广藿香不同部位UPLC指纹图谱及化学模式识别研究

目的建立广藿香药材不同部位的UPLC指纹图谱,研究广藿香不同部位的质量差异。方法采用UPLC法建立广藿香不同部位化学指纹图谱,结合相似度评价、热图聚类分析(CA)、主成分分析(PCA)及正交偏最小二乘法-判别分析(OPLS-DA)对18批广藿香药材、茎、叶的UPLC指纹图谱进行分析,并测定广藿香酮含量。结果18批广藿香药材及不同部位的UPLC指纹图谱均确定了9个相同的共有峰,并通过对照品指认4、6、9号峰分别为毛蕊花糖苷、异毛蕊花糖苷和广藿香酮。CA和PCA结果表明广藿香叶和茎的质量差异大,叶和药材的质量较接近。OPLS-DA发现5种成分是造成不同批次样品质量差异的主要标志物。含量测定结果表明同一批广藿香中的广藿香酮含量均为茎>药材>叶。结论广藿香不同部位的化学成分相似,但含量存在显著性差异,本研究可为广藿香药材的质量控制及资源开发利用提供参考。

一测多评法同时测定蔓荆子配方颗粒中5种化学成分的含量

目的建立一测多评(QAMS)法同时测定蔓荆子配方颗粒中5种成分的含量。方法采用UPLC法,流动相为甲醇-0.2%磷酸溶液,梯度洗脱;流速为0.3 mL·min^(-1);柱温为30℃;检测波长为258 nm;进样量为1μL。以蔓荆子黄素为内参物,建立其与原儿茶酸、对羟基苯甲酸、香草酸和异荭草素的相对校正因子,并在不同色谱仪和色谱柱上考察各成分相对校正因子的稳定性和耐用性。结果原儿茶酸、对羟基苯甲酸、香草酸、异荭草素、蔓荆子黄素浓度分别在0.4221~42.2064μg·mL^(-1),0.7958~79.5760μg·mL^(-1),0.5038~50.3840μg·mL^(-1),0.5464~54.6370μg·mL^(-1),0.5918~59.1766μg·mL^(-1)范围内线性关系良好;平均加样回收率分别为101.41%,96.65%,101.12%,97.90%,99.64%,RSD均小于3.0%(n=9);相对校正因子分别为1.42,1.75,1.15,0.64。QAMS计算10批蔓荆子配方颗粒5种成分的含量与外标法(ESM)测定值间均无明显差异。结论建立的QAMS法简单可行、结果准确,可为提升蔓荆子配方颗粒质量控制方法提供参考。

High Level Expression of Grass Carp Reovirus VP7 Protein in Prokaryotic Cells

Sequences analysis revealed Grass carp reovirus (GCRV) s10 was 909 nucleotides coding a 34 kDa protein denoted as VP7, which was determined to be a viral outer capsid protein (OCP). To obtain expressed OCP in vitro, a full length VP7 gene was produced by RT-PCR amplification, and the amplified fragment was cloned into T7 promoted prokaryotic expression vector pRSET. The recombinant plasmid,which was named as pR/GCRV-VP7,was then transformed into E.coli BL21 host cells. The data indicated that the expressed recombinant was in frame with the N-terminal fusion peptide. The over-expressed fusion protein was produced by inducing with IPTG, and its molecular weight was about 37kDa, which was consistent with its predicted size. In addition, the fusion protein was produced in the form of the inclusion body with their yield remaining steady at more than 60% of total bacterial protein. Moreover,the expressed protein was able to bind immunologically to anti-his-tag monoclonal antibody (mouse) and anti-GCRV serum (rabbit). This work provides a research basis for further structure and function studies of GCRV during entry into cells.

