Genome-wide identification and expression analysis of growth-regulating factors in Dendrobium officinale and Dendrobium chrysotoxum.

Background: Dendrobium, one of the largest genera in Orchidaceae, is popular not only for its aesthetic appeal but for its significant medicinal value. Growth-regulating factors (GRFs) play an essential role in plant growth and development. However, there is still a lack of information about the evolution and biological function analysis of the GRF gene family among Dendrobiumspecies. Methods: Growth-regulating factors from Dendrobium officinale Kimura et Migo and Dendrobium chrysotoxum Lindl. were identified by HMMER and BLAST. Detailed bioinformatics analysis was conducted to explore the evolution and function of GRF gene family in D. officinale and D. chrysotoxum using genomic data, transcriptome data and qRT-PCR technology. Results: Here, we evaluated the evolution of the GRF gene family based on the genome sequences of D. officinale and D. chrysotoxum. Inferred from phylogenetic trees, the GRF genes were classified into two clades, and each clade contains three subclades. Sequence comparison analysis revealed relatively conserved gene structures and motifs among members of the same subfamily, indicating a conserved evolution of GRF genes within Dendrobiumspecies. However, considering the distribution of orthologous DoGRFs and DcGRFs, and the differences in the number of GRFs among species, we suggest that the GRF gene family has undergone different evolutionary processes. A total of 361 cis-elements were detected, with 33, 141, and 187 related to plant growth and development, stress, and hormones, respectively. The tissue-specific expression of GRFs showed that DoGRF8 may have a significant function in the stem elongation of D. officinale. Moreover, four genes were up-regulated under Methyl-jasmonic acid/methyl jasmonate (MeJA) treatment, showing that DoGRFs and DcGRFs play a crucial role in stress response. These findings provide valuable information for further investigations into the evolution and function of GRF genes in D. officinale and D. chrysotoxum.

Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with "short" and "long" inverse agonists.

A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the "short" inverse agonist 6. With the increase in the size of the 6-position substituents, the "short" inverse agonist 6 first reversed its function to agonists and then to "long" inverse agonists. The cocrystal structures of RORγt complexed with the representative "short" inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the "long" inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of "short" inverse agonist 6 and "long" inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that "short" or "long" inverse agonists led to different behaviors of helixes H11, H11', and H12 of RORγt. The "short" inverse agonist 6 destabilizes H11' and dislocates H12, while the "long" inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.

N-Butylphthalide Improves Cognitive Function in Rats after Carbon Monoxide Poisoning.

Cognitive impairment is the most common neurologic sequelae after carbon monoxide (CO) poisoning, and the previous investigations have demonstrated that N-Butylphthalide (NBP) could exert a broad spectrum of neuroprotective properties. The current study aimed to investigate the effect of NBP on cognitive dysfunction in rats after acute severe CO poisoning. Rats were randomly divided into a normal control group, a CO poisoning group and a CO+NBP group. The animal model of CO poisoning was established by exposure to CO in a chamber, and then all rats received hyperbaric oxygen therapy once daily, while rats in CO+NBP group were administered orally NBP (6 mg/ 100g) by gavage twice a day additionally. The results indicated that CO poisoning could induce cognitive impairment. The ultrastructure of hippocampus was seriously damaged under transmission electron microscopy, and the expressions of calpain 1 and CaMK II proteins were significantly elevated after CO exposure according to the analysis of immunofluorescence staining and western blot. NBP treatment could evidently improve cognitive function, and maintain ultrastructure integrity of hippocampus. The expression levels of both calpain 1 and CaMK II proteins in CO+NBP group were considerably lower than that of CO poisoning group (P < 0.05). Taken together, this study highlights the molecular mechanism of cognitive dysfunction in rats after CO exposure via the upregulation of both calpain 1 and CaMK II proteins. The administration of NBP could balance the expressions of calpain 1 and CaMK II proteins and improve cognitive function through maintaining ultrastructural integrity of hippocampus, and thus may play a neuroprotective role in brain tissue in rats with CO poisoning.

