Objective:To evaluate the identification rate of separating gel or HB&L pretreatment methods of MALDI-TOF-MS, thereby to provide a new idea for the rapid and accurate identification of pathogens of bloodstream infections in daily clinic practice.Methods:A total of 149 alarmed positive blood culture samples of single bacterial infection by routine laboratory methods were collected between January to December 2020 from the Sixth Medical Center, Chinese PLA General Hospital. Samples were pretreated with the separation gel accelerating tube method or the HB&L microbial culture system, followed by direct MALDI-TOF MS bacterial identification, the identification rates of the two pretreatment methods were compared and results from the traditional method were used as the standard control.Results:Among the 149 positive blood culture samples, 47.0% (70/149) were gram-negative (G -) bacteria and 53.0% (79/149) were gram-positive (G +) bacteria. Identification rate of G -strain level was 78.6% (55/70) by serum separation gel coagulation tube method and 91.4% (64/70) by HB&L microbial culture system, the difference was statistically significant ( P=0.033). Identification rate of G +strain levels was 73.4% (58/79) by serum separation gel coagulation tube method and 87.3% (69/79) by HB&L microbial culture system, the difference was statistically significant ( P=0.028). For G -bacteria in the range of 3.000-2.300, the identification rate was 22.9% (16/70) by serum separation gel accelerating tube method and 38.6% (27/70) by the HB&L microbial culture system, the difference was statistically significant ( P=0.044). For G +bacteria in the range of 3.000-2.300, the identification rate was 19.0% (15/79) by serum separation gel accelerating tube method and 34.2% (27/79) by the HB&L microbial culture system, the difference was statistically significant ( P=0.031). Conclusion:The identification rate of HB&L microbial culture system is higher than that of serum separation gel coagulation tube method. Direct MALDI-TOF MS identification of pathogenic bacteria in positive blood culture samples after pretreatment is feasible in daily clinical practice.

OBJECTIVE:To establish a method for the content determination of loxoprofen silver in Loxoprofen silver cream, and provide reference for the quality control of the preparation. METHODS:HPLC was performed on the column of Diamonsil C18 with mobile phase of methanol-water-glacial acetic acid-triethylamine(55:45:0.1:0.1,V/V/V/V)at a flow rate of 1.0 mL/min,de-tection wavelength was 223 nm,column temperature was 30 ℃,and the injection volume was 20 μL. RESULTS:The linear range of loxoprofen silver was 6.53-130.7μg/mL(r=0.9999);the limit of quantitation was 0.253μg/mL,limit of detection was 0.076μg/mL;RSDs of precision,stability and reproducibility tests were lower than 2%;recovery was 96.79%-103.68%(RSD=2.23%, n=9). CONCLUSIONS:The method is simple,accurate and reproducible,and can be used for the content determination of Loxo-profen silver cream.

