Objective To screen melanocortin 4 receptor (Mc4R) gene mutation by direct DNA sequencing in order to explore the mutation situation of Mc4R gene in simple obese children in Nanjing.Methods One hundred and five simple obese children(obesity group) and 127 healthy children(healthy control group) were examined for mutations of Mc4R gene.Body mass index(BMI)cutoff points for overweight and obesity adopted Chinese children and adolescents,recommended by China Working Group of Obesity,and all children had no other hereditary and metabolic abnormality.Touch-down PCR was performed to amplify the full length Mc4R gene,then direct DNA sequencing was used to analyze the Mc4R gene.The differences of biochemical index levels between obesity group and healthy control group were analyzed ,including alanine transaminase,aspartate transaminas,total protein,albumin,globulin,albumin/globulin,triglyceride,cholesterol,high density lipoprotein,cortisol,insulin and C peptide.Results There were significant differences of biochemical index levels between obesity group and healthy control group,including triglyceride,insulin level after dining 2 h,C peptide and BMI(Pa

MicroRNAs (miRNAs) are recently discovered major regulators of gene expression, which play a pivotal role in a wide spectrum of biological processes including antiviral defence. There is growing evidence that some viruses either encode their own viral miRNAs or subvert cellular miRNAs. The host-and virus-encoded miRNAs and their targets together thus form a novel regulatory layer of interactions between the host and the virus. A better understanding of host-virus interaction mediated by miRNAs would not only enable us to unravel the molecular basis of viral pathogenesis, but also enable us to develop better therapeutic strategies.

AIM:To investigate the regulatory role of hypoxia mimic reagent cobalt chloride ( CoCl2 ) on cave-olin-1 (Cav-1) generation and the influence of Cav-1 on the abilities of migration and invasion of human lung adenocarcino-ma A549 cells.METHODS:The concentrations of Cav-1 and hypoxia-inducible factor ( HIF)-1αin pleural effusion of the patients with lung cancer ( MPE) or tuberculous pleurisy ( TBPE) were detected, and the correlation was also compared. A549 cells were treated with CoCl2 at different concentrations and time in the presence or absence of HIF-1αinhibitor YC-1.The concentrations of Cav-1 and HIF-1αin the cell supernatants were measured by ELISA.The effects of Cav-1 induced by CoCl2 on the migration and invasion of A549 cells were determined by scratch test and Transwell invasion trial, respec-tively.RESULTS:The levels of Cav-1 and HIF-1αin MPE were significantly higher than those in TBPE.There was a highly positive correlation between Cav-1 and HIF-1αlevels in the pleural effusion.CoCl2 induced the generation of Cav-1 and HIF-1αin A549 cells in a concentration-and time-dependent manner, the peak occurred at 200 μmol/L or 24 h, while the concentration over 200 μmol/L or after treated over 24 h, a concentration-or time-dependent inhibition was ob-served.HIF-1αinhibitor YC-1 concentration-dependently inhibited the generation of HIF-1αand Cav-1 induced by CoCl2 in A549 cells.CoCl2 enhanced A549 cells migration and invasion, with 200 μmol/L played the strongest role, which were down-regulated significantly in the presence of YC-1.CONCLUSION:The alteration of hypoxia-induced Cav-1 generation might be involved in the migration and invasion of A549 cells.A possible role for HIF-1αis indicated in Cav-1 generation.

