Objective To sequence the antigen-encoding genes esxA,fbpB and Rv2660c from clinical isolates of Mycobacterium tuberculosis (MTB) in tuberculosis children and study the polymorphisms.Methods A total of 135 isolated strains were amplified for gene sequences of esxA,fbpB and Rv2660c by PCR,and the products were sequenced respectively.The results of sequencing were compared with the corresponding gene sequences of standard strain H37Rv and human T and B cell epitope sequences from immune epitope database (IEDB).The genetic diversities of the gene sequences of esxA,fbpB and Rv2660c were analyzed.Results Among the 135 strains,no gene polymorphisms were found in esxA and Rv2660c.But fbpB had genetic diversities in 66 clinical isolates (48.89％).The 5 single nucleotide polymorphism (SNP) mutations in fbpB affected 8 (14.81％) T cell epitopes and 9 (24.32％) B cell epitopes.The dN and dS values of whole fbpB gene were 0.000 120 and 0.002 126,respectively.The dN and dS values of T epitopes areas were 0.000 067 and 0.002 382 respectively,those of non-T epitopes area were 0.000 408 and 0.000 470,respectively,those of B epitopes areas were 0.000 063 and 0.00 2380,and those of non-B epitopes areas were 0.Conclusion The gene sequences of esxA and Rv2660c are conservative,and that of fbpB contains genetic diversities.Its polymorphisms may influence the function of antigen Ag85B,and thus affect the efficacy of vaccine H56.

Objective To determine the effects of down-regulating serine/threonine/tyrosine kinase 1 (STYK1) on the proliferation,apoptosis and invasion and on MEK/ERK and PI3K/AKT signaling pathways in human colorectal cancer cell line HCT-15.Methods The expression of STYK1 protein was downregulated by RNA interference technique in HCT-15 cells.Then,the STYK1 protein expression was detected by Western blotting.CCK-8 assay was used to measure the effect of STYK1 down-regulation on the proliferation of the cells,flow cytometry was employed to detect the cell apoptosis,and Transwell chamber assay was adopted for cell invasion ability.The expression levels of p-ERK1/2 and p-AKT were detected by Western blotting.Results Compared with the siRNA NC group,STYK1 down-regulation significantly inhibited the proliferation (P ＜ 0.01) and invasion (P ＜ 0.01),induced cell apoptosis (P ＜ 0.01),and suppressed the expression levels of p-ERK1/2 and p-AKT in the HCT cells transfected with STYK1 siRNA (P ＜0.01).Conclusion STYK1 down-regulation inhibits the proliferation and invasion and induces the apoptosis in colorectal cancer cells,which may be related to its inhibition on the MEK/ERK and PI3K/AKT signaling pathways.

Objective To investigate the killing effect of ethacrynic acid (EA) on lung cancer A549 cells derived spheres and explore the underlying mechanism.Methods A549 spheres were cultured in serum-free medium,and the protein expression of CD133,SOX2,EpCAM and ABCG2 was detected by Western blotting.MTT assay was used to evaluate the cell viability of A549 spheres and A549 cells after treated by 1,2,5,10 and 20 mg/mL cisplatin (DDP) for 48 h.The activity of glutathione S-transferase (GST) was measured by colorimetric method after A549 spheres were treated with 10,50,100 and 200 μmol/L EA,respectively.Flow cytometry,Western blotting,real-time PCR and luciferase assay were used to analyze the levels of cellular reactive oxygen species (ROS),formation of A549 spheres,mRNA and protein expression levels of β-catenin,Sox2 and ABCG2,and promoter activity of β-catenin upon 200 μmol/L EA treated cells for 48 h.A549 sphere was infected with β-catenin adenovirus for 24 h,followed by 200 μmol/L EA treatment (in presence or absence of 5 mg/mL DDP) for 24 h.The expression of β-catenin,Sox2 and ABCG2 at mRNA and protein levels was detected by real-time PCR and Western blotting,and cell growth of A549 spheres was evaluated by MTT assay.Results The A549 spheres,with high expression of tumor stem cells markers CD133,SOX2,EpCAM and drug resistance related molecule ABCG2,and resistance to DDP at different doses,were successfully derived.After 200 μmol/L EA had treated A549 sphere for 48 h,the levels of ROS were significantly increased (P ＜ 0.05),and the mRNA and protein levels of β-catenin,Sox2 and ABCG2,and promoter activity of β-catenin were notably decreased (P ＜ 0.05).The treatment of 200 μmol/L EA enhanced the inhibitory effect on proliferation and the promoting effect on apoptosis in A549 spheres induced by 5 mg/mL DDP (P ＜ 0.05).Up-regulation of β-catenin by adenoviral infection partly reversed the effects of 200 μmol/L EA on suppressing the expression levels of β-catenin,Sox2 and ABCG2,compared to the spheres infected with blank adenovirus.Additionally,β-catenin over-expression significantly remitted the inhibitory effect of 200 μmol/L EA and 5 mg/mL DDP on the proliferation in A549 spheres.Conclusion EA exerts inhibitory effect on the proliferation and stemness of A549 spheres through suppressing GST activity and β-catenin expression,and then promotes cell apoptosis.EA might be a novel drug in treatment of lung cancer and cancer stem cells.

