An R2R3-MYB FtMYB11 from Tartary buckwheat has contrasting effects on abiotic tolerance in Arabidopsis.

R2R3-MYB transcription factors play important roles in response to abiotic stresses in planta, such as salt, drought, and osmotic stress. However, the role of FtMYB11 in Tartary buckwheat (Fagopyrum tataricum) in drought and osmotic tolerance has not yet been elucidated. In this study, we found that FtMYB11 was markedly induced by exogenous abscisic acid (ABA), salinity, and mannitol. Further, FtMYB11-overexpressing Arabidopsis showed hypersensitivity to ABA-mediated seed germination and seedling establishment through regulating transcripts of AtCBF1, AtDREB2A, and AtRD20, compared with wild type, indicating that FtMYB11 plays a positive role in ABA signaling. In contrast, transgenic lines overexpressing FtMYB11 were sensitive to mannitol and NaCl treatments, suggesting that FtMYB11 plays a negative role in osmotic tolerance. Intriguingly, the transcripts of ABA biosynthetic enzyme genes were significantly elevated in plants overexpressing FtMYB11 after exposure to osmotic stresses, such as AtABA3 and AtNCED3. In addition, flavonoid biosynthesis genes were also upregulated in transgenic Arabidopsis under ABA, salt, and drought treatments, including AtC4H, AtF3H, AtANS, AtFLS, and At4CL. The drought tolerance assay showed that plants overexpressing FtMYB11 displayed greater tolerance to water deficit through regulating MDA and proline content. Taken together, FtMYB11 has opposite roles in response to abiotic stresses, but it may mediate flavonoid biosynthesis through regulation of related enzyme genes.

[Clinical random trial of thumbtack needling therapy for analgesia after cesarean section].

OBJECTIVE: To observe the analgesic effect of thumbtack needling therapy and the quality of life in puerpera underwent cesarean section. METHODS: One hundred and thirty-five puerpera underwent cesarean section were randomly divided into a medication group, a sham-thumbtack needling group and a thumbtack needling group, 45 cases in each group. In the medication group, the patient control analgesia (PCA) was given. In the thumbtack needling group, on the base of the regimen as the medication group, acupuncture with thumbtack needles was applied to lower abdominal point (Extra), Zusanli (ST36), Sanyinjiao (SP6), Geshu (BL17), Shenshu (BL23) and Dachangshu (BL25). The needles were retained for 48 hours. During the needle retaining, the sites with the needle embedded were pressed and kneaded for 3 times, 1 min each time, at the interval of 4 to 12 h. In the sham-thumbtack needling group, the sham-thumbtack needles were used; the acupoint selection, operation and treatment course were all the same as the thumbtack needling group. The visual analogue scale (VAS) scores of incision pain and ute-rine contraction pain, the incidence of postoperative adverse reactions, the amount of vaginal bleeding and milk amount in lactation were observed at each time point after the operation separately in each group. RESULTS: Compared with the medication group, the VAS score of incision pain was decreased 8, 12 and 24 h after operation (P<0.05), the VAS score of uterine contraction pain was decreased 8, 12, 24 and 48 h after operation (P<0.05), the lactation score was increased in 24 to 48 h after operation (P<0.05) in both the sham-thumbtack needling group and thumbtack needling group. Compared with the sham-thumbtack needling group, the VAS score of incision pain was decreased 8, 12 and 24 h after operation (P<0.05), the VAS score of uterine contraction pain was decreased 24 and 48 h after operation (P<0.05) in the thumbtack needling group. Compared with those from 2 to 24 h after operation, the vaginal bleeding amount was decreased and the lactation score increased from 24 to 48 h after operation (P<0.05) in all of the three groups. CONCLUSION: The conventional PCA combined with thumbtack needling therapy obtained better analgesic effects on incision dynamic pain and uterine contraction pain in patients after cesarean section as compared with either the simple PCA or the combined treatment with sham-thumbtack needling and medication, and both the thumbtack needling and the sham-thumbtack needling therapy can increase the milk amount of lactation from 24 to 48 h after operation.

Standards for Collection, Preservation, and Transportation of Fecal Samples in TCM Clinical Trials.

