Enhancing the Activity of an Alcohol Dehydrogenase by Using "Aromatic Residue Scanning" at Potential Plasticity Sites.

An alcohol dehydrogenase LkADH was successfully engineered to exhibit improved activity and substrate tolerance for the production of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol, an important precursor of ticagrelor. Five potential hotspots were identified for enzyme mutagenesis by using natural residue abundance as an indicator to evaluate their potential plasticity. A semi-rational strategy named "aromatic residue scanning" was applied to randomly mutate these five sites simultaneously by using tyrosine, tryptophan, and phenylalanine as "exploratory residues" to introduce steric hindrance or potential π-π interactions. The best variant Lk-S96Y/L199W identified with 17.2-fold improvement in catalytic efficiency could completely reduce up to 600 g/L (3.1 M) 2-chloro-1-(3,4-difluorophenyl)ethenone in 12 h with >99.5 % ee, giving the highest space-time yield ever reported. This study, therefore, offers a strategy for mutating alcohol dehydrogenase to reduce aromatic substrates and provides an efficient variant for the efficient synthesis of (S)-2-chloro-1-(3,4-difluorophenyl)ethanol.

[Discrimination of cultivation modes of Dendrobium nobile based on content of mineral elements and ratios of nitrogen stable isotopes].

This study explored the feasibility of mineral element content and ratios of nitrogen isotopes to discriminate the cultivation mode of Dendrobium nobile in order to provide theoretical support for the discrimination of the cultivation mode of D. nobile. The content of 11 mineral elements(N, K, Ca, P, Mg, Na, Fe, Cu, Zn, Mn, and B) and nitrogen isotope ratios in D. nobile and its substrate samples in three cultivation methods(greenhouse cultivation, tree-attached cultivation, and stone-attached cultivation) were determined. According to the analysis of variance, principal component analysis, and stepwise discriminant analysis, the samples of different cultivation types were classified. The results showed that the nitrogen isotope ratios and the content of elements except for Zn were significantly different among different cultivation types of D. nobile(P<0.05). The results of correlation analysis showed that the nitrogen isotope ratios, mineral element content, and effective component content in D. nobile were correlated with the nitrogen isotope ratio and mineral element content in the corresponding substrate samples to varying degrees. Principal component analysis can preliminarily classify the samples of D. nobile, but some samples overlapped. Through stepwise discriminant analysis, six indicators, including δ~(15)N, K, Cu, P, Na, and Ca, were screened out, which could be used to establish the discriminant model of D. nobile cultivation methods, and the overall correct discrimination rates after back-substitution test, cross-check, and external validation were all 100%. Therefore, nitrogen isotope ratios and mineral element fingerprints combined with multivariate statistical analysis could effectively discriminate the cultivation types of D. nobile. The results of this study provide a new method for the identification of the cultivation type and production area of D. nobile and an experimental basis for the quality evaluation and quality control of D. nobile.

Mass Measurements of Neutron-Deficient Yb Isotopes and Nuclear Structure at the Extreme Proton-Rich Side of the N=82 Shell.

High-accuracy mass measurements of neutron-deficient Yb isotopes have been performed at TRIUMF using TITAN's multiple-reflection time-of-flight mass spectrometer (MR-TOF-MS). For the first time, an MR-TOF-MS was used on line simultaneously as an isobar separator and as a mass spectrometer, extending the measurements to two isotopes further away from stability than otherwise possible. The ground state masses of ^{150,153}Yb and the excitation energy of ^{151}Yb^{m} were measured for the first time. As a result, the persistence of the N=82 shell with almost unmodified shell gap energies is established up to the proton drip line. Furthermore, the puzzling systematics of the h_{11/2}-excited isomeric states of the N=81 isotones are unraveled using state-of-the-art mean field calculations.

[Effect of Plant Density and Harvest on Yield and Quality in Blumea balsamifera].

