基于FISH和GISH技术的芝麻栽培种和野生种染色体组特征比较分析

为揭示胡麻属物种进化特征,探究胡麻属基因组结构演变及物种进化,促进野生资源的开发利用,以芝麻栽培种豫芝11号和2n=26类型野生种S.alatum 3651为试验材料,采用荧光原位杂交(FISH)和基因组荧光原位杂交(GISH)技术对芝麻栽培种和野生种染色体组特征进行分析。结果表明,芝麻栽培种和野生种均为2n=2x=26类型;rDNA-FISH杂交结果显示,栽培种的13对染色体中,3对染色体(第7、8、9对染色体)的短臂端部携带45S rDNA特异信号,显示为随体特异染色体;2对染色体(第5、11对染色体)的短臂携带5S rDNA特异信号,5S rDNA与45S rDNA信号位于不同染色体上。野生种的13对染色体中,有2对染色体(第4、7对染色体)携带45S rDNA杂交信号,1对染色体(第4对染色体)携带5S rDNA特异信号,5S rDNA与45S rDNA信号位于同一染色体不同位置,表明栽培种与野生种的染色体组特征差异较大。GISH杂交结果显示,分别以栽培种和野生种的基因组DNA作探针与自身染色体杂交,每条染色体上携带不同强弱的杂交信号,而与对方染色体杂交,均表现出极少的杂交信号。芝麻栽培种和野生种具有相同的染色体数目,但染色体rDNA数量、分布及其GISH信号均存在明显差异,说明栽培种和野生种亲缘关系较远。

黄褐棉45S rDNA的FISH定位及核型分析

黄褐棉是棉属5个四倍体棉种之一,利用荧光原位杂交技术将45S rDNA定位在黄褐棉2、4、9号染色体,2号染色体上的45S rDNA特别大,信号位于随体并覆盖了染色体的短臂,比二倍体和四倍体棉种的45S rDNA都要大得多;另外的2对信号很小,形状与陆地棉中的弱信号类似。黄褐棉的核型公式为:2n=4x=52=50m(2SAT)+2sm,属于2B类型,第2对染色体为亚中着丝粒染色体,其余都为中部着丝粒染色体。黄褐棉的核型、随体数、45S rDNA与其他四倍体棉种区别很大,黄褐棉是一个非常特殊的四倍体棉种。

植物45S rDNA的染色体位置的CPD染色和FISH分析

采用PI和DAPI组合(CPD)染色结合45SrDNA探针的荧光原位杂交(FISH)对分属6个科的16种植物的45S rDNA的染色体位置进行了分析。在所有供试植物中,共检测到53个45S rDNA位点。大多数45S rDNA位点分布在染色体的短臂;位于染色体臂内和染色体末端的位点的比例大体相当;多数位于染色体臂内的45S rDNA位点有次缢痕形成,但rDNA重复单位簇所处的位置存在差异。根据45S rDNA所处的染色体臂的不同、距着丝粒远近的差异、形成次缢痕与否以及rDNA重复单位簇相对于次缢痕的位置等特征,将植物的45S rDNA位点划分为12种染色体分布类型。基于我们的结果和其他的报道对45S rDNA位点、核仁组织区(NOR)、次缢痕和随体相互之间的关系进行了分析。

16轮Blow-CAST-Fish的弱密钥攻击

Blow-CAST-Fish的轮函数F由作为算法子密钥的四个S盒构成。如果S盒产生碰撞,则可构造F函数的输入差分为非零、输出差分为零的差分特征,进而对算法进行差分分析以获取其他子密钥。研究单个S盒的碰撞,构造F函数的差分特征,并测试使特征成立的弱密钥比例,给出了特征概率为2-49,弱密钥比例为2-52.4的14轮差分特征,并以此为基础,攻击16轮Blow-CAST-Fish。

低轮Blow-CAST-Fish算法的差分攻击

对作为Blow-CAST-Fish算法子密钥的4个S盒的碰撞性进行分析,构造输入差分为非零、输出差分为零的轮函数F的差分特征,通过对算法进行差分分析,获取相关子密钥,并测试使特征成立的弱密钥概率。在此基础上,成功利用特征概率为2-61、弱密钥概率为2-12的6轮差分特征攻击8轮Blow-CAST-Fish算法。

