A polycatenated DNA scaffold for the one-step assembly of hierarchical nanostructures

A unique DNA scaffold was prepared for the one-step self-assembly of hierarchical nanostructures onto which multiple proteins or nanoparticles are positioned on a single template with precise relative spatial orientation. The architecture is a topologically complex ladder-shaped polycatenane in which the "rungs" of the ladder are used to bring together the individual rings of the mechanically interlocked structure, and the "rails" are available for hierarchical assembly, whose effectiveness has been demonstrated with proteins, complementary DNA, and gold nanoparticles. The ability of this template to form from linear monomers and simultaneously bind two proteins was demonstrated by chemical force microscopy, transmission electron microscopy, and confocal fluorescence microscopy. Finally, fluorescence resonance energy transfer between adjacent fluorophores confirmed the programmed spatial arrangement between two different nanomaterials. DNA templates that bring together multiple nanostructures with precise spatial control have applications in catalysis, biosensing, and nanomaterials design.     

Cell- and stimulus-dependent heterogeneity of synaptic vesicle endocytic recycling mechanisms revealed by studies of dynamin 1-null neurons

Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a phosphoinositide phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.     

Fabrication of ZnPc/protein nanohorns for double photodynamic and hyperthermic cancer phototherapy

Multifunctionalization of carbon nanotubules is easily achieved by attaching functional molecules that provide specific advantages for microscopic applications. We fabricated a double photodynamic therapy (PDT) and photohyperthermia (PHT) cancer phototherapy system that uses a single laser. Zinc phthalocyanine (ZnPc) was loaded onto single-wall carbon nanohorns with holes opened (SWNHox), and the protein bovine serum albumin (BSA) was attached to the carboxyl groups of SWNHox. In this system, ZnPc was the PDT agent, SWNHox was the PHT agent, and BSA enhanced biocompatibility. The double phototherapy effect was confirmed in vitro and in vivo. When ZnPc-SWNHox-BSA was injected into tumors that were subcutaneously transplanted into mice, the tumors almost disappeared upon 670-nm laser irradiation. In contrast, the tumors continued to grow when only ZnPc or SWNHox-BSA was injected. We conclude that carbon nanotubules may be a valuable new tool for use in cancer phototherapy.     

冠状动脉支架研究

治疗心血管狭窄的冠状动脉支架白出现以来得到不断的发展。在回顾支架研制发展过程的基础上，从支架材料选择、支架结构设计和力学性能分析以及支架的加工与测试等方面系统分析了理想的支架具有的特性。对实现支架关键技术特性所涉及的显著影响再狭窄的支架杆厚度和支架侧窗设计、管状切割支架制作过程中主要工艺参数以及预防再狭窄功能化支架设计等方面进行了分析。由于体外试验难以准确反映体内环境，有限元分析的方法为支架设计验证和改进提供了有效的手段。讨论了使用药物洗脱支架的优点和其内在的局限性，介绍了治疗再狭窄的新的支架设计。最后描述了作者所在实验室率先提出的利用介电泳的原理实现载药纳米颗粒白组装于裸支架的新的载药方式。     

Au纳米颗粒HBV DNA基因探针的制备及其在目视化检测HBV DNA中的应用

制备金(gold,Au)纳米颗粒乙型肝炎病毒脱氧核糖核酸(hepatitis B virus deoxynucleic acid,HBV DNA)基因探针,研究其在目视化检测HBV DNA中的应用。Au纳米颗粒通过金-硫(Au-S)共价键结合烷氢硫醇修饰的寡核苷酸,制备检测探针。用荧光标记法检测Au纳米颗粒表面寡核苷酸的覆盖率和与其互补的寡核苷酸的杂交效率。检测探针与固定在尼龙膜上的捕捉探针构成双探针,用斑点杂交法检测HBV DNA,加入银离子(Ag+)-对苯二酚液染色观察结果。制备的Au纳米颗粒粒径为(12±5)nm,分散良好,在520nm有最大吸收峰,经寡核苷酸修饰后,最大吸收峰改变为524nm。Au纳米颗粒表面寡核苷酸的最大覆盖率为(132±10)条,最大杂交效率为(22±3)%。在尼龙膜上用斑点杂交法可检出低至10fmol的合成靶DNA,可目视化检出乙肝患者血清中HBV DNA的PCR产物。Au纳米颗粒HBV DNA基因探针可用于目视化检测HBV DNA,此方法具有敏感性高、特异性好、简单、价廉的特点,可望在许多领域,尤其是在多基因检测芯片上有潜在的应用价值。     

Nonciliated cells of the rat efferent ducts endocytose testicular sulfated glycoprotein-1 (SGP-1) and synthesize SGP-1 derived saposins

