As the dilution procedure was applied, a simple, rapid and cost-effective high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for determination of aflatoxin B1, B2, G1, and G2 was successfully by performed in a total 83 samples of 10 traditional Chinese medicines (TCMs), which were collected from 5 different hospital pharmacies and 5 different medical stores in Guangzhou city. Matrix effects of these 10 TCMs were ranged from 80.23% to 115.5% in low, intermediate and high concentration levels, indicating that the negative effect was overcome in this study. Meanwhile, the analysis method was proved to be stable and reliable during the whole analysis using Semen Armeniacae Amarum spiked 3 concentration levels of standard solution as quality control samples and the RSD < 6.6% was obtained. The contamination levels of 83 investigated samples were 13.89% and 17.02% in hospital pharmacies and medical stores, respectively. The result was presented to provide relevant reference and supplement to those researchers in TCMs analysis and screening.
Aflatoxin B1 ; analysis ; Aflatoxins ; analysis ; Chromatography, High Pressure Liquid ; methods ; Drug Contamination ; Medicine, Chinese Traditional ; Quality Control ; Tandem Mass Spectrometry ; methods

OBJECTIVETo explore the characteristics of changes in neutrophil gelatinase-associated lipocalcin (NGAL), kidney injury molecule-1 (KIM-1) and interleukin-18 (IL-18) in Phytolaccae Radix-induced kidney injury in rats and the significance of the combined detection.

METHODWistar rats were divided into three groups: high and low dose (crude drug 40, 20 g x kg(-1) x d(-1)) Phytolaccae Radix decoction groups and the control group, and orally administrated with distilled water or equal volume of Phytolaccae Radix decoction for 35 consecutive days. Their blood and urine samples were collected on day 7, 14, 21, 28, 35,42. The anatomical analysis was conducted for each group. The contents of serum total protein (TP), albumin (ALB), blood urea nitrogen (BUN), creatinine (CR) and urinary TP and ALB were detected-by means of biochemical analyzer. The concentrations of urinary NGAL, KIM-1 and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA). The morphological changes of renal pathology were observed by light or electron microscopy. The curve areas of various serum or urine indexes and the combined detection were compared by receiver operating characteristic curve (ROC curve).

RESULTRats were given Phytolaccae Radix decoction at the doses of 40, 20 g crude drug/kg daily for 35 consecutive days to induce kidney injury characterized by the degeneration of renal tubular epithelial cell and protein cast. The injury was partially reversible during the recovery period. Compared with the control group, the content of serum BUN, CR and urinary TP in each dose group mostly showed a downward trend. On day 21, the content of urinary ALB obviously increased till the end of administration. The contents of urinary NGAL, KIM-1 and IL-18 began increasing on day 7. Since day 14, high and low dose groups showed significant difference (P<0.01). The high dose group even showed notable changes during the recovery period. According to ROC analysis, the curve areas of NGAL, KIM-1 and IL-18 were 0.846, 0.837 and 0.863 (P <0.01), respectively, much higher than that of BUN and CR. The area of the combined detection was up to 0.947.

CONCLUSIONUrinary NGAL, IL-18 and KIM-1 could forecast and indicate the occurrence and development of renal injury to some degree, and show higher sensitivity and site specificity. The combined detection could further improve the test efficiency.

Acute-Phase Proteins ; genetics ; metabolism ; Animals ; Cell Adhesion Molecules ; genetics ; metabolism ; Drugs, Chinese Herbal ; adverse effects ; Female ; Humans ; Interleukin-18 ; genetics ; metabolism ; Kidney ; drug effects ; injuries ; metabolism ; Kidney Diseases ; etiology ; genetics ; metabolism ; Lipocalins ; genetics ; metabolism ; Male ; Proto-Oncogene Proteins ; genetics ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo construct a luciferase reporter gene vector of p-selectin gene promoter and determine its transcriptional activity for screening the effect of drugs on the transcriptional activity of p-selectin promoter.

METHODSPrimers were designed based on human p-selectin promoter sequence from UCSC software. The p-selectin promoter from human genome DNA was then amplified. After digestion of pGL3-Basic vector and p-selectin promoter with Kpn I and Xho I, p-selectin promoter was inserted into pGL3-basic vector. The recombinant plasmid, namely pGL3-p-selectin-promoter, was transiently cotransfected into 293F cells with pRL-SV40 as the control vector, and the activity of the dual luciferase was detected. The transcription activity of serially truncated segments of the p-selectin promoter reporter gene was quantified by luciferase expression. 293F cells transfected with pGL3-p-selectin-promoter reporter gene and dual luciferase were stimulated with LPS, TNF-α and As2O3, and the transcriptional activity of p-selectin promoter were assessed.

RESULTSpGL3-p-selectin-promoter was constructed successfully as verified by restriction digestion and sequence analysis. The luciferase activity was higher in pGL3-p-selectin-promoter/pRL-SV40 group than in pGL3-basic/pRL-SV40 group (0.8573±0.4703 vs 0.03955±0.05894). pGL3- 1826 bp was actively transcribed compared with pGL3-1092 bp and pGL3-3738 bp. LPS, TNF-α and As2O3 significantly enhanced the transcriptional activity of p-selectin promoter.