白缘(鱼央)5S rDNA的扩增分析及其在染色体上的FISH定位

本文分别对雌雄白缘(鱼央)(鱼央)的5SrDNA进行了PCR扩增和序列分析,并采用双色荧光原位杂交(FISH)技术,以白缘(鱼央)的5SrDNA序列和小麦的45SrDNA为探针,对其在白缘(鱼央)雌雄中期染色体上进行了FISH定位研究。结果表明,白缘(鱼央)5SrDNA序列无雌雄差异;5SrDNA的保守区序列为117bp;5SrDNA和45SrDNA分别被定位于白缘(鱼央)的性染色体和第5号染色体上。同时从GenBank中获得了22种鱼的5SrDNA,运用DNAman软件构建了23种鱼的系统发育树,对白缘(鱼央)的进化地位进行了初步研究。本研究为阐明白缘(鱼央)在鱼类系统进化中的位置、重复序列在脊椎动物性染色体上的分布状况以及与性别决定与分化的关系,提供了资料积累和分析依据。

海岛棉基于双色FISH的核型分析

以45S r DNA作探针对海岛棉体细胞中期染色体进行荧光原位杂交(Fluorescence in situ Hybridization,FISH),检测出6个强的荧光信号(4大2小),结合DAPI图像观察发现信号分布在各自染色体随体位置,称为NOR(Nucleolar Organizer Region)信号。以二倍体A基因组棉种亚洲棉基因组DNA(g DNA)为探针对海岛棉进行FISH,结果发现52条染色体中有无杂交信号的各占一半,荧光信号分布在较长的A亚组染色体上,直接区分开海岛棉A、D亚组染色体,直观证实了海岛棉的异源双二倍体起源。以45S r DNA和A组棉种亚洲棉g DNA作探针对海岛棉进行双色FISH时,结果发现海岛棉染色体上既检测到了6个强的NOR信号又检测出了g DNA杂交信号,且清楚观察到2个NOR信号分布在A亚组上,另4个在D亚组上。基于该双色FISH图像的核型分析表明,海岛棉的核型公式为:2n=4x=52=40m(2 SAT)+12sm(4 SAT),属于2A类型,且有3对随体,其中1对定位于A9号染色体,属于中部着丝点(m)类型;另2对分别定位于D8和D12号染色体上,属于近中部着丝点(sm)类型;且发现海岛棉A亚组染色体的相对长度的并非全部大于D亚组,两亚组染色体间在长度上存在交叉,说明基于FISH的核型分析比传统方法更为准确。

野生黄瓜5S rDNA-FISH技术体系建立

目的通过改进黄瓜中期染色体制片和rDNA-FISH技术的关键因素,对野生种(Cucumis sativus var.hardwickii)PI 183967进行核型分析为继续研究黄瓜各条染色体的准确鉴定和进一步开展细胞学研究奠定基础。方法使用8-羟基喹啉0.002mol·L-1预处理2h,可以得到形态清晰的中期染色体,分裂相最高比例为17.2%。染色体制片以改良酶解空气干燥制片法较好,流程简单,可以获得大量清晰、分散的有丝分裂中期染色体,且背景干净。荧光原位杂交过程中染色体制片采用胃蛋白酶37℃处理30min,70℃变性3min时,探针使用缺口平移法地高辛进行标记,75~100℃变性10min,能获得杂交信号清晰的染色体图像。结果在黄瓜野生种PI 183967中只有一对5SrDNA信号,位于5号染色体短臂上,相对远离着丝点位置。结论野生黄瓜PI 183967的5号染色体可以用5SrDNA鉴定。