Objective To investigate whether 17β-estradiol (E2) can stimulate the proliferation,migration,and secretion of trefoil factor family 1 (TFF1) in papillary thyroid cancer K-1 cells and explore the molecular mechanisms.Methods ELISA was used to detect the content of TFF1 in the supernatant of K-1 cells after the treatment of E2,propylpyrazoletriol (PPT,ERα agonist) or diarylpropionitrile (DPN,ERβ agonist).The expression of ERα and ERβ in the untreated cells was measured by Western blotting.ERα siRNA and ERβ siRNA by RNA interference were designed and synthesized,and the change of TFF1 was measured by ELISA again after the transfection.The interaction between TFF1 promoter and ER was evaluated by chromatin immunoprecipitation analysis (CHiP).The proliferation and migration were detected in the K-1 cells after E2 treatment by MTT assay and Transwell chamber test respectively.Festults After E2 treatment,the TFF1 content in the supernatant of K-1 cells was increased gradually,reached peak at 24 h,and then declined slowly.PPT treatment enhanced the secretion of TFF1 but DPN decreased it in the K-1 cells.Transfection of ERα siRNA obliterated the inductive effect of E2 on the secretion of TFF1,but that of ERβ siRNA increased the inductive effect in the K-1 cells.Western blotting showed that the expression level of ERα was higher than that of ERβ in the K-1 cells.ChIP results confirmed that ERα protein was bound to the promoter of TFF1 gene in K-1 cells.E2 treatment promoted cell proliferation and improved cell migration in the K-1 cells.Conclusion E2 induces the expression and secretion of TFF1 in K-1 cells through ERα-dependent manner,and thus promotes the proliferation and migration of the cells.

Ovarian pregnancy is a rare ectopic pregnancy.In recent years,the development and popularization of assisted reproductive technology increase the incidence of ovarian pregnancy.This article focused on the ultrasound features of ovarian pregnancy following in vitro fertilization and embryo transfer (IVF-ET) by comparing those of pregnant corpus luteum and tubal pregnancy,and analyzed the advantages of transabdominal ultrasonography (TAS) and transvaginal ultrasonography (TVS) in the diagnosis.

Objective To evaluate the efficacy of less invasive stabilization systems (LISS) fixation and double plating on the treatment of AO-type C3 distal femoral fractures via anterior midline approach.Methods From June 2014 to March 2017,18 cases of AO-type C3 distal femoral fractures treated in our department were recruited in this study.They were 12 males and 6 females,at a mean age of 19-62 years.There were 11 cases due to traffic accidents and 7 due to falls from heights.Six of them had open fractures and 12 had closed fractures.They all were treated with LISS fixation and double plating with anterior midline incision.Kolment and Wulff criteria were used to evaluate knee joint function after internal fixation at the last time of follow-up.Results For all the subjects,the average operative time was 145 (110 to 200) min,and they were followed up for 17.5 (12 to 25) months.They all achieved primary wound healing,and had an average time of 22 (20 to 26) weeks for fracture healing.No nonunion,infection,osteomyelitis or injuries of nerves and arteries were observed.According to the results of Kolment and Wulff criteria for knee joint function evaluation,6 of them got excellent,9 good,2 fair and 1 poor outcomes,with a rate of 83.33％ for good and excellent outcomes.Conclusion The treatment of type C3 distal femoral fractures via anterior middle approach of knee joint can fully reveal the articular surface of the femoral condyle.And the operative vision is clear,making it easy for anatomical reduction of articular surface fractures.Combined with LISS double locking plates fixation treatment,it can avoid the injury of soft tissue,and the fracture also be rigid fixation.Postoperative early functional exercise should be allowed to obtain good clinical results.

