Biochemical characterization of the Helicobacter pylori Cag Type 4 Secretion System protein CagN and its interaction partner CagM

Highly virulent Helicobacter pylori strains contain the cag pathogenicity island (cagPAI). It codes for about 30 proteins forming a type IV secretion system (T4SS) which translocates the pro-inflammatory protein CagA into epithelial host cells. While CagA and various other Cag proteins have been extensively studied, several cagPAI proteins are poorly characterized or of unknown function. CagN (HP0538) is of unknown function but highly conserved in the cagPAI suggesting an important role. cagM (HP0537) is the first gene of the cagMN operon and its product is part of the CagT4SS core complex. Both proteins do not have detectable homologs in other type IV secretion systems. We have characterized the biochemical and structural properties of CagN and CagM and their interaction. We demonstrate by circular dichroism, Multi-Angle Light Scattering (MALS) and small angle X-ray scattering (SAXS) that CagN is a folded, predominantly monomeric protein with an elongated shape in solution. CagM is folded and forms predominantly dimers that are also elongated in solution. We found by various in vivo and in vitro methods that CagN and CagM directly interact with each other. CagM self-interacts stably with a low nanomolar K_D and can form stable multimers. Finally, in vivo experiments show that deletion of CagM reduces the amounts of CagN and other outer CagPAI proteins in H. pylori cells.     

Microglia contributes to plaque growth by cell death due to uptake of amyloid β in the brain of Alzheimer's disease mouse model

Pathological hallmarks of Alzheimer's disease (AD) include extracellularly accumulated amyloid β (Aβ) plaques and intracellular neurofibrillary tangles in the brain. Activated microglia, brain‐resident macrophages, are also found surrounding Aβ plaques. The study of the brain of AD mouse models revealed that Aβ plaque formation is completed by the consolidation of newly generated plaque clusters in vicinity of existed plaques. However, the dynamics of Aβ plaque formation, growth and the mechanisms by which microglia contribute to Aβ plaque formation are unknown. In the present study, we confirmed how microglia are involved in Aβ plaque formation and their growth in the brain of 5XFAD mice, the Aβ‐overexpressing AD transgenic mouse model, and performed serial intravital two‐photon microscopy (TPM) imaging of the brains of 5XFAD mice crossed with macrophage/microglia‐specific GFP‐expressing CX3CR1^GFP/GFP mice. We found that activated microglia surrounding Aβ plaques take up Aβ, which are clusters developed inside activated microglia in vivo and this was followed by microglial cell death. These dying microglia release the accumulated Aβ into the extracellular space, which contributes to Aβ plaque growth. This process was confirmed by live TPM in vivo imaging and flow cytometry. These results suggest that activated microglia can contribute to formation and growth of Aβ plaques by causing microglial cell death in the brain. GLIA 2016;64:2274–2290     

Using Geographic Information Systems to investigate variations in accessibility to ‘extended hours’ primary healthcare provision

There are ongoing policy concerns surrounding the difficulty in obtaining timely appointments to primary healthcare services and the potential impact on, for example, attendance at accident and emergency services and potential health outcomes. Using the case study of potential access to primary healthcare services in Wales, Geographic Information System (GIS)‐based tools that permit a consideration of population‐to‐provider ratios over space are used to examine variations in geographical accessibility to general practitioner (GP) surgeries offering appointment times outside of ‘core’ operating hours. Correlation analysis is used to explore the association of accessibility scores with potential demand for such services using UK Population Census data. Unlike the situation in England, there is a tendency for accessibility to those surgeries offering ‘extended’ hours of appointment times to be better for more deprived census areas in Wales. However, accessibility to surgeries offering appointments in the evening was associated with lower levels of working age population classed as ‘economically active’; that is, those who could be targeted beneficiaries of policies geared towards ‘extended’ appointment hours provision. Such models have the potential to identify spatial mismatches of different facets of primary healthcare, such as ‘extended’ hours provision available at GP surgeries, and are worthy of further investigation, especially in relation to policies targeted at particular demographic groups.     