Objective: To study the effects of different plant density and harvest time on the yield and quality of Miao medicine Blumea balsamifera, in order to provide a theoretical basis for Good Agriculture Practice( GAP). Methods: Two factors trails were used to research the influence of plant density and harvest time on the yield and quality of Blumea balsamifera. The plant density were located at 50 cm × 90 cm,50 cm × 60 cm,50 cm × 30 cm and 30 cm × 30 cm, and the harvest time were set up at mid-October, mid-November and mid-December. The experiments were designed with randomized block design. Results: Yield and quality were both affected by the plant density and harvest time. Yield per unit area and volatile oil yield per unit area were increased significantly with the increasing of plant density. Yield per unit area and volatile oil yield per unit area were peaked at the plant density of 111 112 plants / hm~2 which could obtain high-yield at the mid-December, which were 1 546. 68 kg / hm~2 and 96. 6 L / hm~2,respectively. The content of total flavonoids were peaked at the plant density of 22 223 plants / hm~2 and 111 112 plants / hm~2,which were 2. 50 and 2. 53 mg/g,respectively. Harvest time had no significant effect on the flavonoids content. Conclusion: The suitable plant density of transplanted root tillers of Blumea balsamifera is 111 112 plants / hm~2 and the optimum harvest time is December.

[Comparative Analysis of Main Morphological Characters and Interrelationship in Blumea balsamifera].

Objective: To investigate the main morphological characters and interrelationship of Blumea bdsamifera, and to provide the guidance for selection and breeding of Blumea balsamifera. Methods: 19 main morphological characters and their interrelationship were analyzed by using correlation analysis,multiple stepwise regression analysis and factor analysis. Results: Variation coefficients of the morphological characters were big in Blumea balsamifera. Correlation coefficients for morphological characters were significant( P <0. 05 or P < 0. 01). Using factor analysis,19 morphological characters were classified into six principal divisors. Ten morphological characters, including length / wide of leaf,trunk diameter, leaf number of second branch, leaf number of first branch, number of trunk leaf, weight of strong leaf, weight of young leaf,weight of old leaf, number of first branch, leaf number of whole plant, were the main factors which influenced the weight of whole plant leaf. Conclusion: The variation of every morphological character of Blumea balsamifera is abundant in different population, and a certain correlation is existed among morphological characters. The morphological characters, length / wide of leaf, trunk diameter, number of first branch, number and weight of leaf, are the main factors which influenced the weight of whole plant leaf.

[Content of mineral elements of Gastrodia elata by principal components analysis].

OBJECTIVE: To study the content of mineral elements and the principal components in Gastrodia elata. METHOD: Mineral elements were determined by ICP and the data was analyzed by SPSS. RESULT: K element has the highest content-and the average content was 15.31 g x kg(-1). The average content of N element was 8.99 g x kg(-1), followed by K element. The coefficient of variation of K and N was small, but the Mn was the biggest with 51.39%. The highly significant positive correlation was found among N, P and K . Three principal components were selected by principal components analysis to evaluate the quality of G. elata. P, B, N, K, Cu, Mn, Fe and Mg were the characteristic elements of G. elata. CONCLUSION: The content of K and N elements was higher and relatively stable. The variation of Mn content was biggest. The quality of G. elata in Guizhou and Yunnan was better from the perspective of mineral elements.

[Effects of Different Planting Direction and Layer Combination on Gastrodia elata f. elata in Bionic Wild Cultivation].

OBJECTIVE: Combination of different planting direction and layer were set to choose the best technology of cultivation of Gastrodia elata f. elata. METHODS: To improve the yield and quality of Gastrodia elata f. elata, randomized block design experiments were carried out to investigate the yield and quality, and to analyze their economic effectiveness in bionic wild cultivation. RESULTS: Length, width, thickness and weight of southern direction's Gastrodia elata f. elata developed better than the northeast direction. The three planting layer levels on growth effect of Gastrodia elata f. elata was the 3rd layer > the 2nd layer > the 1st layer. In six treatments, combination of southern direction-the 3rd layer was the best technology of cultivation of Gastrodia elata f. elata, which had the best growth condition, the highest yield significantly higher than other treatments, and the best economic benefits. CONCLUSION: Southern direction associated with the 3rd layer is the best combination to planting Gastrodia elata f. elata in bionic wild cultivation. The planting ways not only improve the yield and quality, but also save land.