粗穗披碱草1H^(t)S染色体特异荧光原位杂交标记开发

小麦-粗穗披碱草1H^(t)S.1BL罗伯逊易位系高抗小麦条锈病和叶锈病,是小麦遗传改良的优异基因源。我们前期利用中国春ph1b基因突变体对该易位系进行诱导,获得了一批诱导后代材料,为了从中准确鉴定小麦-粗穗披碱草1H^(t)S小片段易位系,需要建立能够覆盖粗穗披碱草1H^(t)S染色体的荧光原位杂交(FISH)标记。本研究利用21个小麦及其近缘种探针、61个基于中国春基因组序列新开发的小麦1BS染色体探针以及40个根据简单三碱基重复序列新开发的探针对小麦-粗穗披碱草1H^(t)S.1BL易位系进行非变性FISH分析。结果显示,B74、B76和B77等36个探针(33个为新开发的)在1H^(t)S染色体上具有杂交信号,并且杂交信号可分为仅在染色体末端、仅在着丝粒处、同时在着丝粒处和近着丝粒处、覆盖1H^(t)S约3/4染色体臂、覆盖绝大部分1H^(t)S共五种类型。因此,部分探针单独使用或多个探针联合使用,其信号能覆盖1H^(t)S染色体,能够满足小麦-粗穗披碱草1H^(t)S小片段易位系鉴定,这可为小麦背景中粗穗披碱草染色质追踪提供新的检测手段。

Prevalence of bile reflux in gastroesophageal reflux disease patients not responsive to proton pump inhibitors

AIM:To determine the prevalence and characteristics of bile reflux in gastroesophageal reflux disease(GERD) patients with persistent symptoms who are non-responsive to medical therapy.METHODS:Sixty-five patients(40 male,25 female;mean age,50 ± 7.8 years) who continued to report symptoms after 8 wk of high-dose proton pump inhibitor(PPI) therapy,as well as 18 patients with Barrett's esophagus,were studied.All patients filled out symptom questionnaires and underwent endoscopy,manometry and combined pH-metry and bilimetry.RESULTS:There were 4 groups of patients:22(26.5%) without esophagitis,24(28.9%) grade A-B esophagitis,19(22.8%) grade C-D and 18(21.6%) Barrett's esophagus.Heartburn was present in 71 patients(85.5%) and regurgitation in 55(66.2%),with 44(53%) reporting simultaneous heartburn and regurgitation.The prevalence of pathologic acid reflux in the groups without esophagitis and with grades A-B and C-D esophagitis was 45.4%,66.6% and 73.6%,respectively.The prevalence of pathologic bilirubin exposure in these 3 groups was 53.3%,75% and 78.9%,respectively.The overall prevalence of bile reflux in non-responsive patients was 68.7%.Pathologic acid and bile reflux was observed in 22.7% and 58.1% of non-esophagitic patients and esophagitic patients,respectively.CONCLUSION:The high percentage of patients poorly responsive to PPI therapy may result from poor control of duodenogastroesophageal reflux.Many patients without esophagitis have simultaneous acid and bile reflux,which increases with increasing esophagitis grade.

A Qualitative Appraisal of China’s Efforts in Fishing Capacity Management

This paper provides a critical review of some major efforts made by China to manage the fishing capacity of its marine capture fisheries. It begins by reviewing the key concepts and issues closely related to fishing capacity management. Following on this, a brief introduction to China’s marine capture fisheries is offered, especially the trends of its marine landings and marine fishing fleets and the status of fish stocks in waters under its jurisdiction. Next comes the main concern of the paper, i.e., some major actions recently initiated by China to deal with the overcapacity problem confronting it. The paper ends with conclusions and suggestions.

Modalities of testing Helicobacter pylori in patients with nonmalignant bile duct diseases

