热休克蛋白B1抑制HBV复制的研究

目的 探讨热休克蛋白B1 (heat shock protein B1,HSPB1)对乙型肝炎病毒(HBV)复制的影响.方法 采用Western blot检测正常肝细胞系(HepRG)和HBV稳定复制细胞系(HepAD38和HepG2.2.15)中HSPB1的蛋白水平.在HBV稳定复制细胞系(HepAD38和HepG2.2.15)中转染pCMV6-HSPB1质粒及对照质粒,采用Western blot验证HSPB1过表达水平,实时荧光定量PCR和Southern blot检测HSPB1过表达对HBV复制中间体表达的影响;qRT-PCR检测HSPB1过表达对HBV3.5 kb mRNA表达的影响;ELISA检测HSPB1过表达对HBV s抗原(HBsAg)和e抗原(HBeAg)分泌的影响.在肝癌细胞系Huh-7中共转染pCH9/3091和pCMV6-HSPB1质粒,采用Western blot验证HSPB1过表达水平,实时荧光定量PCR和Southern blot检测HSPB1过表达对瞬时转染的HBV复制中间体表达的影响.结果 HSBP1在HepAD38和HepG2.2.15中的表达明显低于HepRG.HSPB1在HepAD38和HepG2.2.15细胞中成功过表达,在过表达HSPB1的HepAD38和HepG2.2.15细胞中HBV复制中间体水平以及3.5 kb mRNA水平均降低,细胞培养上清中HBsAg和HBeAg的分泌水平也降低.HSPB1在转染pCH9/3091的Huh-7细胞中成功过表达,HSPB1过表达抑制了瞬时表达的HBV的复制水平.结论 HSPB1可以抑制HBV复制.     

肝细胞核因子3β调控小鼠体内HBV转录和复制的实验研究

目的 应用复制型HBV小鼠模型观察肝细胞核因子3β(HNF3β)对小鼠体内HBV转录和复制的调控作用.方法 通过尾静脉高压注射法将复制型HBV重组质粒pHBV4.1和大鼠HNF3β表达质粒pCMVHNF3β导入小鼠体内,用Northern吸印杂交检测小鼠肝组织中HBV 3.5kb、2.4/2.1kb、0.7kb mRNA的转录情况,Southern吸印杂交检测HBV DNA复制中间体水平.结果 经小鼠尾静脉高压注射法体内转染质粒pHBV4.1后,小鼠肝组织中检测到HBV 3.5kb、2.4/2.1kbmRNA转录信号和HBV DNA复制中间体信号;与共转染pHBV4.1和空载质粒pCMVpA对照组相比,共转染pHBV4.1和pCMVHNF3β使HNF3β过量表达,明显降低了小鼠体内HBV RNA转录和DNA复制水平.结论 肝富集转录因子HNF3β对小鼠体内HBV转录和复制具有抑制作用,有可能成为今后抗HBV生物治疗新的靶点.     

HBV对HepG2.2.15细胞α干扰素的JAK-STAT信号转导途径分子及MxA mRNA表达的影响

目的 研究乙型肝炎病毒(HBV)对α干扰素(IFN-α)JAK-STAT信号转导途径分子及MxA mRNA表达的影响.方法 逆转录聚合酶链反应(RT-PCR)检测IFN-α处理的HepG2.2.15细胞在不同处理时间点的STAT1、STAT2、IS-GF3γ mRNA表达水平,并比较分析干扰素抗病毒蛋白MxAmRNA在HepG2和HepG2.2.15细胞中的表达.结果 RTPCR显示经IFN-α处理后HepG2.2.15细胞STAT1、STAT2、ISGF3γ mRNA表达水平明显升高,表明转染HBV全基因组并能分泌完整HBV病毒子的HepG2.2.15对IFN-α有应答.抗病毒蛋白MxA mRNA在HepG2.2.15细胞中未检测到表达,而在HepG2细胞中却能表达.结论 HBV或其抗原成分并不影响IFN-α JAK-STAT信号转导途径分子mRNA表达,但影响抗病毒蛋白如MxA mRNA的表达.     