Objective To investigate the normal waveform,lantency and amplitude of auditory event-related potentials (ERPs) of exogenous components in children at different ages,and summarize the developmental rules and clinical perspectives of exogenous ERPs according to the International Federation of Clinical Neurophysiology (IFCN) Recommended Standards in 2009.Methods One hundred and thirty-six children aged from 3 to 17 years old (admitted in the departments from healthcare,orthopedics,general surgery,and respiratory and digestive diseases,and those recovered from respiratory infection and gastrointestinal infection),and healthy adults (from undergraduates of Chongqing medical university) were recruited in this study from 2008 to 2010.They were divided into 4 groups in accordance with their ages,that is,3 ～5,＞ 5 ～7,＞ 7 ～9,and ＞ 9 ～17 years old groups,and adult group (22 ～29 years old).ERPs were recorded from the midline site (Cz) with classical auditory Oddball paradigm.Each subject was tested twice or more to determine waveform repeatability.The overlapped graphs of ERPs under target and standard stimuli were obtained in the potentiometer.The exogenous ERPs,including P1,N1,P2 and N2,lantency and amplitude were measured.Results The latencies of exogenous ERPs under target and standard stimuli were shortened in a non-uniform manner with the increasing of age.No matter under target or standard stimuli,over 95％ of mean latencies of exogenous ERPs were within the range of 2SD.In addition to N1 component,the amplitude of P1,P2 and N2 components induced by target stimulus were in a trend of increased first and then decreased with the increasing of age.Compared with the children,the amplitude was much lower in the adults and adolescents (P ＜ 0.05).The N2 component induced by target stimulus was absent among 95％ healthy adults.The Standard deviations of amplitudes of exogenous components were great in all aged groups,indicating significant individual differences.Conclusion The latencies of exogenous ERPs are shortened with the increasing of age.Target stimulus will produce more stable results than the standard stimulus,and the ERPs under target stimulus can reflect the primary auditory cortex function of temporal lobe,and are independent on patients' cooperation.

Objective To investigate the application of radial endobronchial ultrasound (R-EBUS)-guided bronchoscopy in the diagnosis of peripheral pulmonary lesions (PPLs),assess its safety and diagnostic value,and explore the influencing factors.Methods Clinical data of 140 patients who underwent R-EBUS transbronchial biopsy (TBB) and brush biopsy in our department of respiratory endoscopy from January 2015 to March 2017 were collected and retrospectively analyzed in the study.Eighty-three cases were diagnosed as PPLs.The detection rate,ability to locate the peripheral lesions,and influencing factors of EBUS were analyzed.The incidence rate of complications was observed to assess its safety.Results Of the 83 PPLs patients,they were 55 males and 28 females,and at a mean age of 59.81 ± 11.85 years.The total success rate of EBUS-guided bronchoscopic diagnosis was 59.04％ (49/83),and the diagnostic rate was 50.94％ (27/53) for malignant lesions,and 73.33％ (22/30) for benign diseases.The rate of EBUS-TBB (56.92％,37/65) was significantly higher than that of brush biopsy (30.12％,25/83,Chi square =10.76,P =0.001).EBUS-guided diagnostic methods had an accuracy of 60.00％ (45/75),while the EBUS-guided bronchoscopy had a rate of 50.0％ (4/8),but no significant difference was seen between the 2 methods (Chi square =0.03,P =0.87).The sizes of the lesions ranged from 10.0 to 52.4 mm,and the diagnostic yield for PPLs ≤20 mm in diameter (36.84％,7/19) was lower than that for those ＞20 mm (65.62％,42/64,Chi square =5.02,P =0.003).There was no significant difference (Chi square =10.05,P =0.07)in the diagnostic yield for different sites,with that of right upper lobe of 46.15％ (12/26),right middle lobe 100.00％ (8/8),right lower lobe 53.85％ (7/13),left upper lobe 37.50％ (3/8),left lingula lobe 63.64％ (7/11),left lower lobe 70.59％ (12/17).But the detection rate was obviously lower in the upper lobe (22/45,48.89％) than the middle/lower lobe (27/38,71.05％,Chi square =5.02,P =0.003).Of all the 83 patients,74 ones (89.16％) were successfully identified using radial probe EBUS,but the lesions were not found in 9 patients.When the radial probe position was within the target lesion,the diagnostic yield was 81.58％ (31/38),notably higher than that when the probe was positioned adjacent to the lesion (50.00％,18/36,Chi square =8.24,P =0.004).Mild bleeding was observed when performing biopsy under bronchoscope,and no pneumothorax,or other serious complications were observed.Conclusion Radial EBUS is a safe and feasible nethod to accurately identify PPLs and improve its diagnostic rate.EBUS-guided bronchoscopy has higher positive detection rate for PPLs,and is cost saving.The relationship of the probe and the site of lesion is the main factor influencing the diagnostic rate.