Effect of psychological intervention on the side effects of ziprasidone in the treatment of schizophrenia / 中国生化药物杂志

Objective To explore the effects of ziprasidone combined with psychological intervention on drug related adverse reactions in the treatment of schizophrenia. Methods 62 patients with schizophrenia collected in Shaoxing seventh people's hospital from January 2016 to January 2017 were divided into study group (n=31) and control group (n=31) by single and double digital methods. The control group were received ziprasidone, the study group were received ziprasidone combined with psychological intervention. PANSS score and drug related adverse reactions in the two groups were recorded. Results Before treatment, PANSS score in the two groups has no statistical difference. PANSS score in the study group were decreased significantly than that in the control group (P<0.05). Insomnia or sleepiness, gastrointestinal reactions, headache, dizziness and other drug related adverse reactions in the study goup were significantly lower than those in the control group (P<0.05). Conclusion In schizophrenia treatment, ziprasidone combined with psychological intervention can effectively improve the therapeutic effect on the basis of reducing drug-related adverse reactions.

Effect of psychological intervention on the side effects of ziprasidone in the treatment of schizophrenia / 中国生化药物杂志

Objective To explore the effects of ziprasidone combined with psychological intervention on drug related adverse reactions in the treatment of schizophrenia. Methods 62 patients with schizophrenia collected in Shaoxing seventh people's hospital from January 2016 to January 2017 were divided into study group (n=31) and control group (n=31) by single and double digital methods. The control group were received ziprasidone, the study group were received ziprasidone combined with psychological intervention. PANSS score and drug related adverse reactions in the two groups were recorded. Results Before treatment, PANSS score in the two groups has no statistical difference. PANSS score in the study group were decreased significantly than that in the control group (P<0.05). Insomnia or sleepiness, gastrointestinal reactions, headache, dizziness and other drug related adverse reactions in the study goup were significantly lower than those in the control group (P<0.05). Conclusion In schizophrenia treatment, ziprasidone combined with psychological intervention can effectively improve the therapeutic effect on the basis of reducing drug-related adverse reactions.

[Evaluation of genetic diversity and population structure of Bletilla striata based on SRAP markers].

Bletilla striata has been used as traditional Chinese medicine for several centuries. In recent years, the quality and quantity of wild B. striata plants have declined sharply due to habitat deterioration and human over-exploitation. Therefore, it is of great urgency to evaluate and protect B. striata wild plant resource. In this study, sequence-related amplified polymorphism (SRAP) markers were applied to assess the level and pattern of genetic diversity in twelve populations of B. striata. The results showed a high level of genetic diversity (PPB = 90.48%, H = 0.349 4, I = 0.509 6) and moderate genetic differentiation among populations (G(st) = 0.260 9). Based on the unweighted pair-group method with arithmetic average (UPGMA), twelve populations gathered in three clusters. The cluster 1 included four populations. There are Nanjing, Zhenjiang, Xuancheng and Hangzhou. The seven populations which come from Hubei Province, Hunan Province, Jiangxi Province and Guizhou Province belonged to the cluster 2. The cluster 3 only contained Wenshan population. Moreover, Mantel test revealed significant positive correlation between genetic distances and geographic distances (r = 0.632 9; P < 0.000 1). According to the results, we proposed a series of conservation consideration for B. striata.

[Molecular identification of wild populations of Dendrobium officinale based on target region amplification polymorphism].

In this study, TRAP molecular markers were used in identification of wild populations and hybrids of Dendrobium officinale, based on the sequences of genes encoding phosphoenolpyruvate carboxylase (PEPC), UDP-glucose pyrophosphorylase (UGP) and phenylalanine ammonia-lyase (PAL). Seven polymorphic target region amplification polymorphism (TRAP) primers were selected from 54 primer combinations and used in the identification of wild populations. Moreover, hybrids had female polymorphic bands, male polymorphic bands and heterozygous bands, which suggest that seven TRAP markers are able to identify the hybrids from their parents. Furthermore, the UPGMA dendrogram revealed that when sample from Guangnan in Yunnan province was used as one parent, reciprocal hybrids grouped with them in first, and then grouped with the other parent. The results indicated that the hybrids were closer to D. officinale from Guangnan population. This study identified the wild populations and hybrids of D. officinale by TRAP molecular markers, which is useful in selection of good varieties for artificial cultivation and early identification of hybrids. The study provides a method in the control of stability of germplasm and quality of D. officinale.