Sertoli cell sulfated glycoprotein-1 (SGP-1) is a heavily glycosylated and sulfated 70 kDa protein that is secreted into the lumen of the seminiferous tubule where it binds to spermatozoa. Recent light and electron microscope immunocytochemistry has suggested that the testicular SGP-1 detaches from the surface of spermatozoa in the lumen of the efferent ducts to be endocytosed within the endocytic apparatus of the epithelial nonciliated cells. The finding of SGP-1 mRNA together with anti-SGP-1 immunogold labeling of the lysosomal compartment suggest that these cells synthesize an efferent duct form of SGP-1. In the present study, a number of different experimental approaches (ligation, tunicamycin treatment and a combination of both) in combination with quantitative electron microscope immunogold labeling and Western blot analysis were performed in order to test this hypothesis. The number of gold particles and the profile area of the early (endosomes, pale multivesicular bodies) and late (dense multivesicular bodies, secondary lysosomes) endocytic apparatus were estimated in each of the experimental groups and expressed as the number of gold particles per μm² (labeling densities). The data revealed that ligation produced a significant reduction of anti-SGP-1 immunogold labeling of the early endocytic apparatus but not of the late endocytic apparatus. Tunicamycin treatment on the other hand produced a significant reduction of immunogold labeling of both the early and late endocytic apparatus. The combination of both treatments resulted in a more effective reduction of the labeling densities of these two endocytic compartments. These results thus indicate that the nonciliated cells of the efferent ducts are involved both in the endocytosis of the Sertoli-derived SGP-1 and in the synthesis of an efferent duct form of SGP-1 that is targeted from the Golgi apparatus to secondary lysosomes after its glycosylation. In order to determine the biosynthetic pathway of SGP-1 within the efferent ducts, an I. V. injection of ³⁵S-cysteine followed by immunoprecipitation and SDS-PAGE revealed that SGP-1 was initially biosynthesized as a 55 kDa protein. This protein appears to be post-translationally modified to a 65 kDa form after 1 hour, which preceded the appearance of the 70 kDa form, and smaller peptides of about 15 kDa characteristic of saposins after 3–4 hours. Western blot analysis of ligated efferent ducts showed an increase in the biosynthesis of the 70 kDa form of SGP-1 when compared to untreated controls, however, it has yet to be established if this protein is secreted or retained in an intracellular compartment. These results thus further substantiate our hypothesis on the endocytosis of the Sertoli-derived SGP-1 by the nonciliated cells and the synthesis of an efferent duct form of SGP-1 as well as provide evidence for the presence of 15 kDa saposins and its 65 kDa precursor in secondary lysosomes of these cells. © 1993 Wiley-Liss, Inc.     

纯钛表面载阿司匹林微球与聚多巴胺复合涂层促进成骨分化北大核心

背景:钛作为骨替代材料已在口腔种植领域中得到广泛应用,但其生物惰性会影响植入早期与骨组织形成稳定的结合,因此探索通过表面改性来提高钛的成骨活性是很有必要的。目的:探讨钛表面载阿司匹林的壳聚糖微球与聚多巴胺复合涂层对体外成骨细胞活性的影响。方法:取纯钛片,表面分别构建聚多巴胺涂层和载阿司匹林的壳聚糖微球与聚多巴胺复合涂层,采用扫描电镜和接触角检测对改性前后钛片的表面微观形貌和亲水性进行表征,检测纯钛表面阿司匹林纳米微球涂层的体外缓释性能。将大鼠骨髓间充质干细胞分别接种于纯钛片与两种改性钛片上培养,采用细胞骨架染色观察钛片表面细胞的铺展形态,CCK-8实验测定细胞的增殖活性,碱性磷酸酶染色和免疫荧光染色评估钛片表面细胞的成骨分化能力。结果与结论:①扫描电镜显示,纯钛表面相对光滑,聚多巴胺改性后出现沉积物及颗粒状突起,阿司匹林微球呈圆球形且粒径分布均匀;聚多巴胺涂层组与阿司匹林微球涂层组钛片的亲水性均明显优于纯钛组(P<0.05);阿司匹林在微球的包裹下呈现缓慢持续释放;②细胞骨架染色显示,纯钛表面的细胞伸展不充分,聚多巴胺涂层组钛片表面的细胞伸出少量伪足,阿司匹林微球涂层组钛片表面的细胞伸展良好;③CCK-8实验结果显示,3组钛片均无明显细胞毒性,且随着细胞培养时间的延长,阿司匹林微球涂层组钛片表面的细胞增殖速率高于其他两组(P<0.05);④阿司匹林微球涂层组钛片表面细胞的碱性磷酸酶活性最高,免疫荧光显示该组细胞中成骨相关蛋白碱性磷酸酶的荧光强度最强;⑤结果表明,纯钛表面载阿司匹林的壳聚糖微球缓释涂层可以增强大鼠骨髓间充质干细胞的增殖和黏附,并促进其成骨向分化。