Objective To assess the expression pattern of protease-activated receptor 2 (PAR2) in human keratinocytes and to characterize its biological functions in the regulation of skin barrier.Methods Primary human keratinocytes and human N/TERT keratinocytes were used as the subject of this study.The expression and distribution of PAR2 in the keratinocytes were analyzed by using immunoflorescence staining and Western blot.Two different PAR2 agonists,trypsin and a PAR2-activating peptide (AP),as well as a PAR2-antagonistic peptide (H2N-FSLLRY-COOH) and a control peptide were used to induce the activation of PAR2 in the keratinocytes.Then,a fluorescence-based calcium mobilization assay was performed to evaluate the biological function of PAR2.Data were statistically analyzed by one-factor analysis of variance.Results Under normal culture conditions,PAR2 was weakly expressed in keratinocytes,and the expression was unaffected by culture medium composition or culture duration.Calcium mobilization was induced by trypsin of 50-250 nmol/L and the PAR2-activating peptide in a dose-and time-dependent pattern.The maximal activation of PAR2 was observed in keratinocytes treated with the PAR2 agonist HAN-SLIGKV-COOH of 75-250 μmol/L.The PAR2-antagonistic peptide (H2N-FSLLRY-COOH) obviously suppressed the increase in calcium mobilization induced by trypsin,while the control peptide PAR-RAP showed no inductive effect on the PAR2 activation based on the absence of calcium mobilization.The substrate-induced calcium release was complete within 250 seconds,and peaked at 50 seconds after the initial trypsin or PAR-AP stimulation.Moreover,the activation of PAR2 was accompanied by an increase in ERK phosphorylation and elicitation of MAPK signaling pathway in keratinocytes.Conclusions Human keratinocytes positively express PAR2,which can be activated by trypsin and PAR2-activating peptides,and the activation of PAR2 may influence the physiological function of keratinocytes by inducing intracellular calcium release.

OBJECTIVE: To study the expression levels of glial cell line-derived neurotrophic factor family receptor α-1 (GFRα1) and enhancer of zeste homolog 2 (EZH2) in the intestinal tissue of children with Hirschsprung's disease (HSCR), as well as the role of EZH2 in the regulation of GFRα1 gene expression and the pathogenesis of HSCR. METHODS: The samples of colon tissue with spasm from 24 children with HSCR after radical treatment of HSCR were selected as the experimental group, and the samples of necrotized colon tissue from 18 children with neonatal necrotizing enterocolitis after surgical resection were selected as the control group. Real-time PCR and Western blot were used to measure the expression levels of GFRα1 and EZH2 in colon tissue in both groups. Human neuroblastoma SH-SY5Y cells were divided into an EZH2 over-expression group and a negative control group. The cells in the EZH2 over-expression group were transfected with pCMV6-EZH2 plasmid, and those in the negative control group were transfected with pCMV6 plasmid. The expression levels of EZH2 and GFRα1 were measured after transfection. RESULTS: Compared with the control group, the experimental group had significant reductions in the mRNA and protein expression levels of GFRα1 and EZH2 in colon tissue (P<0.05), and the protein expression of EZH2 was positively correlated with that of GFRα1 (r=0.606, P=0.002). Compared with the negative control group, the EZH2 over-expression group had significant increases in the expression levels of EZH2 and GFRα1 after SH-SY5Y cells were transfected with EZH2 over-expression plasmid (P<0.05). CONCLUSIONS Low expression of EZH2 in the colon tissue of children with HSCR may be one of the causes of inadequate expression of GFRα1 and onset of HSCR.
Child ; Colon ; Enhancer of Zeste Homolog 2 Protein ; genetics ; Glial Cell Line-Derived Neurotrophic Factor Receptors ; genetics ; Hirschsprung Disease ; genetics ; Humans ; Infant, Newborn ; RNA, Messenger