乙型肝炎病毒对SOX4表达的影响及其机制探讨

目的 观察乙型肝炎病毒(HBV)对SOX4表达的影响,并探讨其机制.方法 RT-PCR和Western印迹法检测HepG2和HepG2.2.15细胞中SOX4 mRNA和蛋白表达的差异,将HBV感染性克隆pHBV1.3及其空载体分别与含SOX4基因启动子的报告质粒pGL3-SOX4共转染HepG2细胞,测定荧光色素酶的活性,RT-PCR和Western印迹法分别检测SOX4 mRNA和蛋白表达的情况.结果 HepG2.2.15细胞中SOX4 mRNA和蛋白的表达水平明显高于HepG2,HBV能够激活SOX4基因启动子的活性,并在mRNA和蛋白水平上调SOX4的表达.结论 HBV能够在转录和翻译水平上调SOX4的表达.     

6-甲基香豆素对乙型肝炎病毒转录和复制的影响

目的 探讨6-甲基香豆素对乙型肝炎病毒(HBV)转录和复制的影响.方法 MTT法检测6-甲基香豆素在HepG2.2.15和HepG2-NTCP细胞中的细胞毒性.使用含0.1％DMSO的生理盐水(阴性对照),6-甲基香豆素(5、10、20μmol/L)及恩替卡韦(25 nmol/L,阳性对照)处理HepG2.2.15和HepG2-NTCP细胞6 d.RT-qPCR检测细胞中HBV DNA和HBV RNA的水平,ELISA检测细胞上清液中乙型肝炎表面抗原(HBsAg)和乙型肝炎e抗原(HBeAg)水平.雄性HBV转基因小鼠每2天腹腔注射含0.1％DMSO的生理盐水(阴性对照),6-甲基香豆素(5 mg/kg)和恩替卡韦(0.02 mg/kg,阳性对照)1次,每6天采血及称重1次.药物处理24 d后,处死小鼠并解剖取肝脏.RT-qPCR检测小鼠血清和肝脏组织中HBV DNA水平及肝脏组织中HBV RNA水平,ELISA检测小鼠血清中丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、HBsAg和HBeAg水平.结果 与对照组相比,6-甲基香豆素可以呈浓度依赖性的降低HepG2.2.15和HepG2-NTCP细胞中HBV DNA和HBV RNA及细胞分泌的HBsAg和HBeAg水平(P<0.05).6-甲基香豆素在不影响小鼠体重及AST、ALT的同时,可以显著降低HBV转基因小鼠血清中HBV DNA、HBsAg和HBeAg水平及小鼠肝脏组织中HBV DNA和HBV RNA水平(P<0.05).结论 在HepG2.2.15细胞、HepG2-NTCP细胞和HBV转基因小鼠模型中6-甲基香豆素均具有较强的抑制HBV转录和复制的作用.     

siRNA抑制乙型肝炎病毒在HepG2.2.15细胞中的复制与表达

目的 探讨靶向HBV S基因的siRNA表达质粒在HepG2.2.15细胞中对HBV病毒的复制与表达的抑制效应及其抗病毒活性.方法 设计并构建了两个靶向HBV S基因的siRNA表达质粒S1和S2,随机设计的用于对照的非同源siRNA表达质粒S3.首先在HepG2.2.15细胞中通过实时荧光定量PCR评估该siRNA表达质粒对HBV mRNA表达的抑制效应,接着在HepG2.2.15细胞中通过ELISA方法进一步枪测它们的抗病毒活性细胞上清液中HBV标志性蛋白HBsAg、HBeAg的表达水平.结果 在siRNA表达质粒转染HepG2.2.15细胞后48 h,HBV S基因mRNA表达量下降64%～88%,HBsAg和HBeAg的表达水平分别降低了60%～82%和56%～78%.S1+S2联合使用转染细胞中显著抑制HBV病毒的复制与表达的效率,而对照组S3无抑制效果.结论 发现靶向HBV S基因的siRNA表达质粒能够在HepG2.2.15细胞中有效的抑制HBV病毒的复制与表达,并能够降低细胞上清液中HBsAg和HBeAg的表达水平.S1+S2联合使用转染细胞中可以显著抑制HBV的复制与表达的效率.RNAi可能成为防治HBV感染有效的全新的抗病毒防御策略.     

