A vertebrate case study of the quality of assemblies derived from next-generation sequences

The unparalleled efficiency of next-generation sequencing (NGS) has prompted widespread adoption, but significant problems remain in the use of NGS data for whole genome assembly. We explore the advantages and disadvantages of chicken genome assemblies generated using a variety of sequencing and assembly methodologies. NGS assemblies are equivalent in some ways to a Sanger-based assembly yet deficient in others. Nonetheless, these assemblies are sufficient for the identification of the majority of genes and can reveal novel sequences when compared to existing assembly references.     

豇豆病毒病病原的分子鉴定

为澄清浙江省豇豆病毒病病原及其分类,采用马铃薯Y病毒属通用引物Sprimer扩增了浙江豇豆线状病毒基因组3′－末端序列。同时又根据已知CMV RNA3序列设计引物pCMV-44-63/1200-1181,扩增了混合的CMV－ZJ运动蛋白基因全序列。外壳蛋白氨基酸序列分析表明,产中仅存在一种线状病毒（CV－ZJ）,该病毒与一泰国豇豆蚜传花叶病毒（CABMV）具有最高的同源性（98.6%）,与花生条纹病毒（PStV）、石斛花叶病毒（DeMV0．）、赤豆花叶病毒（AZMV）、黑眼豆豆花叶病毒（BICMV）和菜豆普通花叶病毒（BCMV）分离物间的同源性为88.5%－94.4%,而与其它CABMV分离物的同源性均低于70％。进化树分析表明,PStV、AZMV、DeMV、BlCMV和BCMV为同种异名,应统称为BCMV,而CABMV为另一种病毒;CV－ZJ和泰国CABMV分离物应分类为BCMV豇豆株系。CMV－ZJ的运动蛋白序列分析表明,该分离物属于CMV亚组Ⅰ,与其它分离物氨基酸序列同源性为93.1%－97.8%。     

吡格列酮抑制大鼠心肌肥厚的实验研究

目的: 以体外实验和体内实验探讨噻唑烷二酮(Thiazolidinedione,TZD)类药物吡格列酮对大鼠心肌肥厚的影响.方法: 体外原代培养新生大鼠的心肌细胞,以血管紧张素Ⅱ刺激建立心肌肥大模型,分别给予不同浓度的吡格列酮处理.采用RT-PCR法检测心肌肥大特征性基因心钠素(ANP)和脑钠素(BNP)mRNA的表达,并以3H-亮氨酸掺入测定心肌细胞蛋白合成速率.体内实验中通过不完全结扎大鼠腹主动脉引起压力负荷增加,导致左心室肥厚.从术前1周起用灌胃器经口给予吡格列酮(20 mg*kg-1*day-1)直至术后4周.处死动物,计算心脏重/体重比值,测量左心室壁厚度和心肌细胞的平均直径,RT-PCR法测定心肌细胞中BNP及炎性细胞因子的mRNA表达.结果: 心肌细胞肥大模型出现后,心肌细胞的ANP和BNP mRNA的表达以及蛋白合成速率增加.经不同浓度的吡格列酮处理后,这些变化得以减轻,并呈一定的剂量依赖性.吡格列酮抑制左心室心肌白细胞介素-1β,心调理素-1的mRNA表达,同时减轻压力负荷升高引起的大鼠心脏重/体重比值、左心室壁厚度和心肌细胞平均直径的增加.结论: 吡格列酮在体外和体内实验研究中显示对大鼠心肌肥厚有保护作用,可能对防治以心肌肥厚为特征的心血管疾病有一定的应用前景.     