The CrylA Crystal Protein from Bacillus thuringiensis is associated with DNA, but the role and sequences of these DNA molecules are unknown. CrylA bipyramidal crystals from B. thuringiensis strain 4.0718 was selectively dissolved and associated DNA was extracted from protoxin. The DNA was digested with Nde I to obtain 3 to 5 kb fragments and then the fragments were subcloned into pMD18-T vector, screening of recombinants were done by PCR-RFLP and sequencing. The ORF of cry1Ac gene was amplified by primers designed and then subcloned. The 3.5 kb BamH I and Sal I fragments of pMDX35 was inserted into the pET30a vector, giving 8.9 kb recombinant plasmid, pETX35. ETX35 strain were obtained by transformed pETX35 into B121 (DE3). A 141 kD fusion protein was superexpressed as inclusion bodies. Quantitative protein analysis indicated that the amount of 141 kD protein was above the level of 51.36% of total cellular protein. Plasmid pHTX42 constructed from shuttle vector pHT304 was transformed B. thuringiensis acrystalliferous strain XBU001 with electroporation to obtain the recombinant HTX42. The recombinant protein was found with a molecular mass of 130 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Scanning analysis indicated that the expressed protein accounted up to 79.28% of total cellular proteins and accumulated in the cells mounted up to 64.13% of cellular dry weight. Under Atomic Force Microscopy (AFM), typical bipyramidal crystals from HTX42 strain were found with a size of 1.2 microm x 2.0 microm. Bioassay showed that these inclusion bodies of ETX35 strain and crystals from HTX42 strain were highly toxic against the larvae of Plutella xylostella. On such a base, constructing insecticidal recombinant and analyzing the source, structure, and function of the 20 kb DNA can be further achieved.
Animals ; Bacillus thuringiensis ; genetics ; Bacterial Proteins ; biosynthesis ; genetics ; pharmacology ; Cloning, Molecular ; Endotoxins ; biosynthesis ; genetics ; pharmacology ; Hemolysin Proteins ; biosynthesis ; genetics ; pharmacology ; Microscopy, Atomic Force ; Moths ; Plasmids ; Recombinant Proteins ; biosynthesis ; pharmacology

OBJECTIVETo prepare streptavidin-tagged hepatitis C virus (HCV) fusion protein and explore its application for the detection of antibody against HCV infection.

METHODSA recombinant plasmid pET-11d-C44P-SA was constructed, which coding a novel HCV diagnostic antigens (C44P) and streptavidin (SA) fusion protein, and the fusion protein was generated with BL21 (DE3) E Coli and identified by Western Blot analysis. Then the fusion protein was purified through the Ni-NTA affinity chromatography and over 90% purity has been achieved. Anti-HCV ELISAs were developed when the fusion protein was used in the biotin-pre-coated microplate or ordinary microplate, and then the sensitivity and specificity of the ELISA were evaluated with confirmed human sera panels.

RESULTSThe fusion protein was expressed in high yields and purified successfully, the ELISA detection of anti-HCV with human sera panel indicated that its sensitivity and specificity is higher when SA-tagged HCV antigen (C44P-SA) coated in biotin-pre-coated microplate, compared to C44P or C44P-SA coated in ordinary microplate.

CONCLUSIONThe sensitivity and specificity of anti-HCV ELISA can be improved when a novel HCV diagnostic antigen fused to SA combined with the biotin- pre-coated microplate. This study laid a foundation for improving the performance of HCV diagnostics.

Antibodies, Viral ; immunology ; Enzyme-Linked Immunosorbent Assay ; Hepatitis C Antigens ; genetics ; immunology ; metabolism ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Streptavidin ; genetics ; immunology ; metabolism

BACKGROUNDSeverity scoring systems are useful tools for measuring the severity of the disease and its outcome. This pilot study was to verify and compare the prognostic performance of the Simplified Acute Physiology Score II (SAPS II) and Glasgow Coma Scale (GCS) in neuro-intensive care unit (N-ICU) patients.

METHODSA total of 1684 patients consecutively admitted to the N-ICU at Xuanwu Hospital between January 1, 2005 and December 31, 2011 were enrolled in this study. The data-base included admission data, at 24-, 48-, and 72-hour SAPS II and GCS. Repeated measure data analysis of variance, Logistic regression analysis, the Hosmer-Lemeshow goodness-of-fit statistic, and the area under the receiver operating characteristic were used to evaluate the performance.

RESULTSThere was a significant difference between the SAPS II or GCS score at four time points (F = 16.110, P = 0.000 or F = 8.108, P = 0.000). The SAPS II scores or GCS score at four time points interacted with the outcomes with significant difference (F = 116.771, P = 0.000 or F = 65.316, P = 0.000). Calibration of the SAPS II or GCS score at each time point on all patients was good. The percentage of a risk estimate prediction corresponding to observed mortality was also good. The 72-hour score have the greatest consistency. Discriminations of the SAPS II or GCS score at each time were all satisfactory. The 72-hour score had the greatest discriminative power. The cut-off value was 33 (sensitivity of 85.2% and specificity of 74.3%) and 6 (sensitivity of 70.6% and specificity of 65.0%). The SAPS II at each time point on all patients showed better calibration, consistency and discrimination than GCS. The binary Logistic regression analysis identified physiological variables, GCS, age, and disease category as significant independent risk factors of death. After the two variables including underlying disease and type of admission were excluded, we built the simplified SAPS II model. A correlation was suggested between the simplified SAPS II score at each time point and outcome, regardless of the diagnosis.