Objective To evaluate the usefulness of the short-segment nerve conduction studies (SSCSs,inching test) of the peroneal nerve across the fibular head in order to diagnose and localize the site of entrapment of the nerve.Methods Fifty-four patients with suspected peroneal nerve palsy and 30 controls were studied.The symptoms all occurred in unilateral leg,involving 25 left legs and 29 right legs.Both long segment motor nerve conduction studies (LSMCs) and SSCSs were performed in all patients and controls.SSCSs were obtained at 2 cm intervals,starting 6 cm proximal (P6,P4 and P2) and 4 cm distal (D2 and D4) ending to the fibular head prominence (P).Results When nerve conduction of the entire 10 cm segment across the fibular head was tested by the conventional method,only 40 showed reduction in amplitude or slowing of motor conduction velocity or both.However,with SSCSs,54 peroneal nerves were all discovered abnormal.The results of comparison of conventional methods suggested that there were significant differences in conduction velocity between the fibular head-Ankle segment and the knee-fibular head segment in the case group,the latter ((33.63 ± 9.29) m/s) being slower than the former ((47.92 ± 4.04) m/s;t =9.776,P =0.000),while there was no obvious abnormality in the control group.The results of the control group detected by SSCSs showed that there was no significant difference between the left and right sides of the mean amplitude of the stimulation points and the mean time of segmental nerve conduction.Therefore,we set the exception criteria as the segmental nerve conduction time is longer than the corresponding control group (i + 2 s),and the CMAP amplitude of proximal stimulation point decreased by more than 20％ than the adjacent distal segment.In accordance with this standard,we found that the lesions were located in P6 to P4 in 2/54 (3.7％) legs,P4 to P2 in 4/54 (7.4％) legs,P2 to P in 43/54 (79.6％) legs,P to D2 in 12/54 (22.2％) legs,D2 to D4 in 3/54 (5.6％) legs,respectively.Consequently,the P2 to P segment was most vulnerable to damage.Conclusions SSCSs are more sensitive in detecting the entrapment of the peroneal nerve across the fibular head than the LSMCs.SSCSs could precisely localize the entrapment lesions,might be a useful tool especially for the detection of mild entrapment which has normal LSMCs findings of the peroneal nerve across the fibular head.

Objective To observe the availability and safety of ventricular septal defect (VSD) occluder in infants and young children with large patent ductus arteriosus (PDA) associated with severe pulmonary hypertension.Methods Five patients (1 male and 4 fomale) of large PDA aged 5 months to 3 years,weighted from 5.1 to 15 kg,body surface area (BSA) 0.37-0.58 m2 underwent transcathter intervention with concentric VSD occluders from June 2008 to May 2009.Arterial ducta were tube-like and their diameters were 5.7 to 8.5 mm,with ulmonary vascular resistance from 4.8 to 5.7 Wood Unit,Qp/Qs 3.4-4.6.Three patients were given Bosentan after intervention.Results The large PDAs were successfully closed with VSD occluders,including 1 concentric perimembranous VSD occluder and 4 muscular VSD occluders.They all discharged 4 to 5 days with hidrosis and weight improved.Echocardiogram indicated VSD occluder was stable,no residue shunt and no stricture of left pulmonary artery and descending aorta were found.According to tricuspid and pulmonary regurgitation,pulmonary arterial pressure decreased differently and returned to normal after 6 months follow-up.Conclusion VSD occluder is available and effective to close large PDA associated with severe pulmonary hypertension in inrants and young children,but more cases and long-term follow-up are necessary.

Acinetobacter baumannii ; classification ; genetics ; isolation & purification ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Phylogeny

OBJECTIVETo study the effects of Compound Danshen Dripping Pill (CDDP) on the structure and functions of sternohyoid muscle in metabolic syndrome (MS) rats, and to study whether it has therapeutic effects on obstructive sleep apnea-hypopnea syndrome (OSAHS).

METHODSTwenty-one healthy male SD rats were randomly divided into three groups, i.e., the normal control group (n = 6), the MS group (n = 8), and the CDDP group (n = 7). Rats in the normal control group were routinely fed. High lipid forage was given to rats in the rest two groups. Nine weeks later, CDDP (at the dose of 375 mg/kg) was additionally given to rats in the CDDP group by gastrogavage, and then rats in the CDDP group and the MS group were fed with the same high lipid forage for 12 successive weeks. The content of malondialdehyde (MDA) and the activities of superoxide dismutase (SOD) in the sternohyoid muscle were detected in the three groups. The capillary density, capillary-to-fiber ratio (C/F), the section area of type I muscle fiber were detected using myosin-ATPase histochemical assay. The contractile changes of isometric stemohyoid muscles were determined under electric stimulation by different frequencies.

RESULTSThe contents of MDA were obviously lower in the CDDP group than in the MS group, while the activities of SOD, the capillary density, C/F, the section area of type I muscle fiber, the tension of stemohyoid muscle at 10 -60 Hz, and the 1-5 min tension percentages of the stemohyoid muscle were higher in the CDDP group than in the MS group (P < 0.05, P < 0.01).