Production and characterization of a fusion peptide derived from the rabies virus glycoprotein (RVG29).

Gene therapy targeting the brain holds great promise in curing nervous system degenerative diseases in clinical applications. With this in mind, in a previous study a 29 amino-acid peptide derived from the rabies virus glycoprotein (RVG29) with a nonamer stretch of arginine residues (RVG29-9R) at its carboxy-terminus was exploited as a ligand for brain-targeting gene delivery. Importantly, the report demonstrated that the RVG29-9R vector was able to cross the blood-brain barrier. RVG29-9R is currently synthesized by commercial companies with high associated costs. In this study, in order to reduce the costs of producing RVG29-9R, we have expressed and purified 6mg of a recombinant peptide (RVG29-9R-6His) from 0.4g of cultured Escherichia coli. We assessed the physiochemical properties of RVG29-9R-6His, its cytotoxicity, and the in vitro transfection efficiency in Neuro 2a cells (which express the acetylcholine receptor). Our results reveal that the RVG29-9R-6His peptide recognized Neuro 2a cells in a dose-dependent manner and it was also able to bind plasmid DNA and deliver it into the Neuro 2a cells effectively. Therefore, our study has demonstrated that the recombinant RVG29-9R-6His peptide retains the functions of RVG29-9R and so may provide an economically viable and alternative production method for the manufacture of RVG29-9R.

An inactivated recombinant rabies CVS-11 virus expressing two copies of the glycoprotein elicits a higher level of neutralizing antibodies and provides better protection in mice.

The rabies virus (RABV) G protein is the primary contributor to the pathogenicity and protective immunity of RABV. In this study, we generated a recombinant rCVS-11-G strain containing two copies of the G protein derived from the pathogenic wild-type (wt) CVS-11 strain and based on its infectious clone. Compared with the wtCVS-11 strain, the rCVS-11-G strain possessed a larger virion and 1.4-fold more G protein, but it exhibited a similar growth property to the rCVS-11 strain, including passaging stability in vitro. qPCR results showed that the two G genes were over-expressed in BHK-21 cells infected with the rCVS-11-G strain. However, the rCVS-11-G strain presented an 80 % lower LD50 than the wtCVS-11 strain when intracranially (i.c.) inoculated in adult mice. Adult mice that were either intracranially (i.c.) or intramuscularly (i.m.) inoculated with rCVS-11-G strain developed more acute neurological symptoms and greater mortality than those inoculated with the wtCVS-11 strain. Furthermore, the rCVS-11-G strain was more easily and rapidly taken up by neuroblastoma cells. These data indicated that the rCVS-11-G strain might have increased neurotropism because of the over-expression of the pathogenic G protein. The inactivated rCVS-11-G strain induced significantly higher levels of virus neutralization antibodies and provided better protection from street rabies virus challenge in mice. Therefore, the rCVS-11-G strain may be a promising inactivated vaccine strain due to its better immunogenicity.

[Research on life history and phenological period of wild-stimulated cultivated Gastrodia elata f. elata in Guizhou].

In order to get to know the imitation of wild Gastrodia elata in life history and phenology period, by G. elata f. elata forest wild simulated cultivation in Dafang county, Guizhou province, observing and recording its morphological characteristics of each growth and development stage. This experiment summarized the law of its life history over 24 months, amplified the characteristics of each 5 phenology periods over the sexual and asexual reproduction of wild simulated cultivated G. elata f. elata in Guizhou. Which the results could clear the process of wild simulated cultivated G. elata f. elata in Guizhou, and provide a theoretical support for the standard technical of the simulated wild G. elata.

