In silico screening of off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography for four saponins isolation.

Being restrained by the limited peak capacity, one-dimensional chromatography usually leads to an unsatisfactory separation with low purity of compounds in a complex mixture. To obtain more highly pure targets for standard reference and to discover new substances for structural elucidation, two-dimensional chromatography is more and more prevalent in many fields. As few metrics on assessment of the preparative capability of two-dimensional chromatographic separations are reported, a methodology of in silico screening of various two-dimensional chromatographic separations with a minimal number of experiments was demonstrated in this work, which was based on three descriptors including the occupation rate of peaks and system homogeneity of a two-dimensional separation space, and the minimal distance of all nearest-neighbor distances of peaks. Combining the advantages of counter-current chromatography and liquid chromatography, we elaborated the methodology by employing off-line comprehensive two-dimensional counter-current chromatography with liquid chromatography to be in silico screened for separation of four saponins from Panax notoginseng at an analytical scale to simulate the case of preparative scale transfer. The predictive results were presented by two-dimensional contour plots and verified by experiments. The result showed that the experimental results were in general accord with the predictive results.

Restoring VTA DA neurons excitability accelerates emergence from sevoflurane general anesthesia of anxiety state.

Preoperative anxiety is common and often comes with a higher probability of worse recovery. However, the neurological mechanism of the effect of preoperative anxiety on general anesthesia and subsequent awakening remains unknown. In this study, we report an anxious state results in delayed awakening in anxiety model mice from sevoflurane general anesthesia. More profound inhibition of DA neurons in the VTA contributes to delayed awakening. Optogenetic stimulation of VTA DA neurons can reverse the delay. The results indicate that VTA DA neurons may be involved in the delay in awakening from general anesthesia caused by anxiety.

[Current researches and prospects of seedling propagation and cultivation modes of Jinxianlian].

In the wake of on-the-spot investigation into Chinese major production bases in Fujian, Zhejiang, Jiangxi, Taiwan, Guangxi and Yunnan provinces, and based on relevant literature, the paper systematically elaborates the current researches of botanical origin, major cultivation type, seedling propagation technique as well as cultivation mode of Jinxianlian. The way of seedling breeding mainly includes aseptic seed culture, in vitro propagation, artificial seed and bioreactor propagation, etc. And the planting model mainly includes protected cultivation modes, bionic wild cultivation modes and pot cultivation modes, etc. Further discussions have also been conducted to tackle significant problems existing in the production process of Jinxianlian, based on personal studies of the authors. It has made considerable contributions for the betterment of Jinxianlian's development and improvement.

[Current status and sustainable development countermeasures of Anoectochilus roxburghii].

The current status of the domestic manufacturing and sales markets of Anoectochilus roxburghii were investigated and analyzed in the study. Some problems in the A. roxburghii industry were revealed and a variety of sustainable development countermeasures were also proposed. The main problems of A. roxburghii industry are the lack of protection for wild resources, the lag in the speed of variety breeding, the insufficient research on the quality systems, the low level of industry and product innovation capability, as well as the relatively low market cognition and brand competence. Therefore, strengthening the protection for breeding resources, establishing a dynamic monitoring system, promoting the variety breeding, constructing a propagation system for improved varieties, enhancing the quality of medicinal herbs, accelerating the adjustment of product structure, upgrading the industry technology, strengthening brand competence and expanding the market, will be the effective methods to realize the sustainable development of A. roxburghii industry.

Prevalence of and risk factors for the occurrence of symptomatic osteoarthritis in rural regions of Shanxi Province, China.

AIM: To determine the prevalence of symptomatic osteoarthritis (OA) in rural regions of Shanxi Province, China, and to identify factors increasing the prevalence of OA. METHOD: Residents over 16 years of age of targeted towns and villages in rural regions of Shanxi Province were sampled using a stratified multi-stage cluster method. Those exhibiting symptoms of rheumatism were referred to rheumatologists and those in whom rheumatism was suspected were X-rayed within 10 days of interview. OA was diagnosed by consensus (two or three rheumatologists). Factors associated with the presence of OA were identified. RESULTS: A total of 7126 permanent residents were surveyed and 1734 (24.3%) had OA. Knee OA was the most prevalent form of OA (13.8%), followed by lumbar (7.4%), cervical (3.4%), hand (3.3%), shoulder (3.0%), elbow (2.9%), ankle (0.7%), hip (0.6%), wrist (0.5%), thoracic (0.5%) and foot OA (0.5%). All of knee, ankle, shoulder and hand OA exhibited a gender bias. Advanced age, a sweet tooth, poor home ventilation, poor home heating, separation, divorce, or death of a partner, low-grade occupation, low educational level, high body mass index and the presence of concomitant cardiovascular disease, were associated with the presence of OA. CONCLUSION: Symptomatic OA is very prevalent in rural regions of Shanxi Province. Many factors increase the prevalence of the condition. Primary and secondary prevention programs seeking to improve living conditions, to reduce obesity, and to effectively treat concomitant cardiovascular disease, are required.

