The complete chloroplast genome of Wurfbainia neoaurantiaca (Zingiberaceae: Zingiberea) from Yunnan Province, China.

Wurfbainia neoaurantiaca is a medicinal plant endemic to Yunnan Province, China. In this study, its complete chloroplast genome was assembled and characterized. The total genome size of W. neoaurantiaca was 158,484 bp in length, consisting of a large single-copy region (LSC), a small single-copy region (SSC) and two inverted repeat regions (IRs) with 88,605 bp, 15,285 bp and 29,822 bp, respectively. Its GC content was 36.08%. The chloroplast genome encoded 113 unique genes, including 79 protein-coding, 30 tRNA, and four rRNA genes. The result of the phylogenetic analysis indicated that W. neoaurantiaca was related to W. villosa var. xanthioides and supported de Boer's classification that W. compacta, W. longiligularis, W. neoaurantiaca, W. villosa, W. villosa var. xanthioides and Amomum krervanh belonged to the Wurfbainia Clade.

Comparative transcriptomic analysis reveals genes regulating the germination of morphophysiologically dormant Paris polyphylla seeds during a warm stratification.

We previously analyzed the expression of genes associated with Paris polyphylla var. yunnanensis seed maturation and dormancy release; however, we were unable to clarify the relationship between gene expression levels and these processes. To reveal the molecular mechanisms underlying P. polyphylla var. yunnanensis seed dormancy release during a warm stratification, the transcriptomes of dormant and germinating P. polyphylla var. yunnanensis seeds were separately analyzed by RNA sequencing and were also compared with the transcriptomes of stem-leaf and root tissues harvested during the seed maturation stage. The RNA sequencing of five tissues generated 234,331 unigenes, of which 10,137 (4.33%) were differentially expressed among the analyzed tissues. The 6,619 unigenes whose expression varied among mature dormant, sprouted, and germinated seeds included 95 metabolic and 62 signaling genes related to abscisic acid, gibberellin, auxin, brassinosteroid, cytokinin, ethylene, jasmonic acid and salicylic acid. Additionally, 243 differentially expressed genes were annotated as known seed dormancy/germination-related genes. Among these genes, 109 were regulated by hormones or involved in hormone signal transduction. Finally, 310 transcription factor unigenes, including 71 homologs of known seed dormancy/ germination-related genes, were observed to be differentially expressed during a warm stratification. These results confirm that multiple hormones and transcription factors influence P. polyphylla var. yunnanensis seed dormancy release and germination during a warm stratification. This study identified candidate genes (e.g., ABI5) that should be cloned and functionally characterized regarding their effects on the release of P. polyphylla var. yunnanensis seed morphophysiological dormancy.

Quality evaluation of Panax quinquefolium from different cultivation regions based on their ginsenoside content and radioprotective effects on irradiated mice.

Ginsenosides are one of major types of bioactive compounds in American ginseng (AG) and utilized to assess the quality of various AG samples. The contents of ginsenosides showed cultivation region-related variation, which is possibly associated with AG's pharmacological effect difference. Therefore, to reveal the quality difference of AGs in different cultivation regions, AG samples from seven cultivation regions were evaluated via analyzing their contents of nine ginsenosides and the biochemical parameters in AG-treated irradiated mice. Pre-administration of AG decoctions could reversely modulate the irradiation-induced changes of antioxidant enzymatic activity, cytokine level and hormone level in irradiated mice, which demonstrated that AG had the radioprotective effects due to its antioxidative, immunomodulatory and anti-inflammatory properties. However, this radioprotection effect varied among different cultivation regions of AGs. Collectively, Beijing and Canada-cultivated AGs had the best radioprotection. Heilongjiang and Jilin-originated AGs had the similar pharmacological effects while USA, Shandong and Shaanxi-grown AGs had closer pharmacological effects. This biochemical measurements-based PCA and heatmap clustering of AGs from seven cultivation regions was nearly consistent with ginsencoside content- and the previous serum metabolome-based analyses. However, the pearson correlation analysis revealed that only Rb3 and Rd were significantly correlated with some of assayed biochemical parameters in irradiated mice pretreated with different cultivation regions of AG extracts.

Tumor ablation using low-intensity ultrasound and sound excitable drug.

