Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions

Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.Many dsDNA viruses, including tailed phages and herpes viruses, initially assemble a DNA-free procapsid with assistance of a network of scaffold proteins. Accompanying the exit of scaffolding proteins during subsequent ATP-driven DNA packaging, the icosahedral shell of the procapsid undergoes dramatic conformational changes and matures into a typically larger and more angular shell of the infectious phage (1–6). However, structural details, including those of capsid intermediates, are limited to the phage HK97 system (5, 7–9), for which recombinantly produced procapsid and nonphysiological conversion products were analyzed.The packaging of the 39.937-kbp DNA genome of the short-tail Escherichia coli bacteriophage, T7, is a model for understanding basic principles common to dsDNA tailed phages and herpes viruses. The T7 system is also of interest because it has been used for popular biotechnologies, such as recombinant protein expression (10) and protein display on the capsid surface (11). The T7 capsid contains 415 copies of the major shell protein gp10 (12) that form a T = 7L icosahedral lattice. From low-resolution cryo-EM 3D reconstructions the tertiary topology of gp10 can be divided into four regions: N-arm, E-loop, A-domain, and P-domain, which together place the gp10 protein in the HK97 fold category (2, 13, 14). The T7 procapsid, capsid I, contains 110–140 molecules of scaffolding protein, gp9 (4, 15, 16). After scaffolding protein expulsion the spherical T7 capsid I expands to more angular intermediates, which are collectively called capsid II (2, 4, 14, 16–18).Two DNA-free capsid IIs are purified in quantity sufficient for structural studies by cryo-EM (16). Both are produced during the normal process of wild-type T7 DNA packaging in vivo. One has an unusually low density during buoyant density centrifugation in a metrizamide density gradient (1.086 g/mL; metrizamide low density, or MLD, capsid II) and the other has a density as expected for hydrated proteins (1.28 g/mL; metrizamide high density, or MHD, capsid II) (16). The low density of MLD capsid II is caused by impermeability to metrizamide (789 Da) (16). The MLD capsid II particles are produced before MHD capsid II particles based on kinetic studies (16).The DNA packaging of T7 phage starts at capsid I state where the DNA is packaged by the ATPases (gp18 and gp19) to pass through the portal (gp8) apparatus (19). By analyzing kinetics of in vivo-produced capsids, MLD capsid II was found to be the first postcapsid I capsid. MLD capsid II appears with the kinetics of an intermediate (16) but is obviously no longer in the DNA packaging pathway because it has detached from the DNA molecule that it was packaging. MLD capsid II is not produced when a nonpermissive host is infected with a T7 amber mutant defective in DNA packaging (summarized in ref. 16). Thus, MLD capsid II is an intermediate that has been altered during either cellular lysis or subsequent purification. MHD capsid II also has the appearance kinetics of an intermediate of packaging, but one that occurs later (16). Whereas MLD capsid II has the internal core stack including proteins gp8, gp14, gp15, and gp16 (16), MHD capsid II does not have the internal core stack proteins, which were presumably lost when packaged DNA exited the capsid (16).The existence of these various capsids provides an opportunity to obtain a high-resolution (3–4 Å) analysis of structural dynamics that occur in vivo. Here we report cryo-EM structures of the shells of the following bacteriophage T7 capsids: capsid I (4.6 Å), MLD capsid II (3.5 Å), MHD capsid II (6.6 Å), and phage (3.6 Å). The two capsid II shells are the first postprocapsid, in vivo-generated shells (for any packaging system) to be subjected to high-resolution structural analysis, to our knowledge. The results reveal (i) an HK97-fold shell protein with an intracapsomere, noncovalent topological linking and another intercapsomere, joint interaction, neither interaction having been found for other dsDNA tailed phages; (ii) details of the interaction of gp9 scaffolding protein with the inner surface of the capsid I shell; (iii) a novel refolding and externalization of the N terminus of major capsid protein, gp10; and (iv) a subtle, surprising contraction of the gp10 shell in transit from MLD capsid II to phage. Based on these observations, we propose a general procapsid assembly and maturation pathway for dsDNA viruses.     

微创拔牙后延期种植与即刻种植的效果评价

目的 探讨和比较微创拔牙后延期种植与即刻种植的临床效果。方法 选择南京同仁医院2013年4月—2018年4月收治的上、下颌单个前牙及前磨牙需要行微创拔牙后种植牙的患者86例,随机分为对照组（40例）和实验组（46例）。对照组微创拔牙后进行延期种植,实验组微创拔牙后进行即刻种植。比较2组患者的种植成功率,记录3个月和永久修复当天患者的种植体稳定性、种植体周围探诊深度、美学效果以及种植体边缘骨水平以及术后满意度。采用SPSS 20.0软件包对数据进行统计学分析。结果 实验组和对照组种植成功率均为100%,差异无统计学意义（P>0.05）;2组在种植体植入3个月后和永久修复当天的ISQ值差异不显著 (P>0.05);实验组种植体周围探诊深度小于对照组,但差异无显著性（P=0.80）;实验组总满意度显著大于对照组（P=0.044）;2组修复后1年,牙龈乳头指数均在1~3级,软组织外形良好,均达到了良好的牙龈美学效果,2组差异无统计学意义（P=0.66）;对照组PES得分7.65±1.32,实验组为8.25±1.19,2组比较差异显著（P<0.05）;2组的近中侧骨吸收量和远中侧骨吸收量相比无显著差异（P>0.05）。结论 微创拔牙后2种植修复方式均能获得良好的临床效果,种植体都具有良好的稳定性,但即刻种植的满意度更高,美学效果更佳,具有良好的临床应用前景。     

