A temperature control system for quantitive polymerase chain reaction (PCR) is presented in the paper with both software and hardware configuration. The performance of the control system has been improved by optimizing the software and hardware design according to the system's properties. The control system has been proven to have a good repeatability and reliability as well as high control precision.
Equipment Design ; Microcomputers ; Polymerase Chain Reaction ; instrumentation ; Software ; Software Design ; Temperature

According to the designed specific primers of gene fragment based on the Salvia miltiorrhiza transcriptome data, with the method of reverse transcription polymerase chain reaction (RT-PCR), this study cloned full-length cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase gene from Salvia miltiorrhiza bge.f.alba, this sequence is named as SmHDS and its GenBank registration number is KJ746807. SmHDS, 2 529 bp long, contains an ORF of 2 229 bp, encodes 742 amino acids, including 5' UTR 170 bp and 3' UTR 130 bp. Using bioinformatics software, having made a homology analysis of the obtained sequence, we can have a conclusion that SmHDS have a close genetic relationship with HDS of Salvia miltiorrhiza. Analysis result of prokaryotic expression revealed that in Escherichia coli, SmHDS expressed target proteins which in size are comparable with the protein predicted. Meanwhile, the 4 factors which can influence the protein expression were optimized, the 4 factors are inducing temperature, inducing time, IPTG concentrations and density of inducing host bacterium (A600). The optimal expression conditions of SmHDS were 30 degrees C until the A600 is 0.6, and add IPTG to a final concentration of 0.2 mmol x L(-1), and the induction time of 20 h. It provides theoretical basis for the further study of the function of 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase in the biosynthesis of tanshinone compounds.
Cloning, Molecular ; DNA, Complementary ; genetics ; Diterpenes, Abietane ; biosynthesis ; Enzymes ; biosynthesis ; genetics ; Escherichia coli ; metabolism ; Plant Proteins ; biosynthesis ; genetics ; Salvia miltiorrhiza ; enzymology ; genetics

Soxhlet extraction is a common method of sample preparation. However, there has been no discussion about the efficiency of Soxhlet extraction from different batches and the factors that cause content fluctuation. In this study, Panax ginseng was selected as a model sample. Soxhlet extraction by means of a water bath, which has always been neglected, was identified as a novel key factor in the poor repeat-ability in different batches of Soxhlet extraction, as it can affect the siphon times and reflux time, which have been positively correlated with the ginsenoside contents. By substituting round bottom flasks in the same column, the relative standard deviation of the most fluctuated compound, ginsenoside Rb1, was decreased from 24.6% to 5.02%. Scanning electron microscopy analysis confirmed that the breakdown of the surface of the ginseng powder in the Soxhlet extraction led to a better dissolution of ginsenosides, indicating that chloroform may promote the extraction of ginsenosides by disrupting the cell structure. Moreover, 70% methanol was regarded as the better solvent for extracting the ginsenosides. Overall, this work offers a practical and effective protocol for improving the accuracy and repeatability of Soxhlet extraction methodology for ginsenosides and other analytes.

AIMTo study the transfection and anti-hepatitis B virus (HBV) effect of the co-modified hepatocytes-targeting cationic liposomes encapsulating anti-HBV antisense oligonucleotides (asON) , and to investigate the transfection mechanisms of the liposomes.

METHODSDipalmitoylphosphatidylcholine (DPPC) and 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) were used as the lipids, beta-sitosterol-beta-D-glucoside (sito-G) and Brij 35 were used to modify the liposomes. Flow cytometry (FCM), fluorescence microscopy and enzyme-linked immunosorbent assay (ELISA) were utilized to evaluate the transfection improvement of the asON encapsulated in the liposomes in primary rat hepatocytes and the antigens inhibition activity in HepG 2.2.15 cells. The transfection mechanisms were evaluated based on the influence of wortmannin, nigericin, and asialofetuin on the antigens inhibition in HepG 2.2.15 cells by ELISA.