Objective By detecting the variation of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) DNA methylation in preeclampsia-like mouse models generated by different ways, to explore the roles of multifactor and multiple pathways in preeclampsia pathogenesis on molecular basis. Methods Established preeclampsia-like mouse models in different ways and divided into groups as follows: (1) Nw-nitro-L-arginine-methyl ester (L-NAME) group: wild-type pregnant mouse received subcutaneous injection of L-NAME;(2) lipopolysaccharide (LPS) group:wild-type pregnant mouse received intraperitoneal injection of LPS; (3) apolipoprotein C-Ⅲ (ApoC3) group: ApoC3 transgenic pregnant mouse with dysregulated lipid metabolism received subcutaneous injection of L-NAME;(4)β2 glycoprotein I (β-2GPI) group:wild-type pregnant mouse received subcutaneous injection ofβ-2GPI. According to the first injection time (on day 3, 11, 16 respectively), the L-NAME, LPS and ApoC3 groups were further subdivided into:pre-implantation (PI) experimental stage, early gestation (EG) experimental stage, and late gestation (LG) experimental stage.β-2GPI group was only injected before implantation. LCHAD gene methylation levels in placental were detected in different experimental stage. Normal saline control groups were set within wild-type and ApoC3 transgenic pregnant mice simultaneously. Results (1) CG sites in LCHAD DNA:45 CG sites were detected in the range of 728 bp before LCHAD gene transcription start site, the 5, 12, 13, 14, 15, 16, 19, 24, 25, 27, 28, 29, 30, 31, 32, 34, 35, 43 CG sites were complex sites which contained two or more CG sequences, others were single site which contained one CG sequence. The 3, 5, 6, 11, 13, 14, 18, 28 sites in L-NAME, LPS, ApoC3 and β-2GPI groups showed different high levels of methylation; the 16, 25, 31, 42, 44 sites showed different low levels of methylation; other 32 sites were unmethylated. (2) Comparison of LCHAD gene methylation between different groups:the methylation levels of LCAHD gene at 3, 11, 13, 14, 18 sites in L-NAME, LPS, ApoC3 andβ-2GPI groups were significantly higher than those in the normal saline control group (P<0.05); and the methylation levels of 42, 44 sites in these groups were significantly lower than those in the normal saline control group (P<0.05). (3) Methylation of LCHAD gene at the same site between different experimental stages: ① The 3, 11, 18 sites of EG experimental stage was significantly lower than PI and LG experimental stage in L-NAME group (P<0.05);the 3, 11, 18 sites of PI experimental stage was significantly lower than EG and LG experimental stage in LPS group (P<0.05);these sites of PI experimental stage was significantly higher than EG and LG experimental stages in ApoC3 group (P<0.05).②The methylation of site 5 in L-NAME and LPS groups were significantly higher than that of the normal saline control group (P<0.05), and the LG experimental stages were significantly higher than other stages, but in ApoC3 group , only PI and EG stages were significantly higher than the normal saline control group (P<0.05).③At site 6 in L-NAME group which showed high methylation level was significantly higher than the same site in other groups which showed low methylation level (P<0.05).④At 13, 14 sites, earlier preeclampsia onset caused a lower methylation level in L-NAME group, but PI experimental stage was significantly higher than EG and LG experimental stages in LPS group (P<0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P<0.05). ⑤ At site 28, earlier preeclampsia onset caused a higher methylation level in L-NAME group, but PI experimental stage was significantly lower than EG and LG experimental stages in LPS group (P<0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P<0.05).⑥The 16, 25, 31 sites in ApoC3 group were significantly higher than other groups (P<0.05). ⑦ At site 42 in β-2GPI group was unmethylated, but it in other groups showed low methylation level, the methylation level of site 42 inβ-2GPI group was significantly lower than that in other groups (P<0.05). Conclusions The methylation of 6 and 42 CG sites may be related to LCHAD gene expression in placenta of L-NAME and β-2GPI induced preeclampsia-like models respectively;LCHAD gene expression and DNA methylation may not have obviouscorrelation in LPS and ApoC3 induced preeclampsia-like models. Differences exist in LCHAD DNA methylation in preeclampsia-like models generated by different ways, revealed a molecular basis to expand our understanding of the multi-factorial pathogenesis of preeclampsia.