肝细胞内相关mRNA抑制HBV复制与表达的功能验证

目的 验证肝细胞内抑制乙型肝炎病毒(HBV)复制与表达的相关mRNA.方法 通过慢病毒介导的方式将过氧化物酶体增殖物激活受体α(PPARα)、核因子I/B(NFIB)及白细胞介素-8(IL-8)基因转入HepG2.2.15及HBV全基因组1.3倍体HepG2(HBV1.3P-HepG2)细胞模型中,以空白对照(NC)组为对照,转染48 h后采用qPCR检测mRNA表达及HBV DNA复制水平,化学发光法检测细胞上清液中HBsAg和HBeAg的表达,免疫荧光法检测细胞内HBsAg的表达.结果 在HepG2.2.15和HBV1.3P-HepG2细胞模型中,PPARα能够促进HBV DNA复制和HBsAg表达,其HBV DNA的表达分别是NC组的1.46倍和1.27倍(P<0.05).NFIB可以抑制HBV DNA复制和HBsAg表达,其HBV DNA的表达分别是NC组的0.76和0.55倍(P<0.05).IL-8能够促进HBV DNA复制和HBsAg表达,其HBV DNA的表达分别是NC组的1.54倍和1.62倍(P<0.05).结论 肝细胞内PPARα和IL-8能够促进HBV复制与表达,NFIB可以抑制HBV复制与表达,可为后续研究提供实验室依据.     

乙肝病毒X蛋白增强小鼠体内HBV的转录及复制

目的 观察小鼠体内乙型肝炎病毒(HBV)X蛋白(HBx)对HBV基因转录和复制水平的影响.方法 应用高压射体内转染法将野生型HBV重组质粒、HBx表达缺失的HBV重组质粒及HBx表达质粒DNA通过尾静脉快速注射入小鼠体内,应用Southern和Northem印迹分别检测小鼠肝组织中HBV DNA复制中间体及HBV mRNA水平.结果 野生型HBx表达缺失的HBV重组质粒注射组小鼠肝组织中均检测到HBV的复制与转录;HBx表达缺失的HBV重组质粒注射组HBV基因转录和复制水平较野生型HBV重组质粒注射组、HBx表达缺失HBV重组质粒与HBx表达质粒共注注射组弱,生理盐水注射组未检测到HBV的转录与复制.结论 HBx的表达缺失可导致体内HBV基因转录和复制水平的下降,而外源HBx的表达可逆转这种因HBx表达缺失引起的HBV复制及转录的减弱,提示HBx增强小鼠体内HBV基因转录和复制.     

肝富集转录因子在HBV基因转录与复制中的作用研究

目的观察各种肝富集转录因子对乙型肝炎病毒（HBV）基因转录和病毒复制水平的影响,探讨其在HBV嗜肝性机理中的作用。方法用复制型HBV重组质粒pHBV4．1与肝富集转录因子HNF1、HNF3、HNF4、HNF6、C／EBP或RXRa／PPARa的表达质粒,分别共转染非肝源细胞株NIH3T3、HeLa、293T、SWl353、CV-1和COSl。用Northern吸印杂交检测HBV3．5kb、2．4／2．1kb、0．7kb mRNA的转录情况,Southern吸印杂交检测HBV DNA复制中间体水平。结果用pHBV4．1转染NIH3T3细胞后,在没有肝富集转录因子表达质粒共转染时未能检出3．5kb HBV RNA转录,也无HBV DNA复制;当用肝富集转录因子共转染时,HNF4和RXRα／PPARα的表达能够激活3．5kb HBV RNA转录和HBV DNA复制,而HNF1、HNF3、HNF6和C／EBP未能激活HBV复制。在HeLa、293T、SW1353、CV-1和COS1细胞中,HNF4、RXRα／PPARα对HBV的转录和复制也具激活作用。进一步用突变型HBV重组质粒研究发现,C启动子HNF4结合位点的突变明显减弱了HNF4和RXRα／PPARα对HBV复制的激活作用。结论肝富集转录因子HNF4和RXRα／PPARα可支持HBV在非肝源细胞中的转录与复制;肝特异性基因转录是HBV嗜肝性的决定因素之一。     