CONCLUSIONSThe GCS scoring system tends to be a little weaker in the predictive power than the SAPS II scoring system in this Chinese cohort of N-ICU patients. The advantage of SAPS II scoring system still exists that it dose not need to take into account the diagnosis or diseases categories, even in the special N-ICU. The simplified SAPS II scoring system is considered a new idea for the estimation of effectiveness.

APACHE ; Adult ; Aged ; Aged, 80 and over ; China ; Female ; Glasgow Coma Scale ; Humans ; Intensive Care Units ; statistics & numerical data ; Male ; Middle Aged ; Young Adult

OBJECTIVETo discuss the central modulating mechanism of Sanyinjiao (SP 6) and the amplitude of low-frequency fluctuation (ALFF) differences of the functional brain regions between SP 6 and sham acupoint.

METHODSSixteen volunteers accepted right Sanyinjiao (SP 6) (SP 6 group) or right sham (sham group) acupuncture for two times after 24 h sleep deprivation (SD), there was two weeks between two acupunctures. The needle was retained for 4 minutes and the brain fMRI scanning was performed by 3.0 TMR every time. The ALFF consequent data was processed by REST. One-sample t-test and two-pair t-test were performed by SPM5.

RESULTSSP 6 group mainly caused ALFF change in inferior parietal lobule, posterior cingulated while sham group in precuneus, posterior cingulated. SP 6 group increased ALFF in left fusiform temporal gyrus, medial frontal gyrus with no decreased regions compared with sham group.

CONCLUSIONSRetaining needle dose influence the activity of the brain region during resting-state in both group, especially the mood-related regions. And its mechanism is probably that the relevant effect is carried out by regulating function to target organs of complex brain network comprised of relevant functional center and related brain region. There are differences between SP 6 and Sham acupoint.

Acupuncture Points ; Acupuncture Therapy ; Brain ; diagnostic imaging ; physiopathology ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Radiography ; Sleep Deprivation ; diagnostic imaging ; physiopathology ; therapy ; Young Adult

OBJECTIVEThe aim of this study was to investigate the efficiency and safety of ibutilide for cardioversion of persistent atrial fibrillation (AF) during radiofrequency ablation.

METHODSEighteen patients (16 males) with persistent atrial fibrillation were enrolled in this study. All patients underwent circumferential pulmonary vein ablation guided by a Carto three-dimensional mapping system. In addition, linear ablation at the top of the left atrium and the isthmus of mitral valves and complex fractionated atrial electrogram (CAFE) ablation were performed. All patients were still in either atrial fibrillation or atrial flutter after ablation, the patients were treated with 1 mg intravenous ibutilide injection within 10 minutes after unsuccessful ablation. Intravenous injection was stopped in case of sinus rhythm (SR) restoration or occurrence of severe adverse reactions such as ventricular tachycardia. Cardioversion rate within 30 min and adverse reactions within 4 h were observed. Patients were divided into either conversion group or non-conversion group according to whether AF was converted to sinus rhythm within 30 minutes after injection.

RESULTSEleven patients (61.11%) converted to SR after ibutilide injection. There were no significant differences in gender, age, body mass index, left atrium and left ventricular function between conversion group and non-conversion groups. The average conversion time was (13.80 ± 7.64) min, left atrium scar area ratio was significantly larger in non-conversion group (12.40 ± 11.03)% than in conversion group (5.12 ± 3.83)%, P < 0.05. Ibutilide significantly prolonged the average wavelength of the AF wave (171.8 ± 29.5) ms vs. (242.0 ± 40.0) ms at baseline, P < 0.01. The QT interval at 30 min after ibutilide injection (0.39 ± 0.21) s was significantly longer than before injection (0.51 ± 0.08) s, P < 0.05. There was no serious arrhythmias or other adverse reactions post ibutilide injection.

CONCLUSIONSIbutilide is highly effective and safe agent for cardioversion in patients underwent unsuccessful ablation. Left atrium scar area ratio is an important determinant for the conversion rate in this cohort.

Adult ; Aged ; Anti-Arrhythmia Agents ; therapeutic use ; Atrial Fibrillation ; surgery ; Catheter Ablation ; methods ; Female ; Humans ; Male ; Middle Aged ; Sulfonamides ; therapeutic use

OBJECTIVETo compare the therapeutic effects between acupuncture and electric stimulation on post-stroke dysphagia on the basis of rehabilitation training.

METHODSNinety-seven patients with post-stroke dysphagia were randomly divided into an acupuncture group (group A, n = 32), an electric stimulation group (group B, n = 35) and a rehabilitation training group (group C, n = 30). In group C, the conventional therapy (conventional therapy of neurologic internal medicine and rehabilitation training) was applied. In group A, the combination of conventional therapy and acupuncture was applied. The acupoints of Fengchi (GB 20), Futu (LI 18), three-needles on the forehead, etc. were selected. In group B, the combination of conventional therapy and electric stimulation was adopted. Watian drinking water experiment, stethocatharsis function scoring and video fluoroscopic swallowing study (VFSS) were used to evaluate swallowing function of patients.

