The ERF-VII transcription factor SmERF73 coordinately regulates tanshinone biosynthesis in response to stress elicitors in Salvia miltiorrhiza.

Here, we investigate the role of SmERF73, a group VII ETHYLENE RESPONSE FACTOR stress response transcription factor, in the regulation of post-modification of the skeleton precursors of diterpene tanshinones in Salvia miltiorrhiza. Most genes found to be involved in tanshinone biosynthesis are located on chromosome 6, and five of these genes comprise a large gene cluster in S. miltiorrhiza. We found that SmERF73 overexpression in S. miltiorrhiza coordinately up-regulated the transcription of seven tanshinone biosynthetic genes, four of which were located in the tanshinone gene cluster, consequently increasing tanshinone accumulation, while SmERF73 silencing reduced corresponding gene transcription and tanshinone accumulation. SmERF73 recognizes GCC-box promoter elements of four tanshinone-associated genes (DXR1, CPS1, KSL1 and CYP76AH3) and activates their expression. Moreover, SmERF73 and its targets were up-regulated by stress elicitors; SmERF73 appears to be at least partly mediated by the jasmonic acid (JA) signaling pathway via interaction with SmJAZ3. SmERF73 coordinately regulates tanshinone biosynthetic gene expression, suggesting a potential link between tanshinone production and plant stress responses.

[Expression analysis and subcellular localization of an ERF transcription factor from Salvia miltiorrhiza].

OBJECTIVE: A SmERF1 gene was isolated from Salvia miltiorrhiza, and expression patterns to different stress condition were analysed in the root tissues of S. miltiorrhiza. METHOD: The cDNA of SmERF1 gene from S. miltiorrhiza was isolated by RTPCR, and the phylogenetic tree using the neighbour-joining tree method in Mega 5 was obtained. To confirm the protein is likely to localize in the nucleus, the SmERF1 coding region was fused to the N-terminus of the GFP gene under the control of the CaMV 35S promoter and transferred into onion epidermal cells using the particle bombardment method. Semi-quantitative RT-PCR analysis revealed different expression pattern of SmERF1 gene in response to exogenous ABA, MeJA and SA. RESULT: The phylogenetic tree analysis revealed that SmERF1 is most similar to AP2/ERF VII subgroup members. The transient expression of the SmERF1::GFP fusion protein indicated that the SmERF1 was exclusively localized to the nucleus. The transcript of SmERF1 highly accumulated when the plants were treated with MeJA, while accumulated slightly in response to exogenous ABA, salicylic acid. CONCLUSION: These results suggest hormone such as ABA, MeJA and SA signaling pathways can be involved in the activation and inhibition of the SmERF1.

Draft genome sequences of two Streptococcus pyogenes strains involved in abnormal sharp raised scarlet fever in China, 2011.

A scarlet fever outbreak caused by Streptococcus pyogenes occurred in China in 2011. To determine the genomic features of the outbreak strains, we deciphered genomes of two strains isolated from the regions with the highest incidence rates. The sequences will provide valuable information for comprehensive study of mechanisms related to this outbreak.

[Establishment and analysis of in vitro culture system for transgenic Salvia miltiorrhiza hairy roots].

OBJECTIVE: To establish a culture system for transgenic Salvia miltiorrhiza hairy roots. METHOD: Investigated the success rate of different explants, different infection time and different co-culture time to induce hairy roots of S. miltiorrhiza. Co-cultured explants were sterilizated with 400 g x L(-1) Cef water for 5 min, inoculated on MS solid medium supplied with 400 mg x mL(-1) cef and 2.5 g x L(-1) Hyg, and then transfered to the 67-V liquid medium with 2.5 g x L(-1) Hyg after complete sterilization. GFP fluorescence detection was performed to detect positive hairy root lines. PCR method to detect rolC gene which is the specific gene of hairy root. Biomass was determinated in different growth periods of root lines. HPLC was conducted to measure the content of dihydrotanshinone I of transgenic hairy roots. RESULT: Leaf base of S. miltiorrhiza was used as a perfect explant to Induce hairy roots, the success rate can reach 93.3%. Inducing efficiency was up to 63.3% after Agrobacterium infection for 10 min. Co-culture for 2-3 d can reach the best induced effect. It is a high credibiliy to use PCR method combined with detection of GFP fluorescence to identified positive transformants. There is a close contact between biomass increases and secondary metabolite accumulation of transgenic hairy roots. CONCLUSION: Successfully in vitro culture system has been established in transgenic S. miltiorrhiza, and this research can lay foundations for the further genetic engineering applications.