Objective To analyze the normal reference values of left ventricular longitudinal peak systolic strain (LS) and strain rate (LSr) by two-dimensional speckle tracking imaging (2D-STI) in healthy children at different ages.Methods Clinical ultrasound data of 330 healthy children who taking physical examination in our hospital from September 2015 to November 2016 were collected and retrospectively.They were divided into 7 groups according to their ages,that is,neonate group (0 to 28 days old),infant group (28 days to 1 year old),toddle group (1 to 3 years old),pre-school group (3 to 6 years old),school age group (6 to 9 years old),pre-adolescence group (9 to 13 years old) and adolescence group (13 to 18 years old).2D-STI was performed in the 3 apical projections of left ventricle (LV).The regional peak systolic longitudinal strain and strain rate of LV were measured.Results ① Significant differences were found in the LS and LSr values from 6 segments of the same wall (P ＜ 0.05),and the LS and LSr values were gradually increased from basal segment to apical segment.②There were no significant differences in the LS,LSr and global LS (GLS) values of 18 segments between sexes (P ＞ 0.05).③ Except the basal segments of posterior wall,inferior wall and posterior septum,LS value was gradually elevated with the increase of age (LS ＜LSinfant ＜ LStoddler,pre-shool and school age ＜ LSpre-adolescence and adolescence),So was GLS value.④LS in 18 segments had a positive correlation with age,and the value of apical segment in lateral wall was most remarkably correlated (r =0.551,P ＜0.01);Regional LS had a negative correlation with heart rate,and that of middle segment in lateral wall was the most apparent (r =-0.625,P ＜ 0.01);GLS was positively influenced by age (r =0.665,P ＜ 0.01) and negatively affected by heart rate (r =-0.625,P ＜ 0.01).⑤The heterogeneities were found in the correlations of regional LSr with age and heart rate in segments and in the LSr among different age groups.Conclusion Normal reference values of LV peak LS and LSr are established for healthy children at different ages,and they possess their own characteristics.

Objective To compare the predictive value of 5 scoring systems for hemorrhagic transformation risk after intravenous thrombolysis in patients with acute ischemic stroke (AIS) in different therapeutic windows.Methods A single-center and retrospective study was performed for 243 AIS patients who underwent intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) in different therapeutic windows in our department during January 2014 and December 2016.Five scoring systems,including HAT model (hemorrhage after thrombolysis),MSS model (multicenter stoker survey),GRASPS model (glucose at presentation,race,age,sex,systolic blood pressure at presentation,severity of stroke at presentation),SEDAN model (baseline blood sugar,early infarct signs,hyperdense cerebral artery sign on admission CT,age,NIHSS on admission),and SITS model (safe implementation of thrombolysis in strokemonitoring study) were used to evaluate the risks for hemorrhagic transformation.The relationships between the 5 scoring systems and incidence rate of hemorrhagic transformation were analyzed among the patients in different therapeutic windows.The predictive values of the 5 scoring systems were compared using the areas (AUC) under the receiver operating characteristic (ROC) curve.Results When the AIS patients were treated by intravenous thrombolysis within 3 h,the AUC of GRASPS and HAT models were 0.698 and 0.619,respectively,higher than those of the other 3 systems.When the therapeutic window was between 3 to 4.5 h,HAT model and SEDAN model had highest AUC (0.719,0.744) than the other 3 systems (P ＜0.05).When the windows were ＞4.5 ～6 h,the HAT model had the highest AUC (0.676).Conclusion The 5 scoring systems show better predictive value for hemorrhagic transformation after intravenous thrombolysis.For the therapeutic window within 4.5 h,HAT model presents best predictive value than the other 4 scoring systems.