RNA silencing is increasingly employed as an experimental strategy to probe for gene function in several organisms. The purpose of the present study was to test the effect of gene silencing by dsRNA in budding yeast Saccharomyces cerevisiae. GRE3 gene encoding an NADPH-dependent aldose reductase was chosen as an example. A recombinant plasmid psiLENT-GRE3 was constructed based on the pESC-LEU backbone and used to transform S. cerevisiae YPH499. The down regulation of GRE3 gene expression by inducing 1 kb RNA duplex and a 136 bp loop was investigated using reverse transcription- PCR. The results showed that double-stranded RNA mediated gene silencing could be used as a functional tool to decrease the expression level of a specific gene in S. cerevisiae, which would contribute to the understanding of RNA interference in budding yeast.

The contamination of extrinsic harmful contaminants including mycotoxins, heavy metals and pesticides, etc, brings serious risks to traditional Chinese medicines (TCMs), further to human health. Due to their unique photoluminescence, chemiluminescence, electrochemical and electrochemiluminescence properties, semiconductor quantum dots (QDs) nanoparticles are widely used to immobilize bioprobes and biosensors, etc. In this review, the luminescence characteristics and specific ligands of QDs probles which are used to determine contaminants were summed up. Then, the applications of QDs-coated novel probes in the determination of mycotoxins, heavy metals and pesticides were discussed in detail. In addition, the contamination levels and characteristics of extrinsic harmful residues in TCMs were investigated. Further, the maximum levels of those contaminants in TCMs were compared with those set by various countries. Finally, the future development trends and problems of QDs-coated probes in the determination of those extrinsic residues in TCMs were prospected.
Drug Contamination ; prevention & control ; Humans ; Medicine, Chinese Traditional ; methods ; Nanotechnology ; instrumentation ; methods ; Quantum Dots ; Safety ; Time Factors

A simple and cost-effective indirect competitive enzyme-linked immune sorbent assay (ic-ELISA) was developed to rapidly screen the content of aflatoxin B1 (AFB1) in lotus seeds, and the results were confirmed by ultra-fast liquid chromatography-tandem mass spectrometry( UFLC-MS/MS). Matrix-matched calibration expressed a good linearity ranging from 0. 171 to 7. 25 µg · L(-1) for AFB, with R2 > 0.978. The medium inhibitory concentration( IC50 ) for AFB1 was 1.29 µg · L(-1), the recovery for AFB1 was 74.73% to 126.9% with RSD < 5%, and the limit of detection (IC10) was 0.128 µg · L(-1). The developed ic-ELSIA method was applied to rapid analysis of AFB, in 20 lotus seeds samples and the results indicated that the contents of AFB, in samples 1-15 were in the range of 1. 19- 115. 3 µg · kg(-1) and in 40% of the samples exceeded the legal limit(5 µg · kg(-1)), while the contamination rate of AFB, in samples 16-20 was 40%. Pearson correlation coefficient(r) reached 0.997 for AFB1 content in the samples detected by ic-ELSIA and UFLC-MS/MS methods. The results proved that the developed ic-ELISA method is simple, sensitive and reliable, and can be used for rapid and high-throughput screening of AFB1 in lotus seeds
Aflatoxin B1 ; analysis ; Drug Contamination ; Enzyme-Linked Immunosorbent Assay ; methods ; Loteae ; chemistry ; Seeds ; chemistry

With the development of low-carbon economy and renewable resource, fermentation of the pentose sugar xylose to produce ethanol becomes a very hot topic. The recombinant Saccharomyces cerevisiae can be constructed by expressing heterologous xylose isomerase (XI). Because Thermus thermophilus XI (TthXI) does not need cofactor, it has been developed for establishing the utilization pathway of xylose in S. cerevisiae. In this article, we reviewed the progress on xylose isomerase. We first introduced the primary properties, sequence and structure characters of xylose isomerase, and discussed its thermostability. The molecular modification of xylose isomerase, including of substrate specificity and thermostability were discussed in detail. Meanwhile, combined with our own research, we also discussed how to improve the xylose isomerase activity at room temperature. Finally, we suggested perspectives of xylose isomerase.
Aldose-Ketose Isomerases ; chemistry ; genetics ; metabolism ; Catalysis ; Enzyme Stability ; Hot Temperature ; Recombinant Proteins ; biosynthesis ; genetics ; Saccharomyces cerevisiae ; genetics ; metabolism ; Substrate Specificity

