Plastid phylogenomics and species discrimination in the "Chinese" clade of Roscoea (Zingiberaceae).

Roscoea is an alpine or subalpine genus from the pan-tropical family Zingiberaceae, which consists of two disjunct groups in geography, namely the "Chinese" clade and the "Himalayan" clade. Despite extensive research on the genus, Roscoea species remain poorly defined and relationships between these species are not well resolved. In this study, we used plastid genomes of nine species and one variety to resolve phylogenetic relationships within the "Chinese" clade of Roscoea and as DNA super barcodes for species discrimination. We found that Roscoea plastid genomes ranged in length from 163,063 to 163,796 bp, and encoded 113 genes, including 79 protein-coding genes, 30 tRNA genes, four rRNA genes. In addition, expansion and contraction of the IR regions showed obvious infraspecific conservatism and interspecific differentiation. Plastid phylogenomics revealed that species belonging to the "Chinese" clade of Roscoea can be divided into four distinct subclades. Furthermore, our analysis supported the independence of R. cautleoides var. pubescens, the recovery of Roscoea pubescens Z.Y. Zhu, and a close relationship between R. humeana and R. cautloides. When we used the plastid genome as a super barcode, we found that it possessed strong discriminatory power (90%) with high support values. Intergenic regions provided similar resolution, which was much better than that of protein-coding regions, hypervariable regions, and DNA universal barcodes. However, plastid genomes could not completely resolve Roscoea phylogeny or definitively discriminate species. These limitations are likely related to the complex history of Roscoea speciation, poorly defined species within the genus, and the maternal inheritance of plastid genomes.

Efficacy of Percutaneous Intraarterial Facial/Supratrochlear Arterial Hyaluronidase Injection for Treatment of Vascular Embolism Resulting From Hyaluronic Acid Filler Cosmetic Injection.

BACKGROUND: Vascular embolism is a serious complication of hyaluronic acid (HA) filler cosmetic injection, and hyaluronidase injection has been proposed as the treatment. Until now, there has been a lack of adequate clinical evidence regarding the benefits of treatment for HA filler-induced vascular embolism by percutaneous facial or supratrochlear arterial hyaluronidase injection. OBJECTIVES: The authors sough to evaluate the efficacy of percutaneous facial or supratrochlear arterial hyaluronidase injection as a rescue treatment for HA filler-induced vascular embolism. METHODS: We included 17 patients with vascular embolism after facial HA filler injection. Intraarterial injection of 1500 units hyaluronidase was performed via facial artery for 13 cases with skin necrosis and via supratrochlear arterial for 4 cases with severe ptosis and skin necrosis but no visual impairment. Simultaneously, general symptomatic treatment and nutritional therapy were performed. RESULTS: After hyaluronidase injection, facial skin necrosis in all cases was restored and ptosis in the 4 cases was also significantly relieved. Patients were subsequently followed-up for 1 month to 1 year. The skin necrosis in 16 patients completely healed, and only 1 patient had small superficial scars. CONCLUSIONS: It is effective to alleviate skin necrosis and ptosis resulting from HA filler embolism via percutaneous facial or supratrochlear arterial hyaluronidase injection.

[DNA super-barcoding of several medicinal species in Gentiana from Yunnan province].

Gentiana is an important but complicated group in Gentianaceae. The genus covers numerous medicinal plants which are difficult to be identified. In the present study, several medicinal species in Gentiana from Yunnan province, including G. rigescens, G.rhodantha, and G. delavayi, were sequenced using the Illumina HiSeq 2500 system. Three complete chloroplast genome sequences were obtained after assembly and annotation. According to several published genome sequences of G. crassicaulis, the DNA super-barcoding of species in Gentiana was preliminarily carried out. The results revealed that chloroplast genomes of the three species were conservative with short lengths(146 944, 148 992, and 148 796 bp, respectively). The genomes encoded 114 genes, including 78 protein-coding genes, 30 tRNA genes, 4 rRNA genes, and 2 pseudogenes. Furthermore, these medicinal species in Yunnan province were identified using DNA super-barcoding based on chloroplast genomes. The results showed that the Gentiana species could be gathered into monophyletic branches with a high support value(100%). It indicated that DNA super-barcoding possessed obvious advantages in discriminating species in complicated genera. This study is expected to provide a scientific basis for the identification, utilization, and conservation of Gentiana species.

[Phylogeography of Paris poliphylla var. yunnanensis based on chloroplast gene trnL-trnF sequences].