[Residue decline dynamics and safety utilization of carbendazim in cultivation of Anoectochilus roxburghii].

The paper aimed to study the residue decline dynamic and standards for safety utilization of carbendazim in roots, stems, leaves of Anoectochilus roxburghii and in growth media. Samples extracted with methanol were purified by liquid-liquid extraction and analysed by HPLC. The results showed that average rate of recovery was 82.9% - 95.7% and RSD were 2.0% - 6.3% with add of carbendazim in respectively diverse concentration, which meets inspection requirement of pesticide residue. Two kinds of dosages of carbendazim were treated, varying from recommended dosage (1.0 kg x hm(-2)) to 1.5 times recommended dosage (1.5 kg x hm(-2)). Results of two years test showed that the half-life period of carbendazim were 7.01 - 8.51 d in the growth media of A. roxburghii, 3.58 - 4.27 d in stems and 3.50 - 3.91 d in leaves, 4.93 - 5.71 d in roots. Providing max recommended residue of carbendazim in the cultivation of A. roxburghii is 0.5 mg x kg(-1), sprayed 4 times a year with the dosage of 1.0 kg x hm(-2), 28 days is proposed for the safety interval of the last pesticide application's and harvest's date.

Fast determination of two atractylenolides in Rhizoma Atractylodis Macrocephalae by Fourier transform near-infrared spectroscopy with partial least squares.

Rhizoma Atractylodis Macrocephalae (RAM) is a commonly used food and traditional Chinese medicine (TCM), which traditionally strengthens the spleen, benefits vital energy, eliminates dampness, and promotes hidroschesis. Its primary effective constituents are polysaccharides and volatile oil, whose main components are atractylenolide I and III. Fourier transform near-infrared spectroscopy (FT-NIR) is widely used in TCM research. However, determination of atractylenolides in RAM using FT-NIR has not been described. In this study, a new method for the determination of atractylenolides I and III in RAM by NIR was established. The spectral characteristics of atractylenolides I and III were obtained by second derivative multiple scattering correction, and its chart to the original absorbance spectra. Additionally, in combination with the partial least squares (PLS) algorithm, the calibration process was performed for the quantitation of the samples. The root mean square error of cross-validation of the PLS models for atractylenolides I and III was 0.0387 and 0.0358, and the determination coefficient of quantitative models was 96.63 and 96.16, respectively. This study demonstrated that NIR spectroscopy can be used to analyze quickly and efficiently the contents of atractylenolides I and III in RAM.

[Constituents of Dendrobium devonianum and their antioxidant activity].

OBJECTIVE: To study the chemical constituents of Dendrobium devonianum and identify the material basis components of its function, and then provide the basis for development and utilization of D. devonianum. METHOD: The constituents were separated and purified on the chromatography of silica gel, Sephadex LH-20 and RP-18 silica gel, and then their structures were elucidated based on the spectra data. ABTS method was used to evaluate the free radical scavenging activity of the phenolic compounds among them. RESULT: Nine compounds were isolated and identified as 2,3,4,9-tetrahydro-1H-pyrido[3,4-b] indole-3-carboxylic acid (1), 2'-deoxythymidine (2), adenosine (3), N-trans-p-coumaroyl tyramine (4), N-trans-p-feruloyl tyramine (5), 3-methoxy-4-hydroxybenzaldehyde (6), 4-hydroxybenzaldehyde (7), 3,4-dihydroxybenzoic acid methyl ester (8), and 4-hydroxy-3,5-dimethoxybenzoic acid (9). Compound 5 showed good antioxidant activity with IC50 1. 61 mmol. L-1, Compound 9 showed weak antioxidant activity with IC50 35.72 mmol. L-1. CONCLUSION: All these compounds were isolated from D. devonianum for the first time. Among them, compounds 5 and 9 had some antioxidant activity.

[The efficacy of enhancing murine zona pellucid 3 DNA vaccine immunized through intranasal vaccination by IL-15].