[Habitat survey and biological characteristics study of Polygonum multiflorum germplasms in Guizhou region].

OBJECTIVE: To provide reference in selecting premium provenance and improve the cultivation techniques of Polygonum multiflorum. METHODS: Field survey, routine field-observation and sampling fixed plant for analysis in lab were adopted. RESULTS: The growing adaptability of Polygonum multiflorum was very strong, which growed flourishly in the condition with adequate light, ample rainfall, rich heat and fertile soil; Along with the lower of latitude, the vegetative period was prolonged and reproductive stage was delayed, which prolonged the time of roots' nutrition acquisition. Time for root shoot ratio increasing continuously of low latitude germplasms was higher than that of higher latitude germplasms. CONCLUSION: Polygonum multiflorum germplasms have different biological characteristics because of different regions and habitats, which can provide useful reference for selecting premium provenance and adjusting measures to local conditions in different areas.

[Study on germination characteristics of Disporum cantoniense].

OBJECTIVE: To study the seed germination characteristic and optimal germination condition of wild Disporum cantoniense. METHODS: Used wild Disporum cantoniense seed as the test materials, the rate of water absorption of the seed was determined. The germination rates under different conditions, along a temperature gradient (15, 20, 25 and 30 degres C), in light or dark, on top or between wet filter papers, and keeping or removing the seed coat, were determined respectively using petri dish method. At the same time germination trends were observed. RESULTS: The thousand seed weight was 33.24 g, and the seed water-absorbing reached saturation pot after soaking for 30 h. Higher germination rates were respectively recorded at 25 degrees C, between filter papers, and in dark after 24 h soaking in the pretreatment solution. CONCLUSION: The optimal condition for the germination of the seed of wild Disporum cantoniense is as follow: keeping testa, seed soaking for 24 h in seed germination agent and being incubated between wet filter papers in dark at 25 degrees C.

[Germination characters of Polygonum capitatum seed].

OBJECTIVE: To study the influence of the Polygonum capitatum seed germination by different temperatures, cultivating methods and salt stresses, which can provide reference for seed germination inspection and field seeding. METHODS: Cultivating seeds in Petri dishes and count germination rate. RESULTS: The highest germination rate occurs at 15 degrees C; In salt stress condition, with the increase of the salt concentration seed germination rate declines and under the condition of low salt concentration germination rate shows no obvious difference with the controlling group; at the same temperature, germination rate is higher when cultivated with light than under dark, and seeds cultured in the filters have higher germination rate than those which cultured on the filter. CONCLUSION: Germination experiment should carry out at 15 degrees C, with light and between paper. Seeds germinate potential should calculate after the germination experiment progressing 20 d. When planting, salt concentration of the planting seedling field can not above 1 - 2 g/L, otherwise the germination will be affected.

[Emergency prophylactic effects of the avian influenza virus immunized serum on the infected mice].

OBJECTIVE: To evaluate emergency prophylactic effects of the avian influenza virus immunized serum on experimentally infected mice. METHODS: Serum HI antibody titers of 30 mice were detected at day 1 to 19 after being inoculated with 0.2 ml immune serum to estimate half life of immune serum. Ten mice clinical symptom was recorded to estimate the serum security after mice injected 1.5 ml immune serum. Seventy mice were randomly divided into 7 groups according to random number table and inoculated with 0.2 ml, 0.1 ml and 0.05 ml immune serum respectively via intraperitoneal injection on day 8, 4 and 1 prior to challenged with 10 LD(50) influenza virus intranasal. Mice were observed continually for 14 days to calculate the morbidity, mortality, average survival days and compare the lung index and viral titers in lung. RESULTS: Serum HI antibody titers of mice which inoculated with 0.2 ml immune serum maintained 2(6) in 15 days after injection, but drawdown after day 17, the mice injected 1.5 ml immune serum were all alive and none onset. The survival rate of mice which injected 0.2 ml serum on the day 8, 4, 1 before challenge was 80%, 100% and 100%, and the average survival period was 13.1 days, 14.0 days and 14.0 days respectively. The survival rate of mice which injected 0.1 ml and 0.05 ml serum on day 1 before challenge was 100% and 50%, and the average survival days were 14.0 days and 11.7 days respectively. The mice lung index of experimental groups (0.0096 +/- 0.0033 - 0.0145 +/- 0.0060) was smaller than that of viral control group (0.0199 +/- 0.0025), with a statistical significance (P value 0.0022 - 0.0470, < 0.05). The viral titers in lung were significantly decreased by 2 titer as compared to the viral controls. CONCLUSION: The avian influenza virus immunized serum might contain the emergency prophylactic effects and could be developed as an agent for possible human-avian influenza pandemic.

