Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

Dengue ; epidemiology ; Dengue Virus ; genetics ; isolation & purification ; Evolution, Molecular ; Genes, Viral ; Humans ; Viral Envelope Proteins ; genetics

Ecological methodology plus negative binomial regression were used to identify dengue fever (DF) epidemiological status and its relationship with meteorological variables. From 2007 to 2012, annual incidence rate of DF in Guangzhou was 0.33, 0.11, 0.15, 0.64, 0.45, and 1.34 (per 100 00) respectively, showing an increasing trend. Each 1° C rise of temperature corresponded to an increase of 10.23% (95% Cl 7.68% to 12.83%) in the monthly number of DF cases, whereas 1 hPa rise of atmospheric pressure corresponded to a decrease in the number of cases by 5.14% (95% Cl: 7.10%-3.14%). Likewise, each one meter per second rise in wind velocity led to an increase by 43.80% or 107.53%, and one percent rise of relative humidity led to an increase by 2.04% or 2.19%.
Adult ; China ; epidemiology ; Dengue ; epidemiology ; Humans ; Weather ; Young Adult

Objective To analyze and trace the infection source the envelope(E) gene of the new emerged type 3 dengue virus in Guangzhou in 2009. Methods Sera were collected from patients infected with local dengue fever. Dengue virus was cultured and isolated by C6/36 cells. The whole length E gene was amplified from the positive specimen by RT-PCR, thereby sequenced and phylogenetic tree drawn by neighbor-joining method. Both data on epidemiologic and molecular studies were processed and analysed. Results 7 strains of type 3 dengue virus were isolated from samples of the 19 patients. E gene of these strains was amplified. The complete E genes of 7 strains belonged to 1479 nucleotides in length, encoding a polyprotein of 493 amino acids. Data from the phylogenetic analysis showed that 09/GZ/1081, 09/GZ/1483 and 09/GZ/10806 strains fell within the Southeast Asia/South Pacific group. 09/GZ/10616, 09/GZ/11144, 09/GZ/11194 while 09/GZ/13105 strains fell within the India group. Conclusion The type 3 dengue virus identified in Guangzhou area in 2009 was imported and could be devided into two genotypes.

Objective To investigate the epidemiological characteristics of Dengue and the E gene of the new isolated strains.Methods Epidemiological data and serum samples were collected.Serotypes were detected by real-time PCR and virus was isolated in C6/36.E gene of the new isolated strains were sequenced and analyzed by Mega 4.0.Results The cases of Dengue reached at the peak during September and November,with Serotype 1,2 and 4 were involved.Five strains of serotype 1 were isolated,with 4 of them fell into the clad of Asia genotype,and 1 belonged to America/Africa genotype.Conclusion The strains isolated in Guangzhou showed a high identity to the Southeast Asian strains.There seemed high risk of outbreak of Dengue in this area,However,the Dengue virus might have already been localized.

Transgenic Atropa belladonna with high levels of scopolamine was developed by metabolic engineering. A functional gene involved in the rate limiting enzyme of h6h involved in the biosynthetic pathway of scopolamine was over expressed in A. belladonna via Agrobacterium-mediation. The transgenic plants were culturing till fruiting through micropropogating and acclimating. The integration of the h6h genes into the genomic DNA of transgenic plants were confirmed by genomic polymerase chain reaction (PCR) analysis. Analysis of the difference of plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight was carried out using SPSS software. The content of hyoscyamine and scopolamine in roots, stems, leaves and fruits was determined by HPLC. The investigation of the expression levels of Hnh6h by qPCR. Both Kan(r) and Hnh6h genes were detected in five transgenic lines of A. belladonna plants (A8, A11, A12, C8 and C19), but were not detected in the controls. The plant height, crown width, stem diameter, leaf length, leaf width, branch number and fresh weight of transgenic plants did not decrease by comparison with the non-transgenic ones, and furthermore some agronomic characters of transgenic plants were better than those of the controls. The highest level of scopolamine was found in leaves of transgenic A. belladonna, and the content of scopolamine was also higher than that of hyoscyamine in leaves. The contents of scopolamine of leaves in different transgenic lines were listed in order: C8 > A12 > C19 > A11 > A8, especially, the content of scopolamine in transgenic line C8 was 2.17 mg x g(-1) DW that was 4.2 folds of the non-transgenic ones (0.42 mg x g(-1) DW). The expression of transgenic Hnh6h was detected in all the transgenic plants but not in the control. The highest level of Hnh6h expression was found in transgenic leaves. Overexpression of Hnh6h is able to break the rate limiting steps involved in the downstream pathway of scopolamine biosynthesis, and thus promotes the metabolic flux flowing toward biosynthesis of scopolamine to improve the capacity of scopolamine biosynthesis in transgenic plants. As a result, transgenic plants of A. belladonna with higher level of scopolamine were developed.
Atropa belladonna ; genetics ; metabolism ; Atropine ; metabolism ; Gene Expression ; Mixed Function Oxygenases ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Plants, Genetically Modified ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Solanaceae ; enzymology ; genetics

AIM To analyze the constituents absorbed into blood and brain from Zhishe Tongluo Capsules.METHODS Sixteen rats were randomly assigned into four groups and given intragastric administration(3.1 g/kg),after which the cerebral ischemia-reperfusion injury(MACO)model was established,the blood and brain tissues were collected,and UHPLC-Q Exactive Focus MS/MS was adopted in the identification of prototype constituents.RESULTS Total 70 constituents were identified,20 of which were found in the blood,mainly including flavonoids,tanshinones and Ligusticum chuanxiong phthalides,and 7 of them could enter the brain through blood-brain barrier.Compared with the normal administration group,the MACO administration group demonstrated added constituents absorbed into blood containing 3-hydroxybenzoic acid,calycosin-7-glucoside,curcumenol,senkyunolide B,dihydrotanshinone I and cryptotanshinone;removed constituents absorbed into brain containing puerarin,elemicin,sedanolide,and added those containing salvianolic acid A,senkyunolide I,dihydrotanshinone I in the left brain tissues(infarcted side).CONCLUSION The constituents absorbed into blood and brain from Zhishe Tongluo Capsules,along with the enhanced absorptions of phthalides,quinones and phenols in MACO rats in vivo may be the active substances for treating cerebral infarction.

