[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.

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.

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.

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.

[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.

Cloning, overexpression, and characterization of a high enantioselective nitrilase from Sphingomonas wittichii RW1 for asymmetric synthesis of (R)-phenylglycine.

In this study, a high (R)-enantioselective nitrilase gene from Sphingomonas wittichii RW1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The recombinant nitrilase was purified to homogeneity with a molecular weight of 40 kDa. The pH and temperature optima were shown to be pH 8.0 and 40 °C, respectively. The purified nitrilase was most active toward succinonitrile, approximately 30-fold higher than that for phenylglycinonitrile. Using the E. coli BL21/ReSWRW1 whole cells as biocatalysts, the kinetic resolution for asymmetric synthesis of (R)-phenylglycine was investigated at pH 6.0. A yield of 46 % was obtained with 95 % enantiomeric excess (ee), which made it a promising biocatalyst for synthesis of (R)-phenylglycine.

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.

Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

[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.

Adeno-associated viruses serotype 2-mediated RNA interference efficiently inhibits rabies virus replication in vitro and in vivo.

To investigate the potential of adeno-associated viruses serotype 2 (AAV2)-mediated RNA interference (RNAi) as an antiviral agent against rabies, recombinant AAV2 vectors expressing siRNA targeting the nucleoprotein (N) gene of rabies virus (RABV) (rAAV-N796) were constructed and evaluated. When NA cells pretreated with rAAV-N796 were challenged with RABV, there was a 37.8 ± 3.4% to 55.1 ± 5.3% reduction in RABV virus titer. When cells pre-challenged with RABV were treated with rAAV-N796, there was a 4.4 ± 1.4 to 28.8 ± 3.2% reduction in RABV virus titer. Relative quantification of RABV transcripts using real-time PCR and Western blot revealed that the knockdown of RABV-N gene transcripts was based on the rAAV-N796 inoculation titer. When any NA cells were treated with rAAV-N796 before or after challenged with RABV, significant reduction in virus titer was observed in both administrations. Mice treated intracerebrally with rAAV-N796 exhibited 50 ± 5.3 and 62.5 ± 4.7% protection when challenged intracerebrally or intramuscally, respectively, with lethal RABV. When mice treated intramuscularly with rAAV-N796 were challenged intramuscularly with lethal RABV, they exhibited 37.5 ± 3.7% protection. When mice were intracerebrally and intramuscularly with rAAV-N796 24 hr after exposure to RABV infection, they exhibited 25 ± 4.1% protection The N gene mRNA levels in the brains of challenged mice with three different administrations were reduced (55, 68, 32 and 25%, respectively). These results indicated that AAV2 vector-mediated siRNA delivery in vitro in NA cells inhibited RABV multiplication, inhibited RABV multiplication in vivo in the mice brain and imparted partial protection against lethal rabies. So, it may have a potential to be used as an alternative antiviral approach against rabies.

Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo.

Rabies is an acute fatal encephalitis disease that affects many warm-blooded mammals. The causative agent of the disease is Rabies virus (RABV). Currently, no approved therapy is available once the clinical signs have appeared. Aptamers, oligonucleotide ligands capable of binding a variety of molecular targets with high affinity and specificity, have recently emerged as promising therapeutic agents. In this study, sixteen high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Viral titer assays revealed aptamers could specifically inhibit the replication of RABV in cells but did not inhibit the replication of canine distemper virus or canine parvovirus. In addition, the FO21 and FO24 aptamers, with and without PEGylation, were found to effectively protect mice against lethal RABV challenge. When mice were inoculated with aptamers for 24h prior to inoculation with CVS-11, approximately 87.5% of the mice survived. Here, we report aptamers that could significantly protect the mice from a lethal dose of RABV in vitro and in vivo, as demonstrated by the results for survival rate, weight loss and viral titers. These results indicate that FO21 and FO24 aptamers are a promising agent for specific antiviral against RABV infections.

[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.

Small interfering RNAs targeting the rabies virus nucleoprotein gene.

Rabies virus (RABV) infection continues to be a global threat to human and animal health, yet no curative therapy has been developed. RNA interference (RNAi) therapy, which silences expression of specific target genes, represents a promising approach for treating viral infections in mammalian hosts. We designed six small interfering (si)RNAs (N473, N580, N783, N796, N799 and N1227) that target the conserved region of the RABV challenge virus standard (CVS)-11 strain nucleoprotein (N) gene. Using a plasmid-based transient expression model, we demonstrated that N796, N580 and N799 were capable of significantly inhibiting viral replication in vitro and in vivo. These three siRNAs effectively suppressed RABV expression in infected baby hamster kidney-21 (BHK-21) cells, as evidenced by direct immunofluorescence assay, viral titer measurements, real-time PCR, and Western blotting. In addition, liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival, compared to a non-liposome-mediated delivery method. Collectively, our results showed that the three siRNAs, N796, N580 and N799, targeting the N gene could potently inhibit RABV CVS-11 reproduction. These siRNAs have the potential to be developed into new and effective prophylactic anti-RABV drugs.

Isolation of ssDNA aptamers that inhibit rabies virus.

Aptamers, functional nucleic acids, capable of binding a variety of molecular targets with high affinity and specificity, have emerged as promising therapeutic agents. In this study, the cell surface-systematic evolution of ligands by exponential enrichment (Cell-SELEX) strategy was used to generate DNA aptamers which targeted to the intact rabies virus-infected live cells. Through 35 iterative rounds of selection, five high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Virus titer assay and real-time quantitative reverse transcription PCR (qRT-PCR) assay revealed that all five aptamers could inhibit replication of rabies virus (RABV) in cultured baby hamster kidney (BHK)-21 cells; and T14 and F34 aptamers were most effective. The qRT-PCR also showed a dose-dependent inhibitory effect in BHK-21 cells. Collectively, these data show the feasibility of generating functionally effective aptamers against rabies virus-infected cells by the Cell-SELEX iterative procedure. These aptamers may prove clinically useful as therapeutic molecules with specific antiviral potential against RABV infections.

[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.