Phylogeography is a research hotspot in the field of the genetic diversity and core germplasm construction of endangered rare plants. Paris polyphylla var. yunnanensis is a rare plant species mainly distributed in China. Wild individuals have been overexploited for the last few decades because of increasing demand for such medicines. Therefore, it is great significance to study the phylogeography of P. poliphylla var. yunnanensis based on chloroplast gene trnL-trnF sequences. In this study, chloroplast genes trnL-trnF were used in the phylogeography analysis of 15 wild and 17 cultivated populations of P. polyphylla var. yunnanensis. This study revealed that based on the results of neutrality tests and mismatch analysis, the rapid expansion of wild population has not been detected in P. polyphylla var. yunnanensis. After aligning and sorting the obtained cpDNA sequences, a total of 15 haplotypes were detected in all 32 populations. One haplotype was unique to the wild population, and 5 haplotypes were unique to the cultivated population. It can be seen that the haplotype richness of cultivated population was higher than that of wild population. The wild populations of P. polyphylla var. yunnanensis were divided into two groups according to evolutionary relationship of haplotypes and distribution map of haplotypes. The haplotype of branch Ⅰ was mainly distributed in Guizhou, and the haplotype of branch Ⅱ was located in Yunnan and Huidong, Sichuan. Therefore, it's speculated that Guizhou and the west Yunnan region may be glacial refuge in the evolutionary history of wild populations of P. polyphylla var. yunnanensis, and in order to protect the wild resources more effectively, wild populations of P. polyphylla var. yunnanensis in these two areas should be included in the protection zone.

Molecular Markers in Whole Genome Evolution of Brachypodium.

Molecular markers play more and more important role in population genetic and phylogenetic studies; choice of marker systems for a particular study has become a serious problem. These marker systems have different advantages and disadvantages, so it is imperative to keep in mind all the pros and cons of the technique while selecting one for the problem to be addressed.Here, we concisely introduced three molecular marker techniques, namely SSR, ISSR, and RFLP. We elaborated their properties such as reliability, simplicity, cost-effectiveness, and speed, in addition to data analysis of genetic diversity. We have outlined here the whole methodology of these techniques.

Upregulation of SIRT1-AMPK by thymoquinone in hepatic stellate cells ameliorates liver injury.

Thymoquinone (TQ) is a biologically active compound isolated from the seeds of Nigella sativa L. (Ranuculaceae). This study investigated the hepato-protective effect of TQ on liver injury through AMP-activated protein kinase (AMPK) signaling in hepatic stellate cells (HSCs). In vitro, TGF-ß time-dependently attenuated liver kinase B-1 (LKB1) and AMPK phosphorylation, which were blocked by pretreatment with TQ and AICAR (an activator of AMPK). TQ significantly inhibited collagen-Ι, α-SMA, TIMP-1 and enhanced MMP-13 expression, contributing to prevent TGF-ß-induced human HSCs activation. Moreover, TQ induced peroxisome proliferator activated receptor-γ (PPAR-γ) expression, which was inhibited by genetic deletion of AMPK. In vivo, C57BL/6 mice were fed with ethanol diet for 10 days, then administering a single dose of ethanol (5g/kg body weight) via gavage. TQ (20 or 40mg/kg) were given by gavage every day. TQ attenuated the increases in serum aminotransferase and hepatic triglyceride in mice fed with ethanol, while significantly activated LKB1 and AMPK phosphorylation. In addition, TQ enhanced the sirtuin 1 (SIRT1) expression. In conclusion, we demonstrate that AMPK pathway is a key therapeutic target for controlling liver injury and TQ confers hepato-protection against TGF-ß-induced the activation of HSCs and ethanol-induced liver injury.

Tetrandrine regulates hepatic stellate cell activation via TAK1 and NF-κB signaling.

We investigated the anti-fibrotic mechanism of tetrandrine, a bisbenzylisoquinoline alkaloid from the Chinese herb, Stephania tetrandra, on the immortalized HSC-T6 rat hepatic stellate cell line. Tetrandrine (0.39-50µM) dose- and time-dependently inhibited HSC-T6 cell viability within 24h and exhibited almost no cytotoxicity at concentrations lower than 6.25µM in the presence of tumor necrosis factor-α (TNF-α). At a much high concentration (50µM), tetrandrine caused fatal cytotoxity in both HSCs and hepatocytes. TNF-α time-dependently increased α-smooth muscle actin (α-SMA) expression, while a lower concentration of tetrandrine (6.25µM) prior to TNF-α treatment reduced the expression of α-SMA and TNFR-1-associated death domain (TRADD). TNF-α treatment induced TGF-ß-activated kinase-1 (TAK1) and c-Jun N-terminal kinase (JNK) phosphorylation, which were attenuated by tetrandrine. Furthermore, TNF-α treatment activated nuclear factor-κB (NF-κB) nuclear translocation and IκB-α degradation. Tetrandrine treatment prior to TNF-α reduced nuclear phosphorylated and total NF-κB p65, while the cytosolic IκB-α and NF-κB p65 levels significantly increased. In addition, treatment with only tetrandrine induced the cleavage of caspase-3 and PARP within a range of higher concentrations. Tetrandrine-induced apoptosis was confirmed by the TUNEL assay and flow-cytometric analysis. Treatment with only tetrandrine markedly reduced α-SMA expression, except for at lower concentrations of tetrandrine. A higher concentration of tetrandrine (25µM) induced a significant increase in JNK and extracellular signal-regulated kinase (ERK) phosphorylation, NF-κB nuclear translocation and IκB-α degradation. In conclusion, the anti-fibrogenic effects of tetrandrine on HSCs involved a dosage-dependent signaling pathway, based on the tetrandrine concentration, by regulating TAK1, JNK and NF-κB. The present data provides strong evidence for the anti-fibrotic dosage-dependent signaling pathway of tetrandrine.

