A simple and sensitive Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) method was established to provide a new alternative for clinical diagnosis of Avian influenza A H5N1 virus. The method employed a set of six specially designed primers that recognized eight distinct sequences of the target for amplification of nucleic acid under isothermal conditions. In current study, fifty-one experimentally infected animal specimens and viral cultures that had been tested were analyzed by RT-LAMP for NA gene and HA gene, respectively. The amplification process of LAMP was monitored in real-time by the addition of SYBR Green dye. Meanwhile, the result showed high correlation between nested PCR and RT-LAMP. The specificity of the RT-LAMP assay was confirmed by restriction enzyme digestion analysis and sequencing of the amplified product. When the sensitivity of this assay was tested by serial 10-fold dilutions of RNA molecules from specimens, it was found that the RT-LAMP method achieved theoretically a sensitivity of 10 RNA molecules. Thus, we concluded that the RT-LAMP assay has potential usefulness for rapid detection of the Avian influenza A H5N1 virus.
Animals ; Birds ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; Influenza in Birds ; diagnosis ; virology ; Nucleic Acid Amplification Techniques ; methods ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

AIM To study the chemical constituents from Gentianella acuta (Michx.) Hulten.METHODS The 30％ and 90％ ethanol fractions of 70％ ethanol extract from G.acuta were isolated and purified by silica,ODS and preparative HPLC column,then the structures of obtained compounds were identified by spectral data.RESULTS Nine compounds were isolated and identified as sinenoside Ⅰ (1),(+)-lariciresinol-4,4'-0-bis-β-D-glucopyranoside (2),(+)-8-hydroxylariciresinol-4'-O-β-D-glucopyranoside (3),(+)-lariciresinol-4-O-3-D-glucopyranoside (4),(7S,8R)-erythro-7,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-4-O-β-D-glucopyranoside (5),(7S,8R)-erythro-4,9,9'-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-7-O-β-D-glucopyranoside (6),(7S,8R)-erythro-4,7,9-trihydroxy-3,3'-dimethoxy-8-O-4'-neolignan-9'-O-β-D-glucopyranoside (7),balanophonin (8),urolignoside (9).CONCLUSION Compounds 2-9 are isolated from genus Gentianella for the first time.

OBJECTIVEStudying on the routes of vas deferens to provide anatomy basis for surgical operation, especially, reconstruction of long segment loss of vas deferens.

METHODSThe routes of vas deferens were observed and anatomic distances along epididymal, infrainguinal, inguinal, retroperitoneal and ampullar segments of vas deferens, the distances from external ring to extremity of vas deferens were measured respectively in 18 formalin fixed adult cadavers.

RESULTSThe vas deferens have a large curve from external ring to extremity in its route, draw it out from the external ring. Eliminating this curve will allow to shorten this segment of vas deferens for vasovasostomy by 6.1 - 12.9 (9.31 +/- 1.30) cm. The length of each segment of vas deferens, respectively, is epididymal: 3.2 - 5.6 (4.53 +/- 0.79) cm, infrainguinal: 4.5 - 9.5 (7.31 +/- 1.78) cm, inguinal: 4.4 - 7.5 (5.52 +/- 0.74) cm, retroperitoneal: 12.5 - 19.5 (16.75 +/- 1.87) cm and ampullar: 2.9 - 3.8 (3.63 +/- 0.23) cm. There was no significant differences in segment length and the distances from external ring to extremity of vas deferens between the right and left.

CONCLUSIONReconstruction of long segment loss of vas deferens can be performed by mobilization retroperitoneal vas deferens and draw it out from external ring. There were no significant differences in lengths of vas deferens and the distances from external ring to vassal extremity between the left and right in adults. The surgical operations of vas deferens are closely related each segment of vasa.

Taxol, a kind of terpenoid secondary metabolite produced by Taxus brevifolia, is an effective anticancer drug that manufacture relies mainly on the extraction form plants. In order to solve the resource shortage, a lot of work has been done to develop the alternative method. Recently, using synthetic biology to realize heterologous biosynthesis of the precursors of taxol has become a hotspot. Now, the basic framework of taxol biosynthetic pathways has been confirmed, and most enzyme genes involved in taxol biosynthesis have been cloned and identified. The two taxol precursors, taxa-4(5),11(12)-diene and taxa-4(20),11(12)-dien-5α-ol, have been synthesized in Escherichia coli and Saccharomyces cerevisiae. Here this paper reviewed the recent advances in the biosynthetic pathway of taxol and the latest developments of synthetic biology, which aims to provide a guidance for the heterologous biosynthesis of taxol.

There are many valuable medicinal plants in Ginseng genus belonging to Araliaceae. Among them, Panax ginseng, P. quinquefolium and P. notoginseng are the most famous species. With the development of next-generation sequencing (NGS) technologies, sequencing and analysis of transcriptomes have become powerful tools for discovery of novel genes, screening molecular markers and elucidation of specific biosynthetic pathway of secondary metabolites. Their transcriptomes provided abundant genes for further study on functional genomics. Here this paper summarized the recent advances in the transcriptomic studies of these three medicinal plants, including discovery of novel genes and elucidation of metabolic regulation, which will contribute to functional genomics in ginseng species.

