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急性上呼吸道感染【32页】

急性上呼吸道感染【32页】

对症治疗
•鉴 于 本 病 患 者 常 常 同 时 存 在 上 述 多 种 症 状 , 可 用 由 上 述 数 种 药 物 组 成的 复方制剂。 •为 了 避 免 抗 过 敏 药 物 引 起 的 嗜 睡 作 用 对 白 天 工 作 和 学 习 的 影 响 , 有 一些复方抗感冒药物分为白片和夜片,仅在夜片中加入了抗过敏药。 •对 于 无 发 热 的 患 者 应 该 使 用 不 含 解 热 镇 痛 药 成 份 的 复 方 制
诊断依据
流行病学
周围血象
鼻咽部的症 状体征
鉴别诊断
• 急性上呼吸道感染应与变应性鼻炎相鉴别 • 百日咳、猩红热、麻疹、脊髓灰质炎、脑炎等急性传染病的早期常有
上呼吸道卡他症状等,应与本病鉴别 • 在上述传染病流行季节和流行地区对有急性上呼吸道感染症状的患者
应密切观察,进行必要的相关实验室检查
咽充血,软腭、悬雍垂、咽及扁桃体表面有灰白色疱疹及浅表溃疡 周围有红晕,以后形成疱疹
咽及结合膜明显充血
咽部明显充血,扁桃体肿大、充血,表面有黄色脓性分泌物,颌下 淋巴结肿大、压痛,肺部无异常体征
并发症
鼻窦炎 中耳炎 眼结合膜炎 颈淋巴结炎及咽后(或侧)壁脓肿 气管-支气管炎 最主要的并发症为肺炎,少数患者可并发风湿病、肾 小球肾炎和病毒性心肌炎等
世界卫生组织(WHO) 推荐: ——右美沙芬是一种可取代可待因的中枢镇咳药1
• 英国胸科学会(BTS)指南指出: ——阿片类镇咳药可待因和福尔可定疗效并不优于右美沙芬,且不 良反应更多,不推荐用于咳嗽治疗
1. 李建明,临床医药实践,2009,18(6):456-458 2. Morice AH, et al. Recommendations for the management of cough in adults[J].Thorax 2006;61(Suppl I):i1–i24.