Objective We conducted an epidemiologic investigation to determine the source of infection on an avian influenza (H5N1) case who returned from Guangzhou,in Hong Kong.Methods Data related to epidemiologic investigation,medical observation on close contacts,Syndromic Surveillance on poultry salesmen,emergency monitoring,detection of the samples,source tracing on potential Avian influenza virus (H5,H7,H9) infected people,situation on environment pollution by avian influenza virus in the markets etc.were gathered.The determination of infection source was through comparing the different genes between the case and positive environmental samples.Results The infected case witnessed the procedure of how a live duck was killed,in market A in Guangzhou during May 17th to 19th.The case was diagnosed as respiratory tract infection in 2 Third-grade-Class A hospitals in Guangzhou on May 23th and 24th.The diagnosis was made as Avian influenza cases on May 26th after going back to Hong Kong.23 close contacts and 34 markets poultry salesmen did not show any ILI related symptoms.However,2 poultry salesmen from the markets nearby the place where the Avian influenza case stayed,were detected having positive H9 avian influenza antibody,with the H9 positive rate as 6.06％ (2/33).Among the environmental samples in the 2 markets nearby home of the patient,chopping block was found to have carried H5,with positive rate as 9.8％(5/51) while poultry cage was found to carry H9,with the positive rate as 2.0％(1/51).A H5 positive sample was found with clade 2.3.2.1,same to the case,from a chopping block at the market B where the sources of poultry was the same as market A.Conclusion The source of infection seemed to come from the markets in Guangzhou,that calling for the strengthening of poultry market management,for avian influenza prevention.History related to contact of poultry should be gathered when a diagnosis of respiratory tract infection was made.Timely sampling and testing should be made to improve the sensitivity of diagnosis.

OBJECTIVETo study the relation of the detection rates of the novel influenza virus A/H1N1 RNA in clinically confirmed patients in the 2009 pandemic with the age distribution of the patients and the disease course.

METHODSA total of 151 clinical patients with H1N1 infection were enrolled in this study, from whom 833 dynamic throat swab samples were obtained for detecting the H1N1 RNA using real-time PCR. A statistical analysis of the age distribution was performed among the patients with different disease courses. Chi-square for trend test was used to study the correlation between the detection rates of H1N1 RNA and the time of disease onset.

RESULTSThe majority of patients were young with their ages ranging from 10 to 20 years (57.26%) and 20 to 30 years (22.18%). Chi-square for trend test revealed that the positivity rates of the throat swabs in the patients decreased with the prolongation of the disease course (chi(2)=9.784, P=0.002).

CONCLUSIONMost of the H1N1 patients are young within the age range of 10-30 years, and the longest disease course can exceed 10 days. The positivity rates of throat swabs from the H1N1 patients decreases with the prolongation of the disease course.

Adolescent ; Adult ; Age Factors ; Child ; China ; epidemiology ; Female ; Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; Influenza, Human ; epidemiology ; virology ; Male ; Pharynx ; virology ; RNA, Viral ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Young Adult

Synthetic biology of natural products is the design and construction of new biological systems by transferring a metabolic pathway of interest products into a chassis. Large-scale production of natural products is achieved by coordinate expression of multiple genes involved in genetic pathway of desired products. Promoters are cis-elements and play important roles in the balance of the metabolic pathways controlled by multiple genes by regulating gene expression. A detection plasmid of Saccharomyces cerevisiae was constructed based on DsRed-Monomer gene encoding for a red fluorescent protein. This plasmid was used for screening the efficient promoters applying for multiple gene-controlled pathways. First of all, eight pairs of primers specific to DsRed-Monomer gene were synthesized. The rapid cloning of DsRed-Monomer gene was performed based on step-by-step extension of a short region of the gene through a series of PCR reactions. All cloned sequences were confirmed by DNA sequencing. A vector named pEASYDs-M containing full-length DsRed-Monomer gene was constructed and was used as the template for the construction of S. cerevisiae expression vector named for pYeDP60-Ds-M. pYeDP60-Ds-M was then transformed into S. cerevisiae for heterologous expression of DsRed-Monomer gene. SDS-PAGE, Western blot and fluorescence microscopy results showed that the recombinant DsRed-Monomer protein was expressed successfully in S. cerevisiae. The well-characterized DsRed-Monomer gene was then cloned into a yeast expression vector pGBT9 to obtain a promoter detection plasmid pGBT9Red. For determination efficacy of pGBT9Red, six promoters (including four inducible promoters and two constitutive promoters) were cloned by PCR from the S. cerevisiae genome, and cloned into pGBT9Red by placing upstream of DsRed-Monomer gene, separately. The fluorescence microscopy results indicated that the six promoters (GAL1, GAL2, GAL7, GAL10, TEF2 and PGK1) can regulate the expression of DsRed-Monomer gene. The successful construction of pGBT9Red lays the foundation for further analysis of promoter activity and screening of promoter element libraries.
Amino Acid Sequence ; Base Sequence ; genetics ; Cloning, Molecular ; DNA Primers ; biosynthesis ; Gene Expression Regulation, Fungal ; Genetic Vectors ; Luminescent Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Promoter Regions, Genetic ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Synthetic Biology ; Transformation, Genetic