Transcriptome and metabolome analyses reveal novel insights into the seed germination of Michelia chapensis, an endangered species in China.

Michelia chapensis Dandy, a well-known medicinal woody plant endemic to China, is endangered and seriously constricted by seed dormancy-induced low-regeneration in natural conditions. Cold stratification can effectively reduce seed dormancy and promote the seed germination of M. chapensis. However, the molecular events and systematic changes that occurred during seed germination in M. chapensis remain largely unknown. In this study, we carried out transcriptomic and metabolomic analyses to elucidate the potential molecular mechanisms underlying seed germination in M. chapensis under cold stratification. The results showed that the embryo cells became bigger and looser with increasing stratification time. Moreover, the endosperm appeared reduced due to the consumption of nutrients. Seventeen phytohormones were examined by the metabolome targeted for hormones. Compared with the ES (no stratification), the levels of indole-3-acetic acid (IAA) and gibberellin A3 (GA3) were increased in the MS (stratification for 45 days), while the abscisic acid (ABA) was downregulated in both MS and LS (stratification for 90 days). The transcriptome profiling identified 24975 differentially expressed genes (DEGs) in the seeds during germination. The seed germination of M. chapensis was mainly regulated by the biological pathways of plant hormone signal transduction, energy supply, secondary metabolite biosynthesis, photosynthesis-related metabolism, and transcriptional regulation. This study reveals the biological evidence of seed germination at the transcriptional level and provides a foundation for unraveling molecular mechanisms regulating the seed germination of M. chapensis.

Anticancer and antibacterial flavonoids from the callus of Ampelopsis grossedentata; a new weapon to mitigate the proliferation of cancer cells and bacteria.

A new flavonoid angelioue (1) together with five known compounds cuminatanol (2), myricetin (3), epigallocatechin (4), taxifolin (5) and dihydromyricetin (6) was isolated from the callus extract of Ampelopsis grossedentata (Hand.-Mazz.) W. T. Wang and the structures were elucidated based on their detailed spectroscopic data. Among the compounds, the new compound angelioue (1) displayed significant antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) with the MIC value of 6.68 µg mL-1 and MBC value of 53.42 µg mL-1; in contrast the other compounds showed moderate to no antibacterial activity. In addition, known dihydromyricetin (6) exhibited potent cytotoxic activities against mouse breast cancer cells (4T1), human lung adenocarcinoma (A549) and human non-small cell lung cancer (NCI-H1975) tumor cell lines with GI50 values of 17.47, 18.91 and 20.50 µM mL-1, respectively. The compounds 1-5 exhibited low micro-molar inhibitory activities. Moreover, the structure-activity relationships of the most active compounds for antibacterial and cytotoxic activities are discussed. The present findings clearly suggest that the A. grossedentata callus is a good source of bioactive compounds.

Transcriptome analysis provides novel insights into the soil amendments induced response in continuously cropped Codonopsis tangshen.

Codonopsis tangshen Oliv (C. tangshen) is an important Chinese traditional medicinal plant with various health benefits. However, the growth of C. tangshen are seriously affected by continuous cropping, which led to the decrease of the yield and quality. A field experiment was conducted to learn the effects of soil amendments on the growth of C. tangshen under continuous cropping condition, and the biological events which occurred at molecular level were investigated. The results indicated that the content of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoid (Car) was significantly higher in SCPM (silicon-calcium-potassium-magnesium fertilizer), SCPMA (SCPM combined with azoxystrobin) and SCPMAOM (SCPM combined with azoxystrobin and organic manure) treatments. Moreover, the yield and the levels of alkaloid, polysaccharide, flavone and total protein in the treatments of SCPM, SCPMA and SCPMAOM were significantly higher than those in the control, and these indexes were all highest in the SCPMAOM treatment. RNA-sequencing (RNA-Seq) is an economical and efficient method to obtain genetic information for species with or without available genome data. In this study, RNA-Seq was performed to understand how continuously cropped C. tangshen responded to the soil amendments at the transcriptome level. The number of differentially expressed genes (DEGs) were as follows: CK vs. SCPM (719 up- and 1456 down-), CK vs. SCPMA (1302 up- and 1748 down-), CK vs. SCPMAOM (1274 up- and 1678 down-). The soil amendments affected the growth of C. tangshen mainly by regulating the genes involved in pathways of 'photosynthesis,' 'plant hormone signal transduction,' 'biosynthesis of unsaturated fatty acids,' 'phenylpropanoid biosynthesis,' and 'starch and sucrose metabolism,' etc. qRT-PCR was performed to validate the expressions of 10 target genes such as CP26, PsaF, and POX, etc., which verified the reliability of RNA-Seq results. Overall, this study revealed the roles and underlying mechanisms of the soil amendments in regulating the growth of continuously cropped C. tangshen at transcriptome level. These findings are beneficial for improving the continuous cropping tolerance and may be valuable for future genetic improvement of C. tangshen.