The cell membrane is a semi-fluid container that defines the boundary of cells, and provides an enclosed environment for vital biological processes. A sound excitable drug (SED) that is non-cytotoxic to cells is developed to disrupt the plasma membrane under gentle ultrasound insonation, 1MHz, 1W/cm2. The frequency and power density of insonation are within the physical therapy and medical imaging windows; thus the applied ultrasound is safe and not harmful to tissues. The insertion of SEDs into the plasma membrane is not toxic to cells; however, the intruding SEDs weaken the membrane's integrity. Under insonation, the ultrasound energy destabilized the SED disrupted membranes, resulting in membrane rupture and eventual cell death. In a xenograft breast tumor model, the SED alone or the ultrasound alone caused little adverse effects to tumor tissue, while the combined treatment triggered necrosis with a brief local insonation of 3min. The described sono-membrane rupture therapy could be a safe alternative to the currently used high-energy tissue ablation technology, which uses X-rays, gamma rays, electron beams, protons, or high-intensity focused ultrasound.

Transcriptomic analysis of American ginseng seeds during the dormancy release process by RNA-Seq.

American ginseng (Panax quinquefolius L.) is an important herb that is cultivated in China, North American, and South Korea. It is propagated from seed, but the seed has deep dormancy characteristics described as morphophysiological dormancy. Two-stage temperature stratification, a warm (15-20°C) and cold (2°C) stratification period of 6 months, has been used successfully for seed dormancy release. However, little is known about the molecular mechanisms of seed dormancy release in the stratification process. In this study, seed development after pollination and seed development in the dormancy release process were investigated in American ginseng. The transcriptome during seed dormancy release was analyzed using RNA-Seq technology and 78,207 unigenes (mean length 531 bp) were generated. Based on similarity searches of public databases, 54,292 of the unigenes (69.4%) were functionally annotated. Further, three digital gene expression (DGE) libraries were sequenced and differences in gene expression at three stages during seed cold stratification were examined. The greatest number of differentially expressed genes occurred in the 90DCS versus 180DCS libraries, while the lowest number of differentially expressed genes occurred in the 135DCS verus 180DCS libraries. GO enrichment analysis revealed that 59, 29, and 39 GO terms were significantly enriched in the biological process, molecular function, and cell component GO categories, respectively. There were 25,190 genes with KEGG pathway annotation in the three DGE libraries and their enrichment pathways were compared. The gene expressions of 30 selected unigenes were validated using quantitative PCR. This study is the first to provide the transcriptome sequences for seed dormancy release in American ginseng, and demonstrates the successful use of DGE profiling data for analyzing transcriptomic variation during dormancy release. These data provide a basis for future researches of seed dormancy in morphophysiological dormancy seeds in non-model plants.

[Study on distribution and dynamic accumulation of catalpol and total iridoid in fresh Rehmannia glutinosa].

Iridoid glycosides were the main active ingredient of Rehmannia glutinosa, of which catalpol has the highest content. This work will provide theoretical basis for metabolic study and cultivation of iridoids on the basis of the dynamic accumulation of catalpol and total iridoids in the growth of R. glutinosa. The samples of rehmannia 85-5 were gathered in the same filed from July to October. The contents of catalpol and total iridoid glycosides were measured by HPLC and specteophotometric, respectively. The results showed that youngest leaves had the higher content of catalpol and total iridoid glyosides than that of the other two leaf ages in the same growth stage from July to September, while their content of catalpol and total iridoid glycosides were all decreased as the growth of leaves of R. glutinosa. The content of catalpol didn't differ significantly from July to September, whereas it has significantly increased in October in the three leaf stage. In the same stage, the wider the root diameter is, the higher content of the effective components are. In August and September, the total iridoid glycosides have the fastest accumulation. The content of catalpol was increased as the accumulation of total iridoid glycosides.

Mining genes involved in the stratification of Paris polyphylla seeds using high-throughput embryo transcriptome sequencing.