[Isolation and characteristic of SmbHLH1 gene in Salvia miltiorrhiza].

OBJECTIVE: A novel bHLH-like gene, designated SmbHLH1, was isolated from Salvia miltiorrhiza, in order to identify a bHLH gene in related to danshinone biosysnthesis. METHOD: SmbHLH1 was isolated by RT-PCR,and Semi-quantitative RT-PCR was used to detect the gene expression level. RESULT: The full length of SmbHLH1 cDNA has an open reading frame of 999 bp. The deduced amino acid sequence of SmbHLH1 has 332 amino acid residues which forms a 36 kDa polypeptide with a calculated pI of 5.4. SmbHLH1 gene was expressed at high level in root, but low level in stem, leaf and flower of S. miltiorrhiza. The transcripts of SmbHLH1 was suppressed when the plants were treated with exogenous MeJA, Yeast + Ag+. The transcripts of SmbHLH1 constitutively accumulated in response to exogenous ABA and low concentration of salicylic acid. CONCLUSION: SmbHLH is a new member of the S. miltiorrhiza bHLH family, and its possible roles in brassinosteriods signaling responses.

Saposhnikoviae Radix Enhanced the Angiogenic and Anti-Inflammatory Effects of Huangqi Chifeng Tang in a Rat Model of Cerebral Infarction.

Huangqi Chifeng Tang (HQCFT), a traditional Chinese formula of three herbs, has been used to treat cerebral infarction (CI). Saposhnikoviae Radix (SR) was designed as a guiding drug for HQCFT to improve its angiogenic and anti-inflammatory effects. In this study, TTC staining was used to detect the area of CI. H&E staining was used to detect the histopathologic changes in the cerebral tissue. Western blotting was performed to detect the protein expression of NLRP3, caspase 1, IL-1ß, IL-6, TNF-α, MMP-9, VEGF, and VEGFR2 in cerebral tissue. Immunohistochemistry was used to detect the protein expression of MMP-9, VEGF, and VEGFR2. The contents of HIF-1α, NLRP3, caspase 1, IL-1ß, IL-6, and TNF-α in the serum were determined by ELISA. Our study showed that HQCFT and HQCFT-SR could improve the pathological condition and reduce the infarcted area of the brain tissue in a rat model. In addition, HQCFT and HQCFT-SR significantly decreased the expression levels and serum contents of NLRP3, caspase 1, IL-1ß, IL-6, and TNF-α; increased the expression levels of the VEGF and VEGFR2 proteins; and obviously reduced the serum content of HIF-1α. Importantly, the cytokines in brain tissue and serum from the HQCFT group exhibited better efficacy than those from the HQCFT-SR group. HQCFT exerted significant angiogenic and anti-inflammatory effects in rats subjected to middle cerebral artery occlusion (MCAO); these effects can be attributed to the guiding and enhancing effect of SR.

[An anatomical study on lateral ligaments of rectum].

OBJECTIVE: To identify the location and contents of the rectal lateral ligaments and its relationship with the middle rectal artery and pelvic plexus. METHODS: Twenty-nine pelvics of human cadavers were sagittally sectioned into 58 hemipelvic specimens. All of hemipelvics were dissected with sharp technique under direct vision by one surgeon. The lateral ligaments were identified and the distances from the center of its pelvic attachment to sacral promontory and coccyx were measured. Then, the lateral ligaments were transected for histologic examination. RESULTS: Lateral ligaments of rectum were found in all 58 hemipelvics. The lateral ligaments connected the posterolateral aspect of the middle 1/3 of the rectum and mesorectum to the lateral aspect of the bodies of the second, third and fourth sacral vertebrae. The distance from the lateral ligament to sacral promontory was (8.3 +/- 1.6) cm on the right side and (8.4 +/- 1.4) cm on left side. The width of lateral ligaments was (3.2 +/- 0.4) cm on the right side and (3.1 +/- 0.4) cm on the left side. The distances from lateral ligament to the coccyx on the right and left sides were (5.2 +/- 1.4) cm and (5.0 +/- 1.3) cm respectively. The content of the lateral ligaments consisted of loose connective tissue with clusters of small nerves and blood vessels. Middle rectal artery was found in 83% (48/58) of hemipelvics and 47% (27/58) of the middle rectal artery went through the lateral ligaments. The inferior hypogastric nerve plexuses were formed inside the lateral ligament, and separated the ligament into two parts. The lateral segment of the lateral ligament contained the tributaries of internal iliac artery, and the medial segment contained nerve fibers or branches to the rectum, together with the middle rectal artery. CONCLUSION: The lateral ligaments of rectum are located on the posterolateral side of the rectum, much closer to the coccyx than to the sacral promontory, consisting of connective tissue containing multiple small nerves and middle rectal artery. The lateral ligament is a pathway of blood vessels and nerve fibers toward the rectum and lymphatic vessels from the lower rectum toward the iliac lymph nodes.