RESULTSThe co-modification with sito-G and Brij 35 significantly improved the transfection of the liposomes in primary rat hepatocytes and antigens inhibition effect in HepG 2.2.15 cells. Both transfection efficiency and antigens inhibition effect showed to be concentration-dependent with the asON-encapsulating liposomes. In fluorescence microscopy, the transfected cells showed strong fluorescence in primary rat hepatocytes, especially in the nuclei. Wortmannin, nigericin and asialofetuin decreased the antigens inhibition of the asON-encapsulating liposomes to different levels. Cationic liposomes modification with sito-G and Brij 35 could improve the transfection and antigens inhibition effect of the asON. The transfection mechanisms of the co-modified liposomes included endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance asialoglycoprotein receptor (ASGPR)-mediated endocytosis.

CONCLUSIONCo-modified hepatocytes-targeting cationic liposomes would be a specific and effective carrier to transfer asON into hepatocytes.

Androstadienes ; pharmacology ; Animals ; Asialoglycoproteins ; pharmacology ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Cell Survival ; Cells, Cultured ; Endocytosis ; drug effects ; Female ; Fetuins ; Flow Cytometry ; Hepatitis B Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatocytes ; cytology ; drug effects ; metabolism ; Humans ; Liposomes ; Microscopy, Fluorescence ; Nigericin ; pharmacology ; Oligonucleotides, Antisense ; chemistry ; genetics ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Wistar ; Sitosterols ; chemistry ; Transfection ; methods ; alpha-Fetoproteins ; pharmacology

Aged ; Arthroplasty, Replacement, Knee ; methods ; Female ; Femur ; surgery ; Humans ; Osteoarthritis ; surgery ; Osteotomy ; methods

Objective To explore the activation and function of Janus protein-tyrosine kinase (JAK)/ signal transducer and activator of transcription (STAT1) signal transduction pathway in kidney,lung and brain of MRL/lpr mice.Methods MRL/lpr mice with systemic lupus erythematosus (SLE) were studied at the age of 12 weeks up.Non-SLE MRL/lpr mice were used as controls.We used phosphospecific antibodies to detect STAT1 activation in kidney,lung and brain by immunohistochemistry and Western blots.Gene expression of the STAT induced feedback inhibitors of cytokine signaling 1 (SOCS-1) was investigated by SYBR green I real-time reverse transcriptase polymerase chain reaction (PCR).Results Phosphorylation of STAT1 protein was markedly activated in these three organs,although renal and pulmonary STAT1 activation were much more evidently activated.SOCS-1 gene expression increased in all three organs,while renal SOCS-1 gene expres- sion increased less than lung and brain.Conclusion The activation of JAK/STATI signal transduction path- way may be pathogenic in the organ involvement and progression of SLE.The pathogenesis of lupus nephritis may also be associated with the down-regulation of SOCS-1 feedback inhibition.

Objective To establish a LC-MS/MS method for pharmacokinetics study of methylergonovine in healthy Chinese volunteers after single and multiple-dose administration of methylergonovine maleate tablets. Methods 6-hydroxyflavone was used as internal standard.The separation was achieved on a Waters Xterra C18 (2. 1 mm × 100 mm, 3. 5 μm) with a mobile phase consisting of 0. 05％ ammonium acetate and methanol-acetonitrile (80:20) solution. At a flow rate of 0. 3 m L·min-1 within 12 min. Methylergonovine and 6-hydroxyflavone were measured by ESI in positive electron mode using multiple reaction monitoring (MRM) . The extracted ions monitored following MRM transitions were m/z 340. 3 →223. 2 for methylergonovine and m/z 239. 2 →129. 1 for 6-hydroxyflavone. Plasma samples were pretreated by liquid-liquid extraction. Results The calibration curve was linear within the range of 0. 1-20. 0 ng·m L-1. The Lower limit of quantitation was 0. 1 ng·m L-1 and CV％ of intra-and inter-day were less than 15％. The plasma samples were stable at room temperature (25 ℃) for 4 h, at-70℃ for 4 months and during three freeze-thaw cycles. There was no accumulationafter ultiple-dose of methylergonovine maleate tablets.Conclusion The method was proved to be accurate and sensitive suitable for the pharmacokinetics of methylergonovine in volunteers after oral administration of 0. 125, 0. 25, 0. 5 mg methylergonovine maleate tablets.