AIM: To observe the efficacy of enhancing murine zona pellucid 3(mZP3) DNA vaccine immunized through intranasal vaccination by IL-15 as adjuvant and chitosan as delivery vactor. METHODS: C57BL/6 mice were delivered with chitosan-loaded mZP3 DNA vaccine or IL-15+pcD-mZP3 vaccine by intranasal route, and to detect specific IgG in serum and sIgA in vaginal fluid by indirect ELISA, and to perform fertility experiment that all female C57BL/6 mice were mated(one male for each female) with normal males of similar age. To do histology analysis of immunized mice ovary and lungs for morphology of ovary. RESULTS: The results of ELISA showed IL-15 can promote IgG antibody level of immunized mice, and the fertility rate was decreased to some degrees. CONCLUSION: IL-15 and chitosan can enhance the antibody levels induced by mZP3 DNA vaccine, and reduce murine fertility rate to a certain extent.

Isolation of laccase gene from Bacillus subtilis and analysis of its physicochemical properties.

The present study reports the cloning and sequencing of lac2 from Bacillus subtilis. The gene is composed of 1542 bp and encodes a 514-amino acid protein. The gene has 86% homology with a published laccase with GeneID 936023. The lac2 gene was deposited in GenBank as a new nucleotide sequence. This new sequence was cloned into the multiple cloning site of pPIC9K to generate pPIC9K-lac2, which was then transformed into Pichia pastoris GS115 via electroporation. The recombinant GS115 (pPIC9K-lac2) was grown initially in BMGY medium and transferred to BMMY to induce gene expression for 48 h. The recombinant Lac2 protein shows laccase activity with α-naphthol and guaiacol as substrates. The optimal pH is between 3.2 and 4.7, and the optimal temperature is 25°C for enzyme reaction.

DW-MRI ADC values can predict treatment response in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy.

The purpose of this study was to evaluate the importance of diffusion-weighted magnetic resonance imaging (DW-MRI) apparent diffusion coefficient (ADC) values to predict treatment response to neoadjuvant chemotherapy (NCT) in patients with locally advanced breast cancer (LABC). Thirty-two patients with LABC underwent 2-4 cylces of NCT (docetaxel and epirubicin). The DW-MRI scans were performed within one week prior to chemotherapy and after the first course of treatment, respectively. Accordingly, tumor volumes, changes in tumor ADC values, and their degree of correlation were analyzed. The overall response (OR) was observed in 62.5% (95% CI, 45.7-79.3%) of patients after 2 cycles of NCT. The clinical complete response (CR) rate and pathological CR (pCR) rate were 15.6 and 9.4%, respectively. The stable disease (SD) rate was 34.4% (11 patients), and progressive disease (PD) was observed in only one patient (3.1%). After the first cycle of NCT, the ADC values in the CR + PR group significantly increased (P < 0.001). The initial ADC values before chemotherapy in the OR group were significantly lower than those in the SD + PD group (P < 0.001). The initial ADC values and the changes in tumor volume after chemotherapy were negatively correlated (r = -0.58, P = 0.02). The lower the initial tumor ADC value was the more obvious the decrease in tumor volume after chemotherapy. The changes in ADC values of tumors after chemotherapy and the changes in tumor volume were positively correlated (r = 0.96, P < 0.001). After chemotherapy, the greater the change in ADC value, the more the tumor volume was reduced. Using the initial ADC values of breast cancer tumors and the early changes in ADC values after NCT, we may be able to predict tumor response to chemotherapy. Tumors with low initial ADC values may be sensitive to chemotherapy; tumors with significantly increasing ADC values early after chemotherapy may be sensitive to chemotherapy.

Epidemiologic study of soft tissue rheumatism in Shantou and Taiyuan, China.

BACKGROUND: Soft tissue rheumatism is a group of common rheumatic disorders reported in many countries. For investigating the prevalence rate of soft tissue rheumatism in different population in China, we carried out a population study in Shantou rural and Taiyuan urban area. METHODS: Samples of 3915 adults in an urban area of Taiyuan, Shanxi Province, and 2350 in a rural area of Shantou, Guangdong Province were surveyed. Modified International League of Association for Rheumatology (ILAR)-Asia Pacific League of Association for Rheumatology (APLAR) Community Oriented Program for Control of Rheumatic Diseases (COPCORD) core questionnaire was implemented as screening tool. The positive responders were then all examined by rheumatologists. RESULTS: Prevalence rate of soft tissue rheumatism was 2.0% in Taiyuan, and 5.3% in Shantou. Rotator cuff (shoulder) tendinitis, adhesive capsulitis (frozen shoulder), lateral epicondylitis (tennis elbow), and digital flexor tenosynovitis (trigger finger) were the commonly seen soft tissue rheumatism in both areas. Tatarsalgia, plantar fasciitis, and De Quervain's tenosynovitis were more commonly seen in Shantou than that in Taiyuan. Only 1 case of fibromyalgia was found in Taiyuan and 2 cases in Shantou. The prevalence of soft tissue rheumatism varied with age, sex and occupation. CONCLUSIONS: Soft tissue rheumatism is common in Taiyuan and Shantou, China. The prevalence of soft tissue rheumatism was quite different with different geographic, environmental, and socioeconomic conditions; and varying with age, sex, and occupation. The prevalence of fibromyalgia is low in the present survey.