Diversity and chemical fingerprinting of endo-metabolomes from endophytes associated with Ampelopsis grossedentata (Hand.-Mazz.) W. T. Wang possessing antibacterial activity against multidrug resistant bacterial pathogens.

BACKGROUND: Serious infections caused by bacteria and their resistance to antibiotics are one of the biggest healthcare threats to mankind. Therefore, the present study aimed to isolate endophytes from medicinal plant Ampelopsis grossedentata, an endemic species of Western Hubei, China and to investigate its antibacterial efficacy and chemical diversity of the secondary metabolites. METHODS: The antibacterial potential of the endophytes was evaluated by disc diffusion method against a panel of eleven type strains and some multidrug resistant pathogenic bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were estimated by broth microdilution using iodonitrotetrazolium chloride assay. Further, the chemical diversity of the metabolites was estimated using LC-Q-TOF-MS/MS and GC-MS fingerprinting. RESULTS: Four endophytic fungi were isolated from the tender shoot of A. grossedentata; they were identified as Fusarium graminearum TC-1, Phomopsis mali TC-3, Pestalotiopsis maculans TC-5 and Alternaria alternata TC-11. Among the endophytes screened, A. alternata TC-11 exhibited significant antibacterial activity with the zones of inhibition ranging from 13.72 ± 0.30 to 21.76 ± 0.53 mm against all the tested type strains and multidrug resistant bacterial pathogens. Further, it showed significant antibacterial activity with MIC values ranging from 0.37 to 3.00 µg/mL. The combined LC-Q-TOF-MS/MS and GC-MS analyses of active extract revealed that alternarian acid, altertenuol, dimethyl sulfone, docosane, dodecane, duclauxin, ergosta-4,6,8(14),22-tetraen-3-one, ethyl 6-cyano-5-oxo-1-phenyl-7-thiophen-2-yl-[1,2,4]triazolo[4,3-a]pyrimidine-3-carboxylate, heptacosane, linoleic acid, neodecanoic acid, oxiranylmethyl ester, pentadecane, verrulactone E, 2,6,11-Trimethyldodecane and 4-[(E,4R,6R)-11-(furan-3-yl)-6-hydroxy-4,8-dimethylundec-8-enyl]-2-hydroxy-2H-furan-5-one were the most abundant compounds present which were responsible for the significant antibacterial activity. CONCLUSIONS: To our knowledge, this is the first report of fungal endophytes isolated from the tender shoot of A. grossedentata with bacteriostatic and bactericidal activities. Our finding provides a new insight into the antibacterial potential of endophytes and envisages the possibility of using them for drug discovery.

Evaluation and comparison of three assays for molecular detection of spinal muscular atrophy.

BACKGROUND: Spinal muscular atrophy (SMA) is mainly caused by deletions in SMA-related genes. The objective of this study was to develop gene-dosage assays for diagnosing SMA. METHODS: A multiplex, quantitative PCR assay and a CNVplex assay were developed for determining the copy number of SMN1, SMN2, and NAIP. Reproducibility and specificity of the two assays were compared to a multiple ligation-dependent probe amplification (MLPA) assay. To evaluate reproducibility, 30 samples were analyzed three times using the three assays. A total of 317 samples were used to assess the specificity of the two assays. RESULTS: The multiplex quantitative PCR (qPCR) assay had higher reproducibility. Intra-assay CVs were 3.01%-8.52% and inter-assay CVs were 4.12%-6.24%. The CNVplex assay had ratios that were closer to expected (0.49-0.5 for one copy, 1.03-1.0 for two copies, and 1.50-1.50 for three copies). Diagnostic accuracy rates for the two assays were 100%. CONCLUSIONS: The multiplex qPCR assay was a simple, rapid, and cost-effective method for routine SMA diagnosis and carrier screening. The CNVplex assay could be used to detect SMAs with complicated gene structures. The assays were reliable and could be used as alternative methods for clinical diagnosis of SMA.

