A new molecular method for simultaneously rapid detection and differentiation of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium and Mycobacterium paratuberculosis was established by using denaturing high-performance liquid chromatography (DHPLC) combined with multiplex nucleic acid amplification. These 4 important pathogenic mycobacteria were identified by separation of 4 specific PCR-amplified target fragments by DHPLC analysis. A total of 51 Mycobacterium strains and 22 other bacterial species were tested to confirm the specificity of the multiplex PCR-DHPLC assay. The sensitivity of the assay was as low as 10~2-10~3 gene copies. This method rapidly identify the positive clinical samples from human and bovine with higher detection ratio than traditional culture method and was able to identify simultaneously four pathogenic Mycobacterium, which provided a new molecular tool for rapid detection of tuberculosis and paratuberculosis in human and animals.

An enhancer-like element VV16 from Vaccinia virus genome DNA was obtained by using the plasmid with CAT reporter gene. Sequence analysis showed the element of 112bp is a part of the DNA-dependent RNA polymerase, polyA polymerase and DNA polymerase (RPO30 gene). It contains 4 AT-rich regions. Detection of β-galactosidase activity showed that VV16 in the positive direction can increase the activity 9.0 times and VV16 in the negative direction can increase 4.1 times. The RNA dot blotting confirmed the enhancing activity of the element are on the transcription level. DNA deletion experiment indicated the sequences of 10bp at the 5′ end and 12bp at the 3′ end in the element are important to its function and the sequence from nt76 to nt82 is essential to its activity.

OBJECTIVETo investigate the relationship between phospholipase D (PLD) activation and neutrophil priming induced by cardiopulmonary bypass (CPB), and try to clarify whether CPB-induced systemic inflammatory response can be attenuated by inhibiting neutrophilic PLD activation.

METHODSNeutrophils were isolated from arterial blood of 8 patients undergoing valve replacement before operation and 30 min after initiation of CPB respectively. Both the preoperative and CPB-stirred neutrophils were subdivided into 5 groups by receiving different experimental interventions: (1) bacterial lipopolysaccharide (LPS, 10 ng x ml(-1)), (2) N-formylmethionylphenylalanine (fMLP, 1 micromol x L(-1)), (3) LPS+fMLP, (4) 1-butanol (0.5%)+LPS+fMLP, (5) vehicle. Elastase and myeloperoxidase (MPO) release was measured for the parameters of neutrophil activation, neutrophil PLD activity was determined by quantitation of choline produced from the stable product of phosphatidylcholine catalyzed by PLD.

RESULTS(1) Preoperative neutrophils treated with LPS+fMLP presented significantly higher PLD activity (13.48+/-2.61 nmol choline x h(-1) x mg(-1)) and released more elastase and MPO than cells treated with vehicle (PLD activity 3.70+/-0.49 nmol choline x h(-1) x mg(-1)), P<0.01), LPS (P<0.01) and fMLP respectively. In 1-butanol+LPS+fMLP group, PLD activity of preoperative neutrophils was lower than that in LPS+fMLP group (P<0.01), besides the release of elastase and MPO decreased sharply below both LPS+fMLP and fMLP groups (P<0.01). In LPS group, PLD activity was higher (P<0.01), while elastase and MPO release did not differ from control. fMLP group presented PLD activity, elastase and MPO release higher than control (P<0.01); nevertheless, lower than LPS+fMLP group (P<0.01). (2) CPB-stirred neutrophils presented prominent PLD activity increment, and even the control level was 3.59-fold of the pre-operative control (P<0.01). PLD activity in LPS+fMLP group was higher than that in other groups. Notably, PLD activity was even nonstatistically lower in 1-butanol+LPS+fMLP group than that in LPS or fMLP group. CPB-stirred neutrophils in LPS+fMLP group released more elastase and MPO than control, LPS, and 1-butanol+LPS+fMLP groups did (P<0.01); however, neither of the release was statistically different from that of fMLP group.

CONCLUSIONSCardiopulmonary bypass enables neutrophil priming accompanied with significant increase in PLD activity. Inhibition of neutrophil PLD activation attenuates its priming and may alleviate CPB-induced systemic inflammatory reaction.