Resveratrol Regulates Activated Hepatic Stellate Cells by Modulating NF-κB and the PI3K/Akt Signaling Pathway.

In the present study, we investigated whether resveratrol could suppress the hepatic fibrogenesis in activated hepatic stellate cells. The immortalized rat hepatic stellate cells, t-HSC/Cl-6, were treated with resveratrol 1 h prior to lipopolysaccharide (LPS, 1 µg/mL). Resveratrol decreased t-HSC/Cl-6 cell viability at much lower concentrations within 24 h. Resveratrol pretreatment also decreased the LPS-induced protein expression of α-SMA and collagen I. In addition, resveratrol significantly reduced the protein expression of Toll-like receptor 4 (TLR4) and myeloid differentiation primary response gene 88 (MyD88), and the expression of phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated serine/threonine kinase B (Akt). Moreover, resveratrol markedly blocked the translocation of nuclear factor (NF)-κB in LPS-activated HSCs. Furthermore, resveratrol inhibited HSCs activation through stimulating LXRß, but did not influence LXRα. Overall, we conclude that the antifibrotic effect of resveratrol is the result of blocking NF-κB activation and PI3K/Akt phosphorylation, which inhibits HSC activation to obstruct liver fibrosis. Thus, resveratrol may be a natural agent for preventing hepatic fibrosis.

The antifungal effect of silver nanoparticles on Trichosporon asahii.

BACKGROUND/PURPOSE: Silver nanoparticles are receiving increasing attention in biomedical applications. This study aims at evaluating the antifungal properties of silver nanoparticles against the pathogenic fungus Trichosporon asahii. METHODS: The growth of T. asahii on potato dextrose agar medium containing different concentrations of silver nanoparticles was examined and the antifungal effect was evaluated using minimum inhibitory concentration. Scanning and transmission electron microscopy were also used to investigate the antifungal effect of silver nanoparticles on T. asahii. RESULTS: Silver nanoparticles had a significant inhibitory effect on the growth of T. asahii. The minimum inhibitory concentration of silver nanoparticles against T. asahii was 0.5 µg/mL, which was lower than amphotericin B, 5-flucytosine, caspofungin, terbinafine, fluconazole, and itraconazole and higher than voriconazole. Silver nanoparticles obviously damaged the cell wall, cell membrane, mitochondria, chromatin, and ribosome. CONCLUSION: Our results demonstrate that silver nanoparticles have good antifungal activity against T. asahii. Based on our electron microscopy observations, silver nanoparticles may inhibit the growth of T. asahii by permeating the fungal cell and damaging the cell wall and cellular components.

[Effects of fungal elicitors on the secondary metabolite steroidal saponin in Paris polyphylla var. yunnanensis].

To compare the effects of inoculated or non-inoculated with arbuscular mycorrhizal (AM) fungi on the steroidal saponin component in root of Paris polyphylla var. yunnanensis. By pot experiments, steroid saponin component in root of P. polyphylla var. yunnanensis was determined and compared by HPLC. The results showed there was difference in the effects of different AM fungal on the secondary metabolite steroid saponin in P. polyphylla var. yunnanensis. After elicitors treatment, AM fungal did not change the chemical backgrounds of P. polyphylla var. yunnanensis, but can improve partly the content of chemical compositions in roots. In conclusion, there was selectivity between AM fungal and P. polyphylla var. yunnanensis. Glomus intraradices was the most appropriate strain for inoculation P. polyphylla var. yunnanensis.

Cross-species amplification and development of new microsatellite loci for Taxus wallichiana (Taxaceae).

