a class of non‐coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3′ UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR‐16 (miRNA‐16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR‐16. Results. In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR‐16, caprin‐1 (cytoplasmic activation/proliferation‐associated protein‐1) and HMGA1 (high‐mobility group A1), and we also studied cyclin E which had been previously recognized as an miR‐16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR‐16 interacts with the 3′ UTR of the three target mRNAs. We showed that miR‐16, in MCF‐7 and HeLa cell lines, down‐regulates the expression of caprin‐1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. Conclusions. Taken together, our data demonstrated that miR‐16 can negatively regulate two new targets, HMGA1 and caprin‐1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.     

Isolation and antimicrobial evaluation of isomeric hydroxy ketones in leaf cuticular waxes of Annona squamosa

A novel natural compound, 11-hydroxy-16-hentriacontanone, has been isolated from the leaf cuticular wax of Annona squamosa along with its known isomer 10-hydroxy-16-hentriacontanone in a ratio of 67:33. This isomeric mixture of hydroxy ketones constituted together 16.5% of the total cuticular waxes. The new compound was characterised using spectral and chromatographic techniques. The major component was found to be 16-hentriacontanone (palmitone), which constituted up to 48% of the total cuticular wax, together with a homologous series of hydrocarbons, fatty aldehydes, fatty alcohols, fatty acids and sterols as minor components. The antimicrobial activity of the isomeric hydroxy ketones was tested against selected Gram-positive and Gram-negative bacterial strains, and also some selected fungal strains, and compared with palmitone. The antibacterial activity of palmitone was significantly higher than that of the isomeric hydroxy ketones, but their antifungal activities were comparable.     

Spectroscopic and molecular modelling analysis of the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride and bovine serum albumin

Several spectroscopic approaches namely fluorescence, time‐resolved fluorescence, UV‐visible, and Fourier transform infra‐red (FT‐IR) spectroscopy were employed to examine the interaction between ethane‐1,2‐diyl bis(N,N‐dimethyl‐N‐hexadecylammoniumacetoxy)dichloride (16‐E2‐16) and bovine serum albumin (BSA). Fluorescence studies revealed that 16‐E2‐16 quenched the BSA fluorescence through a static quenching mechanism, which was further confirmed by UV–visible and time‐resolved fluorescence spectroscopy. In addition, the binding constant and the number of binding sites were also calculated. The thermodynamic parameters at different temperatures (298 K, 303 K, 308 K and 313 K) indicated that 16‐E2‐16 binding to BSA is entropy driven and that the major driving forces are electrostatic interactions. Decrease of the α‐helix from 53.90 to 46.20% with an increase in random structure from 22.56 to 30.61% were also observed by FT‐IR. Furthermore, the molecular docking results revealed that 16‐E2‐16 binds predominantly by electrostatic and hydrophobic forces to some residues in the BSA sub‐domains IIA and IIIA. Copyright © 2015 John Wiley & Sons, Ltd.     

Biochemical characterization of the ribosomal decoding site

Prior to the emergence of crystal structures of the ribosome, different ribosomal functions were identified with specific regions of ribosomal RNA by biochemical and genetic approaches. In particular, three universally conserved bases of 16S rRNA, G530, A1492 and A1493, were implicated in the interaction of the incoming aminoacyl-tRNA with the 30S subunit and mRNA. The conserved region surrounding A1492 and A1493 was called the "decoding site", based on the results of chemical probing experiments and antibiotic resistance mutations. Crystallographic studies from the Ramakrishnan laboratory have now shown that G530 loop, A1492 and A1493 undergo localized conformational changes to form an RNA structure that positions these three bases to inspect the accuracy of the codon-anticodon match with high stereochemical precision, using A-minor interactions. Some results from the pre-X-ray era may provide clues to further aspects of the decoding process.     

跨膜蛋白16A：钙激活氯通道的最新进展

钙激活氯离子通道(calcium-activated chloride channels,CaCCs)介导了众多生理过程,包括跨上皮离子与液体分泌、心肌和神经兴奋、感觉传导、平滑肌收缩和受精过程等,但目前对于其分子基础等重要问题尚未研究清楚．综述了最新报道的CaCCs分子基础跨膜蛋白16A(TMEM16A)的发现过程、基因结构和功能、离子通道电生理特性、相关病理与药理功能的一些热点问题,并展望了该研究领域的发展趋势．     

An open conformation determined by a structural switch for 2A protease from coxsackievirus A16

