OBJECTIVETo establish a method of methyl-DNA immunoprecipitation (MeDIP)-real time quantitative PCR (qPCR) for detecting the promoter methylation level in cell-free seminal DNA (cfsDNA).

METHODSWe obtained cfsDNA samples from 6 normozoospermia men (the NZ group) and 6 post-vasectomy patients (the PV group), and mixed the samples from different individuals of each group, respectively. Then we made DNA fragments by ultrasonication, separated the methylated DNA fragments by MeDIP, and determined the methylation level of the promoters in cfsDNA by qPCR.

RESULTSThe methylation levels of the promoters PRAME, PEG10, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4, DAZ1 and CLPB were 14.93, 2.64, 0.69, 2.66, 17.50, 21.10, 5.98, 2.28, 13.50 and 3.86%, respectively, in the NZ group, obviously lower than 121.25, 73.62, 16.25, 42.90, 76.74, 112.40, 59.79, 25.85, 91.90 and 64.53% in the PV group. The results of MeDIP-qPCR for the methylation of PRAME, MORC1, GML, HOXA5, DNMT3L, SNURF, MSH4 and DAZ1 were coincident with the results of genome-wide promoter methylation microarray.

CONCLUSIONMeDIP-qPCR can quantitatively measure the promoter methylation level in cfsDNA, and effectively determine the testis- and epididymis-specific methylated promoters in human semen.

Adult ; DNA ; chemistry ; DNA Methylation ; Epididymis ; metabolism ; Epigenesis, Genetic ; Humans ; Male ; Polymerase Chain Reaction ; Promoter Regions, Genetic ; Semen ; chemistry ; Testis ; metabolism

LC-MS/MS method was used to simultaneously determine anti-oxidative active catechins EGCG, ECG, EGC and EC in plasma of rats treated with tea polyphenols (TP). The integrated plasma concentration (C') of TP was calculated by means of self-defined weighing coefficient based on percent AUC of individual components, thereby assessing integrated pharmacokinetic (PK) parameters of TP via log C'-T curve. The anti-free radical effects of TP were estimated using inhibitory rate of drug-containing serum collected at different times from rats against in vitro lipid peroxidation of mouse liver homogenate. The obtained E-T curves were used to calculate anti-free radical pharmacodynamic (PD) parameters of TP. E-logC and E-log C' plots and linear regression were carried out in order to obtain the correlation coefficient (R2). The results indicated that the log C'-T curves of TP, which could be best described by three-compartment model, corresponded to elimination rule of iv administration of drugs. The integrated PK parameters showed that TP was distributed in body rapidly and widely, and eliminated from deep compartment slowly. From comparison of R2 values and consistence of C'-T course and E-T course, it was evident that TP integrated PK behaviors correlated much better with its PD behaviors than individual active components, and thus demonstrated that integrated PK parameters could characterize to maximal extent holistic disposition of Chinese herbal drugs and reflect residence properties of holistic effective substances in biological body.
Animals ; Antioxidants ; pharmacokinetics ; pharmacology ; Area Under Curve ; Catechin ; analogs & derivatives ; pharmacokinetics ; Chromatography, Liquid ; Free Radical Scavengers ; blood ; pharmacokinetics ; pharmacology ; Free Radicals ; metabolism ; Injections, Intravenous ; Lipid Peroxidation ; drug effects ; Male ; Mice ; Polyphenols ; blood ; pharmacokinetics ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tandem Mass Spectrometry ; Tea ; chemistry

BACKGROUNDUnplanned extubation is associated with adverse outcomes in intensive care unit. The massive burn patient differs from other critically ill patients in many ways. However, little is known about the unplanned decannulation (UD) in Burn Intensive Care Unit. This paper describes the special features of the circumstances and outcome of UD of tracheotomy tube in massive burn patients.

METHODSA case series study was performed between January 1999 and December 2008 and UD of tracheotomy tube was analyzed retrospectively. A total of 21 patients with 29 UD events were identified. Demographic data, diagnosis, intervention, UD events and outcome of UD patients were collected. Differences in proportions were compared using the chi-square (χ(2)) or Fisher's exact test.