BACKGROUND: Paris polyphylla var. yunnanensis is an important medicinal plant. Seed dormancy is one of the main factors restricting artificial cultivation. The molecular mechanisms of seed dormancy remain unclear, and little genomic or transcriptome data are available for this plant. RESULTS: In this study, massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform was used to generate a substantial sequence dataset for the P. polyphylla embryo. 369,496 high quality reads were obtained, ranging from 50 to 1146 bp, with a mean of 219 bp. These reads were assembled into 47,768 unigenes, which included 16,069 contigs and 31,699 singletons. Using BLASTX searches of public databases, 15,757 (32.3%) unique transcripts were identified. Gene Ontology and Cluster of Orthologous Groups of proteins annotations revealed that these transcripts were broadly representative of the P. polyphylla embryo transcriptome. The Kyoto Encyclopedia of Genes and Genomes assigned 5961 of the unique sequences to specific metabolic pathways. Relative expression levels analysis showed that eleven phytohormone-related genes and five other genes have different expression patterns in the embryo and endosperm in the seed stratification process. CONCLUSIONS: Gene annotation and quantitative RT-PCR expression analysis identified 464 transcripts that may be involved in phytohormone catabolism and biosynthesis, hormone signal, seed dormancy, seed maturation, cell wall growth and circadian rhythms. In particular, the relative expression analysis of sixteen genes (CYP707A, NCED, GA20ox2, GA20ox3, ABI2, PP2C, ARP3, ARP7, IAAH, IAAS, BRRK, DRM, ELF1, ELF2, SFR6, and SUS) in embryo and endosperm and at two temperatures indicated that these related genes may be candidates for clarifying the molecular basis of seed dormancy in P. polyphlla var. yunnanensis.

[Comparison of rattan microscopic characteristics of four Dai medicinal materials from Uncaria genus].

OBJECTIVE: To establish microscopic identification standards for four species from Uncaria genus. METHODS: The microscopic characteristics of transvers section and powder of these four species rattan were observed. RESULTS: As for these four species from Uncaria genus, the microscopic identification of rattan transvers section could be made by the existence of cork cambium and pericycle in the cortex, and the location of phloem fiber and stone cells, while the powder identification could be made according to the existence of scalariform vessel and calcium oxalate crystal, and the quantity of stone cells. CONCLUSION: The microscopic characteristics of rattan can provide information for identification of these four species from Uncaria genus.

[Effects of seedling quality on growth of Rehmannia glutinosa and oligosaccharide content].

OBJECTIVE: To study the effect of seedling quality on growth, yield and quality of Rehmannia glutinosa at harvest and build a basis for its GAP. METHOD: The seedling quality of R. glutinosa in main producing regions was surveyed to understand the current status of seedling quality. Field experiments with different varieties and seedling quality were conducted to measure dry matter accumulation with different growth of R. glutinosa and oligosaccharide content, and economic yield at harvest. RESULT: The seedling was randomly selected by farmers in R. glutinosa producing regions. Seedling quality could significantly improve on seedling emergence rate, and promote seedling growth, especially with early stage R. glutinosa, finally increase yield at harvest. At harvest, 63% and 50% of yield with A and B seedling could be improved for variety of 85-5, and 50% and 47% of yield could be increased for variety of Beijing No. 1, compared to the C seeding. CONCLUSION: In cultivation, the seedlings with the diameter > 1.5 cm should be transplanted firstly.

[Study on seed quality of Achyranthes bidentata].

OBJECTIVE: To study the seed quality of Achyranthes bidentata from different sources and establish a standard of seed quality grading. METHOD: Seed weight and moisture content determination, germination test, purity analysis were carried out by 100 grains method, high-temperature drying, double deck filter paper and GB/T354. 3-Seed Testing for Crops methods respectively. Eight collections were planted in several field plots. RESULT: The 1 000 grains weight of 33 collections is 0.69 to 3.54 g with a variance coefficient 24.83%, the germination rate between 19.02% to 99% with a variance coefficient 38.24%. Seed moisture content and purity showed no significantly difference. K-means cluster analysis showed that 33 collections were divided into 3 grades: the first grade including 23 collections, the second and third grade including 5 collections respectively. Eight collections showed different field growth and yield characters. CONCLUSION: There are significant differences in seed quality of A. bidentata, so the classification management is needed in medicinal materials market. Germination test is an effective method before cultivating.

Cancer treatment using an optically inert Rose Bengal derivative combined with pulsed focused ultrasound.