自组装铁蛋白在纳米疫苗领域的应用进展

自组装铁蛋白在纳米疫苗领域的应用进展

生物技术进展2019年㊀第9卷㊀第3期㊀240~245CurrentBiotechnology㊀ISSN2095 ̄2341进展评述Reviews㊀收稿日期:2018 ̄12 ̄26ꎻ接受日期:2019 ̄02 ̄22㊀基金项目:国家重点研发计划项目(2017YFD0500706ꎻ2016YFD0500108)ꎻ国家自然科学基金项目(31670156)资助ꎮ㊀作者简介:魏珍珍ꎬ硕士研究生ꎬ研究方向为病毒微生物ꎮE ̄mail:646122815@qq.comꎮ∗通信作者:易咏竹ꎬ副研究员ꎬ研究方向为病毒微生物ꎮE ̄mail:Yiyongzhu@126.com自组装铁蛋白在纳米疫苗领域的应用进展魏珍珍1ꎬ㊀刘兴健2ꎬ㊀王㊀朋1ꎬ㊀张志芳2ꎬ㊀易咏竹3∗1.江苏科技大学生物技术学院ꎬ江苏镇江212003ꎻ2.中国农业科学院生物技术研究所ꎬ北京100081ꎻ3.中国农业科学院蚕业研究所ꎬ江苏镇江212018摘㊀要:自组装蛋白在真核细胞及原核细胞中是普遍存在的ꎬ其对生命体的正常运转具有重要意义ꎬ甚至关系到生命体的进化ꎮ常见的自组装蛋白包括病毒颗粒(virusparticles)㊁血清白蛋白(serumalbumin)㊁丝蛋白(silkprotein)及铁蛋白(ferritin)ꎮ其中ꎬ铁蛋白可形成粒径均一㊁生物相容性良好的纳米材料ꎬ还具有独特的理化性质ꎬ如pH敏感㊁高温耐受㊁大多数变性剂耐受ꎬ即可通过调节pH来控制铁蛋白的自组装特性ꎮ铁蛋白是存在于大多数生物体内的天然蛋白ꎬ在肿瘤的诊断成像及治疗㊁药物载体和纳米疫苗等领域具有广阔的应用前景ꎮ重点探讨了铁蛋白的仿生合成及其在纳米疫苗领域的应用进展ꎬ以期为新型动物纳米疫苗的研发提供参考ꎮ关键词:自组装蛋白ꎻ重组铁蛋白ꎻ纳米疫苗DOI:10.19586/j.2095 ̄2341.2018.0139ApplicationProgressofSelf ̄assembledFerritininNano ̄vaccineWEIZhenzhen1ꎬLIUXingjian2ꎬWANGPeng1ꎬZHANGZhifang2ꎬYIYongzhu3∗1.CollegeofBiotechnologyꎬJiangsuUniversityofScienceandTechnologyꎬJiangsuZhenjiang212003ꎬChinaꎻ2.BiotechnologyResearchInstituteꎬChineseAcademyofAgriculturalSciencesꎬBeijing100081ꎬChinaꎻ3.SericulturalResearchInstituteꎬChineseAcademyofAgriculturalSciencesꎬJiangsuZhenjiang212018ꎬChinaAbstract:Self ̄assembledproteinsareubiquitousineukaryoticandprokaryoticcellsꎬandtheyareimportantforlivingorganismstomaintainthenormaloperationꎬandevenrelatedtotheevolutionoflivingorganisms.Commonself ̄assembledproteinsincludevirusparticlesꎬserumalbuminꎬsilkproteinandferritin.Amongthemꎬferritincanformnanomaterialswithuniformparticlesizeandgoodbiocompatibility.ItalsohasuniquephysicalandchemicalpropertiesꎬsuchaspHsensitivityꎬhightemperaturetoleranceꎬandresistancetomostdenaturantsꎬsoastocontroltheself ̄assemblycharacteristicsofferritinbypHregulation.Ferritinisanaturalproteinfoundinmostlivingorganismsꎬandithasabroadapplicationprospectintumordiagnosticimagingandtherapyꎬdrugcarrierandnano ̄vaccine.Thebionicsynthesisofferritinanditsapplicationinnano ̄vaccineweremainlydiscussedinordertoprovidereferencesfortheresearchanddevelopmentofnovelanimalnano ̄vaccine.Keywords:self ̄assembledproteinꎻrecombinantferritinꎻnano ̄vaccine㊀㊀自组装蛋白在真核细胞及原核细胞中是普遍存在的ꎬ蛋白质亚基间会自发组装构成高度有序的结构ꎬ这是维持机体正常运转的保证ꎬ也是机体进化的推动力[1]ꎮ由自组装蛋白形成的纳米材料ꎬ不仅具有生物相容性良好以及粒径均一㊁稳定的特性ꎬ还在细胞成像㊁病灶检测和药物缓释等方面具有广阔的应用前景ꎮ到目前为止ꎬ研究最多的自组装蛋白纳米颗粒包括病毒颗粒(virusparticles)㊁血清白蛋白(se ̄rumalbumin)㊁丝蛋白(silkprotein)及铁蛋白(fer ̄ritin)ꎮ其中ꎬ病毒颗粒侵染宿主细胞并在宿主细胞内的自组装行为ꎬ是自然界中典型的生物纳米. All Rights Reserved.材料的形成方式ꎬ主要用于特异性检测以及病毒侵染宿主细胞的机制和路径的研究[2ꎬ3]ꎬ经基因修饰后还可用于研制借助病毒释放基因的药物等方面的研究[4]ꎻ血清白蛋白是脊椎动物血浆中含量最高的蛋白质ꎬ其分子的弹性良好ꎬ结构改变后也极易恢复ꎬ不同来源的血清白蛋白的空间构造均十分保守[5]ꎬ在药物传递系统领域拥有潜在的应用前景[6]ꎻ丝蛋白是一类线状蛋白的生物高分子材料ꎬ可抗紫外线ꎬ也可抗蛋白水解酶ꎬ其柔韧性好㊁抗疲劳度高ꎬ有着与钢材类似的张力强度ꎬ还具有良好的热㊁酸㊁碱稳定性和生物相容性ꎬ在生物材料[7]和药物载体[8]领域应用广泛ꎮ而铁蛋白是存在于大多数生物体内的天然蛋白ꎬ具有独特的理化性质:①铁蛋白对pH不耐受ꎬ较为敏感ꎬ在酸性条件(pH2.0)下铁蛋白外壳会解体成亚基ꎬ而当pH回升到生理条件(pH7.4)时ꎬ各亚基又重组形成完整的铁蛋白[9ꎬ10]ꎻ②铁蛋白的天然高级结构不受多种变性剂的影响ꎬ一般蛋白质在1~4mol/L的低浓度盐酸胍或者脲溶液中就会发生变性ꎬ而铁蛋白在6mol/L的盐酸胍或8mol/L的脲溶液中才会发生蛋白质解聚ꎬ即铁蛋白对变性剂的耐受性高[11]ꎻ③铁蛋白对高温具有较高的耐受性ꎬ大多数蛋白质在温度高于生理条件后极易变性ꎬ但铁蛋白在高温(70ħ~80ħ)时可维持10min以上不会发生变性ꎬ且其高级结构维持完好[12]ꎮ基于铁蛋白独特的理化性质ꎬ本文主要对铁蛋白的仿生合成及其在肿瘤的诊断成像及治疗㊁药物载体和纳米疫苗领域的应用进展进行了综述ꎬ阐述了天然铁蛋白的结构及修饰㊁人工制备重组铁蛋白的研究进程ꎬ分析了重组铁蛋白在各领域中的应用ꎬ以期为研发对机体无害㊁适应不同生物体的新型疫苗提供参考ꎮ1㊀铁蛋白的结构及其修饰在生命体中ꎬ天然的铁蛋白主要由水合氧化铁核和蛋白质外壳2个部分组成ꎬ其结构是高度对称的ꎬ封闭的笼形结构由24个亚基组成ꎮ哺乳动物铁蛋白外壳的分子量约为480kDaꎬ外直径约为12nmꎬ可容纳约4500个铁原子的内腔直径约为8nmꎮ哺乳动物机体中的铁蛋白外壳是由H亚基和L亚基组成的ꎬ但亚铁氧化酶活性中心(ferroxidasecenter)只存在于H亚基上[13]ꎮ许多在机体中发挥重要作用的蛋白质和辅酶的组成成分都含有铁元素ꎻ而广泛存在于机体中的铁蛋白在铁离子代谢中起着至关重要的作用ꎬ可维持铁的稳态ꎬ抵抗氧化应激ꎻ此外ꎬ铁蛋白还可以捕捉游离二价铁将其氧化并形成稳定的铁核ꎬ从而消除过量金属离子的其他毒性作用[14]ꎮ自然界中的铁蛋白都含有铁核ꎬ其组分是水铁矿(5Fe2O3 9H2O)ꎬ也可称之为全铁蛋白(ho ̄loferritin)ꎬ即铁蛋白(ferritin)ꎬ而不含铁内核的铁蛋白ꎬ称为去铁铁蛋白(apoferritin)ꎮ铁蛋白的球形中空结构有3个界面:内表面㊁外表面及亚基间接触面(图1)[15]ꎮ在对铁蛋白进行修饰改造时ꎬ其内表面可将材料包裹于铁蛋白内核ꎬ作为纳米复合材料合成的纳米反应器ꎻ外表面可连接配体ꎬ赋予铁蛋白特殊功能ꎻ亚基间接触面可通过调节溶液pH完成解聚与重组ꎬ开发铁蛋白的新功能ꎮ图1㊀可用于修饰的铁蛋白3个界面[16]Fig.1㊀Threeinterfacesofferritinthatcanbeusedformodification[16].2㊀重组铁蛋白的人工制备随着交叉学科的快速发展㊁生物学与纳米技术的联用ꎬ仿生合成铁蛋白技术也逐渐得到改善ꎮ1991年ꎬ英国巴斯大学首次合成了磁性铁蛋白ꎬ他们以天然马脾铁蛋白为模板ꎬ人工除去了水铁矿(5Fe2O3 9H2O)的天然内核ꎬ并将磁性铁核在马脾铁蛋白的空腔内合成[17]ꎬ这项工作开辟了一个新领域 仿生合成纳米颗粒ꎮ但这同样也存在着问题ꎬ在利用天然马脾铁蛋白外壳作为模板142魏珍珍ꎬ等:自组装铁蛋白在纳米疫苗领域的应用进展. All Rights Reserved.合成纳米颗粒前ꎬ首先要除去蛋白质内的天然水铁矿内核ꎬ而去核的过程需要利用可破坏蛋白质外壳的强还原剂处理铁蛋白ꎬ以致亚铁离子不能全部进入蛋白质外壳的内核中ꎬ而是吸附到蛋白质外壳的表面被氧化ꎬ从而导致合成的铁蛋白聚集[18]ꎮ天然铁蛋白的自组装特性ꎬ使得在大肠杆菌中批量表达重组铁蛋白成为可能ꎮ利用大肠杆菌表达的铁蛋白亚基可以自组装形成24聚体的铁蛋白外壳ꎬ与天然铁蛋白相比ꎬ结构一致㊁分散性好㊁粒径均一ꎬ所以在不破坏铁蛋白外壳完整性的前提下ꎬ可将大肠杆菌作为优良的模式生物来仿生合成铁蛋白纳米颗粒ꎮ2006年ꎬ美国蒙大拿州立大学首次利用大肠杆菌成功获得几乎纯的铁蛋白外壳ꎬ并以这些铁蛋白外壳为模板ꎬ仿生合成了磁性铁蛋白[19]ꎮ这种新技术不仅极大地简化了分离纯化天然铁蛋白外壳的过程ꎬ而且避免了强还原剂对蛋白质外壳的破坏ꎬ保持了蛋白质外壳良好的完整性ꎬ使得整个合成过程高效且快速ꎮ值得注意的是ꎬ虽然利用大肠杆菌可仿生合成与天然铁蛋白结构相似的铁蛋白ꎬ但是二者内核晶型不同ꎬ仿生合成铁蛋白的内核为Fe3O4ꎬ具有超顺磁性ꎬ这也是仿生合成的铁蛋白被称为磁性铁蛋白的原因ꎮ目前ꎬ已能够成功构建基于大肠杆菌的铁蛋白原核表达体系ꎬ利用IPTG诱导表达后ꎬ经过纯化㊁复性等步骤ꎬ即可获得与天然结构相同的铁蛋白纳米颗粒ꎬ其在生物医药领域具有广泛的应用前景[20]ꎮ仿生合成的铁蛋白纳米颗粒与其他纳米颗粒相比ꎬ具有以下优点:①粒径小ꎬ约为12nmꎬ有利于其在病灶组织(如肿瘤)的渗透和积累[21]ꎻ②粒径均一ꎬ在大肠杆菌中能仿生合成理想的粒径均匀且分散性良好的铁蛋白纳米颗粒ꎻ③生物相容性良好ꎬ利用大肠杆菌表达的人重组铁蛋白纳米颗粒制成的生物技术药物ꎬ应用于机体后ꎬ不易引起免疫排斥反应ꎬ对机体的毒性有较大程度的降低ꎻ④易于靶向性修饰ꎬ铁蛋白纳米颗粒在合成时可直接通过基因修饰ꎬ在外壳及亚基间接触面上修饰所需肽段等ꎬ使其成为纳米载体ꎮ此外ꎬ仿生合成的磁性铁蛋白纳米颗粒内核为Fe3O4ꎬ具有超顺磁性和过氧化物酶活性的双功能特性ꎮFe3O4的内核直径在4~7nmꎬ具有超顺磁性ꎬ使其成为潜在的MRI造影剂[22]ꎮ而我国科学家于2007年发现ꎬFe3O4磁性纳米颗粒还具有过氧化物酶的活性[23]ꎬ即在显色底物中含有H2O2时ꎬFe3O4磁性纳米颗粒可以将其催化氧化发生颜色反应ꎮ已有研究表明ꎬ铁蛋白的表达量在病变的脑组织和多种类型的肿瘤细胞中都较正常组织细胞多[24]ꎮ目前ꎬ检测脑神经退化性疾病及各种肿瘤的无创伤性的手段即为磁共振成像(magneticresonanceimagingꎬMRI)ꎬ可以对病变组织内的铁含量进行定量检测[25]ꎮ因此ꎬ仿生合成的磁性铁蛋白纳米颗粒在病灶诊断及治疗中具有巨大的应用前景(图2)ꎮ3㊀铁蛋白纳米颗粒的应用3.1㊀铁蛋白纳米颗粒在药物载体领域的应用铁蛋白纳米颗粒在药物载体领域ꎬ不仅可作为载体ꎬ同时还可作为信号分子ꎮ基于铁蛋白纳米颗粒具有的良好的生物相容性和特殊的球形空腔结构ꎬ其可成为铁氰化物㊁荧光素等各类小分子探针的理想载体ꎮ英国诺丁汉大学以无内核的铁蛋白外壳作为纳米材料的载体ꎬ系统地评估了铁蛋白包装对纳米材料稳定性及生物相容性的影响ꎮ实验结果表明ꎬ包装有探针的纳米颗粒不仅具有量子点优异的荧光性质ꎬ同时ꎬ还因为被铁蛋白包裹而降低了相应的毒性ꎻ通过进一步对铁蛋白外壳的修饰ꎬ包裹有量子点的铁蛋白纳米颗粒还可实现靶向细胞识别ꎬ并使得靶向过程可视[28]ꎬ为后期的临床诊断及病灶组织治疗提供了重要的技术支持ꎮ此外ꎬ铁蛋白也可作为信号分子ꎬ在生物传感器中利用其纳米材料的特性ꎬ双向放大电信号ꎬ构建一种电化学免疫检测方法ꎮ如利用金纳米颗粒与rGO ̄AuNPs材料修饰的玻碳电极合成AuNPs ̄Ab2 ̄Ferritin复合物ꎬ通过2次免疫反应可形成AuNPs ̄Ab2 ̄ferritin/Ag/Ab1/rGO ̄Au ̄chi/GCꎬ一种特殊的夹心免疫结构ꎬ该结构能实现检测人血浆硝化铜蓝蛋白(nitratedceruloplasmin)的目的[29]ꎮ3.2㊀铁蛋白纳米颗粒在纳米疫苗领域的应用研究人员基于铁蛋白特殊的空间结构ꎬ对其进行改造ꎬ结果表明ꎬ生物基因改造不会影响铁蛋白亚基间的自组装ꎬ而且24个亚基的基因均可进242生物技术进展CurrentBiotechnology. All Rights Reserved.图2㊀可用于靶向肿瘤并使其可视化的磁性铁蛋白纳米颗粒Fig.2㊀Magneticferritinnanoparticlesthatcanbeusedtotargetandvisualizetumors.注:A:仿生合成磁性铁蛋白[26]ꎻB:磁性铁蛋白的双功能特性ꎻC:常规免疫组化方法ꎻD:磁性铁蛋白检测肿瘤新技术[27]ꎮ行改造ꎬ这一发现使得铁蛋白纳米颗粒成为一个疫苗开发和抗原递呈的平台[30]ꎮ2006年ꎬ美国新世纪医药公司首次利用铁蛋白外壳作为呈递抗原的疫苗研发平台ꎬ在铁蛋白L亚基的N端融合表达HIV ̄1病毒的Tat肽段ꎬ利用铁蛋白的自组装特性生成融合蛋白ꎬ随后进行动物免疫实验ꎬ实验结果表明ꎬ该融合蛋白在动物机体内可激起免疫应答反应[30]ꎮ2013年ꎬ美国国家卫生研究所和过敏与传染病研究所将铁蛋白应用于流感疫苗的研发ꎬ将幽门螺杆菌铁蛋白亚基的N端与流感病毒的血凝素蛋白(hemagglutininꎬHA)基因融合ꎬ当铁蛋白自组装形成融合蛋白时ꎬ由蛋白核心向外伸出引入的血凝素HAꎬ由于铁蛋白具有三重对称轴ꎬ因而可形成8个HA突起ꎬ与流感病毒表面的突起相似(图3)[32]ꎮ将该融合蛋白纳米颗粒作为抗原进行动物免疫实验ꎬ在动物体内成功诱导了中和性抗体ꎬ达到了流感病毒疫苗的作用ꎮ同时ꎬ与传统灭活病毒疫苗相比ꎬ这种流感血凝素融合蛋白纳米颗粒在动物体内产生的中和性抗体水平高10倍以上ꎬ而且存在于铁蛋白表面的HA突起能特异性识别流感病毒HA三聚体蛋白的茎部和头部这2个高度保守的位点ꎮ此外ꎬ这种新型疫苗的免疫范围更广ꎬ能中和绝大多数同型病毒ꎮ通过基因修饰ꎬ铁蛋白自组装纳米图3㊀流感病毒HA的铁蛋白纳米颗粒的分子设计和表征[32]Fig.3㊀ThemoleculardesignandcharacterizationofferritinnanoparticlesfrominfluenzavirusHA[32].注:纳米粒子的负面染色TEM图像ꎮ1~6代表了HA尖峰在图像中的编号ꎮ342魏珍珍ꎬ等:自组装铁蛋白在纳米疫苗领域的应用进展. All Rights Reserved.颗粒还可以融合表达其他病毒抗原作为抗原递呈的制备疫苗平台ꎬ为各类动物病毒病的防治提供了较好的技术支持ꎮ目前ꎬ在制备双组分铁蛋白纳米颗粒ꎬ即同时表达多种抗原的铁蛋白纳米颗粒方面也做了尝试(图4)ꎬ纳米颗粒上的抗原多聚化可以使中和抗体响应得到改善[33]ꎮ在此研究中ꎬ设计了双组分铁蛋白变体ꎬ允许在1个颗粒上以确定的比例和几何图案黏着2种不同的抗原ꎮ双组分铁蛋白专门设计用于三聚体抗原ꎬ每个抗原接受每个颗粒图4㊀双组分铁蛋白纳米粒子的设计ꎬ用于附着不同的三聚体抗原[33]Fig.4㊀Designoftwo ̄componentferritinnanoparticlesforattachmentofdifferenttrimericantigens[33].注:单组分铁蛋白的示意图ꎮ其具有8个拷贝的三聚体抗原A(黑色)和双组分铁蛋白ꎬ每个三聚体抗原A具有4个拷贝(黑色)和B(灰色)ꎮ4个三聚体ꎬ并用来自HIV ̄1包膜(Env)和流感血凝素(HA)的抗原进行测试ꎮ用具有不同Env㊁HA或2种抗原的双组分铁蛋白颗粒对豚鼠进行免疫ꎬ引发针对各病毒的中和抗体应答ꎮ该结果证明了铁蛋白表面可展示不只1种抗原ꎬ也提供了双组分纳米颗粒自组装原理的证据ꎬ将来可作为三聚体抗原的多聚体免疫原呈递的一般技术ꎮ此研究的成功展开ꎬ为后期新型疫苗的制备开拓了新的思路ꎮ相比于直接在铁蛋白表面表达抗原ꎬ也可在铁蛋白表面或者空腔内连接衍生自卵清蛋白的抗原肽OT ̄1(SIINFEKL)或OT ̄2(ISQAVHAA ̄HAEINEAGR)ꎬ然后再将重组铁蛋白作用于树突细胞ꎬ其可启动和控制抗原特异性免疫应答ꎮ树突细胞在其中起着重要作用ꎬ即将抗原内化ꎬ再加工和呈递给原始T淋巴细胞并诱导其增殖和分化为效应细胞(图5)ꎬ导致抗原特异性靶细胞的选择性杀伤[21]ꎬ同时ꎬIFN ̄γ/IL ̄2和IL ̄10/IL ̄13细胞因子的产生可证实铁蛋白纳米疫苗会增强机体的免疫反应ꎮ基于树突细胞的铁蛋白纳米颗粒疫苗的开发已成为体内直接抗原特异性适应性免疫的非常有前景的一种方法ꎮ图5㊀携带OT肽的铁蛋白蛋白笼纳米颗粒诱导的抗原特异性T细胞增殖和随后的免疫应答[34]Fig.5㊀FerroproteinproteincagenanoparticlescarryingOTpeptideinducedantigen ̄specificTcellproliferationandsubsequentimmuneresponse[34].4㊀展望自组装蛋白广泛存在于机体中ꎬ与其他自组装蛋白相比ꎬ自组装铁蛋白具有独特的解聚与重组方式ꎬ可耐受高热和高浓度变性剂ꎬ同时其独特的高级空间结构也便于进行基因定向修饰ꎬ可在一定程度上对修饰过程实现精准控制ꎮ通过生物手段与化学方法相结合的修饰方法ꎬ如在铁蛋白表面共价连接各类大分子ꎬ可实现特异性修饰特定位点ꎬ还可赋予铁蛋白更多新的性能ꎬ铁蛋白的应用范围也被拓宽ꎻ而通过将标记蛋白与铁蛋白亚基融合表达ꎬ使融合蛋白有序的展示在铁蛋白外壳的外表面ꎬ可提高抗体或药物等目标蛋白的载量和效率ꎬ从而作为一种潜在的新型疫苗ꎮ同时ꎬ基于铁蛋白的纳米颗粒特性ꎬ其也可作为信号442生物技术进展CurrentBiotechnology. 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UCP1蛋白和UCP3基因表达减少与OLETF鼠早期肥胖有关