Chemical Synthesis and Oxide Dispersion Properties of Strengthened Tungsten via Spark Plasma Sintering.

Highly uniform oxide dispersion-strengthened materials W-1 wt % Nd2O3 and W-1 wt % CeO2 were successfully fabricated via a novel wet chemical method followed by hydrogen reduction. The powders were consolidated by spark plasma sintering at 1700 °C to suppress grain growth. The samples were characterized by performing field emission scanning electron microscopy and transmission electron microscopy analyses, Vickers microhardness measurements, thermal conductivity, and tensile testing. The oxide particles were dispersed at the tungsten grain boundaries and within the grains. The thermal conductivity of the samples at room temperature exceeded 140 W/m·K. The tensile tests indicated that W-1 wt % CeO2 exhibited a ductile-brittle transition temperature between 500 °C and 550 °C, which was a lower range than that for W-1 wt % Nd2O3. Surface topography and Vickers microhardness analyses were conducted before and after irradiations with 50 eV He ions at a fluence of 1 × 1022 m-2 for 1 h in the large-powder material irradiation experiment system. The grain boundaries of the irradiated area became more evident than that of the unirradiated area for both samples. Irradiation hardening was recognized for the W-1 wt % Nd2O3 and W-1 wt % CeO2 samples.

[Applylication of new type combined fragments: nrDNA ITS+ nad 1-intron 2 for identification of Dendrobium species of Fengdous].

In this study, 17 kinds of Dendrobium species of Fengdous including 39 individuals were collected from 4 provinces. Mitochondrial gene sequences co I, nad 5, nad 1-intron 2 and chloroplast gene sequences rbcL, matK amd psbA-trnH were amplified from these materials, as well as nrDNA ITS. Furthermore, suitable sequences for identification of Dendrobium species of Fengdous were screened by K-2-P and P-distance. The results showed that during the mentioned 7 sequences, nrDNA ITS, nad 1-intron 2 and psbA-trnH which had a high degree of variability could be used to identify Dendrobium species of Fengdous. However, single fragment could not be used to distinguish D. moniliforme and D. huoshanense. Moreover, compared to other combined fragments, new type combined fragments nrDNA ITS+nad 1-intron 2 was more effective in identifying the original plants of Dendrobium species and could be used to identify D. huoshanense and D. moniliforme. Besides, according to the UPGMA tree constructed with nrDNA ITS+nad 1-intron 2, 3 inspected Dendrobium plants were identified as D. huoshanense, D. moniliforme and D. officinale, respectively. This study identified Dendrobium species of Fengdous by combined fragments nrDNA ITS+nad 1-intron 2 for the first time, which provided a more effective basis for identification of Dendrobium species. And this study will be helpful for regulating the market of Fengdous.

[Study on Chemical Kinetic Effect of Dielectric Barrier Discharge Plasma].

To reveal the mechanism of plasma (assisted the ignition process of methane/air further, schematic of dielectric barrier discharge plasma system with atmospheric air was designed and set up, the emission spectrum of dielectric barrier discharge plasma with atmospheric air was measured, and the active particles produced by the interaction of dielectric barrier discharge plasma with atmospheric air were analyzed with the spectrum technology, the ignition model and calculation methods of sensitivity analysis and reaction path analysis were given, effects of NO and O3 on the ignition delay time were simulated, and the chemical kinetics mechanism of NO and O3 assisted ignition was revealed via sensitivity analysis and reaction path analysis. The results show that main excited particles of N2 and O3 are generated via effect of plasma on the atmospheric air, which are converted into active particles of NO(ξ) and O3 in the end, the life of which are longer than any other active particles, effects of plasma on the ignition is simplified as effects of NO(ξ) and O3 on the ignition; NO and O3 could reduce the ignition delay time significantly, but the amplitude decrease with increase of the initial temperature, this is because the rate of ignition is decided by the oxidation rate of CH3, the oxidized pathway of CH3 is R155 and R156 for auto-ignition and their rates are slower when temperature is low, so the ignition delay time of methane/air is longer; NO could reduce the ignition delay time significantly because of the oxidized pathway of CH3 is changed to R327 CH3O2 + NO = CH3O + NO2, R328 CH3 + NO2 = CH3O + NO for NO(ξ) (assisted ignition process from R155 and R156 for auto-ignition; and the chemical kinetic effect is the dominating factor of O3 on the ignition and which change the reaction path.