Magnetic resonance imaging-guided percutaneous cryoablation of hepatocellular carcinoma in special regions.

BACKGROUND: Local cryoablation guided by CT or ultrasound has been widely applied in the treatment of hepatocellular carcinoma. However, it is still difficult to apply this technique in certain regions such as the diaphragm dome, the first hepatic hilum, and regions adjacent to the gallbladder. This study aimed to evaluate the safety and efficacy of using magnetic resonance imaging (MRI)-guided percutaneous cryoablation as well as the effect of using an open MRI system in guiding and monitoring the treatment of hepatocellular carcinoma in these regions. METHODS: Cryoablation, guided by an open 0.35T MRI scanner and with the assistance of an MRI-compatible optical navigation system, was performed on 32 patients with hepatocellular carcinoma at the diaphragm dome, the first hepatic hilum, and regions adjacent to the gallbladder. Each patient had one or two tumors. The total number of tumors treated was 36. The tumor diameters ranged from 2.5 to 10.0 cm (mean 4.7+/-1.8 cm). The cryosurgical system was MRI-compatible and equipped with cryoprobes 1.47 mm in outside diameter. Under the guidance of MRI in combination with the optical navigation system, the cryoprobes were introduced percutaneously into a tumor at the planned targeting points while critical organs or tissues were avoided. Each cryoablation procedure included two freezing-thawing cycles, and MRI images were acquired dynamically to monitor the ablation of the tumor from time to time during the operation. In order to investigate the therapeutic effects of a cryoablation procedure, AFP measurements and liver-enhanced MRI or CT-enhanced scans were performed at regular times. RESULTS: MRI and optical navigation system-guided cryoablation procedures were successfully performed on all 32 patients (36 tumor sites) and no serious complications occurred. The follow-up period ranged from 5 to 12 months. The 6- and 12-month overall survival rates were 96.8% and 90.6%, respectively. According to the diagnosis of liver-enhanced MRI scans, 10 patients (31.3%) had complete ablation, 18 (56.3%) partial ablation (>80%), 3 (9.4%) stable disease (>50% ablation), and 1 (3.1%) progressive disease (a new tumor site in the liver). The overall efficacy was 87.5%. CONCLUSIONS: MR-guided percutaneous cryoablation using optical navigation is a safe and effective minimally invasive procedure for the treatment of hepatocellular carcinoma at certain special regions, which is difficult to treat with other imaging guidance approaches. With its unique and superb imaging functions, MRI plays an important role in the display, guidance, and monitoring of the cryoablation procedure in treating hepatocellular carcinoma at these special regions. Equipped with an MRI-compatible optical navigation system, MRI-guided therapy makes the cryoablation procedure more precise and safe.

Transgenic RNAi-mediated reduction of LZP3 in Lagurus lagurus oocytes results in decreased fertilization ability in IVF.

Structural differences in oligosaccharides on mammalian zona pellucida 3(ZP3) from different species may determine whether or not spermatozoa being able to bind to ZP. We reported here that by microinjecting the siRNA interference recombinant construct pGenesil-ZP31 encoding a Lagurus zp3 (lzp3) hairpin dsRNA of 21 bp into the inmatured oocytes of Lagurus lagurus, distributed in northern region of Xingjiang, to disturb its fertility. Results of in vitro fertilization after in vitro maturation of the immature oocytes of Lagurus lagurus showed that the fertilization rate of the transgenic oocytes carried pGenesil-ZP31 was decreased greatly (2.82%) compared to the oocytes carried pGenesil-HK (15.71%), suggesting that the transgenic RNAi-mediated silencing of lzp3 in Lagurus lagurus oocytes results in decreased fertilization ability. These results proved that LZP3 of Lagurus lagurus, like other mammalians, is essential for the recognition between oocyte and spermatozoa.

[Oral immunization of Lugurus lugurus with the plasmid nanoemulsion of zona pellucida 3 to enhance infertility effects].