Coxsackievirus A16 belongs to the family Picornaviridae, and is a major agent of hand-foot-and-mouth disease that infects mostly children, and to date no vaccines or antiviral therapies are available. 2A protease of enterovirus is a nonstructural protein and possesses both self-cleavage activity and the ability to cleave the eukaryotic translation initiation factor 4G. Here we present the crystal structure of coxsackievirus A16 2A protease, which interestingly forms hexamers in crystal as well as in solution. This structure shows an open conformation, with its active site accessible, ready for substrate binding and cleavage activity. In conjunction with a previously reported “closed” state structure of human rhinovirus 2, we were able to develop a detailed hypothesis for the conformational conversion triggered by two “switcher” residues Glu88 and Tyr89 located within the bll2-cII loop. Substrate recognition assays revealed that amino acid residues P1′, P2 and P4 are essential for substrate specificity, which was verifi ed by our substrate binding model. In addition, we compared the in vitro cleavage effi ciency of 2A proteases from coxsackievirus A16 and enterovirus 71 upon the same substrates by fl uorescence resonance energy transfer (FRET), and observed higher protease activity of enterovirus 71 compared to that of coxsackievirus A16. In conclusion, our study shows an open conformation of coxsackievirus A16 2A protease and the underlying mechanisms for conformational conversion and substrate specifi city. These new insights should facilitate the future rational design of effi cient 2A protease inhibitors.     

Development and characterization of a clinical strain of Coxsackievirus A16 and an eGFP infectious clone

Coxsackievirus A16(CA16) is one of the major causes of hand, foot, and mouth disease(HFMD) worldwide, which is a common illness that affects children. The frequent occurrence of HFMD outbreaks has become a serious public health problem in Asia. Therefore, it is important to understand the pathogenesis and replication of CA16. In this study, a stable infectious c DNA clone of an epidemic strain of Coxsackievirus A16(CA16) was assembled, and subsequently a reporter virus(e GFP-CA16) was constructed by inserting the e GFP gene between the 5'-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site(ITTLG) at its C-terminus. This was transfected into Vero cells to generate infectious recombinant viruses. The growth characteristics and plaque morphology, in vitro, in mammalian cells were found to be indistinguishable between the parental and recombinant viruses. Although the e GFP-CA16 showed smaller plaque size as compared to recombinant CA16, both were found to exhibit similar growth trends and EC50 of NITD008. In summary, this stable infectious c DNA clone should provide a valuable experimental system to study CA16 infection and host response. The e GFP-CA16 is expected to provide a powerful tool to monitor e GFP expression in infected cells and to evaluate the antiviral activity of potential antiviral agents in the treatment of CA16 infections.     

中国柯萨奇病毒A组16型部分VP1区序列测定及系统进化分析

利用1999～2004年连续6年从中国深圳及北京地区分离的柯萨奇病毒A组16型(Coxsackievirus A16,Cox．A16)毒株的部分VP1区基因序列进行分析和研究,试图找出中国Cox．A16的种系进化规律和分型依据。6株Cox．A16病毒部分VP1区核苷酸和氨基酸同源性均较高,相互间核苷酸同源性为94．5％～98．0％,氨基酸同源性为97．8％～100％。6株中国分离株与Cox．A16国际参考株相比,部分VP1区核苷酸同源性高于78．2％,氨基酸同源性高于为93．3％。基因系统进化分析表明,中国大陆分离的6株Cox．A16与中国台湾流行株Tainan-5079-98(AF177911),与4株日本分离株、瑞典株及美国株GA95—2095(AF081613)、PA94—5753(AF081628)的关系均较近,核苷酸同源性大于93．3％。了解我国Cox．A16流行株的遗传学背景和种系进化关系,对有效地预防和控制Cox．A16流行有重要意义。     

Deficiency of DICER reduces the invasion ability of trophoblasts and impairs the pro‐angiogenic effect of trophoblast‐derived microvesicles