[Ophiopogonin D protects cardiomyocytes against doxorubicin-induced injury through suppressing endoplasmic reticulum stress].

This study aimed to examine whether ophiopogonin D (OP-D) is capable of protecting cardiomyocytes against DOX-induced injury and the mechanisms involved. H9c2 cells were cultured. MTT assay was used to evaluate cell viability and toxicity. Mito-tracker as fluorescence probe was used to measure ROS content raised from mitochondria. The mRNA and protein expression of ATF6alpha, GRP78 and CHOP were analyzed using real-time PCR and Western blotting, respectively. The results showed that a significant endoplasmic reticulum stress (ERS) was induced upon exposure of H9c2 cells to DOX as indicated by the increase in the expression of ERS related proteins, which was paralleled with the accumulation of reactive oxygen species (ROS) and decrease in the viability of H9c2 cells. Whereas, DOX-induced ROS accumulation and up-regulation of ERS related proteins were partially abolished by pretreatment with OP-D. Consequently, a DOX-induced ERS was mitigated by application of OP-D. Similarly, DOX-induced decrease in cell viability was partially attenuated by either inhibiting CHOP or pretreatment with N-acetylcysteine (NAC), an antioxidant. Moreover, cardiac ultrastructural abnormalities seen in mouse receiving DOX injections were obviously ameliorated by pretreatment of OP-D. Taken together, the present study proved that OP-D protects cardiomyocytes against DOX-induced injury, at least in part, through reducing ROS accumulation and alleviating ERS.

Comparative chloroplast genomes of photosynthetic orchids: insights into evolution of the Orchidaceae and development of molecular markers for phylogenetic applications.

The orchid family Orchidaceae is one of the largest angiosperm families, including many species of important economic value. While chloroplast genomes are very informative for systematics and species identification, there is very limited information available on chloroplast genomes in the Orchidaceae. Here, we report the complete chloroplast genomes of the medicinal plant Dendrobium officinale and the ornamental orchid Cypripedium macranthos, demonstrating their gene content and order and potential RNA editing sites. The chloroplast genomes of the above two species and five known photosynthetic orchids showed similarities in structure as well as gene order and content, but differences in the organization of the inverted repeat/small single-copy junction and ndh genes. The organization of the inverted repeat/small single-copy junctions in the chloroplast genomes of these orchids was classified into four types; we propose that inverted repeats flanking the small single-copy region underwent expansion or contraction among Orchidaceae. The AT-rich regions of the ycf1 gene in orchids could be linked to the recombination of inverted repeat/small single-copy junctions. Relative species in orchids displayed similar patterns of variation in ndh gene contents. Furthermore, fifteen highly divergent protein-coding genes were identified, which are useful for phylogenetic analyses in orchids. To test the efficiency of these genes serving as markers in phylogenetic analyses, coding regions of four genes (accD, ccsA, matK, and ycf1) were used as a case study to construct phylogenetic trees in the subfamily Epidendroideae. High support was obtained for placement of previously unlocated subtribes Collabiinae and Dendrobiinae in the subfamily Epidendroideae. Our findings expand understanding of the diversity of orchid chloroplast genomes and provide a reference for study of the molecular systematics of this family.

[DNA barcoding research and its application on medicinal plants of Bletilla H. G. Reichenbach].