[Expression of osteopontin mRNA and its clinical significance in gastric cancer].

OBJECTIVE: To investigate the expression of osteopontin mRNA and its correlation with clinicopathologic features of gastric cancer and elucidate its role in tumor invasion and distant metastasis. METHODS: The expression of OPN mRNA was detected by semi-quantitive RT-PCR. The relationship between the relative content of OPN mRNA and clinicopathologic features of gastric cancer was analyzed. RESULTS: In 66 cancer tissue samples, a 330 bp band was detected in 50 cases, the positive rate of OPN mRNA expression was 75.8% (50/66). The expression in all 20 cases of normal gastric mucosa was negative. The expression was associated with the depth of tumor invasion, diameter, lymph node metastasis and but had no correlation with differentiation grades. The 66 patients were followed up for 10 approximately 27 months (mean 16 months). The OPN mRNA expression positive group (50 cases) had recurrence in 15 patients and the negative group (16 cases) had only 1 case with recurrence (P = 0.05); 10 patients died in OPN mRNA expression positive group but no patient died in OPN staining negative group (P = 0.05). CONCLUSION: OPN mRNA is over-expressed in gastric cancer. It reflects the progression of disease and association with poor prognosis of gastric cancer. OPN may play an important role in the process of distant metastasis in gastric cancer.

MALDI-TOF mass spectrometry-based identification of group A Streptococcus isolated from areas of the 2011 scarlet fever outbreak in china.

There was a dramatic increase in scarlet fever cases in China from March to July 2011. Group A Streptococcus (GAS) is the only pathogen known to cause scarlet fever. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) coupled to Biotyper system was used for GAS identification in 2011. A local reference database (LRD) was constructed, evaluated and used to identify GAS isolates. The 75 GAS strains used to evaluate the LRD were all identified correctly. Of the 157 suspected ß-hemolytic strains isolated from 298 throat swab samples, 127 (100%) and 120 (94.5%) of the isolates were identified as GAS by the MALDI-TOF MS system and the conventional bacitracin sensitivity test method, respectively. All 202 (100%) isolates were identified at the species level by searching the LRD, while 182 (90.1%) were identified by searching the original reference database (ORD). There were statistically significant differences with a high degree of credibility at species level (χ(2)=6.052, P<0.05 between the LRD and ORD). The test turnaround time was shortened 36-48h, and the cost of each sample is one-tenth of the cost of conventional methods. Establishing a domestic database is the most effective way to improve the identification efficiency using a MALDI-TOF MS system. MALDI-TOF MS is a viable alternative to conventional methods and may aid in the diagnosis and surveillance of GAS.

[An outbreak of gastroenteritis caused by contaminated well water in a village, Henan province].

OBJECTIVE: To identify the cause and mode of transmission of a gastroenteritis outbreak in a village, Henan province. METHODS: Gastroenteritis patients were identified through family visits, interviewing the village doctors and reviewing diagnosis and prescription records at the village health clinic. Cases were defined as onset of one of the four symptoms from the village resident during July 20 to August 12, 2010. The symptoms would include diarrhea (≥ 3 times/day), abdominal pain, nausea or vomiting. A retrospective cohort study was conducted to assess the association between drinking raw well water or eating noodles rinsed by raw well water and gastroenteritis. Stools or vomits of the case-patients and the well water samples were tested for bacterial pathogens. RESULTS: Data for 60 case-patients were collected. All cases occurred in the northern part of the village. Persons who used water from a public well in the northern part of the village had an attack rate of 55%, which was 3.5 times of those who did not use the well water (16%) (RR = 3.5, 95%CI: 1.2 - 10). Results from the retrospective cohort study showed that drinking un-boiled water from the well was a risk factor (RR = 1.7, 95%CI: 1.3 - 2.3). Laboratory testing showed that total coliform and E. coli both greatly exceeded the limit considered safe for drinking, indicating there was fecal contamination in the well water. No bacterial pathogens were detected in the patients' stools or vomits. CONCLUSION: The outbreak was mainly caused by drinking contaminated water from the public well in the northern part of the village.