Background: Unlike chemical drugs with a single or a few kinds of active compounds, traditional Chinese medicines (TCMs)uses herbal formulas composed of numerous kinds of chemical constituents. Therefore, TCM clinical trials require unique and stricter standards for collecting, preserving, and transporting fecal samples than those used for chemical drugs. Unfortunately, there are no special standards for processing fecal samples in TCM clinical trials. Methods: We invited interdisciplinary experts within TCM clinical trials and gut microbiome research to help formulate this standard. After more than a year's in-depth discussion and amendments, we achieved a standard via expert interviews, literature research, questionnaire surveys, and public opinion solicitation. This standard has been reviewed and approved by the Standards Office of China of the Association of Chinese medicine. Results: We established a sample information processing method prior to TCM clinical sample collection, which is adapted to the unique features of TCM. The method formulates detailed processing requirements for TCM information in addition to the factors that may disturb the gut microbiome. We also constructed a set of methods for collecting, preserving, and transporting fecal samples that meet the characteristics of TCM. These methods formulate detailed operating specifications on the collection approaches, storage conditions, transportation requirements, and management of fecal samples. Conclusions: This standard guides the information processing prior to sample collection and the standard operating procedures for the collection, preservation, and transportation of fecal samples in TCM clinical trials, which also can be used as a reference by clinicians and researchers in modern medicines.

Chemical profiling of Honghua Xiaoyao tablet and simultaneous determination of its quality markers by liquid chromatography-tandem mass spectrometry combined with chemometrics methods.

Discovering marker components of traditional Chinese medicine formulas is challenging because of the hundreds of components they inherently contain. This study first proposed a reliable and validated method for the comprehensive profiling of chemical constituents in Honghua Xiaoyao tablet by using high-performance liquid chromatography coupled with mass spectrometry. After searching within the in-house library, a total of 55 constituents were unambiguously characterized or tentatively identified through reference standards and by comparing mass spectrometry data with literature values. Quantitative analysis of 14 compounds, which were selected as the quality marker components based on a serum pharmacochemistry study, has been performed by triple-quardrupole mass spectrometry technique. Multiple chemometric methods, including principal components analysis and hierarchical cluster analysis, were subsequently used to analyze the quantitative results, classify samples from three manufacturers, and distinguish the analytical markers. In method validation results, 14 quality marker compounds have shown good linearity (R2 ≥ 0.9965) with a relative wide concentration range and acceptable recovery at 98.39-102.46%. The proposed approach provides the chemical evidence for revealing the material basis of Honghua Xiaoyao tablet, and establishes a reliable statistical analysis-based strategy of quality marker investigation for controlling its quality.

NLRP3 Inflammasome: A Potential Alternative Therapy Target for Atherosclerosis.

Atherosclerosis (AS) is a complex and chronic inflammatory disease that occurs in multiple systems of the human body. It is an important pathological basis for a variety of diseases and a serious threat to human health. So far, many theories have been formed to explain the pathogenesis of atherosclerosis, among which "inflammation theory" has gradually become a research focus. This theory presents that inflammatory response runs through the whole progress of AS, inflammatory cells play as the main executors of AS, and inflammatory mediators are the key molecules of AS. In the inflammatory process of atherosclerosis, the role of NLRP3 in the atherosclerosis has gradually got the attention of researchers. NLRP3 is a kind of signal-transductional pattern recognition receptors (PRRs). After recognizing and binding to the damage factors, NLRP3 inflammasome will be assembled to activate IL-1ß and caspase-1 pathways, resulting in promoting the inflammation process of AS, reducing the stability of the plaques, and finally increasing the incidence of adverse cardiovascular events. Taken above, the article will review the potential benefits of drugs targeting the NLRP3 inflammasome in the therapy of AS.

Pulsed Direct Current Electrospray: Enabling Systematic Analysis of Small Volume Sample by Boosting Sample Economy.