RESULTSPatients with UD were often burned with head and neck (67%) and combined with inhalation injury (62%). The majority of them (76%) were transferred patients, occurred early (55%) and were accidental UD (79%). UD events tended to happen in day shift (90%) and to be associated with the medical procedure that was performing by caregivers at besides (79%). Loose of the stabilizing rope, medical procedure and tracheotomy malposition were the main causes of UD. Early UD and reintubation failure were associated with patients' death.

CONCLUSIONSUD happened to massive burn patients can lead to patient death. Careful management of respiratory tract was essential for massive burn patients.

Adult ; Burns ; mortality ; surgery ; Device Removal ; adverse effects ; mortality ; Female ; Humans ; Intensive Care Units ; statistics & numerical data ; Intubation, Intratracheal ; Male ; Middle Aged ; Retrospective Studies ; Tracheotomy ; adverse effects

BACKGROUNDIn patients with chronic total occlusion (CTO) and multivessel coronary artery disease, the comparison of surgical and the percutaneous revascularization strategies has rarely been conducted. The aim of this study was to compare long term clinical outcomes of drug eluting stent (DES) implantation with coronary artery bypass surgery (CABG) in the patients with CTO and multivessel disease.

METHODSFrom a prospective registry of 6000 patients in our institution, we included patients with CTO and multivessel coronary artery disease who underwent either CABG (n = 679) or DES (n = 267) treatment. Their propensity risk score was used for adjusting baseline differences.

RESULTSAt a median follow-up of three years, propensity score adjusted Cox regression analysis showed that the rate of major adverse cardiac cerebrovascular events (MACCE) was lower in CABG group (12.7% vs. 24.3%, hazard ratio (HR) 1.969, 95%CI 1.219 - 3.179, P = 0.006) mainly due to lower rate of target vessel revascularization in CABG group than in DES group (3.1% vs. 17.2%, HR 16.14, 95%CI 5.739 - 45.391, P < 0.001). The incidence of cardiac death or myocardial infarction (composite end point) was not significantly different between these two groups. On multivariate analysis, the significant predictors of MACCE were only the type of revascularization. Age, left ventricular ejection fraction (LVEF), and complete revascularization were identified as significant predictors of composite end points.

CONCLUSIONSOur study shows that in patients with CTO and multivessel coronary disease, DES can offer comparable long term outcomes in cardiac death and myocardial infraction free survival in comparison with CABG. However, there is an increased rate of MACCE which results from more repeat revascularizations. Obtaining a complete revascularization is crucial for decreasing adverse cardiac events.

Angioplasty, Balloon, Coronary ; methods ; Chronic Disease ; Coronary Angiography ; Coronary Artery Bypass ; methods ; Coronary Artery Disease ; surgery ; therapy ; Coronary Occlusion ; surgery ; therapy ; Drug-Eluting Stents ; Humans ; Prospective Studies

OBJECTIVETo study the effects of prolonged 85% oxygen exposure on lung vascular development and the expression of angiopoietin-1 (Ang-1) in the neonatal rat lungs.

METHODSNinety-six Sprague-Dawley rat pups were randomly exposed to air (control group) and 85% oxygen (experimental group) 6 hrs after birth. The rats were sacrificed 3, 7 and 14 days after exposure and their lungs were sampled. The lung sections were stained with hematoxylin and eosin for histological evaluation and analysis of vessel volume density. Expressions of angiopoietin-1 (Ang-1) in lung tissue were measured by immunohistochemistry. Expression of Ang-1 protein and mRNA was detected by Western Blot and Real time-PCR.

