OBJECTIVE: To develop the single-tube one-step methylation variable position (MVP) analysis technology-single-tube post-digestion PCR-melting curve analysis (PDP-MCA). METHODS: Based on differentially methylated region (DMR) reported previously as the model, a set of primers with different melting temperatures of products in the two sides of MVP were designed. By using the FastDigest methylation-sensitive restriction enzyme (MSRE), DNA digestion, multiplex amplification, MCA detection and MCA profiles were performed in a single reaction tube. Same samples (peripheral venous blood, semen, and vaginal fluid, 5 samples each type) were tested by single-tube one step MVP and traditional MSRE-PCR MCA technology. To verify the feasibility of this method, the results were compared with that of the traditional technology. The MCA/HRM profiles of different samples were analyzed and compared. RESULTS: When the melting temperature of the fragments had a differential of 2 degrees C, the MCA melting peaks separated well, and MCA detection after multiplex amplification was successful. The single-tube PDP-MCA assay was developed, which integrated multiple reactions (digestion, amplification and detection) into one tube. By this method, the sample-specific profiles and data were analyzed in 2 h, which is similar to that of the traditional method. The rapid classifications of the samples were also realized. CONCLUSION Multiplex MVPs can be analyzed in a single closed-tube. The single-tube PDP-MCA technology is a simple, fast, and automatable method. It can be used for detection of DNA methylation variations.
DNA/isolation & purification* ; DNA Methylation/genetics* ; DNA Primers/genetics* ; Humans ; Multiplex Polymerase Chain Reaction/standards* ; Nucleic Acid Denaturation

OBJECTIVERelationship among three point selection methods based on the midnight-noon ebb-flow theory, namely Najia fa (day- prescription of acupoints), Nazi fa (hour-prescription of acupoints) and Yangzi Shike Zhuxue fa (point-open method based on the mother and son relation as well as the hours and its divisions), were approved in the article, which provided the scientific validity of the correlation among three point selection methods of midnight-noon ebb-flow theory.

METHODSElectric impedance on relevant acupoints of 30 health subjects on the period of the day of Zishi (from 11:00 p.m. to 1:00 a.m.), Maoshi (from 5:00 a.m. to 7:00 a.m.), Wushi (from 11:00 a.m. to 1:00 p.m.) and Youshi (from 5:00 p.m. to 7:00 p.m.) were tested with NQ-1B conductivity meter. And statistical analysis was made on the testing results.

RESULTSThe daily average value of the method of Najia fa in 1 circle of the midnight-noon ebb-flow theory was 0.459omega, the value of the method of Nazi fa was 0.553omega, and the Yangzi Shike Zhuxue fa was 0.533omega. At the same time, it presented a tendency of first raising and then declining from Zishi to Youshi with the peak appears at Maoshi and the valley at Youshi. The differences of comparison of the average electric impedance between Najia fa and Nazi fa, Najia fa and Yangzi Shike Zhuxue fa were considered statistical significant (both P < 0.001). When 2 values of different methods within the same hours were compared, the value difference between Najia fa and Nazi fa in the time period of Wushi and the difference between Najia fa and Yangzi Shike Zhuxue fa in the time period of Wushi were considered statistical significant (P < 0.001, P < 0.01).

CONCLUSIONThe changing tendency of electric impedance of different point selection methods based on midnight-noon ebb-flow theory is similar, however, with weak relevance. Moreover, obvious differences can still be found in point location and time selection.

Acupuncture ; Acupuncture Points ; Adult ; Electric Impedance ; Electrophysiological Phenomena ; Female ; Humans ; Male ; Middle Aged ; Periodicity ; Time Factors ; Young Adult

OBJECTIVETo observe the expression of Ginkgo biloba Tablet (GbT) on scavenger receptor A (SRA) of the aortic wall and changes of serum inflammatory factors in atherosclerotic rats, and to explore its new mechanism for fighting against atherosclerosis (AS).

METHODSTotally 45 male Wistar rats were randomly divided into the control group, the model group, the GbT group, 15 rats in each group. Levels of blood glucose, blood lipids, blood calcium, serum C-reactive protein (CRP), soluble intercellular adhesion molecule-1 (slCAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1) were measured in all rats. The expression of SRA in the aortic wall of atherosclerotic rats was observed by immunohistochemical assay. The correlation between the expression of SRA and levels of in-flammatory factors was also observed.