[Cloning and analysis of the Vibrio harveyi dam gene].

The DNA adenine methylase (dam) gene was cloned by degenerate PCR from Vibrio harveyi strain T4. The gene was 840bp in length and encoded a putative protein of 279 amino acids that shared relatively high homology with the Dam of other Vibrios, especially with that of V. parahaemolyticus (96% in identity). The V. harveyi dam gene was subcloned into plasmid pBR322 and the resulting plasmid pBD was introduced into the E. coli strain ER2925 in which the dam gene had been knocked out. Dpn I, Dpn II, and Sau3A I restriction enzyme analysis of the genomic DNA of ER2925 transformed with pBD indicated that the cloned V. harveyi dam gene could functionally complement the E. coli dam mutant and methylate E. coli chromosome at the GATC sites. The 3251 bp upstream region of V. harveyi dam was obtained by genome walking and analyzed at the sequence level. It was found that this 3251 bp region contained two complete open reading frames (ORF): one was of 1101 bp in length and the other was of 1503 bp in length. The predicted amino acid sequence of ORF1101 shared 91% identity with the 3-dehydroquinate synthase of V. parahaemolyticus. The amino acid sequence of ORF1503 shared 80% identity with V. parahaemolyticus DamX. A truncated ORF was found at the upstream of ORF1101, encoding 169 amino acids that shared 94% identity with the shikimate kinase of V. parahaemolyticus. These three genes, together with dam, were arranged in the order of shikimate kinase-3-dehydroquinate synthase-damX-dam. The region immediate upstream of the V. harveyi dam structural gene was cloned in three fragments of different length: 78bp, 112 bp and 477bp (named P78, P112, and P477, respectively) and tested for promoter activity. The results showed that, while all the three fragments had detectable promoter activities, the activity of P78 appeared to be higher than that of P112 and P477.

[Study on conditions of seed germination of Cistanche].

OBJECTIVE: To study the effect of fluridone concentration, stimulating period, temperature and salt on the seed germination of three species of Cistanche. METHOD: The seeds were cultured in Petri dish, and the germination percentage was counted. RESULT: The highest germination percentage was observed in Cistanche tubulosa, C. deserticola, C. sala seeds pre-treated by 0.1 mg x L(-1) fluridone for 24-29 h. The optimal temperature for the seeds germination of three species of Cistanche was at 20-30 degrees C, and the seeds did not germinate at sub-or supraoptimal temperatures (5 and 35 degrees C). The salt tolerance of C. sala seeds was strong, and the critical value of NaCl concentration was 0.04 mol x L(-1). By contrast, C. tubulosa and C. deserticola seeds were more sensitive to the salt stress, the critical value of NaCl concentration was 0.02 mol x L(-1). CONCLUSION: The optimal germination condition and the method of testing germination percentage of three species of Cistanche seeds are as follow: the seeds are pre-treated by 0.1 mg x L(-1) fluridone for 24 h and then cultured at 20-30 degrees C in salt solution which concentration is lower than 0.02 mol x L(-1).

Influence of light intensity and water content of medium on total dendrobine of Dendrobium nobile Lindl.