CONCLUSIONpGL3-p-selectin-promoter can be transcribed and activated in 293F cells. This study provided an important basis for acquiring transcriptional factors and screening inflammatory factors and drugs.

Genes, Reporter ; Genetic Vectors ; HEK293 Cells ; Humans ; Luciferases ; P-Selectin ; genetics ; Promoter Regions, Genetic ; Transcriptional Activation ; Transfection

OBJECTIVETo evaluate fungal contamination on the surface of Chinese herbal medicines and explore an appropriate method for fast and efficient identification of contaminant fungi.

METHODChinese herbal medicines were first washed and the washing solution was plated onto potato dextrose agar (PDA) to obtain the pure isolates. For molecular identification, two new pairs of specific primers were designed according to ITS region of fungi genome sequences. The strains were identified through polymerase chain reaction (PCR) and sequence analysis.

RESULTFifty fungal strains were obtained from the surface of 15 Chinese herbal medicines with the percent of contaminated samples of 93.3%. Twenty-seven strains among them were successfully identified.

CONCLUSIONFungal contamination on the surface of Chinese herbal medicines is quite common. Although different fungal species were isolated, the genus Aspergillus was the predominant. The primer pairs developed in this study are compatible and can be used to identify fungal species from the surface of Chinese herbal medicines.

Drug Contamination ; Drugs, Chinese Herbal ; Fungi ; genetics ; isolation & purification ; Polymerase Chain Reaction

Intrauterine infection is an important cause of some birth defects worldwide. The most common pathogens include rubella virus, cytomegaloviurs, ureaplasma urealyticum, toxoplasma, etc. General information about these pathogens in epidemiology, consequence of birth defects, and the possible mechanisms in the progress of birth defects, and the interventions to prevent or treat these pathogens' infections are described. The infections caused by rubella virus, cytomegaloviurs, ureaplasma urealyticum, toxoplasma, etc. are common, yet they are proved to be fatal during the pregnant period, especially during the first trimester. These infections may cause sterility, abortion, stillbirth, low birth weight, and affect multiple organs that may induce loss of hearing and vision, even fetal deformity and the long-term effects. These pathogens' infections may influence the microenvironment of placenta, including levels of enzymes and cytokines, and affect chondriosome that may induce the progress of birth defect. Early diagnosis of infections during pregnancy should be strengthened. There are still many things to be settled, such as the molecular mechanisms of birth defects, the effective vaccines to certain pathogens. Birth defect researches in terms of etiology and the development of applicable and sensitive pathogen detection technology and methods are imperative.
Animals ; Congenital Abnormalities ; etiology ; Female ; Humans ; Infant, Newborn ; Placenta Diseases ; complications ; Pregnancy ; Pregnancy Complications, Infectious ; Pregnancy Outcome ; Pregnancy Trimester, First ; Rubella ; complications ; Toxoplasma ; pathogenicity ; Ureaplasma urealyticum ; pathogenicity

In this study,the immunological characteristics of the PhoP protein were explored with a two-component system of Mycobacterium tuberculosis(Mtb).Bioinformatics was used to predict the B and T cell epitopes of the PhoP protein.A re-combinant expression plasmid was constructed by PCR analysis of the phoP sequence and cloning into the prokaryotic expres-sion vector pET-28a(+).Competent Escherichia coli BL21 cells were transformed with the recombinant plasmid and expres-sion was induced with IPTG.The recombinant PhoP protein was purified by affinity chromatography.Serum levels of PhoP-specific antibodies in Mtb-infected mice and tuberculosis(TB)patients were analyzed with an ELISA.BALB/c mice were im-munized with the PhoP recombinant protein by intramuscular injection.Sera of mice were collected and antibody titers were detected with an ELISA and specificity was assessed by West-ern blot analysis.Mouse splenocytes were isolated and the pro-portions of IFN-y-positive cells and cytokine levels were detec-ted with an ELISpot and ELISA,respectively.Bioinformatics i-dentified 24 B cell and 11 T cell epitopes of the PhoP protein.A prokaryotic recombinant vector of PhoP was successfully con-structed and the recombinant PhoP protein was obtained by purification.Specific antibody levels to PhoP in sera of Mtb in-fected mice and TB patients increased significantly,with preci-sion of 99.9％and 82.5％,and specificity of 100％,respectively.PhoP protein immunization successfully induced production of specific antibodies in mice.Stimulated by antigens in vitro,IL-2 and IFN-γ levels were significantly increased in the splenocytes of immunized mice.Immunization with the PhoP protein induce a humoral immune response and Thl-dominated cellular immu-nity,indicating that the PhoP protein was immunogenic with diagnostic efficacy for TB.These results lay a foundation to clari-fy the role of PhoP in Mtb infection and application for diagnosis and prevention of TB.