DICER is a key rate‐limiting enzyme in the canonical miRNAs biogenesis pathway, and DICER and DICER‐dependent miRNAs have been proved to play essential roles in many physiological and pathological processes. However, whether DICER is involved in placentation has not been studied. Successful spiral artery remodelling is one of the key milestones during placentation, which depends mostly on the invasion of trophoblasts and the crosstalk between trophoblasts and endothelial cells. In the present study, we show that DICER knockdown impairs the invasion ability of both primary extravillous trophoblasts (EVT) and HTR8/SVneo (HTR8) cell lines. The decreased invasion of HTR8 cells upon DICER knockdown (sh‐Dicer) was partly due to the up‐regulation of miR‐16‐2‐3p, which led to a reduced expression level of the collagen type 1 alpha 2 chain (COL1A2) protein. Moreover, microvesicles (MVs) can be secreted by HTR8 cells and promote the tube formation ability of human umbilical cord vein endothelial cells (HUVECs). However, conditioned medium and MVs derived from sh‐Dicer HTR8 cells have an anti‐angiogenic effect, due to reduced angiogenic factors and increased anti‐angiogenic miRNAs (including let‐7d, miR‐1‐6‐2 and miR‐15b), respectively. In addition, reduced protein expression of DICER is found in PE placenta by immunoblotting and immunohistochemistry. In summary, our study uncovered a novel DICER‐miR‐16‐2‐COL1A2 mediated pathway involved in the invasion ability of EVT, and DICER‐containing MVs mediate the pro‐angiogenic effect of trophoblast‐derived conditioned medium on angiogenesis, implying the involvement of DICER in the pathogenesis of PE.     

干扰素膜蛋白Tet-on系统稳定细胞系的建立及对CA16病毒的抑制作用

通过Tet-on调控系统,构建受多西环素诱导表达干扰素诱导的跨膜蛋白(interferon-induced transmembrane proteins 1/2/3,IFITM1/2/3)基因的HeLa细胞系,并初步探索了IFITM蛋白对柯萨奇病毒A16(CA16)的抑制作用.首先将调控质粒pTet-on转染进入HeLa细胞,通过G418筛选出阳性克隆细胞系,在此细胞系基础上共同转染反应质粒pTRE2-IFITM1/2/3和伴侣质粒pTK-Hyg,通过潮霉素筛选出单克隆细胞系,加入多西环素后利用Western印迹筛选出可诱导表达IFITM1/2/3蛋白的单克隆细胞系.使用实时荧光定量PCR(RT-qPCR)检测发现,多西环素诱导表达的IFITM蛋白对不同感染复数(multiplicity of infection,MOI)的CA16具有明显的抑制作用,其中IFITM 3对CA16的抑制效果最为明显.Tet调控IFITM1/2/3基因表达HeLa细胞系的成功建立,为进一步研究IFITM基因的功能及其抗病毒机理提供了一个理想的细胞模型.     

5-Fluorouracil or gemcitabine combined with adenoviral-mediated reintroduction of p16INK4A greatly enhanced cytotoxicity in Panc-1 pancreatic adenocarcinoma cells

BACKGROUND: Pancreatic cancer is one of the most lethal of all the common gastrointestinal malignancies. Although surgery offers the best chance for survival, it is not appropriate for all cases. The only adjuvant treatment to show promise is chemotherapy. Hence new treatments are urgently sought. We previously reported that adenoviral (Ad)-mediated delivery of p53 (Adp53) and p16(INK4A) (Adp16) significantly inhibited the growth of pancreatic cancer cell lines and established subcutaneous pancreatic tumours in nude mice (Ghaneh P, et al. Adenovirus mediated transfer of p53 and p16INK4A results in pancreatic cancer regression in vitro and in vivo. Gene Ther 2001; 8: 199-208). In this study we examine whether combining Ad-mediated delivery of p53 or p16(INK4A) with clinically relevant chemotherapeutic drugs has therapeutic potential for pancreatic cancer. METHODS AND RESULTS: Four pancreatic adenocarcinoma cell lines were evaluated for their sensitivity to 5-fluorouracil (5-FU) and gemcitabine and two of these, Suit-2 and Panc-1, were chosen for combination experiments because they showed moderate and poor sensitivity, respectively, to 5-FU and gemcitabine. We found no evidence for enhanced cytotoxicity when either cell line was transduced with Adp53 before or after incubation with chemotherapeutic drugs. In contrast, incubation of Panc-1 cells with either 5-FU or gemcitabine followed by Ad-mediated overexpression of p16(INK4A) resulted in a substantial reduction in cell viability under conditions where the drugs alone had minimal cytotoxicity. Incubation of Suit-2 cells with 5-FU followed by Ad-mediated overexpression of p16(INK4A) also resulted in a significant reduction in cell viability. This, however, was observed only with higher concentrations of 5-FU and viral vector. Cell cycle analysis of Panc-1 cells showed that the combination of cytotoxic drugs and Adp16 resulted in an increase in the sub-G1 population suggesting an increase in apoptosis. Dual labelling of these cells with annexin V and propidium iodide (PI) confirmed that the combination of 5-FU and Adp16 resulted in a significant increase in early apoptotic cells (annexin V positive and PI negative) compared with controls. Moreover, overexpression of p16(INK4A) was associated with a reduction in pRb levels in these cells-high levels of pRb have been proposed to contribute to chemoresistance in pancreatic cancer cells. CONCLUSIONS: We have shown that the currently used chemotherapeutic drugs for pancreatic adenocarcinoma combined with restoration of p16(INK4A) expression hold promise for the adjuvant treatment of this disease. Importantly, the combination facilitated the use of chemotherapeutic drugs at lower concentrations than would otherwise be effective.     