RESULTSCompared with the control group, blood glucose and blood calcium obviously increased (P < 0.05); levels of TG, TC, and LDL-C were significantly elevated (P < 0.01); neointimal areas were significantly thickened, increased intima percentage was significantly enlarged, narrowed lumen index was significantly reduced; levels of CRP, sICAM-1, and sVCAM-1 were significantly elevated in the model group (all P < 0.01). Compared with the model group, blood glucose and blood calcium obviously decreased (P < 0.05); levels of TG, TC, and LDL-C significantly decreased (P < 0.01) in the GbT group. Aortic lumens were obviously narrower in the model group than in the GbT group (P < 0.05). SRA expressed at the aortic wall. The aforesaid 3 indices were significantly improved in the GbT group than in the model group (P < 0.01). Serum levels of CRP, sICAM-1, and sVCAM-1 were significantly decreased in the GbT group than in the model group (P < 0.01). Serum levels of CRP, sICAM-1, and sVCAM-1 were positively correlated with the percentage of SRA positive expression area (r = 0.701, 0.604, 0.581, all P < 0.01).

CONCLUSIONSSerum levels of inflammatory factors in atherosclerotic rats were elevated, and the expression of SRA in the aortic wall was enhanced. The expression of SRA was closely correlated with serum levels of inflammatory factors. GbT could decrease serum levels of inflammatory factors and inhibit the expression of SRA.

Animals ; Aorta ; drug effects ; metabolism ; Atherosclerosis ; drug therapy ; Blood Glucose ; analysis ; C-Reactive Protein ; analysis ; Calcium ; blood ; Drugs, Chinese Herbal ; pharmacology ; Ginkgo biloba ; chemistry ; Intercellular Adhesion Molecule-1 ; blood ; Lipids ; blood ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Scavenger Receptors, Class A ; metabolism ; Tablets ; Vascular Cell Adhesion Molecule-1 ; blood

Objective:To investigate the alteration of delayed memory and its relationship with neurogenesis and angiogenesis in vascular dementia (VD) rats after moxibustion therapy. Methods: Two hundred adult male SPF Wistar rats were chosen for the experiment. Thirty-six rats were randomly selected as the sham operation group. Except for rats in the sham operation group (n=36), the others were made into VD models by bilateral common carotid arteries occlusion (BCCAo). After modeling, the 108 survived rats were randomly divided into 3 groups: a model group, a neural stem cells (NSCs) plus endothelial progenitor cells (EPCs) moxibustion group and a NSCs moxibustion group. Co-transplanted implant was transplanted into the rats in the NSCs plus EPCs moxibustion group, and the rats in the NSCs moxibustion group were transplanted by NSCs only. The NSCs plus EPCs moxibustion group and the NSCs moxibustion group received suspended moxibustion therapy at Baihui (GV 20), Dazhui (GV 14) and Shenting (GV 24), (each group was divided into 3 subgroups by the treatment course as 1, 2 and 3 courses). Every group was measured by Morris water maze to evaluate its delayed memory after 3 treatment courses and the rat’s brain was taken out after perfusion of 4% paraformaldehyde one day after 1, 2 and 3 treatment courses, respectively. Marker protein expression was detected by laser confocal microscope to analyze the effect on neurogenesis and angiogenesis. Results: VD rats showed delayed memory in Morris water maze test 3 d after ischemic injury. After 3 courses of moxibustion therapy, VD-induced delayed memory deficits were improved in the NSCs plus EPCs moxibustion group and the NSCs moxibustion group. The expressions of nestin, doublecortin (DCX) and CD34 increased significantly in the two moxibusiton groups after every treatment course (all P＜0.05), which might contribute to the neurogenesis and angiogenesis in hippocampus. In addition, compared with the rats in the NSCs moxibustion group, the expressions of nestin, DCX and CD34 increased significantly in the NSCs plus EPCs moxibustion group (P＜0.05). Conclusion: Moxibustion can reverse VD-induced delayed memory deficits, which may be related to the promotion of neurogenesis and angiogenesis.