OBJECTIVE: To ascertain the influence of light intensity and water content of medium on the total dendrobine of Dendrobium nobile (D. nobile). METHOD: The principal component analysis combined with total dendrobine accumulation was conducted to assess the yield and quality of D. nobile in all treatments. In the experiment, D. nobile plants were cultivated in greenhouse as tested materials, and complete test of 9 treatments was adopted with relative light intensities 75.02%, 39.74%, 29.93% and relative water content of medium 50%, 65%, 80%. The plants were treated in June and harvested till December. Indexes including agronomic traits, fresh weight and dry weight of stem and leaf, ash content, extract, and dendrobine were measured. RESULTS: Under the light intensity treatments of 75.02% with 50%, 65%, 80% water content of medium, the basal stems of plants were comparatively thicker with more leaves, and the fresh weight and dry weight of stems and leaves were significantly higher than other 6 treatments. Leaves in all treatments contained dendrobine. Under the light intensity treatments of 75.02% with 50%, 65%, 80% water content of medium, dendrobine content of leaves was lower while dendrobine contents of other treatments were more than 0.60%. After comprehensive assessment through the principal component analysis and total dendrobine accumulation, the results showed that 3 treatments with relative light intensity of 75.02% ranked the top three. CONCLUSIONS: In brief, the moderately strong light intensity and water content of medium from low to medium can facilitate the growth and yield of D. nobile plants, while light intensity from moderately weak to weak can enhance the dendrobine content.

Enhancement of ethyl (S)-4-chloro-3-hydroxybutanoate production at high substrate concentration by in situ resin adsorption.

Asymmetric reduction of ethyl 4-chloro-3-oxobutyrate (COBE) by carbonyl reductases presents an efficient way to produce Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important chiral intermediate for the synthesis of hydroxymethylglutaryl-CoA reductase inhibitors such as Lipitor®. In this study, an NADPH-dependent carbonyl reductase (SrCR) from Synechocystis sp. was characterized to demonstrate a broad substrate spectrum, and the highest activity (53.1U/mg protein) with COBE. To regenerate the cofactor NADPH, Bacillus subtilis glucose dehydrogenase was successfully coexpressed with SrCR. Owing to the product inhibition, no more than 400mM of COBE could be completely reduced to (S)-CHBE using the recombinant Escherichia coli/pET-SrCR-GDH. The macroporous adsorption resin HZ 814 was applied to adsorb (S)-CHBE in situ to alleviate the product inhibitio. Consequently, 3000mM (494g/L) of COBE was bioconverted within 8h, resulting in a (S)-CHBE yield of 98.2%, with 99.4% ee and total turnover number of 15,000, revealed great industrial potential of (S)-CHBE production.

[Morphology of seed germination and parasitism in Cistanche tubulosa].

OBJECTIVE: To understand the process of Cistanche tubulosa. METHOD: The process of seed germination and parasitism was observed using stereomicroscope. RESULT: Seedling of C. tubulosa sprouted after forty day without host root's contact in fields, a tube-like-organ formed and grew auger-type from host root, the tuber apex where touches host root swelled and formed haustorium. Haustorium intruded host root epidermis and vascular bundles, and released brown substances. Then, embryo bud with six or more young leaves formed, finally the swelled tuber-like-organ broken and seed coat shed. Due to the parasitism of C. tubulosa, the host root near stem site swelled, but the other part, shrunk and disappered gradually. CONCLUSION: Seed of C. tubulosa could germinate indepently in fields. Tuber-like-organ formatin, haustorium formation and bud formation are key steps of C. tubulosa seedling development.

[Study on inoculation technology of Cistanche tubulosa in the field].

OBJECTIVE: To increase inoculation rate of Cistanche tubulosa in the field by studying inoculation technologies. METHOD: Root-tube inoculation methed was used on field experiments. Inoculation rate of C. tubulosa was compared to different size seeds and inoculation mediums and inoculation time. RESULT AND CONCLUSION: May is suitable inoculation time. The inoculation rate of C. tubulosa is 92.5% while the seed width is more than 0.7 mm and coarse sand is selected during inoculation period.