HBV通过下调PTEN的表达拮抗IFN-α抗病毒活性

目的 探讨乙型肝炎病毒(HBV)感染所致的第10号染色体缺失的磷酸酶及张力蛋白同源的基因(PTEN)下调与α-干扰素(IFN-α)抗病毒活性的相关机制。方法 在适宜条件下培养HepG2细胞和HepG2.2.15细胞,以空白载体(pcDNA3.1)和HBV1.3质粒分别转染HepG2细胞,Western blot法检测PTEN蛋白质的表达;将pcDNA3.1和PTEN过表达(PTEN-OE)质粒分别瞬时转染HepG2.2.15细胞,化学发光法分析细胞培养上清液中HBV相关抗原的表达,实时荧光定量PCR(qRT-PCR)技术分析HBV前基因组RNA(HBV pgRNA)表达;使用合成RNA双链体poly(I∶C)刺激PTEN-OE的细胞,qRT-PCR技术分析IFN-αmRNA的表达,Western blot法分析JAK/STAT信号通路中干扰素调节因子9(IRF9)、黏病毒抗性蛋白1(MxA)的表达。结果 瞬时表达HBV的HepG2细胞和稳定表达HBV的HepG2.2.15细胞中,PTEN蛋白的表达降低;PTEN-OE的HepG2.2.15细胞中,HBV相关抗原及HBV pgRNA的表达...     

Value of D-Dimers in patients with acute aortic dissection

Objective: To evaluatel the value of D-dimers in patients with acute aortic dissection (AAD). Methods: This study consisted of 16 patients with AAD and 27 non-AAD patients. Serum D-dimets were measured by Sta-Liatest D-DI immunoturbidimetric assay. Results: D-dimer level was higher (P ＜ 0.001) in patients with AAD(7.91 ± 5.52 μg/ml) than that in non- AAD group(1.57±1.24 μg/ml). D-dimer was positive (＞0.4 μg/ml) in all patients with AAD and in 10 control group patients (37%). Among patients with acute AAD, D-dimers tended to be higher in Stanford A than in Stanford B (8.67 ± 4.31 μg/ml vs. 3.24±1.27 μg/ml, P ＜0.01). D-dimer values tended to be higher in more extended disease(3.84 ± 1.65 μg/ml, 8.57 ± 3.58 μg/ml and 11.87 ± 5.69 μg/ml in thoracic aorta, thoracic and abdominal aorta, thoracic and abdominal aorta and iliacal arteries, respectively, P ＜ 0.05 for both 8.57 ± 3.58 and 11.87 ± 5.69 vs. 3.84 ± 1.65 ). Including the control group into the analysis, we found a sensitivity of 100%, a negative predictive value of 100%, and a specificity of 66% and a positive predictive value of 64% for D-dimer in diagnosis of AAD in our patients with suspected AAD. Conclusion: D-dimer was elevated in patients with AAD. A negative D-dimer test result could be useful in excluding AAD.     

Research of combined liver-kidney transplantation model in rats

Objective： To set up a simple and reliable rat model of combined liver-kidney transplantation. Methods： SD rats served as both donors and recipients. 4℃ sodium lactate Ringer＇s was infused from portal veins to donated livers,and from abdominal aorta to donated kidneys, respectively. Anastomosis of the portal vein and the inferior vena cava （IVC） inferior to the right kidney between the graft and the recipient was performed by a double cuff method, then the superior hepatic vena cava with suture. A patch of donated renal artery was anastomosed to the recipient abdominal aorta. The urethra and bile duct were reconstructed with a simple inside bracket. Results： Among 65 cases of combined liver-kidney transplantation, the success rate in the late 40 cases was 77.5%. The function of the grafted liver and kidney remained normal. Conclusion： This rat model of combined liver-kidney transplantation can be established in common laboratory conditions with high success rate and meet the needs of renal transplantation experiment.     