CONCLUSIONCDDP could improve oxidative stress induced intramuscularly microcirculation disturbance and changes of muscular fiber structures of the upper airway muscles, and elevate their contractile functions, thus possibly contributing to favorable effects on OSAHS.

Animals ; Capillaries ; Drugs, Chinese Herbal ; pharmacology ; Male ; Metabolic Syndrome ; metabolism ; Muscle Contraction ; drug effects ; Neck Muscles ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; Phenanthrolines ; pharmacology ; Rats ; Rats, Sprague-Dawley

Objective To evaluate the feasibility and effectiveness of combination chemotherapy with etoposide and cisplatin(EP)regimen on the patients with high-risk,chemorefractory and recurrent gestational trophoblastic neoplasia(GTN).Methods Thirty-nine patients with gestational trophoblastic tumors were analyzed retrospectively,25 of 39 patients were of high-risk,9 patients were chemorefractory and 5 patients were recurrent.All 39 patients were administrated with EP regimen,and 10 patients were assisted with surgery.All the patients were followed up.Clinical response,toxicity,the occurrence of secondary tumors of all patients,and the fertility of 30 patients whose fertility function was preserved were investigated. Results Thirty-nine GTN patients underwent a total of 221 cycles of the EP regimen.The average number of courses for each patient was 5.7.The total complete remission rate of the regimen was 74%(29/39). Twenty-five patients with high-risk GTN received a total of 139 cycles and the average number of courses was 5.6.Nineteen patients achieved complete remission and 6 patients showed drug-resistant.The complete remission rate of the high-risk group was 76%(19/25).Nine patients with chemorefractory GTN obtained a total of 55 cycles and the average number of courses was 6.1.Six patients achieved complete remission and 3 patients showed drug-resistant again.The complete remission rate of the chemorefractory group was 6/9. Five patients with recurrent GTN received 27 cycles and the average number of courses was 5.4.Four patients achieved complete remission,1 patient showed drug-resistance and died.Bone marrow toxicity, gastrointestinal reaction and alopecia were the main side effects of the EP regimen,but the bone marrow toxicity was slight and no grade Ⅳ side effect occurred.No fatal effect was found.Eight of 30 patients whose fertility faction was preserved had become pregnant after recovery,with a total of 8 pregnancies.Among them,2 were terminated by induced abortion,and 6 underwent normal term delivery and gained 6 infants who had no congenital malformation.All the 6 children had normal growth and development after childbirth. None of the women developed secondary tumors.Conclusion The EP regimen is effective and safe for the treatment of high-risk,chemorefractory and recurrent GTN.