UCP1蛋白和UCP3基因表达减少与OLETF鼠早期肥胖有关

UCP1蛋白和UCP3基因表达减少与OLETF鼠早期肥胖有关金成吉;王晓梅;李湘;母义明【摘要】Objective To investigate the relationship between the body weight change and the mRNA or protein expression of uncoupling proteins (UCPs) in OLETF rats. Methods Body weight was measured weekly. UCP1 protein expression in brown adipose tissue was detected by Western blot, while UCP2 and UCP3 mRNA expression in skeletal muscle was measured by Northern hybridization. Results OLETF rats gained weight with a faster rate than LETO rats, and the difference in the rate of weight gain was most prominent around 9 weeks of age. UCP1 protein level was 1.9-fold higher in LETO than OLETF rots at 10 weeks of age (P < 0. 05 ). UCP3 mRNA level was 5. 5-fold higher in LETO than OLETF rats at 10 weeks of age ( P < 0. 01 ). Conclusion UCP1 and UCP3 expression may play an important role particularly in the early stage of the development of obesity in OLETF rats.%目的观察肥胖2型糖尿病模型OLETF鼠体质量变化和解偶联蛋白(UCPs)转录基因或蛋白表达的相关性.方法测量OLETF大鼠和对照组LETO大鼠不同周龄阶段体质量,在7、10和25周时用Western blot方法测定棕色脂肪组织(BAT)中的UCP1蛋白,用Northern blot方法测定骨骼肌中UCP2和UCP3 mRNA量.结果 OLETF大鼠体质量增加比LETO大鼠快,9周前后两组体质量增加差异最明显.10周时LETO组UCPI是OLETF组的1.9倍(P<0.05);UCP3 mRNA 量约为OLETF组的5.5倍(P<0.01).结论体质量增加差异明显的阶段UCP1蛋白和UCP3基因表达的减少可能是引起OLETF大鼠肥胖的病因之一.【期刊名称】《基础医学与临床》【年(卷),期】2011(031)003【总页数】5页(P286-290)【关键词】肥胖;UCP1;UCP2;UCP3【作者】金成吉;王晓梅;李湘;母义明【作者单位】大连大学附属中山医院内分泌科,辽宁大连116001;大连大学附属中山医院内分泌科,辽宁大连116001;大连大学附属中山医院内分泌科,辽宁大连116001;中国人民解放军总医院内分泌科,北京100039【正文语种】中文【中图分类】R589.2解偶联蛋白(uncoupling proteins,UCPs)是线粒体内膜上的一种调节质子跨膜转运作用的转运蛋白,提高静息代谢率。

微生物碳酸酐酶在矿化沉积中的研究进展_张小菊

微生物碳酸酐酶在矿化沉积中的研究进展_张小菊

化学与生物工程2011,Vol.28No.3Chemistry &Bioen gineering收稿日期:2010-11-09作者简介:张小菊(1975-),女,湖北恩施人,讲师,研究方向:生物材料。

E ma il:qing ting6175@sina.co m 。

doi:10.3969/j.issn.1672-5425.2011.03.005微生物碳酸酐酶在矿化沉积中的研究进展张小菊,杨 娟,李横江(华中科技大学武昌分校城市建设学院,湖北武汉430064)摘 要:碳酸酐酶是一种含Zn 的金属酶,主要催化CO 2和H CO -3之间的转换反应,微生物是碳酸酐酶的重要来源之一。

对微生物碳酸酐酶在矿化沉积中的研究现状进行了综述,阐述了碳酸酐酶在石刻文物保护、环境生物修复中的应用价值,并对微生物碳酸酐酶的进一步研究进行了展望。

关键词:碳酸酐酶;矿化沉积;石刻文物保护;生物修复中图分类号:Q 939.99 文献标识码:A文章编号:1672-5425(2011)03-0019-03碳酸酐酶(Carbonic anhydrase,CA)是生物体内普遍存在的一种金属酶,其活性中心中含有一个催化活性所必需的锌原子,催化CO 2进行可逆水合反应,在矿化沉积中扮演着重要的角色[1,2]。

生物矿化沉积是一种广泛而复杂的固液之间、有机物和无机物之间的物理化学过程,是以少量有机质为模板,进行分子操作,高度有序地组合成无机材料,构成矿物质点的形态大小、空间排列、结晶取向和同质多晶类型[3]。

目前石质文物的人为破坏作用、微生物破坏作用、风化作用严重,对石质文物进行保护的研究主要集中在石质文物微生物的腐蚀机理[4]、石质文物的防风化、利用生物矿化的原理在石材表面仿生合成保护材料[5~7]等。

已有研究微生物诱导的矿化作用对碳酸钙形成的影响及遗产保护的相关报道[8~10],但利用生物的矿化沉积特别是碳酸酐酶的作用来修复石质文物还研究得较少。

韩江下游赤眼鳟幼鱼鱼体的化学组成及能量密度的研究

韩江下游赤眼鳟幼鱼鱼体的化学组成及能量密度的研究

韩江下游赤眼鳟幼鱼鱼体的化学组成及能量密度的研究林小植;陈蔚辉;范汉金;罗毅平;查广才【摘要】2010年6月在潮州江段采集赤眼鳟(Squaliobarbus curriculus)幼鱼36尾,按体长范围(68~175 mm)分为5个组,测定鱼体化学组成,估算其能量密度.结果显示:赤眼鳟含水量、蛋白质含量、脂肪含量、灰分含量占鲜重的百分比范围为72.79%~75.54%、12.99%~17.06%、8.18%~12.88%和2.35%~3.38%,能量密度范围为6.29~9.11kJ/g蛋白质含量、脂肪含量和能量密度均随体长增加而增加,含水量呈下降趋势.体长与蛋白质含量、脂肪含量、能量密度呈显著的正线性关系,而与含水量呈负线性相关关系(P<0.05);含水量与蛋白质含量、脂肪含量、能量密度呈显著的负线性关系(P<0.05).结果表明可以用赤眼鳟的含水量及体长估计其蛋白质含量、脂肪含量和能量密度,赤眼鳟较高的脂肪含量与韩江中丰富的食物资源及赤眼鳟适应缓流水生活的习性有关.%To observe the variation of body composition of Squaliobarbus curriculus, 36 specimens with body length of 68 ~175 mm and body weight of 4. 78 ~104. 88 g were collected from the lower reaches of Hanjiang river in June, 2010, and divided into 5 groups according to the body length(L). Chemical compositions and energy density were measured. The resuits showed that the contents of water( WAT), protein( PRO), lipid(FAT) and ash ( represented by percentages of them in fresh body weight of fish, respectively) ranged 72. 79% ~ 75.54% , 12. 99%~ 17.06%, 8. 18% ~ 12. 88% and 2. 35% ~3.38%respectively. The energy density (E) ranged 6. 29 ~9. 11 kJ/g. With the increase of L, PRO, FAT and E, WAT decreased. PRO, FAT and E were all positively correlated with L, while WAT was negatively correlated with L (P< 0. 05 ). The contents of PRO, FAT and E were all negatively correlated with the content of WAT ( P < 0. 05 ). The results suggested that the contents of PRO, FAT and E of S. curriculus could be estimated by the content of water or body size. The high content of lipid with the increasing body size in this species may be resulted from its acclimation to the slow flow water environment and abundant food resources.【期刊名称】《淡水渔业》【年(卷),期】2011(041)002【总页数】5页(P25-29)【关键词】韩江;赤眼鳟(Squaliobarbus curriculus);体长;化学组成;能量密度【作者】林小植;陈蔚辉;范汉金;罗毅平;查广才【作者单位】韩山师范学院生物系,广东潮州,521041;韩山师范学院生物系,广东潮州,521041;韩山师范学院生物系,广东潮州,521041;西南大学生命科学学院,淡水生物生殖与发育教育部重点实验室,重庆,400715;韩山师范学院生物系,广东潮州,521041【正文语种】中文【中图分类】Q413鱼体的化学组成及能量密度是鱼类营养学、生理学研究的重要内容之一[1]。

抗菌肽LL-37 抑制肝癌细胞恶性增殖的转录组分析

抗菌肽LL-37 抑制肝癌细胞恶性增殖的转录组分析

DOI:10.16605/ki.1007-7847.2021.06.0169抗菌肽LL-37抑制肝癌细胞恶性增殖的转录组分析吕继龙a,b,c ,李球棣a ,佘东阳a,b,c ,陈宁a,c,d ,丁晓慧a,c*(徐州医科大学a.病原生物学与免疫学教研室/江苏省免疫与代谢重点实验室;b.第二临床医学院;c.基础医学国家级实验教学示范中心;d.第一临床医学院,中国江苏徐州221004)摘要:抗菌肽LL-37与肿瘤的发生发展密切相关,课题组前期研究发现LL-37能够抑制肝癌细胞恶性增殖。

为了给其抑制肝癌发生发展的分子机制研究提供更多的生物学依据,本研究通过高通量RNA 测序技术以及生物信息学方法对LL-37作用前后肝癌细胞中的差异表达基因(differentially expressed gene,DEG)进行了分析,共筛选出753个DEG,其中上调的DEG 374个,下调的DEG 379个;进一步对筛选出的DEG 进行基因本体论(Gene Ontology,GO)及京都基因和基因组数据库(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路富集分析,并构建DEG 编码蛋白互作网络,筛选出10个可能参与LL-37抑制肝癌细胞恶性增殖的潜在关键基因。

经分析这些基因均表达下调,且对机体炎症的激活、转运以及肿瘤细胞的增殖、迁移至关重要。

以上结果为揭示LL-37在肝癌发生发展中的作用及机制提供了数据基础,为探索肝癌诊断和治疗手段提供了新的思路。

关键词:LL-37;肝细胞癌(HCC);转录组测序;差异表达基因(DEG);生物信息学分析中图分类号:Q51,Q811.4,R735.7文献标识码:A文章编号:1007-7847(2022)06-0528-10Transcriptomic Analysis of Malignant Proliferation of Hepatocellular Carcinoma Cells Inhibited by AntimicrobialPeptide LL-37L ÜJi-long a,b,c ,LI Qiu-di a ,SHE Dong-yang a,b,c ,CHEN Ning a,c,d ,DING Xiao-hui a,c*(a.Jiangsu Key Laboratory of Immunity and Metabolism/Department of Pathogenic Biology and Immunology ;b.The SecondClinical Medical College ;c.National Demonstration Center for Experimental Basic Medical Science Education ;d.The FirstClinical Medical College ,Xuzhou Medical University ,Xuzhou 221004,Jiangsu ,China )Abstract:Antimicrobial peptide LL-37is closely related to the occurrence and development of tumors.Our previous study found that LL-37can inhibit the malignant proliferation of hepatocellular carcinoma (HCC)cells.To provide more biological basis for the molecular mechanisms of LL-37inhibiting the occurrence and development of HCC,high-throughput RNA sequencing and bioinformatics methods were used to analyze the differentially expressed genes (DEGs)in HCC cells before and after LL-37treatment.A total of 753DEGs were screened out,among which 374genes were up-regulated and 379genes were down-regulated.The se-lected DEGs were further enriched by Gene Ontology (GO)and Kyoto Encyclopedia of Genes and Genomes (KEGG)pathway analyses,and the protein-protein interaction network encoded by DEGs was also construc-ted.Ten potential key genes that might be involved in inhibition of malignant proliferation of HCC cells by LL-37were screened out.It was found that these genes were all down-regulated and played an important role in the activation of inflammation as well as in the proliferation and migration of tumor cells.Taken to-gether,these results provide data foundation for revealing the role and mechanism of LL-37in the occur-rence and development of HCC,and provide new ideas for exploring the diagnosis and treatment of HCC.收稿日期:2021-06-10;修回日期:2021-09-11;网络首发日期:2022-03-09基金项目:江苏省高等学校大学生创新训练项目(202010313044Y);徐州医科大学优秀人才科研启动经费项目(D2019017);江苏省“双创博士”项目;基础医学国家级实验教学示范中心(徐州医科大学)资助项目作者简介:吕继龙(2000—),男,江苏宿迁人,学生;*通信作者:丁晓慧(1989—),女,河南商丘人,博士,讲师,主要从事肝癌致病机制相关研究,Tel:*************,E-mail:************************。