Adolescent ; Adult ; Cardiopulmonary Bypass ; adverse effects ; Female ; Humans ; Male ; Middle Aged ; Neutrophil Activation ; physiology ; Phospholipase D ; pharmacology ; Systemic Inflammatory Response Syndrome ; etiology ; physiopathology

OBJECTIVETo screen enhancer-like sequences from vaccinia virus genome, to construct an expression vector harboring prokaryotic enhancer-like sequence and study the effect of interferon gene expression.

METHODSEnhancer-like element from vaccinia virus genome was obtained by using the chloramphenicol acetyl-transferase cat gene as reporter gene. An expression vector harboring prokaryotic enhancer-like sequence VV1 from vaccinia virus was constructed. Interferon was expressed and assayed.

RESULTSEighteen enhancing sequences were found. From them two enhancer-like sequences with distance and orientation independence property were screened and named VV1 and VV16 respectively. Quantification test showed that the direct and reverse orientation of VV1 could increase the activity of beta-galactosidase with 10.9 and 3.8 times and those of VV16 could increase by 9.0 times and 4.1 times respectively. The enhancing activity of the element was on transcription level. An expression vector harboring prokaryotic enhancer-like sequence VV1 was constructed. Using this vector the antiviral activity of interferon alpha-2b was increased by 2.6 times in comparison with the original expression plasmid.

CONCLUSIONTwo enhancer-like sequences were screened from vaccinia virus genome. Interferon gene was highly expressed by using an expression vector harboring enhancer-like sequences.

Enhancer Elements, Genetic ; Genetic Vectors ; genetics ; Interferon-alpha ; biosynthesis ; genetics ; pharmacology ; Plasmids ; Recombinant Proteins ; Vaccinia virus ; genetics

OBJECTIVETo investigate the mechanism of hyperlipidemia- and imflammation-induced functional impairment of the endothelium.

METHODSThe experiment was conducted using 3 groups of rats fed for 20 weeks with standard chow (control group), high-fat diet and high-fat diet with daily fenofibrate treatment (10 mg/kg, starting since the fifth week), respectively. After 4 and 20 weeks of feeding, respectively, serum lipid level and NO concentration were measured in the rats, and the epithelial vascular cell adhesion molecule-1 (VCAM-1) expression and cell adhesiveness to the aortic endothelium were observed.

RESULTSCompared with the control group, the rats with hyperlipidemia induced by long-term high-fat diet feeding showed lower NO concentration and increased leukocyte accumulation on the endothelial surface, exhibiting also stronger and more extensive endothelial expression of VCAM-1. In contrast, the hyperlipidemic rats with fenofibrate treatment shoed significantly decreased VCAM-1 expression and leukocyte adhesion with recovery of the NO level.

CONCLUSIONNO deficiency and activation of inflammation are involved in vascular impairment in rats with high-fat diet-induced hyperlipidemia, and fenofibrate can effectively prevent atherosclerosis by restoring NO concentration and down-regulating VCAM-1 expression in these rats.

Animals ; Atherosclerosis ; prevention & control ; Cell Adhesion ; Endothelium, Vascular ; drug effects ; metabolism ; Fenofibrate ; pharmacology ; Hyperlipidemias ; drug therapy ; metabolism ; Inflammation ; Leukocytes ; cytology ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vascular Cell Adhesion Molecule-1 ; metabolism

OBJECTIVETo study the applicability of MultiCalc software to prenatal screening of Down's syndrome in Jiangsu province, China.

METHODSThe gestational age-specific median of maternal serum marker was calculated by means of regression method. Regression functions for adjustment of Multiple of the Median (MoM) by weight were established for our own population.

RESULTSBefore the adjustment by weight, the average level of alpha fetal protein(AFP) was 16% higher and the free beta-human chorionic gonadotrophin (beta-hCG) was 14% higher than those of the Caucasian in MultiCalc software respectively. But when the AFP and free beta-hCG results were converted to weight-adjusted MoM levels, the values were 0.99 and 1.02 respectively. The median of MoM of AFP and the free beta-hCG were 1.00 through the regression model of gestational age and weight adjustment.