Objective To observe the expression of synaptotagmin I(syt I)protein in the prefrontal cortex of adult-onset hypothyroidism rats and the effects of replicated therapy in different doses of thyroid hormone on the syt I protein.Methods All 44 aduh male Sprague-Dawley rats were divided into 4 groups randomly according to their body mass:hypothyroidism group,routine dosage thyroxine treatment group,high dosage thyroxine treatment group and control group.The adult male Sprague-Dawley rats were replicated to the adult-onset hypothyroidism and treatment models with propyhhiouracil(PTU).The levels of serum T3,T4 were assayed by the radioimmunoassay method and the level of the syt I protein in the molecular layer,external granular layer,external pyramidal layer,internal granular layer and internal pyramidal layer in prefrontal cortex was analyzed by immunohistochemistry.Results In the hypothyroidism group,the levels of serum T3 and T4[(0.34±0.04),(43.01±2.95)nmol/L]were significantly lower than those in the control group[(0.65±0.15), (55.20±3.56)nmol/L, F value: 6.026,5.940,4.503,P<0.05 or <0.01 ], the levels of the syt I protein in the molecular layer(0.018±0.010), external granular layer (0.020±0.007), external pyramidal layer(0.013±0.008), internal granular layer(0.011±0.005), internal pyramidal layer(0.024±0.013) of prefrontal lobe were significantly lower compared to the control group[(0.028±0.010,0.031 ± 0.010,0.028 ± 0.010,0.022 ± 0.008,0.038 ± 0.013), F value: 5.697,8.965,14.668,13.597,6.807,P<0.05 or <0.01 ]. In the routine dosage of the thyroxine treatment group, the levels of serum T3,T4 [(0.63 ±0.05), (55.04 ± 3.77)nmol/L] were not significantly different compared to the control group(F value: 3.162,0.367,all P>0.05), and the level of the syt I protein in the molecular layer, external granular layer, external pyramidal layer, internal granular layer and internal pyramidal layer in prefrontal cortex showed a significant improvement of the syt I protein(0.027 ± 0.013,0.025 ± 0.009,0.022 ± 0.008,0.020 ± 0.010,0.033 ± 0.010), which were similar to that of the control group(F value: 0.094,2.208,2.467,0.350,0.693, all P>0.05). In the high dosage thyroxine thyroid hormone treatment group, the levels of serum T3 and T4[ (1.11 ± 0.10), (96.68 ± 6.42)nmoL/L] were higher than the control group(F value: 6.291,12.031, all P<0.01), the expression of the syt I protein(0.028 ± 0.008,0.031 ±0.011,0.026 ± 0.012,0.023 ± 0.011,0.038 ± 0.010) were not significantly different compare to the control group (F value: 0.001,0.019,0.111,0.061,0.001, all P>0.05). Conclusions The expression of the syt I protein in the prefrontal cortex of adult-onset hypothyroidism can be decreased, which can be reversed by routine dosage of thyroxine treatment.

Objective To explore clinical characters of type 1 diabetes mellitus with different serum potassium levels in children and adolescent.Methods One hundred and seventy-five patients with type 1 diabetes mellitus were reviewed,they were divided into 3 groups according to the serum potassium level.The patients whose serum potassium

OBJECTIVETo study the solubilization effect of 2-hydroxypropyl-beta-cyclodextrin(HPCD) on paeonolum at various pH value.

METHODPhase-solubility method was adopted to study the solubilization effect at 25 degrees C and UV spectrohotometer was used to determine paeonolum content.

RESULTSThe apparent solubility of paeonolum was significantly enhanced by increased HPCD concentration. The apparent stability constant of paeonolum compounds was calculated up to 1 425 in which pH was 3 and HPCD concentration was 133.33 mmol x L(-1). The solubility of paeonolum came up to 10 mg x mL(-1).

CONCLUSIONHPCD is an ideal solubilizer for paeonolum.

2-Hydroxypropyl-beta-cyclodextrin ; Acetophenones ; administration & dosage ; chemistry ; isolation & purification ; Drug Stability ; Excipients ; Hydrogen-Ion Concentration ; Paeonia ; chemistry ; Plants, Medicinal ; chemistry ; Solubility ; beta-Cyclodextrins