We had developed pulsed direct current electrospray ionization mass spectrometry (pulsed-dc-ESI-MS) for systematically profiling and determining components in small volume sample. Pulsed-dc-ESI utilized constant high voltage to induce the generation of single polarity pulsed electrospray remotely. This method had significantly boosted the sample economy, so as to obtain several minutes MS signal duration from merely picoliter volume sample. The elongated MS signal duration enable us to collect abundant MS(2) information on interested components in a small volume sample for systematical analysis. This method had been successfully applied for single cell metabolomics analysis. We had obtained 2-D profile of metabolites (including exact mass and MS(2) data) from single plant and mammalian cell, concerning 1034 components and 656 components for Allium cepa and HeLa cells, respectively. Further identification had found 162 compounds and 28 different modification groups of 141 saccharides in a single Allium cepa cell, indicating pulsed-dc-ESI a powerful tool for small volume sample systematical analysis.

Single cell analysis with probe ESI-mass spectrometry: detection of metabolites at cellular and subcellular levels.

Molecular analysis at cellular and subcellular levels, whether on selected molecules or at the metabolomics scale, is still a challenge now. Here we propose a method based on probe ESI mass spectrometry (PESI-MS) for single cell analysis. Detection of metabolites at cellular and subcellular levels was successfully achieved. In our work, tungsten probes with a tip diameter of about 1 µm were directly inserted into live cells to enrich metabolites. Then the enriched metabolites were directly desorbed/ionized from the tip of the probe for mass spectrometry (MS) detection. The direct desorption/ionization of the enriched metabolites from the tip of the probe greatly improved the sensitivity by a factor of about 30 fold compared to those methods that eluted the enriched analytes into a liquid phase for subsequent MS detection. We applied the PESI-MS to the detection of metabolites in single Allium cepa cells. Different kinds of metabolites, including 6 fructans, 4 lipids, and 8 flavone derivatives in single cells, have been successfully detected. Significant metabolite diversity was observed among different cells types of A. cepa bulb and different subcellular compartments of the same cell. We found that the inner epidermal cells had about 20 fold more fructans than the outer epidermal cells, while the outer epidermal cells had more lipids. We expected that PESI-MS might be a candidate in the future studies of single cell "omics".

Depth profiling of nanometer coatings by low temperature plasma probe combined with inductively coupled plasma mass spectrometry.

The development of material science increasingly calls for rapid characterization methods with low limits of detection and high spatial resolution. Here we report a depth profile analysis method for thin layer coatings by combining low temperature plasma (LTP) probe with inductively coupled plasma mass spectrometry (ICP-MS). The LTP probe with diameter of several tens of micrometers served as a tool for mass removal, which is generated by the discharge in a quartz capillary at ambient condition. The sample material is ablated by the LTP probe and converted into an aerosol, and transported by a carrier gas flow to the ICP-MS, where the decomposed and ionized aerosol particles are analyzed with high sensitivity. Scanning electron microscope (SEM) micrographs reveal that the trace after ablation by the LTP probe is a hole with a diameter less than 10 microm. A lateral resolution of approximately 200 microm has been achieved by analyzing an electron component with interval metal stripes. Depth profiling of a 100 nm single layer sample and a multiple layer sample (100 nm Al/250 nm SiO(2)/100 nm Au/50 nm Cr) on a silicon substrate have been successfully performed at ambient condition. The present method offers unique advantages in terms of high spatial resolution, fast analysis speed and ease of implementation. It might be considered a complementary technique to existing depth profiling methods such as GD-MS/OES, AES, and SIMS. In addition, the simple-to-fabricate LTP probe is easily coupled to various other elemental analysis tools for thin layer or direct solid sample analysis in micro area.

Inorganic arsenic modulates the expression of selenoproteins in mouse embryonic stem cell.