AIM:This paper describes the procedure of detection of Helicobacter pylori(H.pylori)in bile specimens in patients suffering from benign diseases of biliary ducts(lithiasis with/without nonspecific cholangitis). METHODS:The group of 72 patients entering the study consisted of 32 male and 40 female(45 % and 55 %, respectively).Bile was obtained during ERCP in 68 patients, and during cholecystectomy in 4 patients.A fast urease test (FUT)to determine the existence of H.pylori in gastric mucosa was carried out for all the patients during the endoscopic examination.The existence of genetic material of H.pylori was determined by detection of ure A gene by the method of nested PCR.The results of this reaction were shown by electrophoresis on 10 g·L^(-1)agarose gel in a band of 256 bp. RESULTS:The majority of the patients included in our study had biliary lithiasis without signs of cholangitis(48 patients, 67 %),whereas other patients were complicated by cholangitis(17 patients,24 %).Seven patients(9 %)had normal ERCP,forming thus the control group.In the group of patients with lithiasis 26 patients(24.2 %)had positive PCR of H.pylori in bile and among the patients with associated cholangitis positive PCR was detected in 9 patients(52.9 %).Among the seven patients with normal ERCP only one(14 %)had positive PCR of H.pylori.A high percentage of H.pylori infection of gastric mucosa was observed(57 patients,79 %).It was also observed that its slightly higher positivity was in the patients with distinct bile pathology:81% FLIT positive patients in the group with choledocholithiasis alone and 76 % in the group with choledocholithiasis associated with cholangitis.Seventy-one percent of the patients with regular findings had positive FUT.CONCLUSION: The prevalence of H. pylori infection both in bile and in gastric mucosa in patients with benign diseases of biliary ducts does not show a statistically significant difference in relation to the prevalence of the same with the patients with normal ERCP. The existence of H. pylori infection possibly does not play a role in pathogenesis of benign biliary diseases.

Serum bile acid profiling reflects enterohepatic detoxification state and intestinal barrier function in inflammatory bowel disease

AIM： To determine free and conjugated serum bile acid （BA） levels in inflammatory bowel disease （IBD） subgroups with defined clinical manifestations. METHODS： Comprehensive serum BA profiling was performed in 358 IBD patients and 310 healthy con- trols by liquid chromatography coupled to electrospray ionization tandem mass spectrometry. RESULTS： Serum levels of hyodeoxycholic acid, the CYP3A4-mediated detoxification product of the second- ary BA lithocholic acid （LCA）, was increased significantly in Crohn＇s disease （CD） and ulcerative colitis （UC）, while most other serum BA species were decreased signifi- cantly. Total BA, total BA conjugate, and total BA glyco- conjugate levels were decreased only in CD, whereas total unconjugated BA levels were decreased only in UC. In UC patients with hepatobiliary manifestations, the conjugated primary BAs glycocholic acid, taurocholic acid, and glycochenodeoxycholic acid were as signifi- cantly increased as the secondary BAs LCA, ursodeoxy- cholic acid, and tauroursodeoxycholic acid compared to UC patients without hepatobiliary manifestations. Finally, we found that in ileocecal resected CD patients, the unconjugated primary BAs, cholic acid and chenode- oxycholic acid, were increased significantly compared to controls and patients without surgical interventions. CONCLUSION： Serum BA profiling in IBD patients that indicates impaired intestinal barrier function and increased detoxification is suitable for advanced diag- nostic characterization and differentiation of IBD sub- groups with defined clinical manifestations.

Vanishing bile duct syndrome in Hodgkin's lymphoma: A case report and literature review

Vanishing bile duct syndrome (VBDS) has been described in different pathologic conditions including infection, ischemia, adverse drug reactions, autoimmune diseases, allograft rejection, and humoral factors associated with malignancy. It is an acquired condition characterized by progressive destruction and loss of the intra-hepatic bile ducts leading to cholestasis. Prognosis is variable and partially dependent upon the etiology of bile duct injury. Irreversible bile duct loss leads to significant ductopenia, biliary cirrhosis, liver failure, and death. If biliary epithelial regeneration occurs, clinical recovery may occur over a period of months to years. VBDS has been described in a number of cases of patients with Hodgkin’s lymphoma (HL) where it is thought to be a paraneoplastic phenomenon. This case describes a 25-year-old man found on liver biopsy to have VBDS. Given poor response to medical treatment, the patient underwent transplant evaluation at that time and was found to have classical stage IIB HL. Early recognition of this underlying cause or association of VBDS, including laboratory screening, and physical exam for lymphadenopathy are paramount to identifying potential underlying VBDS-associated malignancy. Here we review the literature of HL-associated VBDS and report a case of diagnosed HL with biopsy proven VBDS.