Objective By detecting the DNA methylation and gene expression of long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) in trophoblast cells, analyze the correlation of DNA methylation and gene expression in early-onset preeclampsia (EPE), hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome and antiphospholipid syndrome (APS), to investigate the molecular basis of long-chain fatty acid oxidation changes in different preeclampsia and pathological pregnancy. Methods Primary human cytotrophoblast cells and HTR8/Svneo cells were treated with serum from patients with EPE (14 cases), HELLP (12 cases), APS (14 cases), and normal pregnant women (NP, 14 cases). The methylation level of LCHAD gene promoter region through the MassARRAY platform and mRNA expression level by real-time fluorescent quantitative PCR technique were conducted. Results (1) Cytosine-phosphate-guanine (CpG)sites in human LCHAD DNA promoter region:CpG sites were detected in the range of 558 bp before LCHAD gene transcription start site, the detected CpG sites were 11 sites including 8 single sites and 3 complex sites. The position of these sites were at-984,-960,-899,-853,-811,-796,-774,-727,-615,-595,-579 respectively. (2) The sites of-899,-853,-615 and-595 showed increased methylation level in EPE and HELLP groups. The methylation level at-899,-853 and-615 sites in EPE and HELLP groups were significantly higher than those in NP group(P<0.01). The methylation level at-853 site was higher in EPE group than that in HELLP group(P<0.05). The-595 site showed the unmethylated in EPE, HELLP and APS groups. There were significantly difference between the 3 groups and EPE group(P<0.01). (3) The gene expression of LCHAD mRNA in EPE(0.048±0.005), HELLP(0.045±0.006)and APS(0.044±0.004) groups were significantly lower than NP group(0.076 ± 0.009;P<0.01). (4) The correlation of methylation level and gene expression in all groups: the methylation level at-899,-853,-727,-615 and-579 sites were negatively correlated with gene mRNA expression in EPE group (P<0.05). The methylation level at-899,-853 and-615 sites were negatively correlated with gene mRNA expression in HELLP group(P<0.05). Conclusions The variation of LCHAD DNA methylation of trophoblast cells are found among EPE, HELLP syndrome and APS. The different correlation of LCHAD DNA methylation and gene expression are different in pathological groups. LCHAD DNA methylation of EPE and HELLP syndrome were significantly increased and negatively correlated with LCHAD gene mRNA expression. These results further revealed the molecular basis of long-chain fatty acid oxidation in different preeclampsia and pathological pregnancy.

A rapid and sensitive method based on ultrafast liquid chromatography-tandem mass spectrometry was developed and validated for simultaneous determination of Sudan Ⅰ, Sudan Ⅱ, Sudan Ⅲ, and Sudan Ⅳ levels in rat whole blood. Cleanert C18 mixed-mode polymeric sorbent was used for effective solid-phase extraction cleanup. Separation was carried out on a reversed-phase C18 column (100 mm × 2.1 mm, 1.8 μm) using 0.1% (v/v) formic acid in water/0.1% (v/v) formic acid in acetonitrile as the mobile phase in gradient elution. Quantification was performed by an electrospray ionization source in the positive multiple reaction monitoring mode using D5-Sudan I as the internal standard. Calibration curves showed good linearity between 0.2 and 20.0 μg/L, with correlation coefficients higher than 0.9990. The average recovery rates were between 93.05% and 114.98%. The intra- and inter-day relative standard deviations were within 6.2%. The lower limit of quantification was 0.2 μg/L. All the analytes were found to be stable in aseries of stability studies. The proposed method was successfully applied to a pharmacokinetic study of four Sudan dyes after oral administration to rats.

Objective To study the physicochemical properties of aqueous solution of surface active drug montelukast so-dium (MS) ,which could provide experimental basis for further development of micelle or mixed micelle preparations .Methods Critical micelle concentration (CMC) of MS at different temperatures were determined by conductivity measurements .The absorbance and transmittance of MS aqueous solution were measured by UV at different sodium chloride concentration levels . The micelle stability was evaluated via high speed centrifugal .Results The CMC of MS aqueous solution at 25℃ ,30℃ ,35℃were 0 .75 ,0 .82 ,0 .90 mmol/L .The absorbance and transmittance of MS aqueous solution were affected by the sodium chlo-ride concentration and the concentration of MS itself .It was observed that a clear solution was obtained when MS concentration>7 .5 mmol/L and no precipitation was noticed even after high speed centrifugal .Conclusion Montelukast sodium is a surface active drug .Its solubility is related to MS concentration .The solubility is also sensitive to the temperature and the electrolyte concentration .These unique physicochemical properties could be used to develop micelle or mixed micelle pharmaceutical prepa-rations .

OBJECTIVETo investigate whether extracts of Danshen and Chuanxinlian (SL) could promote the function recovery in pre-monocytic cell line THP-1 induced by TNF-alpha.