[Comparison of Bayesian interim analysis and classical interim analysis in group sequential design].

OBJECTIVE: To explore the differences between the Bayesian interim analysis and the classical interim analysis. METHODS: To compare the means of two independent samples between control and treatment, superior hypothesis test was established. In line with the data requirements for group sequential design, Type Iota error of Bayesian interim analysis based on various prior distributions, Power, Average Sample Size and Average Stage were estimated in the interim analysis. RESULTS: In the Pocock and O' Brien & Fleming designs, the Type Iota errors in the Bayesian interim analysis based on the skeptical prior distribution and the handicap prior distribution were controlled at around 0.05. When the powers of these two classical designs were both 80%, Bayesian powers of the skeptical prior distribution and the handicap prior distribution were markedly lower. The powers of the non-informative prior distribution and the enthusiastic prior distribution were distinctly higher than 80%. CONCLUSION: In the Bayesian interim analysis based on the skeptical prior distribution and the handicap Prior distribution, the Type Iota errors can be well controlled. Bayesian interim analyses using these two prior distributions, compared with the analysis adopting the O' Brien & Fleming method, can markedly increase the possibility of ending the clinical trials ahead of time. The Bayesian interim analyses based on these two distributions do not have practical value for group sequential design of the Pocock method.

Perceived empathy of service providers mediates the association between perceived discrimination and behavioral intention to take up HIV antibody testing again among men who have sex with men.

HIV antibody testing is a key measure of HIV prevention for men who have sex with men (MSM). The World Health Organization recommends sexually active and at-risk MSM to take up HIV antibody testing regularly. This study aimed to investigate the prevalence of behavioral intention to take up HIV antibody testing in the next six months among Hong Kong MSM who were ever-testers. An anonymous cross-sectional survey recruited 326 MSM who had taken up HIV antibody testing from gay-friendly venues and internet in Hong Kong. Of the participants, 40.8% had had unprotected anal intercourse with regular or non-regular male sex partners in the last six months; they were at risk of HIV transmission despite experience in HIV antibody testing. Only 37.2% showed a strong intention to take up HIV antibody testing again in the next six months. Adjusted analysis showed that both perceived discrimination toward Hong Kong MSM (AOR = .60, 95% CI: .36-.98) and the CARE Measure assessing perceived empathy of service providers (AOR = 1.05, 95% CI: 1.02-1.08) were significantly associated with intention for retesting. Perceived discrimination, however, became statistically non-significant (AOR = .68, 95% CI: .41-1.14), when both CARE Measure and perceived discrimination entered into the adjusted model. It is warranted to increase HIV retesting rate by removing perceived discrimination and reducing the negative effect of perceived discrimination through enhancement of empathy of service providers.

Impact of the CYP3A5, CYP3A4, COMT, IL-10 and POR genetic polymorphisms on tacrolimus metabolism in Chinese renal transplant recipients.

Tacrolimus is a widely used immunosuppressive drug for preventing the rejection of solid organ transplants. The efficacy of tacrolimus shows considerable variability, which might be related to genetic variation among recipients. We conducted a retrospective study of 240 Chinese renal transplant recipients receiving tacrolimus as immunosuppressive drug. The retrospective data of all patients were collected for 40 days after transplantation. Seventeen SNPs of CYP3A5, CYP3A4, COMT, IL-10 and POR were identified by the SNaPshot assay. Tacrolimus blood concentrations were obtained on days 1-3, days 6-8 and days 12-14 after transplantation, as well as during the period of the predefined therapeutic concentration range. Kruskal-Wallis test was used to examine the effect of genetic variation on the tacrolimus concentration/dose ratio (C 0/D) at different time points. Chi-square test was used to compare the proportions of patients who achieved the target C 0 range in the different genotypic groups at weeks 1, 2, 3 and 4 after transplantation. After correction for multiple testing, there was a significant association of C 0/D with CYP3A5*3, CYP3A4*1G and CYP3A4 rs4646437 T>C at different time points after transplantation. The proportion of patients in the IL-10 rs1800871-TT group who achieved the target C 0 range was greater (p = 0.004) compared to the IL-10 rs1800871-CT and IL-10 rs1800871-CC groups at week 3 after transplantation. CYP3A5*3, CYP3A4 *1G, CYP3A4 rs4646437 T>C and IL-10 rs1800871 C>T might be potential polymorphisms affecting the interindividual variability in tacrolimus metabolism among Chinese renal transplant recipients.