Animals ; Cellular Reprogramming ; Dentate Gyrus ; cytology ; Fibroblasts ; cytology ; Mice ; Neural Stem Cells ; cytology ; transplantation ; Swine

Objective This study aimed to investigate the potential relationship between urinary metals copper (Cu), arsenic (As), strontium (Sr), barium (Ba), iron (Fe), lead (Pb) and manganese (Mn) and grip strength. Methods We used linear regression models, quantile g-computation and Bayesian kernel machine regression (BKMR) to assess the relationship between metals and grip strength.Results In the multimetal linear regression, Cu (β=-2.119), As (β=-1.318), Sr (β=-2.480), Ba (β=0.781), Fe (β= 1.130) and Mn (β=-0.404) were significantly correlated with grip strength (P < 0.05). The results of the quantile g-computation showed that the risk of occurrence of grip strength reduction was -1.007 (95％ confidence interval:-1.362, -0.652; P < 0.001) when each quartile of the mixture of the seven metals was increased. Bayesian kernel function regression model analysis showed that mixtures of the seven metals had a negative overall effect on grip strength, with Cu, As and Sr being negatively associated with grip strength levels. In the total population, potential interactions were observed between As and Mn and between Cu and Mn (Pinteractions of 0.003 and 0.018, respectively).Conclusion In summary, this study suggests that combined exposure to metal mixtures is negatively associated with grip strength. Cu, Sr and As were negatively correlated with grip strength levels, and there were potential interactions between As and Mn and between Cu and Mn.

Objectives:The purpose of this study was to evaluate the prognostic value of the Thrombolysis In Myocardial Infarction (TIMI) and Global Registry of Acute Coronary Events (GRACE) risk scores for in-hospital mortality in Chinese ST-segment elevation myocardial infarction (STEMI) patients. Methods:Present data are obtained from the prospective, multicenter Chinese AMI (CAMI) registry, 107 hospitals from 31 provinces, municipalities or autonomous districts in China took part in this study. From January 2013 to September 2014, 17886 consecutive ST-segment elevation myocardial infarction patients admitted to these 107 hospitals were enrolled. For each patient, TIMI and GRACE risk scores were calculated using specific variables collected at admission. Their prognostic value on the primary endpoint (in-hospital mortality) was evaluated. Results:Mean age of this patient cohort was (61.9±12.4)years, 76.5% (n=13685) patients were males. The in-hospital mortality was 6.4%(n=1 153)and the median length of hospital stay was 10.0 days. The incidence of cardiac arrest at admission were 4.3% (n=764). Coronary reperfusion therapy including fibrinolytic therapy(n=1782), primary percutaneous coronary intervention (n=7763) and emergent coronary artery bypass grafting (n=10) were applied to 9555 (53.4%) patients and the median of time to reperfusion was 300.0 minutes. The predictive accuracy of TIMI and GRACE for in-hospital mortality was similar:TIMI risk score (AUC) [area under the curve:0.7956; 95% confidence interval (95%CI:0.7822~0.8090)] and GRACE risk score (AUC:0.8096; 95%CI:0.7963~0.8230). Conclusions:The TIMI and GRACE risk score demonstrate similar predictive accuracy for in-hospital mortality and there are some disadvantages in risk stratification by these two risk scores for Chinese STEMI patients.

This paper was aimed to compare the acute toxicity of 999 Ganmaoling grain and its different ingredients, and investigate the influence of routine diet on the hepatic toxicity induced by Ganmaoling in mice, so as to provide experimental basis for the clinical safety evaluation. Mice were given a single dose of Ganmaoling grain or its different ingredients respectively by gavage, and then observed for 14 days. LD₅₀ values of Ganmaoling grain or its chemical ingredient and the maximal tolerated dose of its herb ingredient were determined. Mice were divided into starvation and diet group, a single dose of Ganmaoling grain was administered by gavage. LD₅₀ values were estimated after 14 day observation. Mice were divided into starvation and diet group. At the same time,control group was set up for each. A single dose of Ganmaoling grain was given. Serum biochemical indexes were detected, liver weight index was calculated and liver tissue morphological change was observed after 6 h. LD₅₀ values were 4.42, 0.64 g•kg⁻¹ for Ganmaoling grain group and chemical ingredient group, respectively. The maximal tolerated dose of the herb ingredient group was close to 24.24 g•kg⁻¹. The toxic symptom was basically similar in the Ganmaoling grain and the chemical ingredient group. The body weight and food intake were decreased to a certain extent in both groups. There were pathological changes of liver and heart tissue in some of the surviving animals. The animals in the Ganmaoling grain group exhibited a lighter toxicity and recovered faster than that in the chemical ingredient group. LD₅₀ values of Ganmaoling grain were 2.56, 6.93 g•kg⁻¹ for starvation and diet group respectively. TD₅₀ values were 1.29, 6.31 g•kg⁻¹ for starvation and diet group respectively. The toxicity of 999 Ganmaoling was less, which may be related to the reduction of toxicity after the combination of herb and chemical ingredients. Compared with starvation group, the values of LD₅₀ and TD₅₀ of diet group was significantly increased, and toxicity was decreased. From the point of view of safety, it is safer to use Ganmaoling in the absence of hunger or after meal. The above tests provide experimental basis for the clinical safety use of Ganmaoling.