METHODSL extracts (0.125-2 g x L(-1)) were used to incubate THP-1 for 24 h before stimulation with TNF-alpha (20 microg x L(-1)), the adhesion, migration, lipid uptake and secretion of THP-1 were observed.

RESULTSL (0.5-2 g x L(-1)) had obvious effect on decreasing the THP-1 adhesion. The number of passed membrane was much fewer than that of control cells in SL (0.125-2 g x L(-1)). SL (0.125-2 g x L(-1)) reduced the total cholesterol content significantly. The levels of IL-6 in SL (2 g x L(-1)) were significantly decreased,and IL-10 was increased than that before the treatment.

CONCLUSIONSL extracts could promote the function recovery such as adhesion, migration, lipid uptake and secretion of THP-1 induced by TNF-alpha, which probably is one of the mechanisms of inhibit the inflammatory reaction in initiation and development of AS.

Animals ; Cell Adhesion ; drug effects ; Cell Line ; Cell Movement ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Lipid Metabolism ; drug effects ; Male ; Monocytes ; cytology ; drug effects ; metabolism ; secretion ; Rabbits ; Salvia miltiorrhiza ; chemistry ; Tumor Necrosis Factor-alpha ; pharmacology

Objective To observe the effects of olprinone on ischemia/reperfusion (I/R) induced myocardial injury in male (Sprague-Dawley, SD rats) and explore its mechanisms. Methods Rats were subjected to a 30-min coronary arterial occlusion followed by 24-hour reperfusion. The survival rats were randomly divided into sham group (n=6), ischemia reperfusion group (I/R group, n=9), ischemia reperfusion+low dose of olprinone group(IR+olprinone-L group, n=6), ischemia reperfusion+medium dose of olprinone group (IR+olprinone-M group, n=6),ischemia reperfusion +high dose of olprinone group (IR+olprinone-H group, n=6). A MAP heart function analysis system was used to measure hemodynamic parameters; TTC staining method was used to detect the myocardial infarct size;24-hour mortality of SD rats was recorded; western blot was used to detect the levels of Caspase-3, Bax,Bcl-2, LC3B/LC3A,Beclin-1. Results Cardiac function in I/R group was lower than that in sham group, which was significantly improved by pretreatment with olprinone (P＜0.01),but systolic arterial pressure (SAP) diastolic arterial pressure (DAP) mean arterial pressure (MAP) mean pressure developed in left ventricle (Pmean) had no significant difference (P＞0.05). The percentage of myocardial infarct size in olprinone-M and olprinone-H group was lower than that in I/R group (P＜0.05).There was no significant difference in mortality among groups within 24 hours. Compared with sham group, the expressions of Caspase-3 and Bax were obviously up-regulated in I/R group (P＜0.01), whereas caspase-3 was down-regulated in olprinone-M group (P＜0.05) and Bax was inhibited by different doses of olprinone (P＜0.05), but the expression of Bcl-2 increased (P＜0.05); furthermore, the ratio of Bcl-2/Bax decreased in I/R group (P＜0.01) and increased with different degrees in different doses of olprinone (P＜0.05). Meanwhile, compared with sham group, the expression of Beclin-1 was up-regulated in I/R group(P＜0.05),and also increased in olprinone-L and olprinone-M groups(P＜0.05), but the ratio of Bcl-2 /Beclin-1 decreased in different doses of olprinone making statistically significant difference only in olprinone-M group (P＜0.05). Moreover, different doses of olprinone elevated the different ratios of LC3B/LC3A (P＜0.05), and this elevated ratio in olprinone-M group at median among groups. Conclusions Olprinone can strengthen the cardiac function after myocardial ischemia/reperfusion injury, without leading to disorders in hemodynamics; by regulating autophagy with anti-apoptotic protein, olprinone can make autophagy to an appropriate level using the mechanism of autophagy to preventing the myocardium from injury.