内源性儿茶酚胺参与白细胞介素-2对大鼠离体心脏的作用

目的: 研究内源性儿茶酚胺是否参与白细胞介素-2(IL-2)的心脏作用.方法: 采用Langendorff离体心脏灌流模型,观察室性早搏次数、左室发展压(LVDP)、左室舒张末压(LVEDP)、心率(HR)和冠脉流量(CF)的变化.结果: ①50 U/ml IL-2增加离体心脏的室性早搏次数,增加LVDP、LVEDP、HR和CF.②利舍平(reserpine)或普萘洛尔(propranolol)预处理后,50 U/ml IL-2对离体心脏的作用消失;phentolamine预处理后,不改变50U/ml IL-2的离体心脏作用.③200 U/ml IL-2增加离体心脏的室性早搏次数,对LVDP、LVEDP、HR和CF无显著增加作用.④reserpine或propranolol预处理后,200 U/ml IL-2增加离体心脏的室性早搏次数作用不明显,LVDP、HR和CF降低、LVEDP增高.结论: 内源性儿茶酚胺介导了IL-2的致离体心脏心律失常、正性变时和变力作用,较高浓度的IL-2抑制离体心脏的功能.     

Phage Display Selection of Cyclic Peptides That Inhibit Andes Virus Infection

Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β₃ (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table ​(Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).

TABLE 1.

Peptide-bearing phage eluted from ANDV

DNA chip-based expression profile analysis indicates involvement of the phosphatidylinositol signaling pathway in multiple plant responses to hormone and abiotic treatments

The phosphatidylinositol (PI) metabolic pathway is considered critical in plant responses to many environmental factors, and previous studies have indicated the involvement of multiple PI-related gene families during cellular responses.Through a detailed analysis of the Arabidopsis thaliana genome, 82 polypeptides were identified as being involved in PI signaling. These could be grouped into different families including PI synthases (PIS), PI-phosphate kinases (PIPK),phospholipases (PL), inositol polyphosphate phosphatases (IPPase), inositol polyphosphate kinases (IPK), PI transfer proteins and putative inositol polyphosphate receptors. The presence of more than 10 isoforms of PIPK, PLC, PLD and IPPase suggested that these genes might be differentially expressed during plant cellular responses or growth and development. Accordingly, DNA chip technology was employed to study the expression patterns of various isoforms.In total, 79 mRNA clones were amplified and used for DNA chip generation. Expression profile analysis was performed using samples that represented multiple tissues or cellular responses. Tested samples included normal leaf, stem and flower tissues, and leaves from plants treated with various hormones (auxin, cytokinin, gibberellin, abscisic acid and brassinosteroid) or environmental factors (temperature, calcium, sodium, drought, salicylic acid and jasmonic acid).Results showed that many PI pathway-related genes were differentially expressed under these experimental conditions.In particular, the different isoforms of each family were specifically expressed in many cases, suggesting their involvement in tissue specificity and cellular responses to environmental conditions. This work provides a starting point for functional studies of the relevant PI-related proteins and may help shed light onto the role of PI pathways in development and cellular responses.     

Characterizing the spatial distribution of giant pandas (Ailuropoda melanoleuca) in fragmented forest landscapes

Aim To examine the effects of forest fragmentation on the distribution of the entire wild giant panda (Ailuropoda melanoleuca) population, and to propose a modelling approach for monitoring the spatial distribution and habitat of pandas at the landscape scale using Moderate Resolution Imaging Spectro‐radiometer (MODIS) enhanced vegetation index (EVI) time‐series data. Location Five mountain ranges in south‐western China (Qinling, Minshan, Qionglai, Xiangling and Liangshan). Methods Giant panda pseudo‐absence data were generated from data on panda occurrences obtained from the third national giant panda survey. To quantify the fragmentation of forests, 26 fragmentation metrics were derived from 16‐day composite MODIS 250‐m EVI multi‐temporal data and eight of these metrics were selected following factor analysis. The differences between panda presence and panda absence were examined by applying significance testing. A forward stepwise logistic regression was then applied to explore the relationship between panda distribution and forest fragmentation. Results Forest patch size, edge density and patch aggregation were found to have significant roles in determining the distribution of pandas. Patches of dense forest occupied by giant pandas were significantly larger, closer together and more contiguous than patches where giant pandas were not recorded. Forest fragmentation is least in the Qinling Mountains, while the Xiangling and Liangshan regions have most fragmentation. Using the selected landscape metrics, the logistic regression model predicted the distribution of giant pandas with an overall accuracy of 72.5% (κ = 0.45). However, when a knowledge‐based control for elevation and slope was applied to the regression, the overall accuracy of the model improved to 77.6% (κ = 0.55). Main conclusions Giant pandas appear sensitive to patch size and isolation effects associated with fragmentation of dense forest, implying that the design of effective conservation areas for wild giant pandas must include large and dense forest patches that are adjacent to other similar patches. The approach developed here is applicable for analysing the spatial distribution of the giant panda from multi‐temporal MODIS 250‐m EVI data and landscape metrics at the landscape scale.     