BLOOD PRESSURE CHANGE WITH AGE IN SALT-SENSITIVE TEENAGERS

Objective To observe blood pressure change with age in salt-sensitive teenagers whose salt sensitivity were determined by repeated testing.Methods Salt sensitivity was determined through intravenous infusion of normal saline combined with volume-depletion by oral diuretic furosemide in 55 teenagers. After five years, salt sensitivity was re-examined and subject blood pressure was followed up. Blood pressure changes in salt-sensitive teenagers were compared to that of non-salt sensitive teenagers over five years.Results After 5 years, the repetition rate of salt sensitivity determined by intravenous saline loading is 92.7%. In teenagers with salt sensitivity on the baseline, both the systolic blood pressure increments and increment rates were much higher than non-salt sensitive teenagers (12.7±12.1 mmHg vs. 2.8±5.2 mmHg, P＜ 0.01; 12.2%± 12.0% vs. 2.5% ±4.4%, P＜ 0.001,respectively). There was a similar trend for diastolic blood pressure (8.4 ± 6.4 mmHg vs. 3.7 ± 6.4 mmHg, P = 0.052; 13.2% ±10.6 % vs. 6.8%± 10.1%, P = 0.053, respectively).Conclusions Salt sensitivity determined by intravenous saline loading showed good reproducibility. Blood pressure increments with age were much higher in salt-sensitive teenagers than non-salt sensitive teenagers, especially in terms of systolic blood pressure.     

安心颗粒对急诊经皮冠状动脉介入术后生活质量影响的研究

目的：评价使用安心颗粒对急诊经皮冠状动脉介入术（PPCI）术后生活质量的影响.方法：将160例接受PPCI的急性ST段抬高型心肌梗死患者随机分为安心颗粒组（术前顿服安心颗粒8.8g,术后安心颗粒4.4 g/次,每日2次）和对照组（仅接受基础药物治疗）.所有患者均服用阿司匹林、氯吡格雷和阿托伐他汀.分别在入院时、出院前1d、出院后180 d时,应用心肌梗死多维度量表（MIDAS）、中文版SF-36评价量表对患者生活质量评分.并观察术后30 d以内的出血并发症、血小板减少症发生情况.结果：入院时和出院前1d,两组患者的心肌梗死MIDAS、SF-36量表评分比较无差异（P＞0.05）;出院后180 d时,与对照组比较,安心颗粒组MIDAS、SF-36评分明显减低（P＜0.05）;组内与入院时比较,两组出院前1d、出院后180 d时,MIDAS、SF-36评分均降低（P＜0.05）.两组患者在随访期间均无大量出血、少量出血、重度和极重度血小板减少症发生,安心颗粒组有4例、对照组有7例发生不明显出血（P＞0.05）.两组发生轻度血小板减少症的患者数比较无差异（P＞0.05）.结论：PPCI使用安心颗粒,能改善急性ST段抬高型心肌梗死患者的生活质量,且不增加出血风险.     

The comparative studies of the influences of Urapidil and Nicardipine on sino-atrial node function, atrio-ventricular node function and hemodynamics

Objective:To investigate the influences of urapidil and nicardipine on rabbit sinus function,atrio-ventricular node function and hemodynamics.Methods:Thirty-two Angora's rabbits were selected and randomly divided into four groups.U1 group:urapidil 0.25 mg/kg;U2 group:urapidil 0.5 mg/kg;N1 group:nicardipine 10 μg/kg;N2 group:nicardipine 20 μg/kg.All these medicine were administrated within 30 seconds.Measurements were taken before and after the administration of urapidil or nicardipine for the following data:mean blood pressure(MAP),heart rate(HR),sino-atrial conduction time(SACT),maximal sinoatrial recovery time(SNRTmax)corrected sinus node recovery time(CSNRT),index of sinus node recovery time(SNRTI),Wenckebach A-V conduction frequency (WB),and P-R interval.Results:Significant MAP and HR changes were identified in all of the four groups before and after administration of both urapidil and nicardipine.No significant changes could be found in the rest of the parameters.Intergroup analysis showed that SACT and CSNRT of N1 and N2 groups were shorter than those of the U2 group(P＜0.01);the MAP decreased(P＜0.01)and the HR increased drastically(P＜0.01).Conclusions:Neither urapidil(0.25 mg/kg,0.5 mg/kg)nor nicardipine(10μg/kg,20μg/kg)has any significant influence on rabbit sinus function or rabbit atrio-ventricular node function.Nicardipine could be a better choice than urapidil for parafunctional sinus node patients.     

Expression of OPGmRNA and ODFmRNA in the patients of hip fracture due to osteoporosis

Objective：To investigate the gene expression of osteoprotegerin（OPG） and osteoclast differentiation factor（ODF） in the bone tissue of patients with hip fracture due to osteoporosis. Methods：OPGmRNA and ODFmRNA in the bone tissue in 50 cases of osteoporosis sufferers（over 50 years old） with hip fracture（Observer Group） and 30 cases of hip facture sufferers with no osteoporosis（Control group） were analyzed with the Semi-Quantitative RT-PCR method. Results：The mRNA expressed of ODF, OPG were both high in the patients with hip fracture. In the control group, the expression of OPG mRNA was observed, while the expression of ODF mRNA was very slight. Conclusion：Aged patients contained all signals including OPG, ODF that are essential for inducing osteoclastogenesis and promoting bone resorption.     