OBJECTIVETo study the values of brain natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) in the evaluation of cardiac function in children with congenital heart disease (CHD).

METHODSSeventy-one children with CHD were classified to two groups: congestive heart failure (CHF) (n=23 ) and non-CHF (n=48). Thirty-five age-matched normal children were used as the control group. Plasma BNP content was measured using a microparticle enzyme immunoassay (MEIA) on the AxSYM. Plasma NT-proBNP content was measured using an automated electrochemiluminescence immunoassay on a Roche Modular Analytics E170 analyzer. Echocardiographic parameters, including left ventricular end diastolic dimension index (LVEDDI) and left ventricular ejection fraction (LVEF), were measured.

RESULTSPlasma BNP and NT-proBNP contents in the CHF group were significantly higher than those in the non-CHF group (P<0.01). The non-CHF group had higher plasma BNP and NT-proBNP contents than the control group (P<0.01). LogBNP and LogNT-proBNP values were negatively correlated with the LVEF in the CHF group (r=-0.64, r=-0.67 respectively, P<0.01), and they were positively correlated with the LVEDDI (r=0.58, r=0.76 respectively, P<0.01). In the non-CHF group, LogBNP and LogNT-proBNP values were not correlated with the LVEF, but a positive correlation was found between the LogNT-proBNP value and the LVEDDI (r=0.35, P<0.05). Using plasma BNP content > or =149.8 pg/mL and NT-proBNP content > or =820.1 pg/mL as cut-off values for diagnosing CHF respectively, the sensitivities were 87.0 % and 91.3% respectively, the specificities were 91.7% and 97.9% respectively, and the areas under the ROC curves were 0.935 and 0.987 respectively.

CONCLUSIONSBoth BNP and NT-proBNP can be useful in assessment of cardiac function and diagnosis of CHF in children with CHD. NT-proBNP appears to be more sensitive and specific in the diagnosis of CHF than BNP.

Child ; Child, Preschool ; Diastole ; Female ; Heart ; physiopathology ; Heart Defects, Congenital ; blood ; physiopathology ; Heart Failure ; diagnosis ; Humans ; Infant ; Male ; Natriuretic Peptide, Brain ; blood ; Peptide Fragments ; blood ; Ventricular Function, Left

Diagnosis, Differential ; Female ; Humans ; Laparotomy ; methods ; Pregnancy ; Pregnancy, Ectopic ; diagnosis ; surgery ; Retroperitoneal Space ; diagnostic imaging ; Tomography, X-Ray Computed ; Ultrasonography

OBJECTIVEPrevious studies have shown that hydrogen sulfide (H2S) plays key roles in a number of biological processes, including vasorelaxation, inflammation, apoptosis, ischemia/reperfusion and oxidative stress, which are involved in the pathogenesis of myocarditis. This study aimed to examine the expression of cystathionine-γ-lyase(CSE)/H2S pathway in mice with viral myocarditis.

METHODSSix-week-old inbred male mice were randomly assigned to control (n=25) and myocarditis group (n=30). The myocarditis and the control groups were inoculated intraperitoneally with 0.1 mL 10-5.69TCID50/mL CVB3 or vehicle (PBS) alone respectively. Ten mice were sacrificed 4 and 10 days after injection. Blood and heart specimens were harvested for measuring the content of serum H2S and the H2S production rates in cardiac tissues. Heart sections were stained with hematoxylin and eosin. Immunohistochemisty was used to detect the CSE protein expression in the heart.

RESULTSIn the myocarditis group, the serum H2S content and H2S production rates in cardiac tissues were significantly higher than those in the control group 4 and 10 days after injection (P<0.05). The expression of CSE protein in the heart in the myocarditis group was also significantly higher than that in the control group (P<0.05).

CONCLUSIONSCSE and its downstream production H2S increase in mice with acute viral myocarditis. The increased expression of CSE/H2S pathway might be involved in the pathogenesis of viral myocarditis.

Animals ; Coxsackievirus Infections ; etiology ; Cystathionine gamma-Lyase ; analysis ; Enterovirus B, Human ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Hydrogen Sulfide ; metabolism ; Killer Cells, Natural ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Myocarditis ; etiology