Objective To assess the application value of high time-resolved MR angiography (TR-MRA) in postoperative follow-up study of children with congenital heart diseases. Methods Seventy-three patients (median age 6 years, range 1-20 years) with congenital heart diseases who underwent TR-MRA scan after operation were retrospectively analyzed. Twenty-nine cases also were performed conventional contrast-enhanced MRA and forty-four cases were performed phase-contrast MRA. A 3D T1-weighted fast gradient-echo sequence was used for time-resolved three-dimensional MRA (10-20 dynamic data sets, less than three seconds per dynamic data set). The flow dynamics and morphology of pulmonary circulations, lung perfusion and collaterals flow direction were noted. All imaging quality was evaluated by using 5 scales. Left and right pulmonary artery flow volumes were measured and left and right pulmonary artery ratio was noted. SPSS22.0 was used in statistic analysis. The statistical analysis of comparing imaging quality was performed by using paired t-test. The intermodality agreement between TR-MRA and phase contrast in assessing left and right pulmonary perfusion was tested by Kappa coefficient. Results In 73 cases, imaging scores were over 3 and imaging quality was good enough for diagnosis. In 29 cases, there was no statistic difference between TR-MRA and conventional CE-MRA in demonstrating great vessels (P>0.05) except that CE-MRA scores(3.77 ± 0.39)was higher than TR-MRA scores(3.44 ± 0.55)of
inferior vena cava (IVC). There was statistic difference(t=3.68,P=0.01)between two sequences. TR-MRA could qualitatively demonstrate the pulmonary perfusion comparing to the results of PC. In PC sequence, there were 8 cases with symmetric and 36 cases with asymmetric left and right pulmonary perfusion. In TR-MRA sequence, there were 6 cases with symmetric and 38 cases with asymmetric left and right pulmonary perfusion. There was an excellent agreement between PC and TR-MRA (Kappa=0.83,P=0.01). Conclusions TR-MRA not only supplies with high spatial resolution imaging which demonstrates postoperative great arteries anatomy and also with high temporal resolution imaging which can demonstrate the preferential or balanced pulmonary blood flow and collaterals flow direction. TR-MRA is a very important sequence in follow-up study of congenital heart disease.

Revolution energy spectral CT had its structural innovation introduced from the aspects of detector,driving system and CT bulb as well as high voltage generator,technical principle analyzed from the aspects of SSF,multi-model iterative reconstruction, cardiac imaging unlimited as well as material separation and quantitative analysis, and clinical application described in diagnoses of liver cancer, cholecystolithiasis and kidney stone, coronary arteriongraphy and metal artifact elimination.It's pointed out Revolution energy spectral CT was a new method for identifying the focal nature,tumor homology and components of inorganic substance as well as analyzing multi material quantitatively and qualitatively.

Acetaldehyde ; pharmacology ; Animals ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; Flavonoids ; pharmacology ; Hepatocytes ; cytology ; physiology ; Rats