Expression of Outer Capsid Protein VP5 of Grass Carp Reovirus in E.coli and Analysis of its Immunogenicity

Grass carp reovirus (GCRV) is a tentative member of the Aquareovirus genus in the family Reoviridae. The mature virion comprises 11 dsRNA genomes enclosed by two concentric icosahedral proteins shells that is comprised of five core proteins and two outer capsid proteins. The genome sequence and 3D structure demonstrate there is a higher level of sequence homology in structural proteins between GCRV and mammalian orthoreoviruses (MRV) compared to other members of the family. To understand the pathogenesis of GCRV infection, the outer capsid protein VP5, a homology of the μ1 protein of MRV, was expressed in E.coli. It was found that the recombinant VP5 was highly expressed, and the expressed His-tag fusion protein was involved in the formation of the inclusion body. Additionally, specific anti-VP5 serum was prepared from purified protein and western blot demonstrated that the expressed protein was able to bind immunologically to rabbit anti GCRV particle serum and the immunogenicity was determined by ELISA assay. Additional experiments in investigating the functional properties of VP5 will further elucidate the role of the GCRV outer capsid protein VP5 during entry into host cells, and its interaction among viral proteins and host cells during the infection process.

Expression and Identification of Inclusion Forming-related Domain of NS80 Nonstructural Protein of Grass Carp Reovirus

Grass carp reovirus(GCRV),a double stranded RNA virus that infects aquatic animals,often with disastrous effects,belongs to the genus Aquareovirus and family Reoviridea. Similar to other reoviruses,genome replication of GCRV in infected cells occurs in cytoplasmic inclusion bodies,also called viral factories. Sequences analysis revealed the nonstructural protein NS80,encoded by GCRV segment 4,has a high similarity with μNS in MRV(Mammalian orthoreoviruses) ,which may be associated with viral factory formation. To understand the function of the μNS80 protein in virus replication,the initial expression and identification of the immunogenicity of the GCRV NS80 protein inclusion forming-related region(335-742) was investigated in this study. It is shown that the over-expressed fusion protein was produced by inducing with IPTG at 28oC. In addition,serum specific rabbit antibody was obtained by using super purified recombinant NS80(335-742) protein as antigen. Moreover,the expressed protein was able to bind to anti-his-tag monoclonal antibody(mouse) and NS80(335-742) specific rabbit antibody. Further western blot analysis indicates that the antiserum could detect NS80 or NS80C protein expression in GCRV infected cells. This data provides a foundation for further investigation of the role of NS80 in viral inclusion formation and virion assembly.

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.

The first mitochondrial genome for the butterfly family Riodinidae(Abisara fylloides) and its systematic implications

The Riodinidae is one of the lepidopteran butterfly families. This study describes the complete mitochondrial genome of the butterfly species Abisara fylloides, the first mitochondrial genome of the Riodinidae family. The results show that the entire mitochondrial genome of A. fylloides is 15301 bp in length, and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes and a 423 bp A＋T-rich region. The gene content, orientation and order are identical to the majority of other lepidopteran insects. Phylogenetic reconstruction was conducted using the concatenated 13 protein-coding gene （PCG） sequences of 19 available butterfly species covering all the five butterfly families （Papilionidae, Nymphalidae, Peridae, Lycaenidae and Riodinidae）. Both maximum likelihood and Bayesian inference analyses highly supported the monophyly of Lycaenidae＋Riodinidae, which was standing as the sister of Nymphalidae. In addition, we propose that the riodinids be categorized into the family Lycaenidae as a subfamilial taxon.

Tunable wide-angle multi-band mid-infrared linear-to-linear polarization converter based on a graphene metasurface

We present a high-efficiency tunable wide-angle multi-band reflective linear-to-linear(LTL)polarization converter,which is composed of an array of two L-shaped graphene patches with different sizes.In the mid-infrared region,the proposed converter can transform x-polarized wave into y-polarized wave at four resonant frequencies.The polarization conversion ratios of the four bands reach 94.4%,92.7%,99.3%,and 93.1%,respectively.By carefully choosing the geometric parameter,triple-band LTL polarization conversion can also be realized.The three polarization conversion ratios reach 91.50%,99.20%,and 97.22%,respectively.The influence of incident angle on the performances of the LTL polarization converter is investigated,and it is found that our polarization converter shows the angle insensitivity.Also,the dynamically tunable properties of the proposed polarization converter are numerically studied by changing Fermi energy.All the simulation results are conducted by finite element method.