生物质气化焦油催化裂解研究

生物质气化焦油催化裂解研究

239
白云石是一种菱镁矿,分子式一般写作 C a M g(CO 3)2 。 白云石可通过 与生物质干式混合而直接用 在气化反应器中作 为初级催化剂,也可用于二级床中[8]。
生物质燃气经富含 F e 2O3 的白云石催化裂解后,焦油转 化率可达 9 5% 以上,气体产物增加 1 0 %~2 0%,低位热值增 加 l5 %,燃气组分中 H2 增加 4 %[9 ]。若以 H2O 和 O2 为气化 介质,在白云 石的催化作用下,焦油裂解 的同时会发生水气 置换、水蒸气重整、CO2 重整和部分氧化反应。适当增加 H2 O 量有利于提高气体产物中 H2 的含量,降低 C O 和 CH4 的含 量,在 8 40 ℃的温度下可获得 9 6%的焦油裂解率 。 [10 ]
一、焦油的生成机理及危害 生物质气化的 目标是得到尽可能多的可 燃性气体产物。 但在气化的过 程中,粉尘和焦油都是不可 避免的副产物。焦 油生成于气化过程中的热解阶段。当生物质被加热到 2 00 ℃ 以上时,组成 生物质的纤维素、木质素、 半纤维素等成分发 生明显的热分解,生成焦炭、木醋酸、焦油、气体等。在 60 O ℃以上时,液 体产物焦油和木醋酸以气态 的形式存在于所生 成的热解气体 中。焦油性质的不稳定性, 也导致了其组成的 复杂性,目前可分析出的成分有 10 0 多种,还有很多成分难 以确定。焦油对气化系统和用气设备等会产生不利影响[2]: ① 降低气化效率,气化气中焦油产物的能量一般占总能 量的 5 %- 15 %,这部分能量在低温时难以与可燃气体一起被 利用;② 焦油存在于气化的产品气中,在管道内输送过程中 将逐渐冷凝下 来,形成粘稠的液体物质, 附着于管道内壁和 有关设备的壁面上,对设备的安全运行造成威胁;③ 焦油还 会与气流中的 灰尘相结合,在管道内累积 起来,严重时将造 成管道的堵塞;④ 凝结细小液滴的焦油比气体难于燃尽,在 燃烧时容易产 生碳黑,容易造成污染并对 燃气利用设备损害 相当严重。⑤ 焦油成分中含有有毒物质,对人类健康构成威 胁。由此可见 ,可燃气中的焦油是气化气 使用的最大危害, 必须尽量脱除成为干净燃气方可使用。 二、焦油的脱除方法 焦油脱除的方 法大致可分为物理脱除法 和热化学脱除法 两类[3,4]。目前采用较多的物理除焦方法有湿法和干法两种,