右上腹瘢痕粘连下左上腹两新入路腹腔镜胆囊切除术的改进策略

目的右中上腹腹膜炎、大切口手术及放疗后,腹腔镜下见右上腹广泛瘢痕粘连,在解离粘连、建立胆囊切除空间失败后,改从左上腹入路胆囊切除术,依具体情况,探讨建立器械通道和操作空间的可能性和实用性。方法自2001年5月-2015年5月有13例患者(有右上腹腹膜炎、大切口手术、放疗史)例行腹腔镜胆囊切除术(LC),术中发现右上腹广泛粘连,常规LC失败后,改从左上腹肝下脏面入路和肝镰状韧带入路建立手术器械通道,并沿肝脏脏面从上向下、从内向外解剖而建立胆囊切除空间,施行LC术。结果手术时间为70~155 min,平均117 min。13例有右上腹瘢痕粘连的病例,近创伤处粘连重远创则轻,经左上腹两条入路巧妙避开难以分离的瘢痕粘连面,均成功进行探查及切除术,建立器械通道及切除空间顺利,除手术时间较长外,无胃肠、胆管损伤。结论与距离呈负相关是后天性右上腹瘢痕粘连的特点,改从左上腹肝下脏面入路和肝镰状韧带入路,可避开瘢痕粘连轻松建立胆囊LC手术通道;再沿肝下及于胆囊周围由上向下和内侧向外建立胆囊切除空间,两条路径LC为切实可行的手术路径。     

两种内镜下放置小肠减压管的对比研究及临床应用价值

目的比较两种内镜下放置小肠减压管的置管成功率、操作时间、并发症发生率及置管时患者的耐受程度。方法根据入院时间将68例应用经鼻小肠减压管治疗的肠道梗阻患者随机分为两组,即A组和B组,每组34例。A组患者采用在胃镜直视下置管方法,B组患者采用鼻胃镜导丝引导置入法。结果两组患者在置管成功率及并发症发生率方面差异无统计学意义(P0.05),在置管操作时间上差异有统计学意义(P0.05),两组患者置管时耐受程度比较上差异有统计学意义(P0.01)。结论两种内镜下放置小肠减压管方法都有临床应用价值,在操作时间、患者置管时的耐受程度以及特殊患者置管成功率上鼻胃镜导丝引导置入法有明显的优势。     

Neurofibromatosis 2 with peripheral neuropathies: Electrophysiological,pathological and genetic studies of a Taiwanese family

The objective of this study was to assess peripheral nerve involvement and DNA mutation of the neurofibromatosis type 2 (NF2) gene (NF2) in a Taiwanese family with classic NF2. Eleven members (six symptomatic and five asymptomatic) of a family carrying NF2 underwent clinical examination, neuroimaging, and electrophysiological analysis. Mutation and linkage analyses were conducted on DNA samples prepared from peripheral blood (all individuals), a sural nerve biopsy specimen (one symptomatic member), and a tumor specimen (another symptomatic member). Six of the 11 members were diagnosed with classic NF2. DNA sequencing of the tumor specimen demonstrated a frameshift mutation with 756delC on exon 8 of NF2. Three affected subjects showed clinical variability of the neuropathic disorders. Electrophysiological studies demonstrated variation in the disease pattern and severity of peripheral nerve involvement in five affected subjects. The morphometric assessment of the sural nerve biopsy specimen showed a marked reduction in both large myelinated and unmyelinated fibre density and increased density of non‐myelinating Schwann cell nuclei. Apart from numerous pathological nuclei of isolated Schwann cells, multiple profiles of non‐myelinating Schwann cell subunits were apparent in the endoneurium. Schwann cell proliferation in association with first‐hit mutation of the merlin gene might be responsible for the NF2‐associated neuropathy. Sural nerve biopsy showed a progressive neuropathy in the disease. Further, we suggest nonmyelinating Schwann cells are involved in NF2 neuropathy.     