RESULTSAfter treatment, the total effective rate was 96.95 (31/32) in group A and was 94.3% (33/35) in group B, which was superior to that of 66.7% (20/30) in group C (P < 0.01). After treatment, the swallowing function in group A, group B and group C were all improved significantly as compared with that before treatment (all P < 0.05). After treatment, the effects in group A and B were superior to that in group C (both P < 0.05).

CONCLUSIONThe therapeutic effect of the combination of either acupuncture or electric stimulation with rehabilitation training is better than that of simple rehabilitation training. The efficacy on dysphagia is equal between acupuncture and electric stimulation.

Acupuncture Points ; Adult ; Aged ; Deglutition Disorders ; etiology ; therapy ; Electroacupuncture ; Female ; Humans ; Male ; Middle Aged ; Stroke ; complications ; Treatment Outcome

OBJECTIVEHigh short-term successful rate was reported for catheter ablation in patients with paroxysmal atrial fibrillation (AF), we analyzed the long-term outcome (success rate, anticoagulation therapy and embolism event, anti-arrhythmic therapy and death post procedure) of catheter ablation for paroxysmal AF in this study.

METHODSFrom January 2000 to December 2004, 106 consecutive patients with drug-refractory paroxysmal AF underwent catheter ablation and were followed-up for (60.7 + or - 11.8) months. Segmental pulmonary vein isolation (SPVI) was routinely performed by radiofrequency energy under the guidance of circular mapping catheter. The patients were followed up with 24 h-holter, ECG, telephone or letter. Data on recurrence of AF, the anticoagulation medication and the incidence of embolism, anti-arrhythmic therapy were obtained.

RESULTSThere were 9 patients lost to follow up. In the remaining 97 patients [65 males, (54.8 + or - 11.2) years old], 3 cases died from cancer, sinus rhythm was maintained in 68 patients (Group S, 72.3%) and AF recurrence evidenced in 26 patients (Group R, 27.7%). In Group S, 56 patients (82.4%) discontinued anticoagulation medication, and 12 patients continued to take aspirin. There was no embolism event in Group S during follow-up. In Group R, 1 patient continued to take warfarin; 11 patients continued to take aspirin and 2 patients suffered from cerebral embolism. Anticoagulation medication was discontinued in 14 patients (53.8%) and 1 patient suffered form cerebral embolism. The incidence of embolism event in Group R is significantly higher than in Group S (P < 0.01). More patients discontinued anti-arrhythmic medication in Group S than in Group R (80.9% vs. 56.0%, P < 0.05).

CONCLUSIONCatheter ablation is associated with satisfactory long-term success rate, reduced anti-arrhythmia medication, improved quality of life in patients with paroxysmal AF.

Adult ; Aged ; Atrial Fibrillation ; therapy ; Catheter Ablation ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Prognosis ; Retrospective Studies

The receptor-binding domain(RBD) protein of HCoV-NL63 is a major target in the development of diagnostic assay and vaccine, it has a pivotal role in receptor attachment, viral entry and membrane fusion. In this study, we prepared 2 purified recombinant HCoV-NL63 RBD proteins using in E. coli system and identified the proteins by Western blotting. We first optimized codon and synthesized the RL (232-684aa)coding gene, then amplified the RL or RS(476-616aa) coding gene via PCR using different primers . The RL or RS coding gene was cloned into the pM48 expression vector fused with TrxA tag. The RBD (RL and RS) of HCoV-NL63 were expressed majorly as inclusion body when expressed in E. coli BL21pLys S under different conditions. The expressed products were purified by affinity chromatography then analyzed by SDS-PAGE and Western blotting. Our results showed that the recombinant RBD proteins were maximally expressed at 37 degrees C with 0. 8mM IPTG induction for 4h. RL or RS protein with 95 % purity was obtained and reacted positively with anti-sera from mice immunized with the recombinant vaccinia virus (Tiantan strain) in which HCoV-NL63 RL or RS protein was expressed. In conclusion, the purified recombinant RBD proteins(RL and RS)derived from E. coli were first prepared in China and they might provide a basis for further exploring biological role and vaccine development of HCoV-NL63.
Animals ; Coronavirus Infections ; metabolism ; virology ; Coronavirus NL63, Human ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Mice ; Mice, Inbred BALB C ; Protein Engineering ; Protein Structure, Tertiary ; Receptors, Virus ; metabolism ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism