Full-Length Transcriptome Sequencing and Modular Organization Analysis of the Naringin/Neoeriocitrin-Related Gene Expression Pattern in Drynaria roosii.

Drynaria roosii (Nakaike) is a traditional Chinese medicinal fern, known as 'GuSuiBu'. The effective components, naringin and neoeriocitrin, share a highly similar chemical structure and medicinal function. Our HPLC-tandem mass spectrometry (MS/MS) results showed that the accumulation of naringin/neoeriocitrin depended on specific tissues or ages. However, little was known about the expression patterns of naringin/neoeriocitrin-related genes involved in their regulatory pathways. Due to a lack of basic genetic information, we applied a combination of single molecule real-time (SMRT) sequencing and second-generation sequencing (SGS) to generate the complete and full-length transcriptome of D. roosii. According to the SGS data, the differentially expressed gene (DEG)-based heat map analysis revealed that naringin/neoeriocitrin-related gene expression exhibited obvious tissue- and time-specific transcriptomic differences. Using the systems biology method of modular organization analysis, we clustered 16,472 DEGs into 17 gene modules and studied the relationships between modules and tissue/time point samples, as well as modules and naringin/neoeriocitrin contents. We found that naringin/neoeriocitrin-related DEGs distributed in nine distinct modules, and DEGs in these modules showed significantly different patterns of transcript abundance to be linked to specific tissues or ages. Moreover, weighted gene co-expression network analysis (WGCNA) results further identified that PAL, 4CL and C4H, and C3H and HCT acted as the major hub genes involved in naringin and neoeriocitrin synthesis, respectively, and exhibited high co-expression with MYB- and basic helix-leucine-helix (bHLH)-regulated genes. In this work, modular organization and co-expression networks elucidated the tissue and time specificity of the gene expression pattern, as well as hub genes associated with naringin/neoeriocitrin synthesis in D. roosii. Simultaneously, the comprehensive transcriptome data set provided important genetic information for further research on D. roosii.

[Effect of Chinese Herbal Extract HNA-1 on the Thymic Output Function in Simian Immunodeficiency Virus Chronically Infected Chinese Rhesus Macaques].

OBJECTIVE: To observe the effect of Chinese herbal extract HuNan A-1 (HNA-1) on the thymic output function in Simian immunodeficiency virus (SIV) chronically infected rhesus macaques. METHODS: Eight Chinese rhesus macaques had been infected by SIVmac239 for 16 to 21 months, and then they were randomly divided into the treatment group and the control group, 4 in each group. Monkeys in the treatment group were administered with HNA-1 by gastrogavage, once daily for 2 successive months, while those in the control group were administered with equal volume of normal saline by gastrogavage, once daily for 2 successive months. The general condition and body weight of monkeys were observed. Plasma viral loads were detected using real-time fluorescent quantitative PCR assay. CD4 percentages and counts, as well as naive CD subsets were detected using flow cytometry. T-cell receptor excision circles (TREC) were detected using real-time fluorescent quantitative PCR assay. The thymus tissue was pathologically observed using routine HE staining. The correlation between lesions of the thymus tissue, CD4 counts, naive CD counts, and TREC were analyzed. RESULTS: There was no statistical difference in body weight, viral loads, absolute CD ratios between the two groups after treatment (P > 0.05). The altered TREC multiple showed an obvious decreasing tendency in the control group, while it showed an increasing tendency in the treatment group (P < 0.05). In both groups, destroyed structures of the thymus tissue could be seen, filled with pink unstructured material. Increased connective tissues, lowered connective cell density, and confused arrangement could also be seen in the two groups, with no obvious difference. TREC contents were positively correlated with naive CD4 counts after removing extremum (r = 0.926, P = 0.001). Naive CD4 counts were positively correlated with CD4 counts (r = 0.961, P = 0.005). CONCLUSIONS: TREC content determination, as a marker of newly thymic emigrants, could be taken as a testing method for evaluating the thymic output function. Besides, HNA-1 treatment increased the thymic output significantly in SIV chronically infected monkeys. Correlation existed among TREC contents, naive CD4 counts, and pathologies of thymus tissues, especially in late infection stage.

[Oncomelania hupensis snail situation in wetlands surrounding reservoirs in hilly areas in Danling County].

OBJECTIVE: To master the Oncomelania hupensis snail situation in wetlands surrounding reservoirs in hilly areas in Danling County, Sichuan Province, so as to provide the reference for improving the snail control in these areas. METHODS: A medium-sized reservoir (Meiwan Reservoir) and a small-sized reservoir (Zhulinkan Reservoir) were chosen respectively , and the snail situation of the environments surrounding the reservoirs were investigated , the indexes such as the occurrence rate of frames with snails and the density of living snails at the upstream and downstream of the reservoirs as well as the surrounding environments were calculated and compared. Meanwhile , the data of the snail situation of the adjacent villages in the irrigation district at the downstream of the reservoirs year by year were collected to analyze the variation trend. RESULTS: The Meiwan Reservoir was located in the deep hill, the occurrence rate of frames with snails was 4.29%, the highest and average densities of living snails were 28.00 snails/0.1 m2 and 0.38 snails/0.1 m2, respectively. There was a statistically significant difference between the occurrence rates of frames with snails at the upstream and downstream, which were 0.17% and 22.16%, respectively (χ2 = 1,267.40, P < 0.05), and the rate of spillway was the highest (51.69%). The highest and average densities of living snails in the downstream were 7.00 and 656.10 times higher than those in the upstream respectively. The Zhulinkan Reservoir was located in the shallow hill, the occurrence rate of frames with snails was 17.29%, and the highest and average densities of the living snails were 38.00 snails/0.1 m2 and 0.61 snails/0.1 in 2, respectively. There was a statistically significant difference between the occurrence rates of frames with snails at the upstream and the downstream, which were 12.95% and 50.92% respectively (χ2 = 194.68, P < 0.05), and the rate of spillway was the highest (65.74%). The highest and average densities of living snails in the downstream were 5.43 and 10.00 times higher than those in the upstream respectively. After the construction of the two reservoirs, the snail area in the irrigation districts of the adjacent villages at the downstream was 57.26% of that before and after the construction (398,030 m2/695 160 m2). The snail area showed a down-ward tendency with repeatedly rebound since the 1990s. CONCLUSIONS: The wetland surrounding the reservoir in hilly region is suitable for snail breeding, and the snail situation in the shallow hill is more serious than that in the deep hill. There exists a risk for the repeatability of snail situation in this type of area where the snails can easily diffuse.

[Regulation of naotai recipe on the expression of HIF-lα/VEGF signaling pathway in cerebral ischemia/reperfusion rats].

OBJECTIVE: To observe the therapeutic angiogenesis effect of naotai recipe (NR) on local ischemia/reperfusion (I/R) injury of rats by observing signaling pathway of hypoxia-inducible factor-lα (HIF-1α) and vascular endothelial growth factor (VEGF). METHODS: Totally 120 Sprague-Dawley (SD) rats were randomly divided into 4 groups, namely, the normal control group (n =12), the sham-operation group (n =12), the I/R model group (n =48), and the NR group (n =48). Cerebral I/R injury models were established using thread suture method. Rats in the I/R model group and the NR group were sub-divided into 4 sub-groups according to the 1st, 3rd, 5th, and 7th I/R day (n =12). The phenomenon of neovasculization was observed by immunofluorescence staining. The protein and mRNA expression levels of HIF-la, VEGF-A, and VEGFR II receptor were detected by RT-PCR. RESULTS: There were a large amount of labels for neovasculization in the ischemic area of the NR group. Double-immunofluorescence labeling [vWF (red) and BrdU (green)] was observed in the NR group. Compared with the model group, the HIF-1α protein expression was obviously enhanced on the 1 st day of I/R (P <0.01), and the VEGF protein expression started to enhance on the 3rd day in the NR group (P <0.01). The VEGFR protein expression level was the highest in the NR group on the 5th day of I/R (P <0.01). The protein expression of VEGF and HIF-1α started to decrease on the 7th day of I/R. CONCLUSION: NR could strengthen angiogenesis after I/R by elevating the expression of HIF-lα and activating HIF-lα/VEGF signaling pathway.

[Surveillance of schistosome antibodies in population in mountainous schistosomiasis endemic regions in China].

OBJECTIVE: To understand the dynamics of schistosome antibodies in population in mountainous schistosomiasis low endemic regions, China, so as to provide the evaluation reference for immunodiagnosis. METHODS: The people who consecutively received the indirect heamagglutination assay (IHA) for schistosomiasis and Kato-Katz technique were enrolled in this study in Guixiang Village, Danling County, Sichuan Province from 2006 to 2011. The positive rates for schistosome antibodies, antibody titers and eggs per gram (EPG) were analyzed. RESULTS: A total of 353 people were investigated in this study from 2006 to 2011. The positive rates for schistosome antibodies decreased from 20.73% to 13.67%, and the geometric mean reciprocal titer (GMRT) decreased from 1.63 to 1.06. The antibodies tended to be negative in most antibody-positive people and the GMRT decreased gradually. The antibodies remained negative in most antibody-negative people. After chemotherapy in parasitology confirmed patients, the positive rate for schistosome antibodies decreased from 100% to 20%, and the GMRT decreased from 34.29 to 1.58. There was a highly positive correlation between GMRT and EPG (r = 0.94, P < 0.05). CONCLUSION: The positive rate for schistosome antibodies in population in mountainous schistosomiasis low endemic regions is relatively high (13.67%). The standardization of immunodiagnostic methods is necessary and the suitable diagnostic reagents should be developed in this region.

[Effects of 5-Aza-2-deoxycytidine on DNA methylation of anti-oncogenes in non-small cell lung cancer cells].

OBJECTIVE: To observe the expression of SFRP1 gene methylation in non-small cell lung cancer (NSCLC), and study the effect of 5-Aza-2-deoxycytidine (5-Aza-CdR) on DNA methylation and expression of SFRP1, p16 and MGMT genes in the human lung cancer cell line SPC-A-1 cells. METHODS: SP immunohistochemistry and methylation-specific PCR were used to detect the SFRP1 methylation in 60 NSCLC cases, and 21 cases of benign lung diseases were used as control group. SPC-A-1 cells were cultured and treated with 5-Aza-CdR. The promoter methylation status of SFRP1, p16 and MGMT genes were detected by methylation-specific polymerase (MSP) chain reaction, and mRNAs were detected by real-time PCR. RESULTS: The positive rate of SFRP1 gene methylation in NSCLC was significantly higher than that in normal lung tissue (58.3% vs. 14.3%; χ(2) = 12.118, P = 0.001). SFRP1 gene methylation was closely correlated with lymph node metastasis and degree of differentiation in NSCLC (P < 0.05). SFRP1 protein expression was correlated with clinical stage, degree of differentiation and lymph node metastasis in NSCLC (P < 0.05). The positive expression of SFRP1 protein in 30 cases of NSCLC tissue containing SFRP1 gene methylation was significantly higher than that in non-methylated NSCLC (68.6% vs. 24.0%; χ(2) = 9.613, P = 0.002). SFRP1 gene methylation was closely correlated with SFRP1 gene protein expression in NSCLC (P < 0.05). Negative expression of SFRP1 protein was correlated with the differentiation, clinical stage, and lymph node metastasis in NSCLC (all P < 0.05). Without 5-Aza-CdR treatment, the expressions of methylation of SFRP1, p16 and MGMT genes and their mRNA were low. After 5-Aza-CdR treatment at different concentrations, their expressions were significantly elevated (all P < 0.05). CONCLUSIONS: SFRP1 gene methylation is closely associated with carcinogenesis and development of NSCLC. 5-Aza-CdR may reverse the methylation of SFRP1, p16 and MGMT genes, and facilitate the re-expression of the anti-oncogenes.

[Effect of comprehensive control pattern on soil-transmitted nematodiasis in Danling County].

The comprehensive control pattern centering on health education, deworming and improving drinking water and lavatories was adopted to control soil-transmitted nematodiasis in Danling County, Sichuan Province from 2007-2009. In 2009, the infection rate of soil-transmitted nematodes decreased from 57.12% of the baseline survey in 2006 to 10.06%.

[Genetic characterization of two Seoul virus strains isolated from patients and rats in Hebei province].

OBJECTIVE: Complete S and M segments of two Seoul virus (SEOV) strains were obtained to determine their genetic types and characteristics. METHODS: The complete S and M segments from the isolate Li and lung tissue (sample LF18) were amplified by RT-PCR. Genetic analyses were performed by using DNAStar and PHYLIP program package. RESULTS: Their sequences consisted of 1772 nucleotides, and had an open reading frame (ORF, 43 to 1332 nt) encoding a nucleoprotein of 429 amino acids for both two strains. The complete M segment sequences consisted of 3653 nucleotides and had an ORF encoding a GnGc precursor of 1133 amino acids. The GnGc precursor of the two strains had 62 cysteine and 6 N-glycosylation sites. Both two strains shared a high degree of homology with other known SEOV strains including strains L99, Gou3, and vaccine strain Z37, with 87.6% to 99.2% and 83.6% to 97.3% nucleotide identities, respectively. On the S- and M-trees, the two strains LF18 and Li were grouped into the third cluster of SEOV. CONCLUSION: Both LF18 and Li strains belonged to SEOV and shared the congruent genetic characteristics with the vaccine strains Z37. Thus, the bivalent vaccine including the strain Z37 could effectively prevent HFRS which was caused by SEOV in Hebei province.

H2 strain attenuated live hepatitis A vaccines:protective efficacy in a hepatitis A outbreak.

AIM:To investigate the protective efficacy of H2 strain attenuated live hepatitis A vaccines (H2-strain vaccines) in hepatitis A (HA) outbreaks.METHODS:With the permission of their parents, 5551 pre-school and grade 1-3 primary school children were inoculated with 1 dose (10(6.5) TCID(50)) of H2 strain vaccines in a nonrandomized, controlled trial conducted in Fucheng County, Hebei Province in May 1997.Another 6485 children in the same grades and compatible in gender and age were enrolled as controls. Epidemiological and serological survey was conducted to evaluate the protective efficacy of the vaccines. ELISA was used to detect serum IgM anti-HAV.RESULTS:HA outbreak started in early May 1998, peaked in the middle of the same month, and lasted about 80 days. Overall 302 HA cases were found, 192(63.58%) were 5-9 years old. One vaccinee and 25 control cases were found to have hepatitis A, which account for 0.28% (1/356) and 5.92% (25/422) of all vaccinees and controls in the 14 villages, respectively. The protective efficacy of vaccines was 95.27% (95% CI: 85.83%-104.72%). In subjects tested for anti-HAV IgM from 13 villages, 1(0.40%) overt and 11(4.06%) asymptomatic HAV cases were found in 271 vaccinees but 21(6.69%) of overt and asymptomatic ones were found in 314 controls.CONCLUSION:H2 strain vaccines were excellent in preventing overt hepatitis A,but not so effective in preventing asymptomatic hepatitis A virus infection.A booster dose might be needed to get permanent reliable immunity.