金线莲水提物给药前后肿瘤细胞蛋白质组的差异比较

目的:建立利用二维液相色谱法分离肿瘤细胞全细胞裂解液的分析方法,进而研究金线莲水提物的抗肿瘤作用以及肿瘤细胞蛋白质组的差异。方法:将金线莲水提物给药后的肿瘤细胞裂解样品及对照样品用初始缓冲液置换后,进行一维色谱聚焦分离,然后先对二维色谱条件进行优化和重复性分析,再将一维收集的pH4.0～8.5之间的组分分别进行二维无孔硅胶HPLC分离,利用ProteoVue软件将UV图转换成胶图,分析差异。结果:一维色谱聚焦分离pH4.0～8.5之间组分共收集到16个,每个组分的二维UV图转换成PI/UV胶图,结果表明金线莲水提物给药前后肿瘤细胞蛋白质组差异具显著性。结论:二维液相色谱分离法是一种有效的分离肿瘤细胞裂解液的方法,给药后的肿瘤细胞蛋白质组和对照相比差异具显著性。     

双液相萃取法测定三七块根与茎叶中三七总皂苷含量

目的 比较三七根和茎叶中三七总皂苷含量.方法 以三七总皂苷回收率为指标,设计正交试验,优选硫酸铵/聚乙二醇双液相体系的最优萃取条件,在最优条件下用香草醛/高氯酸比色法分别测定三七根和茎叶中三七总皂苷的含量.结果 硫酸铵/聚乙二醇双液相最优萃取条件为聚乙二醇分子量6 000,聚乙二醇浓度24%,硫酸铵浓度12%,pH值2.5,有较高的分配系数7.45,回收率82.4%.此条件下测得三七根和茎叶中三七总皂苷含量分别为11.24%和12.98%.结论 三七茎叶在某些方面可作为三七根的替代资源,可为合理开发利用三七资源提供依据.     

糖尿病的药物治疗

目的近年,随着糖尿病发病率的日益上升,抗糖尿病药物越来越受到重视,本文主要介绍国内外治疗糖尿病的药物以及抗糖尿病药物的研究方向。为临床合理用药提供参考。方法参阅国外大量相关文献,进行归纳和总结。结果按作用机制和化学结构治疗糖尿病的药物主要有胰岛素促泌剂、胰岛素增敏剂、α-葡萄糖苷酶抑制剂、胰岛素及胰岛素类似物、二肽基肽酶-Ⅳ(DPP-Ⅳ)抑制剂、其他口服降糖药。在临床使用中,二甲双胍、格列齐特、阿卡波糖应用广泛,居主导地位。在糖尿病的治疗中,常常联合用药。结论糖尿病的发生、发展与酶功能的紊乱密切相关。与糖尿病关系较为密切的酶,包括α-葡萄糖苷酶、醛糖还原酶、一氧化氮合酶、血管紧张素转换酶、肉碱脂酰转移酶Ⅰ和Ⅱ、蛋白激酶C、二肽基肽酶Ⅳ、蛋白酪氨酸激酶、蛋白酪氨酸磷酸酶,这些酶都可能成为糖尿病治疗的靶点,是研发糖尿病治疗药物的新方向。     

Detecting protein-protein interactions in vesicular stomatitis virus using a cytoplasmic yeast two hybrid system

Protein-protein interactions play an important role in many virus-encoded functions and in virus-host interactions. While a “classical” yeast two-hybrid system (Y2H) is one of the most common techniques to detect such interactions, it has a number of limitations, including a requirement for the proteins of interest to be relocated to the nucleus. Modified Y2H, such as the Sos recruitment system (SRS), which detect interactions occurring in the cytoplasm rather than the nucleus, allow proteins from viruses replicating in the cytoplasm to be tested in a more natural context. In this study, a SRS was used to detect interactions involving proteins from vesicular stomatitis virus (VSV), a prototypic non-segmented negative strand RNA (NNS) virus. All five full-length VSV proteins, as well as several truncated proteins, were screened against each other. Using the SRS, most interactions demonstrated previously involving VSV phosphoprotein, nucleocapsid (N) and large polymerase proteins were confirmed independently, while difficulties were encountered using the membrane associated matrix and glycoproteins. A human cDNA library was also screened against VSV N protein and one cellular protein, SFRS18, was identified which interacted with N in this context. The system presented can be redesigned easily for studies in other less tractable NNS viruses.