PREMISE OF THE STUDY: Previously published and newly developed microsatellite primers for Taxus species were screened for their potential transfer to Taxus wallichiana and to characterize the genetic diversity and population structure of this species. METHODS AND RESULTS: Twenty microsatellite markers that successfully PCR amplified showed polymorphisms when tested on T. wallichiana. Ten of these were obtained by cross-species amplification. The remaining 10 markers were developed in the current study. These markers were characterized on 58 individuals from two T. wallichiana populations from China. Overall, the number of alleles per locus ranged from two to twelve. No significant linkage disequilibrium (LD) was detected between the comparisons of these loci. CONCLUSIONS: The polymorphic microsatellite markers screened in this study will be useful for further investigations of the conservation genetics of the endangered species T. wallichiana.

DNA barcoding for the discrimination of Eurasian yews (Taxus L., Taxaceae) and the discovery of cryptic species.

There is currently international interest in the application of DNA barcoding as a tool for plant species discrimination and identification. In this study, we evaluated the utility of five candidate plant DNA barcoding regions [rbcL, matK, trnH-psbA, trnL-F and internal transcribed spacer (ITS)] in Eurasian yews. This group of species is taxonomically difficult because of a lack of clear-cut morphologically differences between species and hence represents a good test case for DNA barcoding. Forty-seven accessions were analysed, representing all taxa treated in current floristic works and covering most of the distribution range of Taxus in Eurasia. As single loci, trnL-F and ITS showed the highest species discriminatory power, each resolving 11 of 11 lineages (= barcode taxa). Species discrimination using matK, trnH-psbA and rbcL individually was lower, with matK resolving 8 of 10, trnH-psbA 7 of 11 and rbcL 5 of 11 successfully sequenced lineages. The proposed CBOL core barcode (rbcL + matK) resolved 8 of 11 lineages. Combining loci generally increased the robustness (measured by clade support) of the barcoding discrimination. Based on overall performance, trnL-F and ITS, separately or combined, are proposed as barcode for Eurasian Taxus. DNA barcoding discriminated recognized taxa of Eurasian Taxus, namely T. baccata, T. cuspidata, T. fuana and T. sumatrana, and identified seven lineages among the T. wallichiana group, some with distinct geographical distributions and morphologies, and potentially representing new species. Using the proposed DNA barcode, a technical system can be established to rapidly and reliably identify Taxus species in Eurasia for conservation protection and for monitoring illegal trade.

Separation of anthocyanins from Perilla frutescens by high speed countercurrent chromatography.

OBJECTIVE: To develop an efficient method for the separation of anthocyanins from Perilla frutescens. METHODS: Freeze-dried Perilla frutescens was extracted with 1% HCl. After purified by Amberlite XAD-7 column chromatography, the bioactive anthocyanins were separated by high-speed countercurrent chromatography (HSCCC). The structures of the compounds were elucidated by 1H- and 13C-NMR spectroscopy. RESULTS: When the HSCCC separation was performed with a two-phase solvent system composed of n-butanol-tert-butyl methyl ether-acentonitrile-water (2:2:1:5 + 0.1% TFA) by eluting the mobile phase at a flow rate of 3.0 mL/min and a revolution speed of 800 r/min, 10.1 mg malonylshisonin and 8.6 mg shisonin were obtained from 1.0 g of the XAD-7 extract. The purities of malonylshisonin and shisonin were 96.7% and 97.5%, respectively. CONCLUSION: HSCCC is a fast and efficient technique to prepare pure malonylshisonin and shisonin from Perilla frutescens.

[The quantified analysis of fresh mutton tenderness using PLS methods and Fourier transform near-infrared spectroscopy].

Ninety eight representative fresh mutton samples from Neimeng, Ningxia, Gansu, Xinjiang province were selected for this study, the nondestructive measurement of the fresh mutton tenderness by Fourier transform near infrared (FT-NIR) spectroscopy was discussed. Partial least squares(PLS) algorithm was used to build the model between the shear force value of the fresh mutton tenderness measured by the texture machine and the FT-NIR spectra. The influence of different processing method of spectra, factors and wave regions on the determination coefficients (r2), root mean square error of cross validation (RMSECV) and root mean square error of prediction (RMSEP) was studied. The result showed that the shear force value of ninety eight representative fresh mutton samples was 1.673-6.631 kg, and the shear force value above 75% samples was 2-5 kg, almost covering the fresh mutton tenderness of our country's sheep, the r2 of the calibration could reach 86.2% and the RMSECV was up to 0. 445 in the wave number range 11 995-5 446 cm(-1) and 4 601-4 246 cm(-1) with vector normalization when the PLS factors was ten. The correlation coefficient(R), RMSEP and average bias between value measured by the texture machine and predicted value of model based on validation samples were 0.87, 0.524 and 0.385 respectively. The result indicates that FT-NIR spectroscopy is capable of predicting tenderness value of fresh mutton.