Induction of transient ion channel-like pores in a cancer cell by antibiotic peptide

The anticancer activity of anti-bacterial cecropins makes them potentially useful as peptide anti-cancer drugs. We used the cell-attached patch to study the effect of cecropin B (CB; having one hydrophobic and one amphipathic alpha-helix) and its derivative, cecropin B3 (CB3; having two hydrophobic alpha-helices) on the membrane of Ags cancer cells. Application of 10-60 microM CB onto the membrane of the cancer cell produces short outward currents. Comparative study with CB3, which induces no outward currents, shows that the amphipathic group of CB is necessary for the pore formation. The results provide a rationale to study the cell-killing activity of antimicrobial peptides at the single cancer cell level.     

Effects of Overexpression of the Endogenous Farnesyl Diphosphate Synthase on the Artemisinin Content in Artemisia annua L.

Artemisinin is a novel effective antimalarial drug extracted from the medicinal plant Artemisia annua L. Owing to the tight market and low yield of artemisinin, there is great interest in enhancing the production of artemisinin. In the present study, farnesyi diphosphate synthase （FPS） was overexpressed in high-yield A. annua to Increase the artemisinin content. The FPS activity in transgenic A. ennue was twoto threefold greater than that In non-transgenic A. annua. The highest artemisinin content in transgenic A. annua was approximately 0.9% （dry weight）, which was 34.4% higher than that in non-transgenic A. annua. The results demonstrate the regulatory role of FPS in artemisinin biosynthesis.     

Energy and emission benefits of alternative transportation liquid fuels derived from switchgrass: a fuel life cycle assessment

We conducted a mobility chains, or well-to-wheels (WTW), analysis to assess the energy and emission benefits of cellulosic biomass for the U.S. transportation sector in the years 2015-2030. We estimated the life-cycle energy consumption and emissions associated with biofuel production and use in light-duty vehicle (LDV) technologies by using the Greenhouse gases, Regulated Emissions, and Energy use in Transportation (GREET) model. Analysis of biofuel production was based on ASPEN Plus model simulation of an advanced fermentation process to produce fuel ethanol/protein, a thermochemical process to produce Fischer-Tropsch diesel (FTD) and dimethyl ether (DME), and a combined heat and power plant to co-produce steam and electricity. Our study revealed that cellulosic biofuels as E85 (mixture of 85% ethanol and 15% gasoline by volume), FTD, and DME offer substantial savings in petroleum (66-93%) and fossil energy (65-88%) consumption on a per-mile basis. Decreased fossil fuel use translates to 82-87% reductions in greenhouse gas emissions across all unblended cellulosic biofuels. In urban areas, our study shows net reductions for almost all criteria pollutants, with the exception of carbon monoxide (unchanged), for each of the biofuel production option examined. Conventional and hybrid electric vehicles, when fueled with E85, could reduce total sulfur oxide (SO(x)) emissions to 39-43% of those generated by vehicles fueled with gasoline. By using bio-FTD and bio-DME in place of diesel, SO(x) emissions are reduced to 46-58% of those generated by diesel-fueled vehicles. Six different fuel production options were compared. This study strongly suggests that integrated heat and power co-generation by means of gas turbine combined cycle is a crucial factor in the energy savings and emission reductions.