Gallbladder bile composition in patients with Crohn's disease

AIM： To further elucidate the pathogenesis and mechanisms of the high risk of gallstone formation in Crohn＇s disease. METHODS： Gallbladder bile was obtained from patients with Crohn＇s disease who were admitted for elective surgery （27 with ileal/ileocolonic disease and 7 with Crohn＇s colitis）. Fourteen gallstone patients served as controls. Duodenal bile was obtained from ten healthy subjects before and after the treatment with ursodeoxycholic acid. Bile was analyzed for biliary lipids, bile acids, bilirubin, crystals, and crystal detection time （CDT）. Cholesterol saturation index was calculated. RESULTS： The biliary concentration of bilirubin was about 50% higher in patients with Crohn＇s disease than in patients with cholesterol gallstones. Ten of the patients with Crohn＇s disease involving ileum and three of those with Crohn＇s colitis had cholesterol saturated bile. Four patients with ileal disease and one of those with colonic disease displayed cholesterol crystals in their bile. About 1/3 of the patients with Crohn＇s disease had a short CDT. Treatment of healthy subjects with ursodeoxycholic acid did not increase the concentration of bilirubin in duodenal bile. Several patients with Crohn＇s disease, with or without ileal resection/disease had gallbladder bile supersaturated with cholesterol and short CDT and contained cholesterol crystals. The biliary concentration of bilirubin was also increased in patients with Crohn＇s colitis probably not due to bile acid malabsorption. CONCLUSION： Several factors may be of importance for the high risk of developing gallstones of both cholesterol and pigment types in patients with Crohn＇s disease.The skillful technical assistance of Ms Lisbet Benthin and Ms Ingela Arvidsson is acknowledged.

A new iron free treatment with oral fish cartilage polysaccharide for iron deficiency chronic anemia in inflammatory bowel diseases:A pilot study

AIM： To investigate the effect of a new oral preparation, highly concentrated in fish cartilage, in a group of inflammatory bowel diseases （IBD） patients with chronic iron deficient anemia. METHODS： In an open label pilot study, we supplemented a group of 25 patients （11 with Crohn＇s disease and 14 with ulcerative colitis） in stable clinical conditions and chronic anemia with a food supplement which does not contain iron but contains a standardized fraction of fish cartilage glycosaminoglycans and a mixture of antioxidants （Captafer Medestea, Turin, Italy）. Patients received 500 mg, twice a day during meals, for at least 4 mo. Patients were suggested to maintain their alimentary habit. At time 0 and after 2 and 4 too, emocrome, sideremia and ferritin were examined. Paired data were analyzed with Student＇s t test. RESULTS： Three patients relapsed during the study （2 in the 3^rd too, 1 in the 4^th too）, two patients were lost to follow up and two patients dropped out （1 for orticaria, 1 for gastric burning）. Of the remaining 18 patients, levels of serum iron started to rapidly increase within the 2^nd mo of treatment, P 〈 0.05）, whereas serum ferritin and hemoglobin needed a longer period to significantly improve their serum levels （too 4） P 〈 0.05. The product was safe, easy to administer and well tolerated by patients. CONCLUSION： These data suggest a potential new treatment for IBD patients with iron deficiency chronic anemia and warrant further larger controlled studies.

High level of deoxycholic acid in human bile does not promote cholesterol gallstone formation