To identify adulterants from medicinal plants of Bletilla H. G. Reichenbach, the suitable candidate DNA barcoding of Bletilla was evaluated. In this study, the internal transcribed spacer (ITS) of nuclear ribosomal DNA, the LFY homologous gene intron 2 and chloroplast ycfl gene were amplified and sequenced from forty-one samples. The intra-specific and inter-specific divergences of Bletilla were calculated, and the identification efficiency was assessed using Barcoding Gap, NJ tree by K2P distance and BLAST1 method. The result showed the intra-specific divergence of nrDNA ITS and ycJfl (0.022-0.106 and 0.017-0.106) were obviously higher than the inter-specific divergence (0-0.012 and 0-0.015), and four species of Bletilla were also accurately distinguished in NJ trees. Whereas, there was no Barcoding Gap on LFY homologous gene intron 2, thus it cannot effectively identify species of Bletilla. Using NJ tree of nrDNA ITS and ycfl gene, powdery medicine and the adulterants of Bletilla were successfully unidentified. In conclusion, nrDNA ITS and ycfl can be used as a potential DNA barcoding to identify the medicinal plants in Bletilla and its adulterants. There were only three basic differences on nrDNA ITS between "Jujing baiji" and Bletilla striata of Lu'an in Anhui province, and two basic differences in ycfl. Based on morphological and molecular data, "Jujing baiji" could be recognized as the species of Bletilla striata.

[Sequence analysis of LEAFY homologous gene from Dendrobium moniliforme and application for identification of medicinal Dendrobium].

The LEAFY (LFY) homologous gene of Dendrobium moniliforme (L.) Sw. was cloned by new primers which were designed based on the conservative region of known sequences of orchid LEAFY gene. Partial LFY homologous gene was cloned by common PCR, then we got the complete LFY homologous gene Den LFY by Tail-PCR. The complete sequence of DenLFY gene was 3 575 bp which contained three exons and two introns. Using BLAST method, comparison analysis among the exon of LFY homologous gene indicted that the DenLFY gene had high identity with orchids LFY homologous, including the related fragment of PhalLFY (84%) in Phalaenopsis hybrid cultivar, LFY homologous gene in Oncidium (90%) and in other orchid (over 80%). Using MP analysis, Dendrobium is found to be the sister to Oncidium and Phalaenopsis. Homologous analysis demonstrated that the C-terminal amino acids were highly conserved. When the exons and introns were separately considered, exons and the sequence of amino acid were good markers for the function research of DenLFY gene. The second intron can be used in authentication research of Dendrobium based on the length polymorphism between Dendrobium moniliforme and Dendrobium officinale.

Identification of the geographic origin of Dendrobium thyrsiflorum on Chinese herbal medicine market using trinucleotide microsatellite markers.

The stems of Dendrobium thyrsiflorum RCHB.F. ex ANDRÉ can be processed into an important class of Traditional Chinese Medicine named "Huangcao Shihu," which has diverse curative effects, such as nourishing yin and clearing away unhealthy heat, benefiting the stomach, and promoting the production of body fluid. The identification of the geographical origin of D. thyrsiflorum is vital for preserving its natural resource and ensuring the quality of "Huangcao Shihu." In order to identify the origin of D. thyrsiflorum on Chinese herbal medicine market, 14 D. thyrsiflorum-specific microsatellite markers were developed in this study. Assignment tests were performed by the microsatellite marker analysis coupled with three new statistical approaches (partially Bayesian, frequency-based, and fully Bayesian methods) to determine the origin populations of 12 commercial samples of "Huangcao Shihu" collected from a medicine market in Nanjing, Jiangsu Province, China. Their genotypes were compared with those of 136 individuals belonging to five wild D. thyrsiflorum populations from China, Thailand, India, Myanmar, and Laos. Comparisons of the probabilities of 12 unknown individuals originating from each candidate population indicated that most of them appeared to originate from Myanmar and Laos. This suggests that the two countries may be the predominant sources of D. thyrsiflorum on the medicine market in Nanjing. In addition, the 14 microsatellite markers developed in this study may be an effective tool for identification of the origin of commercial available "Huangcao Shihu" and play an important role in its quality control.

[Evaluation of genetic diversity of Dendrobium officinale wild populations based on RAMP markers].