OBJECTIVEWuji pill is a prescription of traditional Chinese medicine(TCM) and was composed of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae and Radix Paeoniae Alba. The aim of this research is to investigate the effects of Wuji pill compound with different compatibility on the levels of enzymic activity of cytochrome P450 CYP3A1/3A2 in rat liver microsomes in vitro, and to confirm the compatibility mechanism of Wuji pill from the point of relationships between compound prescription of TCM and metabolism.

METHODWith testosterone being a probe, the levels of enzymic activity of CYP3A1/3A2 were detected by HPLC, which were suppressed by Wuji pill with different compatibility in vitro.

RESULTThe IC50 of Rhizoma Coptidis, Fructus Evodiae Rutaecarpae, Radix Paeoniae Alba and 1"-9" of different level Wuji pill is: 38.96, 871.96, 15 519.17, 43.17, 60. 47, 276.12, 133.40, 118.08, 88. 47, 64. 36, 35. 13 and 39. 91 mg x L -', respectively. Rhizoma Coptidis and 1-9" of Wuji pill can suppress the enzymic activity of CYP3A1/3A2 significantly, and the capability of Rhizoma Coptidis in Wuji pill of action on CYP3A1/3A2 can be modified by different composition of Fructus Evodiae Rutaecarpae and Radix Paeoniae in Wuji pill, and there are statistical differences among the IC50 of 1#-9# of Wuji pill. While the ratio of Rhizoma Coptidis raises up in Wuji pill, Wuji pill may suppress the enzymic activity of CYP1A2 largely.

CONCLUSIONThe reason why Wuji pill with different compatibility has different pharmacodynamics and pharmacokinetics characteristics is likely to lie in the difference of the capability of Wuji pill with different compatibility on CYP3A1/3A2. [Key words] Wuji pill; CYP3A1/3A2; testosterone; HPLC; different compatibility prescription of traditional Chinese medi-cine

Animals ; Aryl Hydrocarbon Hydroxylases ; antagonists & inhibitors ; metabolism ; Coptis ; chemistry ; Cytochrome P-450 CYP3A ; Drug Compounding ; Drugs, Chinese Herbal ; adverse effects ; pharmacology ; Enzyme Inhibitors ; adverse effects ; pharmacology ; Evodia ; chemistry ; Inhibitory Concentration 50 ; Male ; Membrane Proteins ; antagonists & inhibitors ; metabolism ; Microsomes, Liver ; drug effects ; enzymology ; Paeonia ; chemistry ; Rats ; Rats, Wistar

Objective To evaluate the effectiveness of quantitative midbrain measurements in differentiating progressive supranuclear palsy (PSP) from multiple system atrophy (MSA) and Parkinson's disease (PD).Methods Quantitative midbrain measurements,including midbrain width (MW),midbrain to pons ratio (M/P) and magnetic resonance parkinsonism index (MRPI),were performed in patients with parkinsonism who were diagnosed in the Movement Disorder Clinic of Peking Union Medical College Hospital during the period of January to September 2017.A cross-sectional study was conducted in the series to evaluate the effectiveness of these quantitative measurements.Results Ten PSP-RS,15 MSA-P and 49 PD patients were included in this study.The values of MW,M/P and MRPI in PSP-RS patients were (8.21 ± 1.30) mm,0.49 ±0.06 and 15.26 ±4.53,respectively,with statistically significant difference compared toMSA-P ((10.24±0.77) mm,0.65 ±0.09,7.75 ±2.71) and PD patients ((10.53 ±0.93) mm,0.62±0.06,9.86 ±2.46;F=24.27,18.37,21.47,all P＜0.01).After adjusting for age,disease course and gender,analysis of covariance suggested significant correlation between the quantitative midbrain measurements and diagnosis of these diseases.Receiver operating characteristic curve indicated that MW ≤9.4 mm,M/P≤0.57 and MRPI ＞ 10.77 showed ideal sensitivity and specificity (90.0％ and 92.1％,80.0％ and 93.7％,100.0％ and 82.5％) in differentiating PSP-RS from MSA-P and PD.Conclusion Quantitative measurement of midbrain atrophy is useful in differentiating PSP-RS from MSA-P and PD.