微生物英文文献及翻译—原文

微生物英文文献及翻译—原文

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic e aerobic sequencing batch reactorZhang Bin a ,b ,Chen Zhe a ,b ,Qiu Zhigang a ,b ,Jin Min a ,b ,Chen Zhiqiang a ,b ,Chen Zhaoli a ,b ,Li Junwen a ,b ,Wang Xuan c ,*,Wang Jingfeng a ,b ,**aInstitute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China bTianjin Key Laboratory of Risk Assessment and Control for Environment and Food Safety,Tianjin 300050,PR China cTianjin Key Laboratory of Hollow Fiber Membrane Material and Membrane Process,Institute of Biological and Chemical Engineering,Tianjin Polytechnical University,Tianjin 300160,PR Chinaa r t i c l e i n f oArticle history:Received 30June 2011Received in revised form 10September 2011Accepted 10September 2011Available online xxx Keywords:Ammonia-oxidizing bacteria Granular sludgeCommunity development Granule sizeNitrifying bacteria distribution Phylogenetic diversitya b s t r a c tThe structure dynamic of ammonia-oxidizing bacteria (AOB)community and the distribution of AOB and nitrite-oxidizing bacteria (NOB)in granular sludge from an anaerobic e aerobic sequencing batch reactor (SBR)were investigated.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and characterize these organisms.The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge.Along with granules formation,the AOB diversity declined due to the selection pressure imposed by process conditions.Denaturing gradient gel electrophoresis (DGGE)and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling e washing out action.Furthermore,DGGE analysis revealed that larger granules benefit more AOB species surviving in the reactor.In the SBR were various size granules coexisted,granule diameter affected the distribution range of AOB and NOB.Small and medium granules (d <0.6mm)cannot restrict oxygen mass transfer in all spaces of the rger granules (d >0.9mm)can result in smaller aerobic volume fraction and inhibition of NOB growth.All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.ª2011Elsevier Ltd.All rights reserved.1.IntroductionAt sufficiently high levels,ammonia in aquatic environments can be toxic to aquatic life and can contribute to eutrophica-tion.Accordingly,biodegradation and elimination of ammonia in wastewater are the primary functions of thewastewater treatment process.Nitrification,the conversion of ammonia to nitrate via nitrite,is an important way to remove ammonia nitrogen.It is a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and NOB).Aerobic ammonia-oxidation is often the first,rate-limiting step of nitrification;however,it is essential for the*Corresponding author .**Corresponding author.Institute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China.Tel.:+862284655498;fax:+862223328809.E-mail addresses:wangxuan0116@ (W.Xuan),jingfengwang@ (W.Jingfeng).Available online atjournal homepage:/locate/watresw a t e r r e s e a r c h x x x (2011)1e 100043-1354/$e see front matter ª2011Elsevier Ltd.All rights reserved.doi:10.1016/j.watres.2011.09.026removal of ammonia from the wastewater(Prosser and Nicol, 2008).Comparative analyses of16S rRNA sequences have revealed that most AOB in activated sludge are phylogeneti-cally closely related to the clade of b-Proteobacteria (Kowalchuk and Stephen,2001).However,a number of studies have suggested that there are physiological and ecological differences between different AOB genera and lineages,and that environmental factors such as process parameter,dis-solved oxygen,salinity,pH,and concentrations of free ammonia can impact certain species of AOB(Erguder et al., 2008;Kim et al.,2006;Koops and Pommerening-Ro¨ser,2001; Kowalchuk and Stephen,2001;Shi et al.,2010).Therefore, the physiological activity and abundance of AOB in waste-water processing is critical in the design and operation of waste treatment systems.For this reason,a better under-standing of the ecology and microbiology of AOB in waste-water treatment systems is necessary to enhance treatment performance.Recently,several developed techniques have served as valuable tools for the characterization of microbial diversity in biological wastewater treatment systems(Li et al., 2008;Yin and Xu,2009).Currently,the application of molec-ular biotechniques can provide clarification of the ammonia-oxidizing community in detail(Haseborg et al.,2010;Tawan et al.,2005;Vlaeminck et al.,2010).In recent years,the aerobic granular sludge process has become an attractive alternative to conventional processes for wastewater treatment mainly due to its cell immobilization strategy(de Bruin et al.,2004;Liu et al.,2009;Schwarzenbeck et al.,2005;Schwarzenbeck et al.,2004a,b;Xavier et al.,2007). Granules have a more tightly compact structure(Li et al.,2008; Liu and Tay,2008;Wang et al.,2004)and rapid settling velocity (Kong et al.,2009;Lemaire et al.,2008).Therefore,granular sludge systems have a higher mixed liquid suspended sludge (MLSS)concentration and longer solid retention times(SRT) than conventional activated sludge systems.Longer SRT can provide enough time for the growth of organisms that require a long generation time(e.g.,AOB).Some studies have indicated that nitrifying granules can be cultivated with ammonia-rich inorganic wastewater and the diameter of granules was small (Shi et al.,2010;Tsuneda et al.,2003).Other researchers reported that larger granules have been developed with the synthetic organic wastewater in sequencing batch reactors(SBRs)(Li et al., 2008;Liu and Tay,2008).The diverse populations of microor-ganisms that coexist in granules remove the chemical oxygen demand(COD),nitrogen and phosphate(de Kreuk et al.,2005). However,for larger granules with a particle diameter greater than0.6mm,an outer aerobic shell and an inner anaerobic zone coexist because of restricted oxygen diffusion to the granule core.These properties of granular sludge suggest that the inner environment of granules is unfavorable to AOB growth.Some research has shown that particle size and density induced the different distribution and dominance of AOB,NOB and anam-mox(Winkler et al.,2011b).Although a number of studies have been conducted to assess the ecology and microbiology of AOB in wastewater treatment systems,the information on the dynamics,distribution,and quantification of AOB communities during sludge granulation is still limited up to now.To address these concerns,the main objective of the present work was to investigate the population dynamics of AOB communities during the development of seedingflocs into granules,and the distribution of AOB and NOB in different size granules from an anaerobic e aerobic SBR.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and char-acterize these organisms.Based on these approaches,we demonstrate the differences in both AOB community evolu-tion and composition of theflocs and granules co-existing in the SBR and further elucidate the relationship between distribution of nitrifying bacteria and granule size.It is ex-pected that the work would be useful to better understand the mechanisms responsible for the AOB in granules and apply them for optimal control and management strategies of granulation systems.2.Material and methods2.1.Reactor set-up and operationThe granules were cultivated in a lab-scale SBR with an effective volume of4L.The effective diameter and height of the reactor was10cm and51cm,respectively.The hydraulic retention time was set at8h.Activated sludge from a full-scale sewage treat-ment plant(Jizhuangzi Sewage Treatment Works,Tianjin, China)was used as the seed sludge for the reactor at an initial sludge concentration of3876mg LÀ1in MLSS.The reactor was operated on6-h cycles,consisting of2-min influent feeding,90-min anaerobic phase(mixing),240-min aeration phase and5-min effluent discharge periods.The sludge settling time was reduced gradually from10to5min after80SBR cycles in20days, and only particles with a settling velocity higher than4.5m hÀ1 were retained in the reactor.The composition of the influent media were NaAc(450mg LÀ1),NH4Cl(100mg LÀ1),(NH4)2SO4 (10mg LÀ1),KH2PO4(20mg LÀ1),MgSO4$7H2O(50mg LÀ1),KCl (20mg LÀ1),CaCl2(20mg LÀ1),FeSO4$7H2O(1mg LÀ1),pH7.0e7.5, and0.1mL LÀ1trace element solution(Li et al.,2007).Analytical methods-The total organic carbon(TOC),NHþ4e N, NOÀ2e N,NOÀ3e N,total nitrogen(TN),total phosphate(TP) concentration,mixed liquid suspended solids(MLSS) concentration,and sludge volume index at10min(SVI10)were measured regularly according to the standard methods (APHA-AWWA-WEF,2005).Sludge size distribution was determined by the sieving method(Laguna et al.,1999).Screening was performed with four stainless steel sieves of5cm diameter having respective mesh openings of0.9,0.6,0.45,and0.2mm.A100mL volume of sludge from the reactor was sampled with a calibrated cylinder and then deposited on the0.9mm mesh sieve.The sample was subsequently washed with distilled water and particles less than0.9mm in diameter passed through this sieve to the sieves with smaller openings.The washing procedure was repeated several times to separate the gran-ules.The granules collected on the different screens were recovered by backwashing with distilled water.Each fraction was collected in a different beaker andfiltered on quantitative filter paper to determine the total suspended solid(TSS).Once the amount of total suspended solid(TSS)retained on each sieve was acquired,it was reasonable to determine for each class of size(<0.2,[0.2e0.45],[0.45e0.6],[0.6e0.9],>0.9mm) the percentage of the total weight that they represent.w a t e r r e s e a r c h x x x(2011)1e10 22.2.DNA extraction and nested PCR e DGGEThe sludge from approximately8mg of MLSS was transferred into a1.5-mL Eppendorf tube and then centrifuged at14,000g for10min.The supernatant was removed,and the pellet was added to1mL of sodium phosphate buffer solution and aseptically mixed with a sterilized pestle in order to detach granules.Genomic DNA was extracted from the pellets using E.Z.N.A.äSoil DNA kit(D5625-01,Omega Bio-tek Inc.,USA).To amplify ammonia-oxidizer specific16S rRNA for dena-turing gradient gel electrophoresis(DGGE),a nested PCR approach was performed as described previously(Zhang et al., 2010).30m l of nested PCR amplicons(with5m l6Âloading buffer)were loaded and separated by DGGE on polyacrylamide gels(8%,37.5:1acrylamide e bisacrylamide)with a linear gradient of35%e55%denaturant(100%denaturant¼7M urea plus40%formamide).The gel was run for6.5h at140V in 1ÂTAE buffer(40mM Tris-acetate,20mM sodium acetate, 1mM Na2EDTA,pH7.4)maintained at60 C(DCodeäUniversal Mutation Detection System,Bio-Rad,Hercules,CA, USA).After electrophoresis,silver-staining and development of the gels were performed as described by Sanguinetti et al. (1994).These were followed by air-drying and scanning with a gel imaging analysis system(Image Quant350,GE Inc.,USA). The gel images were analyzed with the software Quantity One,version4.31(Bio-rad).Dice index(Cs)of pair wise community similarity was calculated to evaluate the similarity of the AOB community among DGGE lanes(LaPara et al.,2002).This index ranges from0%(no common band)to100%(identical band patterns) with the assistance of Quantity One.The Shannon diversity index(H)was used to measure the microbial diversity that takes into account the richness and proportion of each species in a population.H was calculatedusing the following equation:H¼ÀPn iNlogn iN,where n i/Nis the proportion of community made up by species i(bright-ness of the band i/total brightness of all bands in the lane).Dendrograms relating band pattern similarities were automatically calculated without band weighting(consider-ation of band density)by the unweighted pair group method with arithmetic mean(UPGMA)algorithms in the Quantity One software.Prominent DGGE bands were excised and dissolved in30m L Milli-Q water overnight,at4 C.DNA was recovered from the gel by freeze e thawing thrice.Cloning and sequencing of the target DNA fragments were conducted following the estab-lished method(Zhang et al.,2010).2.3.Distribution of nitrifying bacteriaThree classes of size([0.2e0.45],[0.45e0.6],>0.9mm)were chosen on day180for FISH analysis in order to investigate the spatial distribution characteristics of AOB and NOB in granules.2mg sludge samples werefixed in4%para-formaldehyde solution for16e24h at4 C and then washed twice with sodium phosphate buffer;the samples were dehydrated in50%,80%and100%ethanol for10min each. Ethanol in the granules was then completely replaced by xylene by serial immersion in ethanol-xylene solutions of3:1, 1:1,and1:3by volume andfinally in100%xylene,for10min periods at room temperature.Subsequently,the granules were embedded in paraffin(m.p.56e58 C)by serial immer-sion in1:1xylene-paraffin for30min at60 C,followed by 100%paraffin.After solidification in paraffin,8-m m-thick sections were prepared and placed on gelatin-coated micro-scopic slides.Paraffin was removed by immersing the slide in xylene and ethanol for30min each,followed by air-drying of the slides.The three oligonucleotide probes were used for hybridiza-tion(Downing and Nerenberg,2008):FITC-labeled Nso190, which targets the majority of AOB;TRITC-labeled NIT3,which targets Nitrobacter sp.;TRITC-labeled NSR1156,which targets Nitrospira sp.All probe sequences,their hybridization condi-tions,and washing conditions are given in Table1.Oligonu-cleotides were synthesized andfluorescently labeled with fluorochomes by Takara,Inc.(Dalian,China).Hybridizations were performed at46 C for2h with a hybridization buffer(0.9M NaCl,formamide at the percentage shown in Table1,20mM Tris/HCl,pH8.0,0.01% SDS)containing each labeled probe(5ng m LÀ1).After hybrid-ization,unbound oligonucleotides were removed by a strin-gent washing step at48 C for15min in washing buffer containing the same components as the hybridization buffer except for the probes.For detection of all DNA,4,6-diamidino-2-phenylindole (DAPI)was diluted with methanol to afinal concentration of1ng m LÀ1.Cover the slides with DAPI e methanol and incubate for15min at37 C.The slides were subsequently washed once with methanol,rinsed briefly with ddH2O and immediately air-dried.Vectashield(Vector Laboratories)was used to prevent photo bleaching.The hybridization images were captured using a confocal laser scanning microscope (CLSM,Zeiss710).A total of10images were captured for each probe at each class of size.The representative images were selected andfinal image evaluation was done in Adobe PhotoShop.w a t e r r e s e a r c h x x x(2011)1e1033.Results3.1.SBR performance and granule characteristicsDuring the startup period,the reactor removed TOC and NH 4þ-N efficiently.98%of NH 4þ-N and 100%of TOC were removed from the influent by day 3and day 5respectively (Figs.S2,S3,Supporting information ).Removal of TN and TP were lower during this period (Figs.S3,S4,Supporting information ),though the removal of TP gradually improved to 100%removal by day 33(Fig.S4,Supporting information ).To determine the sludge volume index of granular sludge,a settling time of 10min was chosen instead of 30min,because granular sludge has a similar SVI after 60min and after 5min of settling (Schwarzenbeck et al.,2004b ).The SVI 10of the inoculating sludge was 108.2mL g À1.The changing patterns of MLSS and SVI 10in the continuous operation of the SBR are illustrated in Fig.1.The sludge settleability increased markedly during the set-up period.Fig.2reflects the slow andgradual process of sludge granulation,i.e.,from flocculentsludge to granules.3.2.DGGE analysis:AOB communities structure changes during sludge granulationThe results of nested PCR were shown in Fig.S1.The well-resolved DGGE bands were obtained at the representative points throughout the GSBR operation and the patterns revealed that the structure of the AOB communities was dynamic during sludge granulation and stabilization (Fig.3).The community structure at the end of experiment was different from that of the initial pattern of the seed sludge.The AOB communities on day 1showed 40%similarity only to that at the end of the GSBR operation (Table S1,Supporting information ),indicating the considerable difference of AOB communities structures between inoculated sludge and granular sludge.Biodiversity based on the DGGE patterns was analyzed by calculating the Shannon diversity index H as204060801001201401254159738494104115125135147160172188Time (d)S V I 10 (m L .g -1)10002000300040005000600070008000900010000M L S S (m g .L -1)Fig.1e Change in biomass content and SVI 10during whole operation.SVI,sludge volume index;MLSS,mixed liquid suspendedsolids.Fig.2e Variation in granule size distribution in the sludge during operation.d,particle diameter;TSS,total suspended solids.w a t e r r e s e a r c h x x x (2011)1e 104shown in Fig.S5.In the phase of sludge inoculation (before day 38),H decreased remarkably (from 0.94to 0.75)due to the absence of some species in the reactor.Though several dominant species (bands2,7,10,11)in the inoculating sludge were preserved,many bands disappeared or weakened (bands 3,4,6,8,13,14,15).After day 45,the diversity index tended to be stable and showed small fluctuation (from 0.72to 0.82).Banding pattern similarity was analyzed by applying UPGMA (Fig.4)algorithms.The UPGMA analysis showed three groups with intragroup similarity at approximately 67%e 78%and intergroup similarity at 44e 62%.Generally,the clustering followed the time course;and the algorithms showed a closer clustering of groups II and III.In the analysis,group I was associated with sludge inoculation and washout,group IIwithFig.3e DGGE profile of the AOB communities in the SBR during the sludge granulation process (lane labels along the top show the sampling time (days)from startup of the bioreactor).The major bands were labeled with the numbers (bands 1e15).Fig.4e UPGMA analysis dendrograms of AOB community DGGE banding patterns,showing schematics of banding patterns.Roman numerals indicate major clusters.w a t e r r e s e a r c h x x x (2011)1e 105startup sludge granulation and decreasing SVI 10,and group III with a stable system and excellent biomass settleability.In Fig.3,the locations of the predominant bands were excised from the gel.DNA in these bands were reamplified,cloned and sequenced.The comparative analysis of these partial 16S rRNA sequences (Table 2and Fig.S6)revealed the phylogenetic affiliation of 13sequences retrieved.The majority of the bacteria in seed sludge grouped with members of Nitrosomonas and Nitrosospira .Along with sludge granula-tion,most of Nitrosomonas (Bands 2,5,7,9,10,11)were remained or eventually became dominant in GSBR;however,all of Nitrosospira (Bands 6,13,15)were gradually eliminated from the reactor.3.3.Distribution of AOB and NOB in different sized granulesFISH was performed on the granule sections mainly to deter-mine the location of AOB and NOB within the different size classes of granules,and the images were not further analyzed for quantification of cell counts.As shown in Fig.6,in small granules (0.2mm <d <0.45mm),AOB located mainly in the outer part of granular space,whereas NOB were detected only in the core of granules.In medium granules (0.45mm <d <0.6mm),AOB distributed evenly throughout the whole granular space,whereas NOB still existed in the inner part.In the larger granules (d >0.9mm),AOB and NOB were mostly located in the surface area of the granules,and moreover,NOB became rare.4.Discussion4.1.Relationship between granule formation and reactor performanceAfter day 32,the SVI 10stabilized at 20e 35mL g À1,which is very low compared to the values measured for activated sludge (100e 150mL g À1).However,the size distribution of the granules measured on day 32(Fig.2)indicated that only 22%of the biomass was made of granular sludge with diameter largerthan 0.2mm.These results suggest that sludge settleability increased prior to granule formation and was not affected by different particle sizes in the sludge during the GSBR operation.It was observed,however,that the diameter of the granules fluctuated over longer durations.The large granules tended to destabilize due to endogenous respiration,and broke into smaller granules that could seed the formation of large granules again.Pochana and Keller reported that physically broken sludge flocs contribute to lower denitrification rates,due to their reduced anoxic zone (Pochana and Keller,1999).Therefore,TN removal efficiency raises fluctuantly throughout the experiment.Some previous research had demonstrated that bigger,more dense granules favored the enrichment of PAO (Winkler et al.,2011a ).Hence,after day 77,removal efficiency of TP was higher and relatively stable because the granules mass fraction was over 90%and more larger granules formed.4.2.Relationship between AOB communities dynamic and sludge granulationFor granule formation,a short settling time was set,and only particles with a settling velocity higher than 4.5m h À1were retained in the reactor.Moreover,as shown in Fig.1,the variation in SVI 10was greater before day 41(from 108.2mL g À1e 34.1mL g À1).During this phase,large amounts of biomass could not survive in the reactor.A clear shift in pop-ulations was evident,with 58%similarity between days 8and 18(Table S1).In the SBR system fed with acetate-based synthetic wastewater,heterotrophic bacteria can produce much larger amounts of extracellular polysaccharides than autotrophic bacteria (Tsuneda et al.,2003).Some researchers found that microorganisms in high shear environments adhered by extracellular polymeric substances (EPS)to resist the damage of suspended cells by environmental forces (Trinet et al.,1991).Additionally,it had been proved that the dominant heterotrophic species in the inoculating sludge were preserved throughout the process in our previous research (Zhang et al.,2011).It is well known that AOB are chemoau-totrophic and slow-growing;accordingly,numerous AOBw a t e r r e s e a r c h x x x (2011)1e 106populations that cannot become big and dense enough to settle fast were washed out from the system.As a result,the variation in AOB was remarkable in the period of sludge inoculation,and the diversity index of population decreased rapidly.After day 45,AOB communities’structure became stable due to the improvement of sludge settleability and the retention of more biomass.These results suggest that the short settling time (selection pressure)apparently stressed the biomass,leading to a violent dynamic of AOB communities.Further,these results suggest that certain populations may have been responsible for the operational success of the GSBR and were able to persist despite the large fluctuations in pop-ulation similarity.This bacterial population instability,coupled with a generally acceptable bioreactor performance,is congruent with the results obtained from a membrane biore-actor (MBR)for graywater treatment (Stamper et al.,2003).Nitrosomonas e like and Nitrosospira e like populations are the dominant AOB populations in wastewater treatment systems (Kowalchuk and Stephen,2001).A few previous studies revealed that the predominant populations in AOB communities are different in various wastewater treatment processes (Tawan et al.,2005;Thomas et al.,2010).Some researchers found that the community was dominated by AOB from the genus Nitrosospira in MBRs (Zhang et al.,2010),whereas Nitrosomonas sp.is the predominant population in biofilter sludge (Yin and Xu,2009).In the currentstudy,Fig.5e DGGE profile of the AOB communities in different size of granules (lane labels along the top show the range of particle diameter (d,mm)).Values along the bottom indicate the Shannon diversity index (H ).Bands labeled with the numbers were consistent with the bands in Fig.3.w a t e r r e s e a r c h x x x (2011)1e 107sequence analysis revealed that selection pressure evidently effect on the survival of Nitrosospira in granular sludge.Almost all of Nitrosospira were washed out initially and had no chance to evolve with the environmental changes.However,some members of Nitrosomonas sp.have been shown to produce more amounts of EPS than Nitrosospira ,especially under limited ammonia conditions (Stehr et al.,1995);and this feature has also been observed for other members of the same lineage.Accordingly,these EPS are helpful to communicate cells with each other and granulate sludge (Adav et al.,2008).Therefore,most of Nitrosomonas could adapt to this challenge (to become big and dense enough to settle fast)and were retained in the reactor.At the end of reactor operation (day 180),granules with different particle size were sieved.The effects of variation in granules size on the composition of the AOBcommunitiesFig.6e Micrographs of FISH performed on three size classes of granule sections.DAPI stain micrographs (A,D,G);AOB appear as green fluorescence (B,E,H),and NOB appear as red fluorescence (C,F,I).Bar [100m m in (A)e (C)and (G)e (I).d,particle diameter.(For interpretation of the references to colour in this figure legend,the reader is referred to the web version of this article.)w a t e r r e s e a r c h x x x (2011)1e 108were investigated.As shown in Fig.5,AOB communities structures in different size of granules were varied.Although several predominant bands(bands2,5,11)were present in all samples,only bands3and6appeared in the granules with diameters larger than0.6mm.Additionally,bands7and10 were intense in the granules larger than0.45mm.According to Table2,it can be clearly indicated that Nitrosospira could be retained merely in the granules larger than0.6mm.Therefore, Nitrosospira was not present at a high level in Fig.3due to the lower proportion of larger granules(d>0.6mm)in TSS along with reactor operation.DGGE analysis also revealed that larger granules had a greater microbial diversity than smaller ones. This result also demonstrates that more organisms can survive in larger granules as a result of more space,which can provide the suitable environment for the growth of microbes(Fig.6).4.3.Effect of variance in particle size on the distribution of AOB and NOB in granulesAlthough an influence of granule size has been observed in experiments and simulations for simultaneous N-and P-removal(de Kreuk et al.,2007),the effect of granule size on the distribution of different biomass species need be revealed further with the assistance of visible experimental results, especially in the same granular sludge reactors.Related studies on the diversity of bacterial communities in granular sludge often focus on the distribution of important functional bacteria populations in single-size granules(Matsumoto et al., 2010).In the present study,different size granules were sieved,and the distribution patterns of AOB and NOB were explored.In the nitrification processes considered,AOB and NOB compete for space and oxygen in the granules(Volcke et al.,2010).Since ammonium oxidizers have a higheroxygen affinity(K AOBO2<K NOBO2)and accumulate more rapidly inthe reactor than nitrite oxidizers(Volcke et al.,2010),NOB are located just below the layer of AOB,where still some oxygen is present and allows ready access to the nitrite produced.In smaller granules,the location boundaries of the both biomass species were distinct due to the limited existence space provided by granules for both microorganism’s growth.AOB exist outside of the granules where oxygen and ammonia are present.Medium granules can provide broader space for microbe multiplying;accordingly,AOB spread out in the whole granules.This result also confirms that oxygen could penetrate deep into the granule’s core without restriction when particle diameter is less than0.6mm.Some mathematic model also supposed that NOBs are favored to grow in smaller granules because of the higher fractional aerobic volume (Volcke et al.,2010).As shown in the results of the batch experiments(Zhang et al.,2011),nitrite accumulation temporarily occurred,accompanied by the more large gran-ules(d>0.9mm)forming.This phenomenon can be attrib-uted to the increased ammonium surface load associated with larger granules and smaller aerobic volume fraction,resulting in outcompetes of NOB.It also suggests that the core areas of large granules(d>0.9mm)could provide anoxic environment for the growth of anaerobic denitrificans(such as Tb.deni-trificans or Tb.thioparus in Fig.S7,Supporting information).As shown in Fig.2and Fig.S3,the removal efficiency of total nitrogen increased with formation of larger granules.5.ConclusionsThe variation in AOB communities’structure was remarkable during sludge inoculation,and the diversity index of pop-ulation decreased rapidly.Most of Nitrosomonas in the inocu-lating sludge were retained because of their capability to rapidly adapt to the settling e washing out action.DGGE anal-ysis also revealed that larger granules had greater AOB diversity than that of smaller ones.Oxygen penetration was not restricted in the granules of less than0.6mm particle diameter.However,the larger granules(d>0.9mm)can result in the smaller aerobic volume fraction and inhibition of NOB growth.Henceforth,further studies on controlling and opti-mizing distribution of granule size could be beneficial to the nitrogen removal and expansive application of granular sludge technology.AcknowledgmentsThis work was supported by grants from the National Natural Science Foundation of China(No.51108456,50908227)and the National High Technology Research and Development Program of China(No.2009AA06Z312).Appendix.Supplementary dataSupplementary data associated with this article can be found in online version at doi:10.1016/j.watres.2011.09.026.r e f e r e n c e sAdav,S.S.,Lee, D.J.,Show,K.Y.,2008.Aerobic granular sludge:recent advances.Biotechnology Advances26,411e423.APHA-AWWA-WEF,2005.Standard Methods for the Examination of Water and Wastewater,first ed.American Public Health Association/American Water Works Association/WaterEnvironment Federation,Washington,DC.de Bruin,L.M.,de Kreuk,M.,van der Roest,H.F.,Uijterlinde,C., van Loosdrecht,M.C.M.,2004.Aerobic granular sludgetechnology:an alternative to activated sludge?Water Science and Technology49,1e7.de Kreuk,M.,Heijnen,J.J.,van Loosdrecht,M.C.M.,2005.Simultaneous COD,nitrogen,and phosphate removal byaerobic granular sludge.Biotechnology and Bioengineering90, 761e769.de Kreuk,M.,Picioreanu,C.,Hosseini,M.,Xavier,J.B.,van Loosdrecht,M.C.M.,2007.Kinetic model of a granular sludge SBR:influences on nutrient removal.Biotechnology andBioengineering97,801e815.Downing,L.S.,Nerenberg,R.,2008.Total nitrogen removal ina hybrid,membrane-aerated activated sludge process.WaterResearch42,3697e3708.Erguder,T.H.,Boon,N.,Vlaeminck,S.E.,Verstraete,W.,2008.Partial nitrification achieved by pulse sulfide doses ina sequential batch reactor.Environmental Science andTechnology42,8715e8720.w a t e r r e s e a r c h x x x(2011)1e109。