Pulsed high intensity focused ultrasound (HIFU) has been combined with a photo-insensitive Rose Bengal derivative (RB2) to provide a synergistic cytotoxicity requiring the presence of both ultrasonic cavitation and drug. In vitro tests have shown that a short treatment (less than 30 s) of pulsed HIFU with peak negative pressure >7 MPa (~27 W acoustic power at 1.4 MHz) destroys >95% of breast cancer cells MDA-MB-231 in suspension with >10 µM of the compound. Neither the pulsed HIFU nor the RB2 compound was found to have any significant impact on the viability of the cells when used alone. Introducing an antioxidant (N-acetylcysteine) reduced the effectiveness of the treatment. In vivo tests using these same cells growing as a xenograft in nu/nu mice were also done. An ultrasound contrast agent (Optison) and lower frequency (1.0 MHz) was used to help initiate cavitation at the tumor site. We were able to demonstrate tumor regression with cavitation alone, however, addition of RB2 compound injected i.v. yielded a substantial synergistic improvement.

[Soil microbial community composition and diversity in Panax quinquefolius rhizosphere].

The objective of this work was to analyze the soil microbial community diversity and structure in Panax quinquefolius rhizosphere and elucidated the rules of the ecology shift. Two community-based microbiological measurements, community level physiological profiling (CLPP) using Biolog sole C source utilization tests and phospholipid ester-linked fatty acid (PLFA) profiles, were used to evaluate soil microbial community function and composition of different Panax quinquefolius cropping soils. The properties, pH, N, P, K and organic matter were analyzed also. There were many differences in soil properties such as lower pH (5.82) in Jilin soil but higher pH (8.27) in Beijing soil and there were significant higher content of organic matter, available N, P, K in Jilin soil than those in Beijing soil. The analysis of soil microbial ecology showed a similar result in AWCD, Biolog and PLFA pattern. According to the PC1 score in principal analysis of Biolog and PLFA, the soils in 2, 3 and 4 years cultivated P. quinquefolius rhizophere were distinguished from the control and 1 year soils. The result indicated that the metabolism function reduced and the structure shifted in rhizophere soil microbial community after P. quinquefolius cropping. These may be the primary reason of the disease occur severely in P. quinquefolius field.

[Investigation of Paris genus resources in Xishuangbanna].

OBJECTIVE: To explore the species, distributing and resource of Paris genus plants in Xishuangbanna area of Yunnan province. METHOD: Two steps, talking with the folk doctors and field survey, were employed to explore the resource status of Paris plants. Twenty six villages and towns, which are at an elevation of 500 m to 2000 m, were investigated. RESULT: A total of 214 specimens were collected and identified according to morphological characteristics. All investigated spots had Paris genus resources distribution but there always had a small Paris population less than 20. The Paris genus is an understory plants which lives always in a specific environment beside creek in hill valley and with many commensal plants such as bamboo and broadleaf. Three species of Paris, P. polyphylla var. yunnanensis, P. polyphylla var. chinensis, and P. vietnamensis were collected and identified. CONCLUSION: Paris genus resources declined rapidly in recent year, particular the P. polyphylla var. yunnanensis. Developing the breeding and cultivation technologies and enhancing the resources protection are needed urgently.

[Study on seed testing for Salvia miltiorrhiza].

OBJECTIVE: To establish a seed testing methods for Salvia miltiorrhiza. METHOD: Referring to the International Seed Testing Rules made by ISTA and the Seed Testing for Crops (GB/T3543. 1-1995) issued by China. RESULT: The seeds are selected by winnowing; the seed purity is about 50%-60%; 100 grain weight is used to determine the quality of the seed; the seed moisture content is determined by air drying, the drying hour is 3 h. Seed viability is tested by TFC method.

Novel acyl phosphate mimics that target PlsY, an essential acyltransferase in gram-positive bacteria.

PlsY is a recently discovered acyltransferase that executes an essential step in membrane phospholipid biosynthesis in Gram- positive bacteria. By using a bioisosteric replacement approach to generate substrate-based inhibitors of PlsY as potential novel antibacterial agents, a series of stabilized acyl phosphate mimetics, including acyl phosphonates, acyl alpha,alpha-difluoromethyl phosphonates, acyl phosphoramides, reverse amide phosphonates, acyl sulfamates, and acyl sulfamides were designed and synthesized. Several acyl phosphonates, phosphoramides, and sulfamates were identified as inhibitors of PlsY from Streptococcus pneumoniae and Bacillus anthracis. As anticipated, these inhibitors were competitive inhibitors with respect to the acyl phosphate substrate. Antimicrobial testing showed the inhibitors to have generally weak activity against Gram-positive bacteria with the exception of some acyl phosphonates, reverse amide phosphonates, and acyl sulfamates, which had potent activity against multiple strains of B. anthracis.

Genetic relationships among Rehmannia glutinosa cultivars and varieties.