Random amplified microsatellite polymorphism (RAMP) markers were used to access the genetic diversity among 112 samples of nine populations of Dendrobium officinale Kimura et Migo. Using 16 informative primers, 123 bands were amplified and 86 (69.92%) were polymorphic. The polymorphic bands from three to eight could be detected for each RAMP primer, with a mean of 5, indicating abundant genetic diversity among populations. Genetic similarity coefficients ranged from 0.250 to 0.813. UPGMA dendrogram illustrated 9 populations clustered into 3 groups, and the cluster pattern showed correlation with the locations of the D. officinale populations. These results were supported by the previous conclusions that were achieved by other molecular markers, and RAMP is proved to be effective for evaluating the genetic diversity of wild populations of Dendrobium officinale.

Anthraquinones from the roots of Knoxia valerianoides.

Five new 9,10-anthraquinones (1-5) were isolated from an ethanol extract of the roots of Knoxia valerianoides. Their structures including absolute configuration of 1 were determined by spectroscopic analysis. Compounds 4 and 5 showed moderate activity against nitrogen oxide production in macrophages induced by lipopolysaccharide, at 10(- 5) M, with inhibition ratios of 50.4 ± 3.6 and 41.7 ± 2.1%, respectively.

[Development of microsatellites of Dendrobium officinale and its application in purity identification of germplasm].

Simple sequence repeat (SSR) was used to investigate the genetic diversity and structure of Dendrobium officinale. A total of 15 primer pairs with stable and repeatable polymorphism were screened out from 60 SSR primer pairs developed by the method of microsatellite enrichment by magnetic beads. Forty-eight samples of Dendrobium officinale were analyzed in genetic polymorphism. These loci were polymorphic and displayed 3 to 9 alleles per locus with a mean number of 6.1. The observed and expected heterozygosities ranged from 0.60 to 0.85 and from 0.49 to 0.85 respectively. The polymorphic information content (PIC) of each SSR locus varied from 0.437 to 0.829 with an average of 0.702. Fifteen primer pairs were used in Dendrobium cross-species amplification and totally 13 primer pairs were proved to have the transferability in D. officinale related species. In addition, 500 tissue culture plantlets of D. officinale were tested for purity identification by means of PCR amplification with four SSR primer pairs. The results showed that SSR technique is a feasible, simple and inexpensive method for determining adulterants in germplasm identification.

[Analysis and authentication of cpDNA psbA-trnH regions of Dendrobium species of fengdous].

The psbA-trnH regions of Dendrobium species of Fengdous were sequenced by our research group. The psbA-trnH sequences of fifteen Dendrobium species were analyzed with software MEGA 4.0. The results showed that the lengths of sequences varied from 721 to 767 bp. The variable sites were 42 while the informative sites were 11. Genetic distances were calculated using the Kimura 2-parameter model. Genetic distances varied from 0.0013-0.0183 among fifteen species while the average genetic distance was 0.0148. The interspecies differences of psbA-trnH regions were demonstrated. Six indels happened in this fragment, which led to the great difference of sequence lengths among fifteen species. We found that there were no population differences in the psbA-trnH region of various species of Fengdous so far. By using the database of various Dendrobium species of Fengdous and two genetics software, the botanical origin of the inspected species of Fengdous was authenticated successfully by sequencing the psbA-trnH regions. The psbA-trnH region of cpDNA can be used as a candidate marker for authentication of Dendrobium species of Fengdous.

[Molecular identification of heparin from pigs by allele-specific PCR (AS-PCR) and amplification refractory mutation system (ARMS)].

The aim of this study is to develop a convenient and effective method for the identification of heparin from pigs (include Sus scrofa domestica Brisson and Sus scrofa riukiuanus). Based on sequences of D-loop region of pigs and the other animals, two pairs of highly specific primers were designed for distinguishing heparin of pigs from other animals. The primers were employed to amplify D-loop region of DNA templates extracted from pig and seven other animal species that amounted to 49 samples. AS-PCR (allele-specific PCR) and ARMS (amplification refractory mutation system) were all suitable for fast identification of heparin from pig with anneal temperature at 54-56 degrees C in AS-PCR and with wider anneal temperature in ARMS,at 52-58 degrees C. An about 170 bp DNA fragments were amplified from separately pigs and whereas no DNA fragment was amplified from other samples under the same reaction condition.