RESULTSAfter being exposed to 85% oxygen for 14 days, lung tissues had pathological changes as "new" bronchopulmonary dysplasia (BPD). The RAC on day 7 and day 14 in experimental group decreased significantly as compared with the control group [(10.55 ± 0.13) vs. (11.74 ± 0.19), (12.47 ± 0.05) vs. (15.03 ± 0.16), P < 0.05]. The X-ray showed that the diameter of lung vessel was much smaller and the vessels had less branches in experimental group compared with the control group on day 14. The vessel volume density on day 14 in experimental group decreased significantly as compared with the control group [(3.55 ± 0.09) vs. (6.03 ± 0.16), P < 0.05]. Immunohistochemistry and Western blotting showed that the expressions of Ang-1 protein on day 7 and day 14 in the experimental group decreased significantly as compared with the control group [(4.27 ± 0.34) vs. (3.10 ± 0.29), P < 0.05, (5.65 ± 0.49) vs. (3.21 ± 0.28), P < 0.01], [(0.88 ± 0.31) vs. (0.41 ± 0.12), P < 0.05, (0.90 ± 0.29) vs. (0.21 ± 0.06), P < 0.01]. The expressions of Ang-1 mRNA on day 7 and day 14 in the experimental group also decreased significantly as compared with the control group [(0.85 ± 0.14) vs. (0.44 ± 0.21), P < 0.05, (0.87 ± 0.24) vs. (0.24 ± 0.05), P < 0.01].

CONCLUSIONSProlonged exposure of high concentration of oxygen may cause impairment of lung vascular development by inhibiting expression of Ang-1 in neonatal rats, which is likely to contribute to pathogenesis of BPD.

Angiopoietin-1 ; metabolism ; Animals ; Animals, Newborn ; Hyperoxia ; Lung ; blood supply ; metabolism ; Pulmonary Artery ; growth & development ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

BACKGROUNDAdenosine phosphate-mediated platelet aggregation is a prognostic factor for major adverse cardiac events in patients who have undergone selective percutaneous coronary interventions. This study aimed to assess whether an adjusted loading dose of clopidogrel could more effectively inhibit platelet aggregation in patients undergoing selected percutaneous coronary intervention.

METHODSA total of 205 patients undergoing selected percutaneous coronary intervention were enrolled in this multicenter, prospective, randomized study. Patients receiving domestic clopidogrel (n = 104) served as the Talcom (Taijia) group; others (n = 101) received Plavix, the Plavix group. Patients received up to 3 additional 300-mg loading doses of clopidogrel to decrease the adenosine phosphate-mediated platelet aggregation index by more than 50% (the primary endpoint) compared with the baseline. The secondary endpoint was major adverse cardiovascular events at 12 months.

RESULTSCompared with the rational loading dosage, the tailored loading dosage better inhibited platelet aggregation based on a > 50% decrease in adenosine phosphate-mediated platelet aggregation (rational loading dosage vs. tailored loading dosage, 48% vs. 73%, P = 0.028). There was no significant difference in the eligible index between the Talcom and Plavix groups (47% vs. 49% at 300 mg; 62% vs. 59% at 600 mg; 74% vs. 72% at 900 mg; P > 0.05) based on a standard adenosine diphosphate-mediated platelet aggregation decrease of > 50%. After 12 months of follow-up, there were no significant differences in major adverse cardiac events (2.5% vs. 2.9%, P = 5.43). No acute or subacute stent thrombosis events occurred.

CONCLUSIONAn adjusted loading dose of clopidogrel could have significant effects on antiplatelet aggregation compared with a rational dose, decreasing 1-year major adverse cardiac events in patients undergoing percutaneous coronary interventions based on adenosine phosphate-mediated platelet aggregation with no increase in bleeding.