Immunocontraceptive technology is beginning to attract more attention for the control of wild mouse or other pest animals. Oral vaccination of animals and man, to provide effective mucosal and/or systemic immunity, is a good delivery system, but it remains poor bioavailablity, one of the most promising strategies to improve the oral delivery relies on polymeric nanoparticles carriers to encapsulate vaccine. Nanoemulsion was used as the carriers of oral immunization of Lugurus Lugurus zona pellucida 3, the immune response and antifertility potential of this vaccine is valuated. Nanoemulsion-encapsulated plasmid pcDNA3.0-Aat-COMP-lzp3-C3d3 (pACLC DNA vaccine) was prepared and evaluated the encapsulation efficiency by anion exchange chromatography. The mice were orally delivered with nanoemulsion-encapsulated plasmid, to detect specific IgG in serum and IgA in vaginal fluid by indirect ELISA, and to perform fertility experiment, to do histological analysis of immunized mice ovary. The results showed that the specific IgG and IgA antibody is elicited, the fertility rate is deceased at a certain extent. In conclusion, it is feasible that nanoemulsion is used as the carriers of oral immunization.

Inducible expression of human angiostatin by AOXI promoter in P. pastoris using high-density cell culture.

A high-density cell culture method was successfully established in P. pastoris with the alcohol oxidase I (AOXI) promoter in order to produce large quantities of recombinant human angiostatin (AS) which has been reported to have antiangiogenic activity. A preliminary study on fermentation conditions in shaking flasks indicated that adequacy of biomass is beneficial to obtain more products. The fermentation was carried out in a 10 l bioreactor with 5 l modified growth medium recommended by Invitrogen at 30 degrees C. The cells were first grown in glycerol-PTM4 trace salts for 24 h. When the cell density reached A(600) = 125, methanol-PTM4 trace salts was added to induce the expression of AS. During the fermentation, dissolved oxygen level was maintained at 20-30%, pH was controlled at 5 by the addition of 7 M NH(4)OH and the biomass was maintained at about A(600) = 200. After 60 h of induction, the secreted AS was 153 mg/l. The recombinant AS inhibited the angiogenesis on CAM and suppressed the growth of B16 melanoma in C57BL/6J mice (P \0.01).

Recent advances on the GAP promoter derived expression system of Pichia pastoris.

Pichia pastoris is an efficient host for the expression and secretion of heterologous proteins and the most important feature of P. pastoris is the existence of a strong and tightly regulated promoter from the alcohol oxidase I (AOX1) gene. The AOX1 promoter (pAOX1) has been used to express foreign genes and to produce a variety of recombinant proteins in P. pastoris. However, some efforts have been made to develop new alternative promoters to pAOX1 to avoid the use of methanol. The glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP) has been used for constitutive expression of many heterologous proteins. The pGAP-based expression system is more suitable for large-scale production because the hazard and cost associated with the storage and delivery of large volume of methanol are eliminated. Some important developments and features of this expression system will be summarized in this review.

[Constitutive expression of human angiostatin in Pichia pastoris using glycerol as only carbon source].

Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 (pGAP9K-AS)in shaking flask. The result shows that oleic acid is the best (163 mg/L) compared with glycerol (83mg/L), glucose (76 mg/L)and methanol (57 mg/L). Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 (pGAP9K-AS) in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P < 0.01).

[Preparation of plasmid nanoemulsion and its character determination by anion exchange chromatography method].

Nanoemulsion-encapsulated lzp3 DNA vaccine (pCDNA3-Aat-COMP-lzp3-C3d3, lzp3) was prepared, and its quality was evaluated. The interfacial emulsification method was employed to make the nanoemulsion-encapsulated plasmid, and its sizes and distribution were detected by electron microscope. Anion exchange chromatography was used to separate the nanoemulsion from the plasmid based on the charge characteristics of plasmid. The determination method for encapsulation efficiency of the plasmid nanoemulsion was established by anion exchange chromatography. The results indicated that the average size of the plasmid nanoemulsion is (23 +/- 10) nm, and the encapsulation efficiency is 80.5%. The separation and determination conditions of nanoemulsion-encapsulated plasmid was as follows: eluent buffer was 0.05 mol/L Tris-HCl solution at a flow rate of 0.7 mL/min, while detection wavelength for plasmid was 260 nm, the temperature of column was maintained at 30 degrees C, and injection volume was 2mL. On this condition, nanoemulsion and plasmid can be separated on the column of anion exchange chromatography significantly. This method is simple, sensitive and reproducible with respect to good linearity in the range of 0.05-0.80 mg/mL (r = 0.9983), which can be applied to determine the entrapment efficiency of nanoemulsion-encapsulated plasmid.