At least 25 selenoproteins in humans and 24 homologues in rodents have been identified. They play important roles in antioxidation, redox regulation and detoxification. The modulation of the expression of selenoproteins by inorganic arsenic (iAs) exposure may highlight the molecular mechanism for the arsenic toxicity. To investigate the effects of iAs exposure on the expression of selenoproteins, we determined how addition of iAs to culture medium affected all known selenoproteins in the mouse embryonic stem (ES) cells. Separated groups of ES cells were treated with arsenite (iAsIII) (0.25-0.5microM), arsenate (iAsV) (1.0-2.0microM) and co-treatment with sodium selenite (SeIV) (0.5microM). The mRNA levels of all selenoproteins were detected by real time quantitative PCR. The up-regulated selenoproteins were confirmed by immunoblotting analysis and enzymatic activity detection. Results showed that CGR8 cells treated with iAsIII (0.25-0.5microM) and iAsV (2.0microM) displayed significant increases of cellular reactive oxygen species (ROS) generation and nuclear accumulation of the transcription factor NF-E2-related factor 2 (Nrf2). Treatments of iAsIII (0.5microM) or iAsV (2.0microM) for 24h caused significant increases in the expression of the antioxidant selenoproteins (Gpx1, Gpx4, and Tr1), whereas led to significant decreases in the mRNA levels of selenoprotein H and some endoplasmic reticulum (ER) located selenoproteins (15-Sep, SelK, SelM, and SelS). Additionally, supplement of SeIV (0.5microM) could restore most of the down-regulated selenoproteins. These results suggested that iAs exposure modulated not only the antioxidant selenoproteins but also the ER stress associated selenoproteins. Further studies are required to clarify whether these modulated selenoproteins genes are targets for selenium supplement in the defense against the toxicity of iAs.

Effects of selenium on the structure and function of recombinant human S-adenosyl-L-methionine dependent arsenic (+3 oxidation state) methyltransferase in E. coli.

The effects of Se(IV) on the structure and function of recombinant human arsenic (+3 oxidation state) methyltransferase (AS3MT) purified from the cytoplasm of Escherichia coli were studied. The coding region of human AS3MT complementary DNA was amplified from total RNA extracted from HepG2 cell by reverse transcription PCR. Soluble and active human AS3MT was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25 degrees C. Spectra (UV-vis, circular dichroism, and fluorescence) were first used to probe the interaction of Se(IV) and recombinant human AS3MT and the structure-function relationship of the enzyme. The recombinant human AS3MT had a secondary structure of 29.0% alpha-helix, 23.9% beta-pleated sheet, 17.9% beta-turn, and 29.2% random coil. When Se(IV) was added, the content of the alpha-helix did not change, but that of the beta-pleated sheet increased remarkably in the conformation of recombinant human AS3MT. Se(IV) inhibited the enzymatic methylation of inorganic As(III) in a concentration-dependent manner. The IC(50) value for Se(IV) was 2.38 muM. Double-reciprocal (1/V vs. 1/[inorganic As(III)]) plots showed Se(IV) to be a noncompetitive inhibitor of the methylation of inorganic As(III) by recombinant human AS3MT with a K (i) value of 2.61 muM. We hypothesized that Se(IV) interacts with the sulfhydryl group of cysteine(s) in the structural residues rather than the cysteines of the active site (Cys156 and Cys206). When Se(IV) was combined with cysteine(s) in the structural residues, the conformation of recombinant human AS3MT changed and the enzymatic activity decreased. Considering the quenching of tryptophan fluorescence, Cys72 and/or Cys226 are deduced to be primary targets for Se(IV).

Structure-function roles of four cysteine residues in the human arsenic (+3 oxidation state) methyltransferase (hAS3MT) by site-directed mutagenesis.

Cysteine (Cys) residues are often crucial to the function and structure of proteins. Cys157 and Cys207 in recombinant mouse arsenic (+3 oxidation state) methyltransferase (AS3MT) are shown to be related to enzyme activity and considered to be the catalytic sites. The roles of some conserved Cys residues in the N-terminal region of the rat AS3MT also have been examined. However, little is known about the roles of the Cys residues in the middle region. The metabolism of inorganic arsenic in human is different from rat and mouse in some aspects though the AS3MT has a high degree of similarity in these species. In order to determine whether the Cys156 and Cys206 (corresponding to the catalytic sites, Cys157 and Cys207 in the mouse AS3MT) in the hAS3MT act as the catalytic sites and to study the roles of the Cys residues (Cys226 and Cys250) near the catalytic center in the middle region, we designed and prepared four mutants (C156S, C206S, C226S, and C250S) in which one Cys residue replaced by serine by PCR-based site-directed mutagenesis. The native form and cysteine/serine mutants were assayed for enzyme activity, free thiols, and the secondary structures by circular dichroism and Fourier transform infrared. Our data show that, besides C156S and C206S, C250S is another potential important site. C226S seems to have the same action as the wild-type hAS3MT with the consistent K(M) and V(max) values. Meanwhile, selenium can also inhibit the methylation of inorganic arsenic by C226S. All the mutants except C226S are calculated to have dramatic changes in the secondary structures. Cys250 might form an intramolecular disulfide bond with another Cys residue. These findings demonstrate that Cys residues at positions 156, 206, and 250 play important roles in the enzymatic function and structure of the hAS3MT.