Adenosine Diphosphate ; pharmacology ; Aged ; Angioplasty, Balloon, Coronary ; Female ; Humans ; Male ; Middle Aged ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; administration & dosage ; Prospective Studies ; Ticlopidine ; administration & dosage ; adverse effects ; analogs & derivatives

Objective To establish a novel method for the determination of betaine in serum by high performance liquid chromatography with UV detection.Methods Pre-column derivatization of serum was performed directly in acetonitrile without extraction with p-bromophenacyl bromide and 18-crown-6 ether as catalyst.The p-bromophenacyl ester derivatives were then separated by Supelcosil LC-SCX, using an was 0.8 ml/min and the effluent was monitored at 259 nm. Betaine was used for preparation of standard curve and quantification with external standards.Results The linearity of this method was 6.25-200.00 μmol/L,the regression was 0.999 8.The detection limit was 3.0 μmol/L.The within-day imprecision was 1.88%-3.79% ( average 3.24% ), the between-day imprecision was 3.14%-6.76% ( average 4.39% ), the recovery rates were 95.89%-102.86% (average 99.16% ).Conclusion This method is sensitive, rapid, accurate and suitable for the research and routine clinical practice.

OBJECTIVETo investigate bone defect healing by true bone ceramic complex carrying core binding factor a1 (Cbfa1) gene modified rabbit skin fibroblasts.

METHODSTransfect rabbit skin fibroblasts (RSF) with both eukaryotic expression vector pSG5 which could express Cbfa1 gene and pSG5. After being cultured for 48 h, the transfected RSF were seeded into true bone ceramic (TBC) of 2 cm in length and 4 mm in diameter to construct pSG5-Cbfa1/RSF/TBC complex and pSG5/RSF/TBC complex. Forty-eight bone defect model rabbits were randomized into four groups, each has 6 rabbits (12 radius), due to different treatment. group I: with pSG5-Cbfa1/RSF/TBC complex, group II: with pSG5/RSF/TBC complex, group III: with TBC, Group IV: empty control. After being seeded and cultured for about 24 h the complexes were implanted into 2 cm long bone defects in the middle of bilateral radius of rabbits. The radius were inspected by X-ray and then the specimens were collected at the end of the fourth and twelfth weeks after operation. Then, the specimens were decalcified and histologically investigated with Hematoxylin eosin staining and Masson staining methods. Newly synthesized trabecular bone was inspected by image analysis system and the strength of bone defect area treated with graft-implantation was tested with biomechanical method-three point bending test.

RESULTSIn group I, trabecular bone was actively synthesized to generate a great amount of trabecular bone and osteon. Preliminary union and bone defect healing were completed with good biomechanical characteristics. There were no newly synthesized trabecular in the other three groups, and bone defect healing were not discovered. The amount of newly synthesized trabecular bone and the results of biomechanical testing differed significantly between group I and the other three (P < 0.01). The efficacy of group I was significantly better than that of the other three groups.

CONCLUSIONTrue bone ceramic complex composed with Cbfa1 gene modified rabbit skin fibroblasts can effectively heal bone defect in rabbits.

Animals ; Bone Regeneration ; Bone Substitutes ; Bone Transplantation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; genetics ; Disease Models, Animal ; Fibroblasts ; cytology ; metabolism ; Plasmids ; genetics ; Rabbits ; Radius ; injuries ; physiopathology ; surgery ; Random Allocation ; Skin ; cytology ; Tissue Engineering ; methods ; Transfection

AIMTo observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE.

METHODSIn the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia.

RESULTSBlocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs.

CONCLUSIONThe results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.

Animals ; Cell Hypoxia ; Cells, Cultured ; Hydroxyeicosatetraenoic Acids ; pharmacology ; Hypoxia ; physiopathology ; Kv1.5 Potassium Channel ; biosynthesis ; genetics ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; metabolism ; Potassium Channels, Voltage-Gated ; antagonists & inhibitors ; Pulmonary Artery ; physiopathology ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Shab Potassium Channels ; biosynthesis ; genetics ; Vasoconstriction ; drug effects

15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.
Animals ; Cattle ; Down-Regulation ; drug effects ; Endothelium, Vascular ; cytology ; drug effects ; enzymology ; Hydroxyeicosatetraenoic Acids ; pharmacology ; In Vitro Techniques ; Male ; Nitric Oxide Synthase Type III ; metabolism ; Pulmonary Artery ; cytology ; enzymology ; physiology ; Rats ; Rats, Wistar