CONCLUSIONThere was no difference of average weight-adjusted MoM levels between the Jiangsu population and the Caucasian, and the MultiCalc software was applicable to maternal serum screening for Down's syndrome of Jiangsu.

Adolescent ; Adult ; Body Weight ; China ; Chorionic Gonadotropin ; blood ; Down Syndrome ; diagnosis ; Female ; Fetal Diseases ; diagnosis ; Gestational Age ; Humans ; Middle Aged ; Mothers ; Pregnancy ; Pregnancy Trimester, Second ; blood ; Prenatal Diagnosis ; methods ; Reference Values ; Regression Analysis ; Software ; alpha-Fetoproteins ; metabolism

OBJECTIVETo develop an HPLC-DAD-ELSD method for detecting the fingerprint of Astragali Radix and evaluate the quality through similarity calculation and chemical pattern recognition.

METHODSeparation was performed at 25 degreeC on an Agilent Zorbax ODS C18 column(4.6 mm x250 mm,5 microm). Gradient elution was performed with the mobile phases of acetonitrile and water containing 0. 2% formic acid. The flow rate was 0. 8 mL min-1 , and sample size was 10 microL. The UV detection wavelength was set at 280 nm. The drift tube temperature for ELSD was set at 110 degreeC , and the nebulizing gas flow rate was 3.0 L min-1. The similarity calculation and chemical pattern recognition were used for fingerprint analysis.

RESULTThe HPLC-DAD-ELSD method for chromatographic fingerprint of Astragali Radix showed better results of stability, precision and repeatability. The reference chromatographic fingerprint of Astragali Radix was established on the eighteen Astragali Radix samples from different sources. The results of similarity calculation were higher than 0. 83, which was in accordance with the result of chemical pattern recognition analysis.

CONCLUSIONFingerprint and chemical pattern recognition analysis could effectively distinguish Astragali Radix from different source, which could be applied to the quality control of Astragali Radix.

Astragalus Plant ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Temperature

Objective:To investigate the anti-anxious depression mechanism of Baihe Dihuangtang from the NOD-like receptor thermal protein domain 3 （NLRP3） inflammasome. Method:Fifty SD rats were randomly divided into normal group， model group， venlafaxine group （13.5 mg·kg^-1）， Baihe Dihuangtang high and low dose group （16，4 g·kg^-1）， with 10 rats in each group. Chronic restraint stress for 28 days （6 h） combined with subcutaneous injection of corticosterone （30 mg·kg^-1） was used to establish induce an anxious depression model. From the 8th day of modeling， the rats in the normal group and the model group received distilled water， and those in groups with drug intervention were treated with corresponding drugs by gavage for 21 days. Elevated plus maze and open field test were used to evaluate the behavioral changes of rats. Enzyme- linked immunosorbent assay （ELISA） was used to detect serum and hippocampal interleukin-1β （IL-1β）， interleukin-6 （IL-6） and interleukin-18 （IL-18） levels. Western blot were used to detect the relative expression of hippocampal NLRP3， apoptosis-associated speck-like protein containing a CARD （ASC）， and Caspase-1. The pathological changes of the hippocampus were observed by hematoxylin-eosin（HE） staining， the average fluorescence intensity of NLRP3， ASC， and Caspase-1 was detected by immunofluorescence. The ultrastructure of neurons was observed under electron microscopy. Result:Compared with the normal group， the model group showed reduced total entries （TE）， the ratio of open-arm entries （OE%）， the ratio of open-arm times （OT%）， and the autonomous activity score （P<0.01）， significant anxiety and depression-like behaviors， increased levels of IL-1β， IL-6， and IL-18 in the serum and hippocampus （P<0.01）， elevated protein expression of NLRP3， ASC， and Caspase-1 （P<0.01）， activated NLRP3 inflammasomes， and injured hippocampal neurons. Compared with the model group， the high-dose Baihe Dihuangtang group showed improved anxiety and depression-like behaviors （P<0.01）， and decreased levels of IL-1β， IL-6， and IL-18 in the serum and hippocampus （P<0.05，P<0.01）， reduced protein expression of NLRP3， ASC， and Caspase-1 （P<0.01）， and alleviated hippocampal neuron damage. Conclusion:Baihe Dihuangtang can improve neuronal damage in anxious depression by inhibiting the excessive activation of NLRP3 inflammasomes.