The first mitochondrial genome for the butterfly family Riodinidae(Abisara fylloides)and its systematic implications

The Riodinidae is one of the lepidopteran butterfly families.This study describes the complete mitochondrial genome of the butterfly species Abisara fylloides,the first mitochondrial genome of the Riodinidae family.The results show that the entire mitochondrial genome of A.fylloides is 15301 bp in length,and contains 13 protein-coding genes,2 ribosomal RNA genes,22 transfer RNA genes and a 423 bp A+T-rich region.The gene content,orientation and order are identical to the majority of other lepidopteran insects.Phylogenetic reconstruction was conducted using the concatenated 13 protein-coding gene(PCG)sequences of 19 available butterfly species covering all the five butterfly families(Papilionidae,Nymphalidae,Peridae,Lycaenidae and Riodinidae).Both maximum likelihood and Bayesian inference analyses highly supported the monophyly of Lycaenidae+Riodinidae,which was standing as the sister of Nymphalidae.In addition,we propose that the riodinids be categorized into the family Lycaenidae as a subfamilial taxon.

Visualization of Two-dimensional Single Chains of Hybrid Polyelectrolytes on Solid Surface

The polyacidic character of polyoxometalate(POM)clusters endows high ionic conductivity,making these clusters good candidates for solar and fuel cells.Covalent bonding of clusters to polymer chains creates poly(POM)s that are polyelectrolytes with both cluster functions and polymer performance.Thus,solution-processable poly(POM)s are expected to be used as key materials in advanced devices.Further understanding of poly(POM)s will optimize the preparation process and improve device performance.Herein,we report a study of the first linear poly(POM)s by directly visualizing the chains using scanning transmission electron microscopy.Compared with traditional polymers,individual clusters of poly(POM)s can be directly visualized because of the resistance to electron-beam damage and the high contrast of the tungsten POM pendants.Thus,cluster aggregates with diverse shapes were observed.Counting the number of clusters in the aggregates allowed the degree of polymerization and molecular weight distribution to be determined,and studying the aggregate shapes revealed the presence of a curved semirigid chain in solution.Further study of shape diversity revealed that strong interactions between clusters determine the diverse chain shapes formed during solution processing.Fundamental insight is critical to understanding the formation of poly(POM)films from solutions as key functional materials,especially for fuel and solar cells.

Biodiversity of zooplankton in 0-3000 m waters from the eastern Indian Ocean in spring 2019 based on metabarcoding

To increase knowledge on zooplankton diversity in the deep sea,42 zooplankton samples(depths of 0–3000 m)were taken across five stations throughout the northeastern Indian Ocean using a vertical multi-plankton sampler.According to metabarcoding results of the V4 region of the 18S rRNA gene,Copepoda,Hydrozoa and Malacostraca were present at all stations,while Polychaeta,Scyphozoa,and Thaliacea mainly appeared in the Bay of Bengal.A correlation heatmap showed that the abundance of surface species belonging to Copepoda,Gastropoda,Thaliacea,and Sagittoidea responded most closely to temperature,dissolved oxygen and chlorophyll-a,while the abundance of deep-water species belonging to Thecofilosea,Copepoda,Sagittoidea,and Polychaeta was negatively correlated with temperature,suggesting that temperature differences were driving depth-based variation in zooplankton community distributions in the tropical region.Furthermore,Copepods are important for differentiating depth assemblages in the Indian Ocean.Gaetanus minutus,Lucicutia ovalis,Heterostylites major and Metridia asymmetrica prefer the mesopelagic environment.The vertical distribution of zooplankton diversity showed a bimodal pattern.Generally,communities between 200 and 400 m at stations ZS1,ZS3 and ZS4 differed greatly from those of 0–200 m.However,at stations ZS2 and ZS5,communities between 200 and 400 m had high similarity with those of 0–200 m,possibly due to the influence of upwelling.Findings from this study significantly improve our understanding of zooplankton diversity and ecological functions in the deep sea.