Background Arresting the overexpression of vascular endothelial growth factor (VEGF) will be a new approach to the inhibition of neovascularization.RNA interference (RNAi) can inhibit the expression of specific gene,and its application in eye has little interference to other gene expression.Objective This study was to investigate the effect of small interference RNA (siRNA) targeting VEGF on the expression of VEGF and retinal neovascularization in oxygen-induced retinopathy (OIR) model.Methods psi-HITM/enhanced green fluorescent protein (EGFP)/VEGF siRNA was designed and prepared in vitro.Mouse endothelioma (EOMA) were cultured in DMEM without antibiotic and divided into 5 groups.The cells were incubated in DMEM only in the blank control group;while 1 μl of LipofectamineTM 2000 + psi-HITM/EGFP,1 μl LipofectamineTM 2000 + 40,50 or 60 nmol/L of psi-HITM/EGFP/VEGF siRNA was added into DMEM in the negative control group and siRNA groups,respectively.The expression of VEGF mRNA and protein was detected by real time PCR (RT-PCR) and Western blot.The optimal effective concentration of VEGF siRNA was assessed.OIR models were established in 48 7-day-old C57BL/6J mice by raising them at an oxygen concentration of (75±2) ％ for 5 days and then to normal air.The mice were randomized into the model group,null vector group and VEGF siRNA group.1 μl of a mixture of psi-HITM/EGFP or VEGF siRNA (60 nmol/L) and LipofectamineTM 2000 was intravitreally injected,respectively,in the null vector group and VEGF siRNA group.The normal mice were used as the normal control group.Expression of VEGF mRNA and protein in the mouse retinas was detected by RT-PCR and Western blot,respectively,and FITC-dextran stretched retinal preparation was examined to evaluate the neovascularization,and retinal sections were examined to quantify the number of vascular endothelial cell nuclei extending beyond the internal limiting membrane (ILM).Results The in vitro transfection test showed that the expression of VEGF mRNA and protein in the EOMA cells was significantly different among the blank control group,negative control group and 40,50,60 nmol/L VEGF siRNA groups (F =148.890,P ＜ 0.001; F =306.960,P ＜ 0.001),and the expression of VEGF mRNA was lower in different concentrations of VEGF siRNA groups than that in the blank control group (t=73.950,119.890,156.480,all at P＜0.001).Also,the expression of VEGF protein was less in different concentrations of VEGF siRNA groups than that in the blank control group (t =15.452,23.344,42.119,all at P＜0.001).The optimal inhibitory concentration of VEGF siRNA was 60 nmol/L.In vivo study determined that compared to the model group and null vector group,the non-perfusion zones and neovascular net in the retina were decreased,and the number of vascular endothelial cell nuclei extending beyond the ILM was less in the VEGF siRNA group.The relative expression level of VEGF mRNA in the retinas was 1.23±0.18,4.02±0.16,3.98±0.19 and 1.98±0.12 in the normal control group,model group,null vector group and VEGF siRNA group,respectively,with a significant difference among them (F=369.780,P＜0.001),and the relative expression levels of VEGF mRNA in the model group and null vector group were higher than that in the normal control group (t=37.880,37.336,both P＜0.001),and the expression of VEGF mRNA in the VEGF siRNA group declined by 50.8％ (t=10.183,P＜0.001).The difference in the expression levels of VEGF protein also was assayed among the various groups (F=408.980,P＜0.001),and VEGF level in the retina was lowered by 68.0％ in the VEGF siRNA group compared to the model group (t =11.473,P＜0.001).However,VEGF level in the VEGF siRNA group remained at a high level in comparison with the normal control group (t =2.422,P＜0.001).Conclusions Intravitreal injection of VEGF siRNA can attenuate retinal neovascularization by effectively downregulate the expression VEGF mRNA and protein in the retina.

Objective To detect the immunogenicity of VirB9,a protein of type Ⅳ secretion system of Brucella.Methods Full length VirB9 gene was cloned into plasmid pET32a and expressed in Escherichia (E.) coli BL21 (DE3).Expression of recombinant protein was induced by isopropyl beta-D-thiogalactopyranoside (IPTG) and the recombinant fusion protein was purified by affinity chromatography on Ni2+-conjugated chelateing sepharose.The purity of the purified protein was ascertained by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDSPAGE) and the concentration was measured by bicinchoninic acid (BCA) protein assay kit.Animal model was established by immunizing BAL B/c mice with live vaccine strain S19 of Brucella and the mice immunized with phosphate buffered saline (PBS) as control.The blood of immunized mice was acquired after 4 weeks.Antibody against VirB9 in S19 immunized mice was detected by Rose Bengal plate agglutination test and serum tube agglutination test; IgG antibody titers against VirB9 in immunized mice were determined by enzyme linked immunosorbent assay(ELISA).At the 35th day,the immunized mice and control mice were killed and spleens were collected.The splenocytes were harvested and stimulated with each of VirB9,concanvalin A(ConA) or medium in triplicate.Production of gamma interferon (IFN-γ) was determined by enzyme-linked immunospot assay (Elispot).Results The full length of VirB9 gene was cloned into pET32a.The recombinant VirB9 protein was expressed at 43 × 103 in relative molecular mass and the purity of the purified recombinant VirB9 protein was above 97％ in SDS-PAGE and the concentration was 1.6 g/L in BCA protein assay.The antibody of VirB9 was detected in all S19 immunized mice but not PBS immunized mice by Rose Bengal plate agglutination test.The antibody titer in all S19 immunized mice was ＞ 1 ∶ 800 or ＞ 1 ∶ 3200 by tube agglutination test and ELISA,respectively.Meanwhile,the protein stimulated stronger IFN-γresponse in immunized mice than that in the control mice(147 cells Vs 38 cells).Conclusion VirB9 can stimulate humoral and cellular immunity and it might be an appropriate target for developing subunit vaccine against Brucella.