Pharmacological inhibition of TLR4/NF‐κB with TLR4‐IN‐C34 attenuated microcystin‐leucine arginine toxicity in bovine Sertoli cells

This study investigated the pharmacological inhibition of the toll‐like receptor 4 (TLR4) genes as a measure to attenuate microcystin‐LR (MC‐LR) reproductive toxicity. Bovine Sertoli cells were pretreated with TLR4‐IN‐C34 (C34) for 1 hour. Thereafter the pretreated and non‐pretreated Sertoli cells were cultured in medium containing 10% heat‐activated fetal bovine serum + 80 μg/L MC‐LR for 24 hours to assess the ability of TLR4‐IN‐C34 to attenuate the toxic effects of MC‐LR. The results showed that TLR4‐IN‐C34 inhibited MC‐LR‐induced mitochondria membrane damage, mitophagy and downregulation of blood‐testis barrier constituent proteins via TLR4/nuclear factor‐kappaB and mitochondria‐mediated apoptosis signaling pathway blockage. The downregulation of the mitochondria electron transport chain, energy production and DNA replication related genes (mt‐ND2, COX‐1, COX‐2, ACAT, mtTFA) and upregulation of inflammatory cytokines (interleukin [IL]‐6, tumor necrosis factor‐α, IL‐1β, interferon‐γ, IL‐4, IL‐10, IL‐13 and transforming growth factor β1) were modulated by TLR4‐IN‐C34. Taken together, we conclude that TLR4‐IN‐C34 inhibits MC‐LR‐related disruption of mitochondria membrane, mitophagy and downregulation of blood‐testis barrier proteins of the bovine Sertoli cell via cytochrome c release and TLR4 signaling blockage.     

美国和日本医疗器械数据库系统构建思路探析

目的：探讨医疗器械监管先行国家的数据库系统建设思路,为我国医疗器械科学监管提供借鉴和参考。方法：分析美国和日本医疗器械数据库的功能、组织结构、数据信息相关联情况,进而探讨其数据库系统的构建思路,对比分析不同数据库的优缺点。结果与结论：两国数据库的构建基于其监管实践,注重数据库系统各层级的分工与互联。综合考虑我国的监管需求和信息化建设实际情况,建议从整体设计方面参考美国数据库分数据库独立建设,加强互联互通,实现数据库之间或数据库系统内部的有效联通。日本在构建数据库过程中,在语言和监管思路两方面兼顾国际接轨、注重监管要求的长期稳定性的思路和做法,也值得我国借鉴。     

HPLC法测定注射用冷冻干燥用卤化丁基橡胶塞中抗氧剂1010及硫化剂在药液中的迁移

目的：建立HPLC方法,考察药用卤化丁基橡胶塞中的抗氧剂、硫化剂及其在药液中的迁移情况。方法：采用Sunfire-C18（4.6 mm×150 mm,5 μm）色谱柱;流动相：乙腈-四氢呋喃-水（6：3：1）;流速：1.5 mL·min^-1;检测波长：280 nm;柱温：35℃;进样量：20 μL。结果：抗氧剂1010及硫化剂硫磺在浓度0.004~0.5 mg·mL^-1具有良好的线性关系（r ≥ 0.9996）。抗氧化剂1010及硫化剂硫磺提取试验回收率范围分别是102.05%~111.55%及91.75%~94.83%;抗氧化剂1010及硫化剂硫磺迁移试验回收率范围分别为98.92%~110.70%及98.68%~106.63%;各回收率试验RSD均不大于5%（n=9）。结论：本方法灵敏、准确、可靠,可用于药用卤化丁基橡胶塞中抗氧剂及硫化剂的提取及迁移研究。     

肝源性糖尿病临床特征及治疗

目的研究探讨肝源性糖尿病临床特征及治疗方法。方法对58例肝源性糖尿病患者的临床表现、肝功能及血糖检测结果进行探讨分析(实验组),并从同期患者中随机抽取58例原发性2型糖尿病患者作为对照组。两组患者均实施了胰岛素、C肽释放及OGTT测定。结果所有肝源性糖尿病患者均出现了不同程度的腹胀、乏力、纳差等典型的肝病特征,但只有4例出现"三多一少"等糖尿病典型症状。肝源性糖尿病患者空腹血糖含量的平均值为(6.9±2.5)mmol/L,饮食后2 h血糖含量的平均值为(12.9±2.7)mmol/L。OGTT结果的比较表明肝源性糖尿病患者在空腹时血糖水平比原发性2型糖尿病低,且两组间差异具有统计学意义(P0.05),但用餐后1、2、3 h两组患者血糖水平无显著性差异。胰岛素释放结果及C肽释放水平的比较表明,肝源性糖尿病患者在任何时间段均比对照组高,且两组间差异具有统计学意义(P0.05)。结论肝源性糖尿病的临床症状表现不典型,主要特征为饮食后高血糖,治疗该病的首选药物为胰岛素。