AIM:To study whether patients with excess deoxycholic acid (DCA) differ from those with normal percentage of DCA with respect to biliary lipid composition and cholesterol saturation of gallbladder bile. METHODS:Bile was collected during operation through puncturing into the gallbladder from 122 cholesterol gallstone patients and 46 gallstone-free subjects undergoing cholecystectomy.Clinical data,biliary lipids,bile add composition, presence of crystals and nucleation time were analyzed. RESULTS:A subgroup of gallstone patients displayed a higher proportion of DCA in bile than gallstone free subjects. By choosing a cut-off level of the 90th percentile,a group of 13 gallstone patients with high DCA levels (mean 50 percent of total bile acids) and a large group of 109 patients with normal DCA levels (mean 21 percent of total bile acids) were obtained.The mean age of the patients with high DCA levels was higher than that of the group with normal levels (mean age:62 years vs45 years) and so was the mean BMI (28.3 vs.24.7).Plasma levels of cholesterol and triglycerides were slightly higher in the DCA excess groups compared with those in the normal DCA group.There was no difference in biliary lipid composition,cholesterol saturation,nucleation time or occurrence of cholesterol crystals in bile between patients with high and normal levels of DCA. CONCLUSION:Gallstone patients with excess DCA were of older age and had higher BMI than patients with normal DCA.The two groups of patients did not differ with respect to biliary lipid composition,cholesterol saturation,nucleation time or occurrence of cholesterol crystals.It is concluded that DCA in bile does not seem to contribute to gallstone formation in cholesterol gallstone patients.

Mucin and phospholipids determine viscosity of gallbladder bile in patients with gallstones

AIM: An increased viscosity of gallbladder bile has been considered an important factor in the pathogenesis of gallstone disease. Besides lipids and proteins, mucin has been suggested to affect the viscosity of bile. To further clarify these issues we compared mucin, protein and the lipid componEnts of hepatic and gallbladder bile and its viscosity in patients with gallstones. METHODS: Viscosity of bile (mPa.s) was measured using rotation viscosimetry in regard to the non Newtonian property of bile at low shear rates. RESULTS: Biliary viscosity was markedly higher in gallbladder bile of patients with cholesterol (5.00 +/- 0.60 mPa.s, mean +/- SEM, r= 28) and mixed stones (3.50 +/- 0.68 mPa.s; r= 8) compared to hepatic bile (0.92 +/- 0.06 mPa.s, r= 6). A positive correlation between mucin and viscosity was found in gallbladder biles (r = 0.65; P 【 0.001) but not in hepatic biles. The addition of physiologic and supraphysiologic amounts of mucin to gallbladder bile resulted in a dose dependent non linear increase of its viscosity. A positive correlation was determined between phospholipid concentration and viscosity (r = 0.34, P 【 0.005) in gallbladder biles. However, no correlation was found between total protein or the other lipid concentrations and viscosity in both gallbladder and hepatic biles. CONCLUSION: The viscosity of gallbladder bile is markedly higher than that of hepatic bile in patients with gallstones. The concentration of mucin is the major determinant of biliary viscosity and may contribute by this mechanism to the role of mucin in the pathogenesis of gallstones.

Apoptosis of human cholangiocarcinoma cells induced by ESC-3 from Crocodylus siamensis bile

AIM： To investigate the effects of ESC-3 isolated from crocodile bile on the growth and apoptosis induction of human cholangiocarcinoma cells.

DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Accurate species identification is a key component of biodiversity research.DNA barcoding is an effective molecular method used for fish species identification.We aimed to study the DNA barcoding of fish in Zhoushan coastal waters,explore the differences and applicability of two gene fragments(12S rRNA and COI)of DNA barcoding in fish species identification,and established a comprehensive fish barcoding reference database.Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding.A total of 26412S rRNA sequences(belonging to eight orders,31 families,55 genera,and 66 species)and 188 COI sequences(belonging to seven orders,30 families,48 genera,and 58 species)were obtained.The lengths of the 12S rRNA sequences ranged from 165 to 178 bp,and the guanine-cytosine(GC)content was 45.37%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.10%and 26.66%,respectively.The length of the COI sequence ranged 574–655 bp,and the content of GC was 45.97%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.16%and 27.45%,respectively.The minimum interspecific genetic distances of 12S rRNA and COI(1.23%and 1.86%)were both greater than their maximum intraspecific genetic distances(2.42%and 8.66%).Three molecular analyses(NJ tree,ABGD,and GMYC)were performed to accurately identify and delineate species.Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method,and the delimitation results of ABGD and GMYC are consistent with the final species identification results.Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification,and 12S rRNA has good application prospects in the environmental DNA(eDNA)metabarcoding of marine fish.