地理WAT考试(国家部分)重点

地理WAT考试(国家部分)重点
年份 分值
考点
年份 分值 选择:8 2011 大题:11+7
2005 大题:7 选择:6 2006 大题:9 2007 大题:9
俄罗斯 巴西、美国 法国 美国
考点 澳大利亚、巴 西日本、法国 美国
2012 选择:4 2013 选择:8
俄罗斯 日本+澳大利 亚 俄罗斯+美国 美国 巴西 美国、埃及
选择:4 澳大利亚 大题:8+8 2008 大题:9+7 美国+日本、 2014 选择:4 选择:4 2009 大题:6 俄罗斯+巴 西 大题:6 2015 选择:6+4
(1)美国人口最多的城市,最大的港口,世界四大金融机构之 一 。美国首都 ,美国的钢铁工业中心 ;美国最大的 飞机制造基地 ; 美国汽车工业中心 ;美国交通中 心 ;新兴的宇航工业中心 。 城市___,这里的 是大规模的华人社区。以 为依托的 , (2)世界最大的高新技术产业区。 (3)美国 沿岸的最大港口城市和经济、文化中心 。享誉 世界的电影城 和 乐园都在这里。位于美国____工业区,
4.小麦、玉米和乳畜 带分布的特点(地 形和气候)
③ 4.图中显示美国现代农业的突出特点是 A.高度机械化 B.区域专业化 ⑦D.高度工业化 C.高度商品化
地形平坦, 光照充足, 有灌溉水源
6.美国是世界上出口粮食最多的国家,居世界 5.美国农业的显 前列的有_,_,_,_.但咖啡,可可,天然橡胶等 著特点?试分 热带产品需要大量进口原因___。 析其优势? 8、美国80%人口是__,有色人种主要是___。 , 美国人口____稠密()—原因—,_______稀疏。
⑦ F 西班牙

6.农业发展的有利条件? 小麦分布__?有利条件? 葡萄分布__?有利条件? 7.法国葡萄酒世界闻名原因 8.法国工业特点9.巴黎三大象征

恶性Wolffian附件肿瘤1例报道

恶性Wolffian附件肿瘤1例报道

甘肃医药2020年39卷第10期Gansu Medical Journal,2020,Vol.39,No.101病例资料患者,43岁,体检发现盆腔包块1年,伴下腹间断性下坠感1月,于2016年6月30日入院。

1年前在外院体检发现盆腔包块(直径约3.0cm),患者因月经正常、无腹痛等症,未予重视,未进一步检查。

1月前无明显诱因出现间断性小腹下坠感,就诊于我院,我院查超声:子宫多发肌瘤;左侧附件区囊实性肿物,大小5.5 cm×4.0cm,以囊性为主,囊内可见低回声团及条状分隔,边界尚清,未见明显血流信号。

盆腔MRI检查:左侧附件区可见囊实性肿物,约5.4cm×3.3cm×4.0cm大小,子宫体积增大,肌层内可见多发结节。

盆腔MRI诊断:①左侧附件区囊实性肿物,考虑恶性;盆腔少量积液。

②子宫多发肌瘤。

查肿瘤标志物:甲胎蛋白(AFP)、糖类抗原199(CA199)、癌胚抗原(CEA)、糖类抗原125(CA125)均正常。

妇科检查:子宫增大,扪及多个质硬肌瘤;左侧附件区肿物,直径5cm,质硬,活动,无触痛。

入院后于2016年7月6日在全身麻醉下行剖腹探查术,术中探查见:淡黄色腹水约70mL,子宫见多个肌瘤样肿物突起,如孕70余天大小;左侧卵巢常大,在输卵管系膜上有一囊实性肿物约5.5cm,表面光滑,无粘连,右侧附件未见明显异常;盆腹腔内余组织脏器未见明显异常,盆腔及腹主动脉旁淋巴结未扪及肿大。

切除左侧输卵管系膜肿物送快速冰冻示:恶性肿瘤,术中剖视标本:呈囊实性,切面灰白色,见少量浆液性液体,囊内未见明显乳头及结节。

遂行全子宫、双附件、大网膜、阑尾切除术+盆腔及腹主动脉旁淋巴结清扫术。

术中腹腔冲洗液未见恶性肿瘤细胞。

术后病理检查示:送检肿瘤主要排列呈管状结构和实性结构,细胞致密伴异型,核分裂象易见,结合免疫组化结果和部位提示输卵管系膜恶性Wolffian附件肿瘤,肿瘤大小5cm×4.5cm ×4.0cm,未累及周围组织。