Bayesian estimation of the effective reproduction number for pandemic influenza A H1N1 in Guangdong Province, China.

PURPOSE: During the course of a pandemic, it is necessary to understand its transmissibility, which is often summarized by the effective reproduction number. Accurate estimation of the effective reproduction number (R) is of vital significance in real-time decision making for coping with pandemic influenza. METHODS: We used daily case notification data in Guangdong Province, China, in conjunction with Bayesian inference of two different stochastic susceptible, infectious, recovered (SIR) models to estimate the effective reproduction number. The duration of infectiousness was taken from published literature, and the proportion of imported cases was obtained from individual-level data. RESULTS: At the initial epidemic phase, 40% of the first 261 cases were not locally acquired. Explicitly accounting for imported cases and different infectious periods, the possible range of basic reproduction number was preliminarily estimated to be between 1.05 and 1.46. We showed how the daily case reports provided valuable information to estimate the effective reproduction number. We also found the potential delay in reporting had a relatively minor impact on estimating R. CONCLUSIONS: Our proposed models and findings provide a relevant contribution towards establishing a basis for monitoring the evolution of emerging infectious diseases in real time and understanding the characteristics of pandemic influenza A H1N1 in Guangdong Province.

Modeling the initial transmission dynamics of influenza A H1N1 in Guangdong Province, China.

BACKGROUND: The novel influenza A H1N1 (2009) virus, identified in mid-2009, spread rapidly in Guangdong Province. The accurate estimation of epidemiological parameters is of vital significance in decision-making for coping with pandemic influenza. METHODS: We used influenza A H1N1 epidemic data from local cases in Guangdong Province, China, in conjunction with a complex SEIR model (susceptible, exposed, infectious, recovered) to estimate the basic reproduction number. The transmission rate was obtained by fitting the model to the cumulative number of local daily infected cases using the nonlinear ordinary least squares method. The latent period and duration of infectiousness were obtained from the published literature, and the proportion of symptomatic infected cases was obtained from the serological survey conducted by the Center for Disease Control and Prevention of Guangdong Province. We determined the variance of model parameters via a simulation study. RESULTS: The model was in keeping with the observed epidemic data (coefficient of determination=0.982). The basic reproduction number was estimated preliminarily to be R0=1.525 (95% confidence interval 1.448-1.602), with the possible range of true R0 being 1.30-1.85. We estimated the transmission rate ß to be between 0.390 and 0.432. CONCLUSIONS: With the help of the serological survey, useful estimates of key epidemiological parameters for the influenza A H1N1 outbreak in Guangdong Province were obtained. The sensitivity analysis suggests that different latent periods and infectious periods, which specify different mean durations of generation time, have a significant impact on R0. Our proposed model and findings provide a relevant contribution towards understanding the characteristics of influenza A H1N1 in Guangdong Province.

[Analysis of causes of death and potential years of life lost in 7 districts of Guangzhou].

OBJECTIVE: To analyze the causes of death and potential years of life lost (PYLL) in 7 districts of Guangzhou from 2003 to 2005. METHODS: The bureau of Health of Guangzhou provided the data of 70 201 cases of death occurring between 2003 and 2005. The top 10 causes of death were analyzed and the PYLL and crude death rate were calculated using mortality difference dissembling method. RESULTS: Between 2003 and 2005, the top 3 causes of death in Guangzhou were diseases of the circulatory system, neoplasms and respiratory diseases. Aging of the population led to an increased death rate. The major causes of death affecting PYLL consisted of neoplasms, diseases of the circulatory system, injuries and poisoning. CONCLUSION: Priority should be given to the control of diseases of the circulatory system in the health care in Guangzhou. Neoplasms, injuries and poisoning all contribute to a high PYLL.