The chemical constituents were isolated and purified by various chromatographic techniques indluding silica gel, reverse phase silica gel, sephadex LH-20 and pre-HPLC and identified by their physicochemical properties and spectral data. Sixteen phenolic compounds had been isolated and n-butanol extracts which were fractionated from the ethanol extract of Oplopanax horridus roots bark. Their structures were identified as below, including 7 phenylpropanoid compounds, ferulic acid (1), 3-acetylcaffeic acid (2), caffeic acid (3), homovanillyl alcohol 4-O-beta-D-glucopyranoside (4), 3-hydroxyphenethyl alcohol 4-O-beta-D-glucopyranoside (5), 3, 5-dimethoxycinnamyl alcohol 4-O-beta-D-glucopyranoside (6), and 3-dimethoxycinnamyl alcohol 4-O-beta-D-glucopyranoside (7). Three coumarins, scopoletin (8), esculetin (9) and 3'-angeloyl-4'-acetyl-cis-knellactone (10). And 6 lignan compounds, (+)-isolaricires-inol-9'-O-beta-D-glucopyranoside (11), 3, 3'-dimethoxy-4, 9, 9'-trihydroxy-4', 7-epoxy-5', 8-lignan-4, 9-bis-O-beta-D-glucopyranoside (12), (+)-5, 5'-dimethoxylariciresinol 4'-O-beta-D-glucopyranoside (13), (-)-5,5'-dimethoxylariciresinol 4'-O-beta-D-glucopyranoside (14), (-)-pinoresinol 4'-O-beta-D-glucopyranoside (15), and (+)-5, 5'-dimethoxylariciresinol 9'-O-beta-D-glucopyranoside (16). All compounds were isolated and identified for the first time from this plant All the constituents except compounds 4, 6, 12 and 13 were obtained for the first time from the genus Oplopanax.
Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Oplopanax ; chemistry ; Phenols ; chemistry ; isolation & purification ; Spectrometry, Mass, Electrospray Ionization

OBJECTIVEIn this experiment, genes sensitive to mechanical stretch in osteoblast like cells were cloned through subtractive hybridization technique.

METHODSTwo dimensional mechanical stretch with deformation of 12% and frequency of 6 cycles was loaded on human osteoblastic like cell line Saos-2. Complementary deoxyribonucleic acid (cDNA) library of cells was constructed 12 h after loading, acting as tester. cDNA library of cells without loading was constructed, acting as driver. A subtractive cDNA library osteoblastic like cell stimulated with mechanical stretch was constructed through subtractive hybridization technique.

RESULTSOf clones randomly selected from this library, fifteen genes were identified to be the differentially expressed genes. Comparing with the sequences published in GeneBank via Internet, two sequences located in chromosome 9 and 18 respectively were identified to be novel, which were named as stretch sensitive gene 1 and stretch sensitive gene 2.

CONCLUSIONIt is an efficient approach to clone and study genes relative to mechanical stretch through subtractive hybridization technique.

Effects of serotonin (5-HT) on spontaneous discharges of single hypothalamic arcuate neurons were observed in rat brain slices by extracellular recordings. The results showed that (1) of 149 neurons selected randomly and tested for 5-HT application, 33 (22.2%) were excited, 82 (55.0%) were inhibited, and 34 (22.8%) showed biphasic responses or failed to respond; (2) substitution of low Ca(2+)-high Mg(2+) artificial cerebrospinal fluid (aCSF) for normal aCSF abolished the 5-HT-induced inhibitory effect but failed to affect the 5-HT-induced excitatory effect; (3) cyproheptadine, a non-selective 5-HT receptor antagonist, could block either the 5-HT-induced excitatory or inhibitory effects in all neurons tested; and (4) bicuculline, a GABA(A)-receptor antagonist, blocked the 5-HT-induced inhibitory effect. These results imply (1) 5-HT excites arcuate neurons through a mechanism that is insensitive to the decreased extracellular Ca(2+), suggesting a direct postsynaptic action of 5-HT on the 5-HT-receptors located in the membrane of the neurons recorded; and (2) 5-HT might elicit the inhibitory effect through a Ca(2+)-sensitive release of GABA from intercalated GABAergic local neurons that are excited first by 5-HT.
Animals ; Arcuate Nucleus of Hypothalamus ; drug effects ; physiology ; Cyproheptadine ; pharmacology ; In Vitro Techniques ; Neurons ; drug effects ; physiology ; Rats ; Rats, Wistar ; Serotonin ; pharmacology ; gamma-Aminobutyric Acid ; physiology