松毛虫赤眼蜂两性生殖品系和孤雌产雌品系在不同品种柞蚕卵中的寄生和生长发育表现

松毛虫赤眼蜂两性生殖品系和孤雌产雌品系在不同品种柞蚕卵中的寄生和生长发育表现

http : ! www. insect. org. cndoi : 10.16380/j. kcb.2021.01.0091 月 Januay2021, 64(1): 80-89昆虫学报ACTAENTOMOLOGICASINICA松毛虫赤眼蜂两 殖品系和孤雌产雌品系在品种柞 中的寄生和生长 现周金成#,何 胡#,赵 倩,董 辉**基金项目:国家重点研发计划(2017YFD0201805)作者简介:周金成,男,1989年1月生,山东临沂人,博士,讲师,研究方向为害虫生物防治,E-mail : parasitoidsaasp @ 163. com ;何明,女,1995年3月生,辽宁沈阳人,硕士研究生,研究方向为害虫生物防治,E-m/1: 763429223@qq. com#共同第一作者 Authov with equal contribution* 通讯作者 Corresponding authcs, E-mail : biocontrol@ 163. com收稿日期 Received : 2020-07-07 ;接受日期 Accepted : 2020-08-T4(沈阳农业大学植物保护学院,沈阳110866)摘要:【目的】明确两性生殖i 系和孤雌产雌i 系松毛'赤眼蜂Trichogramma dendroliml 在不同i 种柞蚕Aetheroeo peroyi 卵中的寄生及发育表现,为以柞蚕卵为替代寄主更好地规模化繁育赤眼蜂提供依据。

【方法】测定两性生殖i 系和孤雌产雌i 系雌蜂在6个i 种柞蚕卵[抗大(KD )、大四(DS )、高新'GX )、988(NEE )、青大(QD )和特大(TD )]上的寄生率、子代蜂窝蜂数(单窝羽化子代 蜂数)和子代蜂雌性比等生物,以及6个i 种柞的质量指标(单 湿重、质含量、总糖含量和甘油三酯含量);利用主成分分析揭示柞蚕卵质量指标与赤眼蜂生物学指标间的相关性。

一种基于祖先序列重建的ω-转氨酶突变体[发明专利]

一种基于祖先序列重建的ω-转氨酶突变体[发明专利]

专利名称:一种基于祖先序列重建的ω-转氨酶突变体
专利类型:发明专利
发明人:黄俊,蔡婷婷,邱帅,吕常江,樊芳芳,李业,王丰,李元源申请号:CN202111482472.1
申请日:20211207
公开号:CN114134128A
公开日:
20220304
专利内容由知识产权出版社提供
摘要:本发明公开了一种基于祖先序列重建的ω‑转氨酶突变体,涉及分子生物学技术领域。

由来自土曲霉(Aspergillusterreus)的ω‑转氨酶突变所得,所述ω‑转氨酶突变体的氨基酸序列如SEQIDNO.4或SEQIDNO.6所示。

与野生型酶相比,ω‑转氨酶突变体的半衰期均在24h以上,而野生型仅为6.90min,突变体的半失活温度分别为49.00℃和49.03℃,比野生型(37.89℃)提高了约11℃,热稳定性显著提高。

申请人:浙江科技学院
地址:310023 浙江省杭州市西湖区留和路318号
国籍:CN
代理机构:杭州天勤知识产权代理有限公司
代理人:沈金龙
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生物学变异系数

生物学变异系数
2
11.7
29.9
5.9
8
17.7
α淀粉酶<胰腺>
18
尿
α-Amylase <pancreatic>
2
69.5
105
34.75
31.48
88.82
α淀粉酶<胰腺>
19
尿
α-Amylase concentration, random
1
94
46
47
26.2
103.7
α淀粉酶浓度,随机
20
血浆
α-Carotene
3
7.8
26.7
3.9
7
13.4
胶原蛋白C I型前肽<PICP>
95
血清
Collagen type I N propeptide <PINP>
3
7.4
57.3
3.7
14.4
20.5
胶原蛋白I型N前肽<PINP>
96
血清
Collagen type III N propeptide <PIIINP>
1
13.6
27.7
β-Carotene
59
血清
β-Carotene
4
36
39.7
18
13.4
43.1
β-胡萝卜素
60
血清
β-Cryptoxantin
1
36.7
---
18.4
---
---
β-Cryptoxantin
61
血清
β-Globulins
2
10.1

转腺病毒-人骨形态发生蛋白7软骨细胞分泌的透明质酸和Ⅱ型胶原

转腺病毒-人骨形态发生蛋白7软骨细胞分泌的透明质酸和Ⅱ型胶原

转腺病毒-人骨形态发生蛋白7软骨细胞分泌的透明质酸和Ⅱ型胶原张洁;刘巍;朱新辉;周怡【期刊名称】《中国组织工程研究》【年(卷),期】2011(015)028【摘要】BACKGROUND: Chondrocytes are limited in tissue engineering due to their poor self-dividing capacity and defifferentiation.OBJECTIVE: To investigate the expression of bone morphogenetic protein 7 (BMP-7) in chondrocytes transfected by adenovirus BMP-7 and the effects of transfection on chondrocyte secretion of type Ⅱ collagen and hyaluronic acid.METHODS: Adenoviral vector containing BMP-7 was prepared and then transfected into rabbit passage chondrocytes. BMP-7 mRNA and protein expressions in chondrocytes were detected. Changes in type Ⅱ collagen and hyaluronic acid in chondrocytes were determined.RESULTS AND CONCLUSION: At 48 and 72 hours after transfection, RT -PCR and western blot analysis showed that BMP-7 mRNA and protein expressions in the chondrocytes were increased; RT-PCR and ELISA showed the type Ⅱ collagen and hyaluronic acid in the chondrocytes were also obviously increased. These findings suggest that BMP-7 can be successfully transfected into chondrocytes by adenovirus and promote the secretion of type Ⅱ collagen and hyaluronic acid.%背景:软骨细胞自身分裂能力不强和去分化现象限制了其在组织工程中的应用.目的:观察腺病毒-骨形态发生蛋白7转染兔软骨细胞后骨形态发生蛋白7的表达及其对软骨细胞分泌Ⅱ型胶原和透明质酸功能的影响.方法:包装骨形态发生蛋白7腺病毒载体,将其转染至兔第2代软骨细胞.检测骨形态发生蛋白7 mRNA及蛋白的表达;检测软骨细胞中Ⅱ型胶原和透明质酸的变化.结果与结论:转染腺病毒-骨形态发生蛋白7后48,72 h,RT-PCR和Western blot方法显示软骨细胞表达的骨形态发生蛋白7 mRNA和蛋白均明显增加,RT-PCR和ELISA显示软骨细胞分泌的Ⅱ型胶原和透明质酸也显著增加.说明应用腺病毒可成功将骨形态发生蛋白7转染至兔软骨细胞,并能促进软骨细胞分泌Ⅱ型胶原和透明质酸.【总页数】4页(P5153-5156)【作者】张洁;刘巍;朱新辉;周怡【作者单位】南通大学医学院免疫系,江苏省南通市,226019;南通大学附属第二医院关节外科,江苏省南通市,226001;南通大学附属第二医院关节外科,江苏省南通市,226001;南通大学医学院免疫系,江苏省南通市,226019【正文语种】中文【中图分类】R318【相关文献】1.褪黑素对自身免疫性肝炎大鼠肝星状细胞透明质酸、Ⅲ型前胶原分泌及Ⅰ型胶原mRNA表达的抑制作用 [J], 马勇;丁体龙;袁福华;于莉;王莉;张文学2.利多卡因对体外培养软骨细胞分泌Ⅱ型胶原和蛋白多糖的影响 [J], 杨朝晖3.氟浓度对体外培养大鼠软骨细胞糖胺多糖和Ⅱ型胶原分泌的影响 [J], 范玉兰;王宁;施志冲;郑卫东;卢伟;肖萍4.透明质酸对体外培养大骨节病软骨细胞Ⅱ型胶原和聚集蛋白聚糖mRNA表达的影响 [J], 高宗强;郭雄;陈君长;段琛;马玮娟;刘瑞宇;顾其胜5.组织蛋白酶S对RA软骨细胞分泌Ⅱ型胶原的研究 [J], 赵进军;黄琴;任昊;欧阳晴晴;毋静;杨敏因版权原因,仅展示原文概要,查看原文内容请购买。

阔韧带Wolff管附件肿瘤的临床病理观察

阔韧带Wolff管附件肿瘤的临床病理观察

阔韧带Wolff管附件肿瘤的临床病理观察宋汉香;郭乔楠;唐雪峰【摘要】目的:探讨Wolffian管附件肿瘤( Wolfffian adnexal tumor,WAT)的临床病理学特征及免疫组化。

方法对1例发生于阔韧带的WAT进行临床病理特征分析和免疫组织化学观察。

结果肿瘤位于阔韧带,呈带蒂有包膜的囊实性肿块,组织学可见大小腺管紧密排列、筛状及实性区结构。

免疫组织化学显示肿瘤细胞pan-cytokeratin、Vimentin、CK18、CD99阳性表达, alpha-in-hibin灶性阳性;calretinin、CD10、EMA、ER、PR、Syn、CA15-3和CA19-9阴性,Ki-67指数小于5%。

PAS染色腺样结构周围基底膜阳性。

结论WAT主要依靠病理组织学、免疫组化和具有特征性的wolffian管残件结构诊断。

%Objective To explore the clinicopathological and immunohistochemical features of wolffian adnexal tumour ( WAT) . Meth-ods The clinical and pathological features analysis and immunohistochemistry methods were utilized to study the histopathology features of a case Wolffian adnexal tumor. Results One case of unilateral WAT which located in broad ligament display a solid-cystic mass with pedicled and enveloped,its histology showed large and small tubulars,screen structure and solid zone. Its immunohistochemistry staining was positive for pan-cytokeratin,vimentin,CK18 and CD99,positive staining for α-inhibin in a little tumor cell,and negative forcalretinin,CD10,EMA, ER,PR,Syn,CA15-3,CA19-9,Ki-67 index is less than 5%. PAS positive staining for the basement membrane around Gland like structure. Conclusion Wolffian adnexal tumour is diagnosised by itshistopathological,immuno histochemical and it’ s distinctive location where Wolffi-an duct remnants are found.【期刊名称】《局解手术学杂志》【年(卷),期】2015(000)003【总页数】3页(P277-278,279)【关键词】Wolffian管肿瘤;临床病理学特征;免疫组化;诊断及鉴别诊断【作者】宋汉香;郭乔楠;唐雪峰【作者单位】第三军医大学新桥医院病理科,重庆400037;第三军医大学新桥医院病理科,重庆400037;第三军医大学新桥医院病理科,重庆400037【正文语种】中文【中图分类】R361;R730.21Wolff 管附件肿瘤(Wolfffian adnexal tumor,WAT),亦称中肾管残件肿瘤,是一种具备中肾管分化特征的上皮性肿瘤,临床上罕见,因其临床特点与病理组织学形态的非特异性,很容易误诊。

苔藓植物生殖生态学研究

苔藓植物生殖生态学研究

第23卷第11期2003年11月生 态 学 报ACT A ECOLOGICA SINICA V ol.23,No.11N ov.,2003苔藓植物生殖生态学研究王中生1,安树青1,方炎明2(1.南京大学生命科学学院,南京 210093;2.南京林业大学森林资源与环境学院,南京 210037)基金项目:科技部973重点资助项目(2002CB111504);国家自然科学基金资助项目(30270110)收稿日期:2003-06-08;修订日期:2003-09-10作者简介:王中生(1972~),男,安徽桐城人,博士,讲师。

主要从事植物生态学及分子系统学研究。

E -mail :W an gzs @nju Foundation item :T he 973-project of the M inis try of S cience an d Tech nology (No.2002CB111504)an d the National Natu -ral S cience Foundation of China(No.30270110)Received date :2003-06-08;Accepted date :2003-09-10Biography :W ANG Zhong -Sh eng ,Ph .D .,mainly en gaged in th e res earch of plant ecology and molecular sys tematics .E -mail:Wangzs @摘要:近年来苔藓植物生殖生态学研究主要集中于繁育系统、生殖代价与对策,以及不同生殖方式对种群遗传变异的影响等方面。

生殖结构的原始性及其对水分的独特需求,以及雌雄异株比例较高等导致苔藓植物中有性生殖比例偏低;雌配子体很少完成整个有性生殖过程,其“真实的生殖代价”主要指雌性性表达(雌配子发生)的能耗,并且显著低于雄性性表达;基于对资源有效分配的生殖对策而导致雌性偏向及部分孢子体败育。

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2011年潍坊市初中学业水平考试生物试卷注意事项:1.本试题共8页,分Ⅰ、Ⅱ两卷。