Many cultivars of Rehmannia glutinosa are grown in China for medicinal uses, but detailed agronomic and morphological descriptions are available for only a few. Knowledge of genetic relationships among most of the cultivars is also scanty and poorly documented. Here, cultivars, varieties and some sexually produced seeds of R. GLUTINOSA were raised in the field and studied for morphological diversity including shape, color, edges of leaves, color of anther, cornal and root, as well as yield of the medicinal part of the roots. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to determine genetic relationships and ribosome DNA internal transcribed spacer (ITS) sequences were used for analyzing sequence variations and phylogenetic history. The 118 and 1019 polymorphic markers produced by 10 RAPD and 8 AFLP primers discriminated cultivars and varieties satisfactorily. Sixty-eight accessions were clustered in three main groups at 0.69 similarity levels by unweighted pair-group method arithmetic average (UPGMA) cluster analysis using RAPD in combination with AFLP markers. The average polymorphism information content (PIC) and Shannon index were 0.438 and 2.19 in RAPD and 0.476 and 26.68 in AFLP primers, respectively. This indicates that AFLP markers would be more efficient than RAPD for screening large numbers of R. GLUTINOSA accessions. The analysis of ITS sequences indicated that ITS1 - 5.8S-ITS2 of R. GLUTINOSA was informative in its 611 - 614-bp-long sequence and had 106 variable sites. Phylogenetic trees generated based on ITS sequences as well as the dendrogram obtained from two molecular markers identified four accessions: BY3, BY5, BY6 and Wildness6, with great genetic divergence.

[Comparative analysis of character on germplasm resources of Rehmannia glutinosa].

OBJECTIVE: To compare difference in character between wild germplasm and cultivar of Rehmannia glutinosa Libosch. METHOD: Field test and statistical analysis were applied. RESULT: The results showed that the plant height and leave weight of individual plant in cultivar were decreased significantly comparing to wild germplasm, and the output was increased significantly. The leave length was reduced. The leave width, the catalpol content in leave and polysaccharides and reducing sugar content in cultivar was increased not significantly. Whereas the catalpol content and the water extract content in cultivar were equal to wild germplasm. CONCLUSION: The plant height and leave weight of individual plant of R. glutinosa was decreased significantly in cultivar, but the active compounds content not changed obviously.

[Analysis of genetic diversity of wild Rehmannia glutinosa by using RAPD and ISSR markers].

OBJECTIVE: To analyze the genetic diversity of wild Rehmannia glutinosa and evaluate and compare random amplified polymorphic DNA (RAPD) and inter sample sequence repeat (ISSR) for analysis of R. glutinosa accessions. METHOD: Two molecular markers, RAPD and ISSR were used for analyzing 55 wild R. glutinosa accessions. RESULT: Average 16.00 and 19.08 bands were amplified by RAPD primers and ISSR primers respectively, and the percentage of polymorphic bands were 89.58% and 94.32% respectively; Fifty-five R. glutinosa accessions categorized into 7 clusters were identified by unweighted pair-group method, arithmetic average (UPGMA) method. CONCLUSION: A high level of genetic diversity of wild Rehmannia glutinosa was displayed at DNA level, and genetic diversity coefficient of R. glutinosa from different production areas was 0.63-0.98, and ISSR marker can detect higher genetic diversity of R. glutinosa germplasms than RAPD marker.

[Study on seed determination practice in Polygala tenuifolia].

OBJECTIVE: To control the seed quality market, a study on the seed determination practice in Polygala tenuifolia was carried out. METHOD: By studying the thousand grain weight moisture content, viability, genuineness, purity and germination percentage, some indices of seeds were fixed to the standards. The seed determination practice in P. tenuifolia was established. RESULT AND CONCLUSION: The practice could be utilized to control the seed quality of P. tenuifolia.

[Studies on diversity of quality of Forsythia suspensa].

OBJECTIVE: To study on diversity of quality of Forsythia suspense collected from different regions. METHOD: The hundred-seed weight of shucks was analyzed by the method of hundred grain mass. The thousand-seed weight of seeds was analyzed by the method of thousand grain mass. The contents of the active components in shucks and seeds were determined by HPLC. RESULT: The hundred-seed weight of shucks, the thousand-seed weight of seeds and the contents of the active components in the shucks and seeds from different regions were significantly different. CONCLUSION: The quality of F. suspense from different regions is not consistant.