This study aimed to examine the preparation of cationic lipid microbubble (CLM), and evaluate its physical and chemical properties and toxicity, measure the gene transfection efficiency by ultrasound triggered microbobble destruction (UTMD) in combination with CLM. The CLM was prepared by the method of the thin film hydration, and its morphology was observed under the electron microscopy at 1st, 3rd, 7th, 10th, and 14th day after preparation, respectively. The size, Zeta potential and stability of CLM were tested. The acute toxicity of CLM was assessed. The green fluorescent protein gene (EGFP) transfection efficiency was evaluated. The experiment grouping was as follows: naked plasmid group (P group), ultrasonic irradiation plus naked plasmid group (P-US group), naked plasmid plus CLM group (P-CLM group), naked plasmid plus ultrasound and CLM group (UTMD group). The expression of EGFP was detected by fluorescent microscopy and flow cytometry. The results showed that CLMs were spherical in shape, with the similar size and good distribution degree under the light and electron microscopies. The size of CLMs was varied from 250.4±88.3 to 399.0±99.8 nm and the Zeta potential of CLMs from 18.80±4.97 to 20.1±3.1 mV. The EGFP expression was the strongest in the UTMD group, followed by the P-CLM group, P-US group and P group. Flow cytometry results were consistent with those of fluorescent microscopy. The transfection efficiency was substantially increased in the P-US group, P-CLM group and UTMD group as compared with that in the P group, almost 7 times, 10 times and 30 times higher than that in the P group respectively. It is suggested that CLMs prepared by the method of thin film hydration are uniform in diameter, and proved non-toxic. UTMD combined with CLM can significantly increase the transfection efficiency of EGFP to targeted cells.

AIMTo investigate the antiepileptic effect of dizocilpine (MK-801) on amygdala kindling models in rats and the effects of its combination with general antiepileptic drugs.

METHODSTo establish amygdala kindling models in rats and observe the effect of dizocilpine on kindling models and its combination with general antiepileptic drugs (phenobarbital, valproate and nicardipine) at ineffective dose. The influence of dizocilpine on convulsions induced by semicarbazide (SCZ) in mice were also observed.

RESULTSDizocilpine (0.1-0.25 mg.kg-1, i.p.) was shown to dose-dependently inhibit amygdala kindled seizure, shorten the after discharge duration (ADD) and reduce the Racine's stage (P < 0.01). The combination of dizocilpine with phenobarbital, valproate, nicardipine at ineffective dose shortened ADD or reduced Racine's stages (P < 0.01). Dizocilpine (0.1-0.25 mg.kg-1, i.p.) significantly prolonged the latency and reduced the rate of convulsions and death in mice.

CONCLUSIONDizocilpine inhibits the seizure of the amygdala kindling and improve the antiepileptic activity of phenobarbital, valproate and nicardipine, indicating that these combination may provide a new approach for treating epilepsy.

Amygdala ; drug effects ; physiopathology ; Animals ; Anticonvulsants ; pharmacology ; therapeutic use ; Dizocilpine Maleate ; pharmacology ; therapeutic use ; Electric Stimulation ; Epilepsy ; chemically induced ; drug therapy ; Female ; Kindling, Neurologic ; drug effects ; Male ; Mice ; Nicardipine ; pharmacology ; Phenobarbital ; pharmacology ; Random Allocation ; Rats ; Rats, Wistar ; Semicarbazides ; Valproic Acid ; pharmacology

OBJECTIVETo study the risk factors of male infertility.

METHODCase control study including 94 cases and control group with a ratio of 1 to 1.

RESULTSThe risk factors of male infertility were long time heavy smoking habit (OR = 3.45, 95% CI: 1.95 - 6.10), illegal sexual intercourse (OR = 7.29, 95% CI: 2.54 - 20.89), growing vegetable under plastic in higher temperature (OR = 6.73, 95% CI: 1.91 - 23.69), contact with benzene chemicals (OR = 20.53, 95% CI: 4.67 - 90.25) and having Ureaplasma urealyticum (Uu) infection (OR = 5.55, 95% CI: 2.28 - 13.53).

CONCLUSIONMale infertility was resulted from many factors repeatedly acting on men for long time. In order to prevent male infertility, issues as environmental pollution, occupational protection need to be improved while bad working condition and risky behavior should be changed.

Adult ; Benzene ; toxicity ; Case-Control Studies ; China ; epidemiology ; Environmental Pollution ; adverse effects ; Humans ; Infertility, Male ; epidemiology ; etiology ; Male ; Occupational Exposure ; adverse effects ; Risk Factors ; Smoking ; adverse effects ; Ureaplasma Infections ; complications ; Ureaplasma urealyticum