Comparing aminoglycoside binding sites in bacterial ribosomal RNA and aminoglycoside modifying enzymes

Aminoglycoside antibiotics are used against severe bacterial infections. They bind to the bacterial ribosomal RNA and interfere with the translation process. However, bacteria produce aminoglycoside modifying enzymes (AME) to resist aminoglycoside actions. AMEs form a variable group and yet they specifically recognize and efficiently bind aminoglycosides, which are also diverse in terms of total net charge and the number of pseudo‐sugar rings. Here, we present the results of 25 molecular dynamics simulations of three AME representatives and aminoglycoside ribosomal RNA binding site, unliganded and complexed with an aminoglycoside, kanamycin A. A comparison of the aminoglycoside binding sites in these different receptors revealed that the enzymes efficiently mimic the nucleic acid environment of the ribosomal RNA binding cleft. Although internal dynamics of AMEs and their interaction patterns with aminoglycosides differ, the energetical analysis showed that the most favorable sites are virtually the same in the enzymes and RNA. The most copied interactions were of electrostatic nature, but stacking was also replicated in one AME:kanamycin complex. In addition, we found that some water‐mediated interactions were very stable in the simulations of the complexes. We show that our simulations reproduce well findings from NMR or X‐ray structural studies, as well as results from directed mutagenesis. The outcomes of our analyses provide new insight into aminoglycoside resistance mechanism that is related to the enzymatic modification of these drugs. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

柯萨奇病毒A组16型河南省分离株基因组序列分析

为了解河南省手足口病患者标本中分离柯萨奇A组的16型(CoxAl6)病毒基因组特征,对2010年采集的手足口病患者临床标本406份进行RT-PCR扩增和病毒分离鉴定;通过10对引物分段扩增和拼接CoxAl6分离株基因组序列,利用生物信息学软件对序列分析,构建序列遗传发育树。测序获得河南省CoxAl6分离株HN1162/HN/CHN/2010基因组全长序列7 411bp,5′非编码区(5′UTR)、P1、P2、P3、3′非编码区(3′UTR)区域核苷酸序列与GenBank公布的其它分离株相似性分别为87.0%～97.9%、77.0%～95.4%、80.3%～96.9%、77.9%～96.2%、80.5%～100%;VP1区核苷酸相似性为91.4%～96.4%,氨基酸相似性为99.3%～99.7%;遗传发育树分析表明与我国深圳、广州、福建分离株处于同一分支。河南省手足口病患者标本CoxAl6病毒分离株属于C2基因亚型/B-2基因亚型,对加强该病毒变异检测,预防控制手足口病疫情具有重要意义。     

Zafirlukast inhibits the growth of lung adenocarcinoma via inhibiting TMEM16A channel activity

Lung cancer has the highest mortality among cancers worldwide due to its high incidence and lack of the effective cures. We have previously demonstrated that the membrane ion channel TMEM16A is a potential drug target for the treatment of lung adenocarcinoma and have identified a pocket of inhibitor binding that provides the basis for screening promising new inhibitors. However, conventional drug discovery strategies are lengthy and costly, and the unpredictable side effects lead to a high failure rate in drug development. Therefore, finding new therapeutic directions for already marketed drugs may be a feasible strategy to obtain safe and effective therapeutic drugs. Here, we screened a library of over 1400 Food and Drug Administration–approved drugs through virtual screening and activity testing. We identified a drug candidate, Zafirlukast (ZAF), clinically approved for the treatment of asthma, that could inhibit the TMEM16A channel in a concentration-dependent manner. Molecular dynamics simulations and site-directed mutagenesis experiments showed that ZAF can bind to S387/N533/R535 in the nonselective inhibitor binding pocket, thereby blocking the channel pore. Furthermore, we demonstrate ZAF can target TMEM16A channel to inhibit the proliferation and migration of lung adenocarcinoma LA795 cells. In vivo experiments showed that ZAF can significantly inhibit lung adenocarcinoma tumor growth in mice. Taken together, we identified ZAF as a novel TMEM16A channel inhibitor with excellent anticancer activity, and as such, it represents a promising candidate for future preclinical and clinical studies.