BACKGROUNDData on the epidemiology of hypertension in Chinese non-dialysis chronic kidney disease (CKD) patients are limited. The aim of the present study was to investigate the prevalence, awareness, treatment, and control of hypertension in the non-dialysis CKD patients through a nationwide, multicenter study in China.

METHODSThe survey was performed in 61 tertiary hospitals in 31 provinces, municipalities, and autonomous regions in China (except Hong Kong, Macao, and Taiwan). Trained physicians collected demographic and clinical data and measured blood pressure (BP) using a standardized protocol. Hypertension was defined as systolic BP ≥ 140 mmHg and/or diastolic BP ≥ 90 mmHg, and/or use of antihypertensive medications. BP < 140/90 mmHg and < 130/80 mmHg were used as the 2 thresholds of hypertension control. In multivariate logistic regression with adjustment for sex and age, we analyzed the association between CKD stages and uncontrolled hypertension in non-dialysis CKD patients.

RESULTSThe analysis included 8927 non-dialysis CKD patients. The prevalence, awareness, and treatment of hypertension in non-dialysis CKD patients were 67.3%, 85.8%, and 81.0%, respectively. Of hypertensive CKD patients, 33.1% and 14.1% had controlled BP to < 140/90 mmHg and < 130/80 mmHg, respectively. With successive CKD stages, the prevalence of hypertension in non-dialysis CKD patients increased, but the control of hypertension decreased (P < 0.001). When the threshold of BP < 130/80 mmHg was considered, the risk of uncontrolled hypertension in CKD 2, 3a, 3b, 4, and 5 stages increased 1.3, 1.4, 1.4, 2.5, and 4.0 times compared with CKD 1 stage, respectively (P < 0.05). Using the threshold of < 140/90 mmHg, the risk of uncontrolled hypertension increased in advanced stages (P < 0.05).

CONCLUSIONSThe prevalence of hypertension Chinese non-dialysis CKD patients was high, and the hypertension control was suboptimal. With successive CKD stages, the risk of uncontrolled hypertension increased.

Adult ; Aged ; Awareness ; Female ; Humans ; Hypertension ; complications ; epidemiology ; therapy ; Male ; Middle Aged ; Prevalence ; Renal Insufficiency, Chronic ; complications

Objective: The relationship between serum uric acid (SUA) levels and glycemic indices, including plasma glucose (FPG), 2-hour postload glucose (2h-PG), and glycated hemoglobin (HbA1c), remains inconclusive. We aimed to explore the associations between glycemic indices and SUA levels in the general Chinese population. Methods: The current study was a cross-sectional analysis using the first follow-up survey data from The China Cardiometabolic Disease and Cancer Cohort Study. A total of 105,922 community-dwelling adults aged ≥ 40 years underwent the oral glucose tolerance test and uric acid assessment. The nonlinear relationships between glycemic indices and SUA levels were explored using generalized additive models. Results: A total of 30,941 men and 62,361 women were eligible for the current analysis. Generalized additive models verified the inverted U-shaped association between glycemic indices and SUA levels, but with different inflection points in men and women. The thresholds for FPG, 2h-PG, and HbA1c for men and women were 6.5/8.0 mmol/L, 11.0/14.0 mmol/L, and 6.1/6.5, respectively (SUA levels increased with increasing glycemic indices before the inflection points and then eventually decreased with further increases in the glycemic indices). Conclusion An inverted U-shaped association was observed between major glycemic indices and uric acid levels in both sexes, while the inflection points were reached earlier in men than in women.
Aged ; Asian Continental Ancestry Group ; Blood Glucose/analysis* ; China/epidemiology* ; Cohort Studies ; Diabetes Mellitus/blood* ; Female ; Glucose Tolerance Test ; Glycated Hemoglobin A/analysis* ; Glycemic Index ; Humans ; Male ; Middle Aged ; Uric Acid/blood*