答题时间为60分钟。

2.第Ⅰ卷为选择题,共50分;第Ⅱ卷为非选择题,共50分;全卷共100分。

3.答第Ⅰ卷前,需用蓝、黑色钢笔或圆珠笔将自己的姓名和准考证号填写在答题卡上,并用2B铅笔将自己的准考证号、试卷类型、学科填涂清楚。

第Ⅰ卷选择题(共50分)1.为探究温度与蟋蟀鸣叫之间是否存在联系,某同学将蟋蟀放在以温度为变量的环境中饲喂,并在同一时间段内统计蟋蟀的鸣叫次数,该研究过程主要采用了A.观察法 B.实验法C.测量法D.调查法2.在使用显微镜过程中,常有压碎装片的现象发生,下表是对不同学习小组装片压碎情况及原因的调查统计结果。

则通过此表归纳出的装片压碎原因应为:A.用低倍镜头、镜筒下落、粗准焦螺旋B.用低倍镜头、镜筒上升、细准焦螺旋C.用高倍镜头、镜筒下落、粗准焦螺旋 D.用高倍镜头、镜筒上升、细准焦螺旋3.细胞分裂能够产生新细胞,细胞分化可以形成不同的细胞群。

对此,以下描述正确的是A.刚分裂出来的细胞体积与母细胞相同B.所有的细胞长到一定程度后可再进行分裂C.细胞分裂过程中,细胞核内的遗传物质先进行复制D.所有细胞都能在遗传物质的控制下分裂、分化4.管理不善的玉米地里杂草丛生,会影响玉米的产量,这主要是因为A.杂草与玉米互利共生的结果B.杂草寄生在玉米上影响玉米生长C.杂草使玉米的品质受到影响D.杂草和玉米争夺生活条件的结果5.细心观察你会发现,我们周围植物体的形态结构各不相同。

以下植物有根、茎、叶分化的是A.海带 B.水绵 C.发菜D.蕨菜6.下列四组培养在不同实验条件下的相同植物,叶片气孔张开程度最大的应是组7.自然界中动物种类繁多,形态千差万别,请分辨出下列哪种描述是错误的A.鸟类的身体大都呈流线型,有与肺相通的气囊B.哺乳动物都是体表被毛,胎生C.鱼属于脊椎动物,体温会随温度的变化而改变D.蚯蚓身体分节,因此蚯蚓属于节肢动物8.人体的各种运动都是以一定的结构为基础。

以下有关人体结构的叙述,正确的是A.人体内所有的关节都是能够活动的B.关节由关节头和关节窝组成C.关节包括关节面、关节囊和关节腔D.肱二头肌属于肌肉组织9.在寿光的稻田一带,农户有家养慈伦鸡的习惯,这使得传统鸡种得以延续,科研人员利用这些鸡种,经过杂交培育出了适应现代需求的多个寿光大鸡新品种。

A.新品种的培育过程体现了遗传的多样性B..新品种的培育过程体现了生物种类的多样性C.鸡的所有行为都是在遗传因素的基础上,通过环境因素的作用而习得的D.家养的几只母鸡先后出现抱窝现象,这属于学习行为10.“超级细菌”是一种对绝大多数抗生素不再敏感的细菌,它的产生与人类滥用抗生素有关。

“超级细菌”的结构特点是A.没有成形的细胞核 B.同SARS病毒一样无细胞结构C.有同真菌一样的细胞结构 D.没有DNA11.“唾液对淀粉的消化实验”要求在一定温度下进行,这是由于唾液中的淀粉酶在高于和低于37℃条件下催化作用都会逐渐降低。

下图能正确表示唾液淀粉酶的作用与温度关系的是12.给婴幼儿接种脊髓灰质炎疫苗可预防脊髓灰质炎,下列有关该疫苗的说法正确的是A.对人体来说是抗原B.能直接杀死侵入人体的病原体C.对人体来说是抗体D.直接参与构成人体的第二道防线13.当有人不小心被生锈的铁钉扎伤后,医生会给伤者注射破伤风抗毒血清,使可能侵入体内的病原体直接失去致病性。

注射的物质和采取的措施分别是A.抗原、控制传染源B.抗原、保护易感者C.抗体、控制传染源D.抗体、保护易感者14.大棚栽培青州银瓜需进行人工辅助授粉,而在自然条件下银瓜属于A.自花传粉、主要靠昆虫传粉B.异花传粉、主要靠昆虫传粉C.异花传粉、主要靠风传粉D.自花传粉、主要靠风传粉15.为探究种子萌发的条件,有人选取若干绿豆种子平均分成4组,然后放在同一环境中分别按下列方式培养:①组:常温+湿润土壤;②组:冷藏+湿润土壤;③组:常温+干燥土壤;④组:常温+湿润土壤+隔绝空气。

以下说法正确的是A.①和②能验证温度对种子萌发的影响B.②和③能验证水分对种子萌发的影响C.②和④能验证空气对种子萌发的影响D.③和④能验证空气对种子萌发的影响16.参观寿光蔬菜博览会时会发现,有许多植物无土栽培在各种营养液中,这些植物吸收营养物质主要靠根尖的A.分生区B.伸长区C.成熟区D.除根冠外的所有区域17.以下是作物培育过程中所采取的繁殖方式,其中属于有性生殖的是A.利用枝芽繁殖安丘蜜桃B.利用实生苗繁殖培育板栗C.利用组织培养技术繁殖月季D.利用分根法培育菊花18.临朐家蚕属于完全变态发育昆虫,下列那种生物的发育过程与之相同A.蝗虫B.蟋蟀C.果蝇D.青蛙19.以下有关人类生殖与发育的叙述,不正确的是A.男性、女性的性腺分别是睾丸和卵巢B.子宫是女性最主要的性器官C.胎儿通过胎盘和脐带与母体完成物质交换D.受精卵是人类个体发育的起点20.一株基因组成为AA的玉米与一株基因组成为aa的玉米相互授粉,则这两株玉米所结的玉米粒A.胚的基因组成相同,种皮的基因组成不同B.胚的基因组成不同,种皮的基因组成相同C.胚的基因组成相同,种皮的基因组成相同D.胚的基因组成不同,种皮的基因组成不同21.美国科学家米勒等人所做的化学进化模拟实验,支持了生命起源过程的A.无机物→有机小分子阶段B.有机小分子→有机大分子阶段C.有机大分子→独立体系阶段D.独立体系→原始生命阶段22.下列各生物分类单位所包含的生物中,蛋白质分子相似程度最高的应是A.种B.目C.门D.纲23.从一位健康成年男性体内获得了一个精子和三种成熟的血细胞各一个,这四个细胞中含有的Y染色体数目最少可能是A.4条B.2条C.1条D.0条24.某同学在尝试“用鲜奶制作酸奶”和“用葡萄制作葡萄酒”的过程中遇到了一系列问题,其中分析正确的是A.制作酸奶需要密封而制作葡萄酒不需要密封B.制作酸奶和葡萄酒都需要在无氧的条件下进行C.制作酸奶用到一种微生物而制作葡萄酒用到两种微生物D.制作酸奶和葡萄酒用到的“菌”都能进行孢子生殖25.低温以及适当控制氧气和二氧化碳浓度可以延长果蔬的贮藏期,这主要因为该环境A.能使呼吸作用旺盛B.能使呼吸作用减弱C.能够调节光合作用强度D.使细菌等微生物不能生存A.分生区B.伸长区C.成熟区D.除根冠外的所有区域17.以下是作物培育过程中所采取的繁殖方式,其中属于有性生殖的是A.利用枝芽繁殖安丘蜜桃B.利用实生苗繁殖培育板栗C.利用组织培养技术繁殖月季D.利用分根法培育菊花18.临朐家蚕属于完全变态发育昆虫,下列那种生物的发育过程与之相同A.蝗虫B.蟋蟀C.果蝇D.青蛙19.以下有关人类生殖与发育的叙述,不正确的是A.男性、女性的性腺分别是睾丸和卵巢B.子宫是女性最主要的性器官C.胎儿通过胎盘和脐带与母体完成物质交换D.受精卵是人类个体发育的起点20.一株基因组成为AA的玉米与一株基因组成为aa的玉米相互授粉,则这两株玉米所结的玉米粒A.胚的基因组成相同,种皮的基因组成不同B.胚的基因组成不同,种皮的基因组成相同C.胚的基因组成相同,种皮的基因组成相同D.胚的基因组成不同,种皮的基因组成不同21.美国科学家米勒等人所做的化学进化模拟实验,支持了生命起源过程的A.无机物→有机小分子阶段B.有机小分子→有机大分子阶段C.有机大分子→独立体系阶段D.独立体系→原始生命阶段22.下列各生物分类单位所包含的生物中,蛋白质分子相似程度最高的应是A.种B.目C.门D.纲23.从一位健康成年男性体内获得了一个精子和三种成熟的血细胞各一个,这四个细胞中含有的Y染色体数目最少可能是A.4条B.2条C.1条D.0条24.某同学在尝试“用鲜奶制作酸奶”和“用葡萄制作葡萄酒”的过程中遇到了一系列问题,其中分析正确的是A.制作酸奶需要密封而制作葡萄酒不需要密封B.制作酸奶和葡萄酒都需要在无氧的条件下进行C.制作酸奶用到一种微生物而制作葡萄酒用到两种微生物D.制作酸奶和葡萄酒用到的“菌”都能进行孢子生殖25.低温以及适当控制氧气和二氧化碳浓度可以延长果蔬的贮藏期,这主要因为该环境A.能使呼吸作用旺盛B.能使呼吸作用减弱C.能够调节光合作用强度D.使细菌等微生物不能生存第Ⅱ卷非选择题(共50分)26.(8分)请同学们根据自己掌握的安全保护方法,回答下列在学习、生活过程中遇到的实际问题:(1)请你帮助有关部门找出路边食品摊点可能存在的主要食品安全问题(2)在体育运动、野外科学探究活动过程中,若有人发生大静脉或动脉出血,正确的处理方法是(3)当我们在学习、生活过程中遇到心理矛盾、困难和挫折时,正确的做法应该是:(4)青少年沉迷于网络或电子游戏不但会影响学业,而且因长时间近距离注视屏幕会导致眼球中且不能恢复原状而形成近视眼。

(5)为确保用药安全,在服药前应弄清药品说明书上的各项信息,你所知道的信息有:(写出三项)。

27.以下是与人体生理活动相关的一系列问题,请根据所学的生物学知识作出解释:(1)人体内的气体交换包括两个过程,分别是。

(2)给病人输液时,一般采用静脉注射,这是因为静脉一般分布在身体的表层,并且在静脉中有防止血液倒流的;血液中运输氧气的是。

(3)当人的尿液中被检验出红细胞和蛋白质时,说明通透性增大。

(4)原尿中的葡萄糖,经肾小管的重吸收作用回到毛细血管中,需穿过层细胞膜。

(5)请将右图A、B、C、D四条血管与心脏上相应的血管连接起来。

28.(9分)阅读下列文字,回答相关问题:2010年2月,我国科学家首次选育出“抗性淀粉”、蛋白质、膳食纤维含量高的水稻新品种──“降糖稻1号”。

“抗性淀粉”和普通淀粉相比,难以在消化道内降解成葡萄糖,不会使血糖过快升高。

科学家利用糖尿病模型大鼠进行的相关动物试验证明,该稻米对于保持体重稳定增长、降低血糖有明显效果。

(1)上述提到的各种营养物质中,构成组织细胞的基本物质是。

被称为“第七营养素”的是。

(2)在人体消化道内,淀粉变成葡萄糖的过程称为。

人体吸收葡萄糖主要器官的结构特点是。

(3)糖尿病是人类的常见病,产生该病的主要原因是。

(4)进行动物试验是验证“降糖稻1号”降糖功效的重要环节,请你利用上述模型大鼠还原科学家的简要实验步骤:29.(8分)以下是有关人体生命活动调节方面的问题,请分析回答(1)人体的各项生理活动之所以能够协调、统一,主要是通过的调节作用共同完成。

(2)呆小症和侏儒症患者身材都很矮小,二者分别是由于幼年时期分泌不足造成的。

(3)神经元是一种高度特化的细胞,该细胞的集中形成脊髓的灰质和大脑半球表层的灰质;脊髓的白质和大脑皮层以内的白质是由汇集而成。

(4)当你沿海岸散步,忽然踩到一只尖锐的贝壳,你的脚会先自动弹起然后才感觉到疼痛,该过程证明脊髓具有功能。

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