Elucidation of the biosynthetic pathways of natural products is not only the major goal of herb genomics, but also the solid foundation of synthetic biology of natural products. Here, this paper reviewed recent advance in this field and put forward strategies to elucidate the biosynthetic pathway of natural products. Firstly, a proposed biosynthetic pathway should be set up based on well-known knowledge about chemical reactions and information on the identified compounds, as well as studies with isotope tracer. Secondly, candidate genes possibly involved in the biosynthetic pathway were screened out by co-expression analysis and/or gene cluster mining. Lastly, all the candidate genes were heterologously expressed in the host and then the enzyme involved in the biosynthetic pathway was characterized by activity assay. Sometimes, the function of the enzyme in the original plant could be further studied by RNAi or VIGS technology. Understanding the biosynthetic pathways of natural products will contribute to supply of new leading compounds by synthetic biology and provide "functional marker" for herbal molecular breeding, thus but boosting the development of traditional Chinese medicine agriculture.

Understanding of codon usage bias of Fritillaria cirrhosa can provide theoretical basis for heterologous biosynthesis of F. cirrhosa alkaloids by genetic engineering technology. A total of 9 843 full length coding sequences (CDS) from the F. cirrhosa transcriptome data were used for the analysis of codon usage bias. The GC and GC3s contents, effective number of codons(ENC) and relative synonymous codon usage (RSCU) were calculated using the CodonW software. The results show that the codon usage bias value is low in the CDS of F. cirrhosa. A total of 15 codons, including UUG, CUU, AUU, GUU, UCA, CCU, CCA, ACU, ACA, GCA, UAU, CAU, AAU, AGA and GGA, were identified as optimal codons in F. cirrhosa. The optimal codons generally end with A/T at the third codon position. By the transcriptome annotation, we found 26 CDSs possibly involved in the biosynthesis of alkaloids in the F. cirrhosa. The proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae are low in these CDSs. We also proposed a method for the codonoptimization in these target genes. Our work lays the foundation for further study on the biosynthesis of alkaloids of the F. cirrhosa in heterologous species.

This study aimed to provide guidance for the heterogenous gene expression, gene prediction and species evolution by analyzing codon usage bias of Catharanthus roseus.The codon composition and usage bias of 30 437 high-confidence coding sequences from C.roseus were analyzed and the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 25 genes involved in the biosynthesis of terpenoid indole alkaloids (TIAs) in C.roseus were calculated.The results showed that the average GC content of the genes was 42.47%; the average GC content of the third bases in codon was 35.89%.The relative synonymous codon usage (RSCU) of 28 codons were greater than 1 and 26 of them ended with A or T.The above 25 genes involved in TIA biosynthesis contained much more rare condons of E.coli than that of S.cerevisiae.It was concluded that C.roseus mainly prefered the codons ending with A or T and the rule of codon usage was more different to E.coli than S.cerevisiae.Thus, S.cerevisiae may be more suitable host for heterologous expression of these genes.

Catharanthus roseus can produce a variety of terpenoid indole alkaloids (TIA), most of which exhibit strong pharmacological activities. Hence, biosynthesis and regulation of TIA have received recent attention. 3α (S)-strictosidine is an important node in TIA biosynthesis, which is a condensation product of secologanin and tryptamine. The former is produced in iridoid pathway, and the latter is produced in indole pathway. Vindoline and catharanthine, which are produced respectively by 3α (S)-strictosidine via multi-step enzymatic reaction, can form α-3, 4-anhydrovinblastine by the condensation reaction. Then, vinblastine and vincristine are generated from α-3, 4-anhydrovinblastine. Many transcription factors are involved in the regulation of TIA synthesis, such as AP2/ERF and WRKY. Illumination of biosynthetic pathway has laid a foundation for the study of synthetic biology. Today, 3α (S)-strictosidine and vindoline have been synthesized in heterologous hosts Saccharomyces cerevisiae.Research about synthetic biology and the regulation mechanisms will provide a guidance for the production and development of TIA drugs in C. roseus.

Flavonoids are the valuable components in medicinal plants, which possess a variety of pharmacological activities, including anti-tumor, antioxidant and anti-inflammatory activities. There is an unambiguous understanding about flavonoids biosynthetic pathway, that is,2S-flavanones including naringenin and pinocembrin are the skeleton of other flavonoids and they can transform to other flavonoids through branched metabolic pathway. Elucidation of the flavonoids biosynthetic pathway lays a solid foundation for their synthetic biology. A few flavonoids have been produced in Escherichia coli or yeast with synthetic biological technologies, such as naringenin, pinocembrin and fisetin. Synthetic biology will provide a new way to get valuable flavonoids and promote the research and development of flavonoid drugs and health products, making flavonoids play more important roles in human diet and health.