Triterpenoid saponins and monoterpenoid glycosides from Incarvillea delavayi.

Two new triterpenoid saponins, incarvillosides A (1) and B (2), and two new monoterpenoid glycosides, incarvillosides C (3) and D (4), were isolated from the high-polarity fraction of the whole plant of Incarvillea delavayi. By means of spectroscopic data and chemical degradation, the structures were established as (3beta,21beta)-3,19,21,23-tetrahydroxyurs-12-en-28-oic acid 28-O-beta-d-glucopyranoside (1), (2beta,3beta,19alpha)-2,3,19,23-tetrahydroxyolean-12-en-28-oic acid 28-O-beta-d-glucopyranoside (2), (2S,6R)-2,6-dimethyl-1,8-octanediol 1-O-beta-d-glucopyranoside (3), and (2S,6R)-2,6-dimethyl-1,8-octanediol 8-O-beta-d-glucopyranoside (4).

Low selenium status affects arsenic metabolites in an arsenic exposed population with skin lesions.

BACKGROUND: The antagonistic effects between selenium (Se) and arsenic (As) suggest that low selenium status plays important roles in arsenism development. However, no study has been reported for humans suffering from chronic arsenic exposure with low selenium status. METHODS: Sixty-three subjects were divided into 2 experimental groups by skin lesions (including hyperkeratosis, depigmentation, and hyperpigmentation). Total urine and serum concentrations of arsenic and selenium were determined by ICP-MS with collision/reaction cell. Arsenic species were analysed by ICP-MS coupled with HPLC. RESULTS: The mean concentration of As in the drinking waters was 41.5 microg/l. The selenium dietary intake for the studied population was 31.7 microg Se/d, and which for the cases and controls were 25.9 and 36.3 microg Se/d, respectively. Compared with the controls, the skin lesions cases had lower selenium concentrations in serum and urine (41.4 vs 49.6 microg/l and 71.0 vs 78.8 microg/l, respectively), higher inorganic arsenic (iAs) in serum (5.2 vs 3.4 microg/l, P<0.01), higher percentages of iAs in serum and urine (20.2) vs 16.9% and 18.3 vs 14.5%, respectively, P<0.01) but lower percentages of monomethylarsonate (MMA) in serum (15.5 vs 18.8%, P<0.01) ans dimethylarsinate acid (DMA) in urine (65.1 vs 69.8%, P<0.01). Subjects with lower selenium concentrations in serum (<50 microg/l) had a stronger tendency to the risk of skin lesions than individual having higher selenium concentrations [odd ratio (OR), 7.3; 95% confidence interval (95% CI), 1.5-35.7; P=0.014]. This OR estimation was confirmed in those subjects having higher ratios of As/Se in urine and serum, with OR as high as 10.3 and 3.8 respectively. CONCLUSIONS: Lower serum selenium status (<50 microg/l) is significantly correlated to the arsenic-associated skin lesions in the arsenic exposed population. The accumulation of iAs and its inhibition to be biotransformed to DMA occurred in human due to chronic exposure of low selenium status.

A research on determination of explosive gases utilizing cataluminescence sensor array.

In this paper, we propose a model of a sensor array system, which consists of three cataluminescence sensors based on nanosized SrCO3, gamma-Al2O3 and BaCO3 as catalysts, for quantitative analysis of the explosive gases of propane, n-butane and iso-butane in a mixture. Six linear regression equations of the cataluminescence intensity vs. the gas concentrations in the range 2000-10,000 ppm were established from the sensor array system at two working temperatures, as the explosive gases show different sensitivity to the three sensors. The least squares method was employed for solving the simultaneous equations and quantifying the concentrations of the three components. The detection limits (3sigma) of propane, n-butane and iso-butane on SrCO3, gamma-Al2O3 and BaCO3 sensors are 50, 40 and 20 ppm, 80, 60 and 40 ppm, and 20, 10 and 5 ppm, respectively. The concentrations of two artificial samples containing the tertiary mixture were analysed with satisfactory results.