The clinicopathological and immuohistochemical analysis of solid-pseudopapillary tumor of the pancreas： report of 9 cases

Objective：To investigate the clinical features, pathological characteristics and immunophenotype of solid-pseudopapillary tumor of the pancreas（SPTP）. Methods：Nine surgically treated cases of SPTP were retrospectively reviewed. Hematoxylin and Eosin（HE） staining and immunohistochemical staining were used to analyze all cases, and the general clinical data was collected. Results：Six patients were asymptomatic except for a palpable mass. Two patients complained of vague-epigastric pain. One patient appeared jaundice. The tumor was encapsulated and solid tissues alternately with cystic tissues. Histologically, the histological structure of solid portion was pseudopapillary with a fibrovascular core. Tumor cells were uniform and medium-sized which were arranged in sheets ets or nests or pseudopapillary patterns. Immunohistochemical studies demonstrated that SPTP proved positive in vimentin（9/9 cases）, AAT（9/9 cases）, NSE（9/9 cases）, ACT（7/9 cases）, CK20（2/9 cases）, CgA（1/9 cases）, S-100（3/gcases）, PR（4/gcases）, Syn（3/9 cases） and CD56（5/9cases）, negative in CEA and ER. Conclusion：SPTP is a tumor predominantly occurring in young women frequently without special symptoms. This tumor has various characteristical histological patterns with different immunophenotype.     

Dynamic Changes in Serum Estradiol and Progesterone levels in Patients of Premenstrual Syndrome with Adverse Flow of Liver-qi

Objective:To probe into the influence of changes of ovarian hormones on the pathogenesis of the specific sub-type premenstrual syndrome(PMS)and reveal partial microcosmic mechanisms of adverse flow of liver-qi.Methods:Estradiol(E2)and progesterone(P)levels in serum were determined at different phases of menstrual cycle by radioimmunoassay.Results:In the group of PMS with adverse flow of liver-qi.the secretive peak value Of E2 and P at the follicular phase significantly decreased,and the secretive peak value at the luteal phase did not come into being.Conclusions:Low E2 and P secretive peak at the follicular phase and absence of secretive peak at the luteal phase is one of the microcosmic mechanisms of PMS with adverse flow of liver-qi.One of the pathophysiologic mechanisms of specific sub-type PMS is probably the continuous low level of E2and P.     

Real-time Three-dimensional Echocardiography in Assessment of Left Ventricular and Right Ventricular Volumes

Real-time three-dimensional echocardiography (RT3DE)is a new ultrasound technique that enables dynamic threedimensional visualization and quantification of the heart in real time. Investigation of feasibility and methodology of RT3DE in determining left ventricular (LV) and right ventricular (RV) volumes, RT3DE was performed in 35 normal adults using Philips SONOS 7500 system with a 2-4 MHz matrix array transducer. The 60°×60° "pyramid" volume database was obtained and analyzed on a TomTec echo workstation. Both LV and RV volumes were calculated with four 3DE methods (i.e. apical 2, 4, 8, and 16-plane) through manually tracing ventricular endocardial borders in end diastole and end systole. Stroke volumes were then calculated. LV volume was also measured by 2DE Simpson's rule using GE VIVID 7 ultrasound machine.     

SCREENING FOR A 21-CHROMOSOME ABNORMALITY IN PREIMPLANTED EMBRYOS OF ELDERLY WOMEN

Increasing maternal age is the only etiological factor unequivocally linked to Down's syndrome in humans. The occurrence rate of newborns with Down's syndrome is about 1/220 in women over 35 years old. However, the occurrence rate in embryos fertilized in vitro, of the elder woman is unclear. Using FISH we screened the number of chromosome 21 in preimplanted embryos of 5 elderly women (average age, 38.4 years) to study the feasibility and necessity of screening trisomy 21 in embryos in patients over 35 years old at the in vitro fertilization (IVF) center.