以MyD88 TIR二聚化为靶点的抗炎抑制剂筛选模型的建立

MyD88是IL-1R/TLR受体超家族向细胞内转导胞外信号时募集到受体胞浆尾部的重要接头蛋白．由TIR结构域介导的MyD88分子同源二聚化是它招募到受体胞浆尾部的前提,然后二聚化的MyD88再募集下游信号分子,传递信号,引发促炎基因的表达．本研究旨在建立一种模型,以实现活细胞原位的、基于荧光信号变化的MyD88二聚化抑制物的高通量筛选．我们分别构建了MyD88 TIR与GFP和RFP的融合蛋白表达质粒,瞬时转染HeLa细胞,在488 nm激发光下,转染GFP-MyD88 TIR和RFP-MyD88 TIR细胞,检测到绿色荧光与红色荧光间的共振能量转移(FRET)．而当细胞转染GFP-MyD88 TIR和RFP或RFP-MyD88 TIR和GFP,因TIR二聚化不能实现,FRET效率受到严重影响．实验结果提示,依赖双阳性表达GFP-MyD88 TIR和RFP-MyD88 TIR的细胞株,检测不同化合物对于荧光FRET效率的影响,可以建立MyD88 TIR二聚化抑制药物的筛选模型．此外,我们构建了原核表达质粒,利用纯化的His-MyD88 TIR分别与GST或GST-MyD88 TIR蛋白进行体外结合实验,发现GST-MyD88 TIR(而非GST)可以与His-MyD88 TIR相互结合．结果的差异性提示,利用His-MyD88 TIR和GST-MyD88 TIR体外结合实验分析,可以进一步确定抑制物是否直接阻断了TIR的相互作用．结合真核细胞的荧光FRET阻断结果和原核表达的重组蛋白相互作用分析,可确定MyD88 TIR二聚化的抑制物．利用这一模型可以对商品化的小分子库、自行制备的天然产物组分进行广泛的筛选,从中获得有效抑制MyD88二聚化的化合物,参与对MyD88信号通路依赖的慢性炎症、自身免疫性疾病的药物治疗．     

Hybridization between diploid Centaurea pseudophrygia and tetraploid C. jacea (Asteraceae): the role of mixed pollination,unreduced gametes,and mentor effects

We studied hybridization between the diploid Centaurea pseudophrygia and the tetraploid C. jacea by performing crossing experiments and screening natural populations using flow cytometry. The experiments confirm that the studied species exhibit strong reproductive isolation. Interspecific hybrids were formed at a low frequency, including triploids (originating from reduced gametes) and tetraploids (involving unreduced gametes of the diploids). In contrast, hybrids were almost absent among seeds and adult plants of natural mixed populations and among the offspring from experimental pollinations with a mixture of pollen of both ploidy levels. We found that mixed pollination is an important mechanism for preventing hybridization between plants of different ploidy levels and sustaining the reproduction of the tetraploids. A mentor effect (induced selfing in the presence of pollen of different ploidy levels) was observed in both diploids and tetraploids, reinforcing the reproductive isolation between cytotypes. Higher ploidy levels (pentaploid, hexaploid) involving unreduced gametes of the tetraploid species were identified. Notably, pentaploids were discovered for the first time in Centaurea sect. Jacea. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 93–106.     

Surprising negative association between IgG1 allotype disparity and anti-adalimumab formation: a cohort study

Introduction

The human monoclonal antibody adalimumab is known to induce an anti-globulin response in some adalimumab-treated patients. Antibodies against adalimumab (AAA) are associated with non-response to treatment. Immunoglobulins, such as adalimumab, carry allotypes which represent slight differences in the amino acid sequences of the constant chains of an IgG molecule. Immunoglobulins with particular IgG (Gm) allotypes are racially distributed and could be immunogenic for individuals who do not express these allotypes. Therefore, we investigated whether a mismatch in IgG allotypes between adalimumab and IgG in adalimumab-treated patients is associated with the development of AAA.

Methods

This cohort study consisted of 250 adalimumab-treated rheumatoid arthritis (RA) patients. IgG allotypes were determined for adalimumab and for all patients. Anti-idiotype antibodies against adalimumab were measured with a regular radio immunoassay (RIA), and a newly developed bridging enzyme linked immunosorbent assay (ELISA) was used to measure anti-allotype antibodies against adalimumab. The association between AAA and the G1m3 and the G1m17 allotypes was determined. For differences between groups we used the independent or paired samples t-test, Mann-Whitney test or Chi square/Fisher's exact test as appropriate. To investigate the influence of confounders on the presence or absence of AAA a multiple logistic regression-analysis was used.

Results

Adalimumab carries the G1m17 allotype. No anti-allotype antibodies against adalimumab were detected. Thirty-nine out of 249 patients had anti-idiotype antibodies against adalimumab (16%). IgG allotypes of RA patients were associated with the frequency of AAA: patients homozygous for G1m17 had the highest frequency of AAA (41%), patients homozygous for G1m3 the lowest frequency (10%), and heterozygous patients' AAA frequency was 14% (P = 0.0001).

Conclusions

An allotype mismatch between adalimumab and IgG in adalimumab-treated patients did not lead to a higher frequency of AAA. On the contrary, patients who carried the same IgG allotype as present on the adalimumab IgG molecule, had the highest frequency of anti-adalimumab antibodies compared to patients whose IgG allotype differed from adalimumab. This suggests that the allotype of adalimumab may not be highly immunogenic. Furthermore, patients carrying the G1m17-allotype might be more prone to antibody responses.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.     

生物软件在序列分析过程中的运用

介绍了组合使用BLAST、FASTA/BLASTScan3.2,或用多序列比对软件,从数据库中快速提取大数量目标序列,最后用MEGA4快捷编辑整理大数量序列的方法。还介绍了一种生成核酸序列与其氨基酸序列相似性百分率整合表格的方法。简述了对引物设计的基本认识并介绍了多重引物兼容性筛选软件;对构建系统发育树的认识并引出分子进化树构建软件MEGA4的使用和PAUP 4.0常用建树命令模块。期望这些方法和软件的使用能解决生物序列分析过程的常见问题。     

河西走廊天然固沙植被区地表甲虫多样性及其对沙漠化的指示作用

干旱、半干旱区沙漠化强烈影响动植物分布及多样性,地表甲虫是荒漠中主要的动物类群,它们对沙漠化引起的植被和土壤环境变化响应十分敏感。鉴于此,以河西走廊中部张掖绿洲外围的天然固沙植被区作为研究区,依据沙漠化发育程度选择流动沙丘（ASD）、丘间低地（IL）、半固定（SFSD）和固定沙丘（FSD）4种生境,调查了地表甲虫群落组成及影响甲虫分布的植被和土壤环境。研究发现,4种生境地表甲虫群落组成明显不同并存在季节变异,5月ASD与IL、SFSD和FSD生境地表甲虫群落的相异性大于8月。5月和8月SFSD生境地表甲虫活动密度均显著高于其他生境,8月FSD生境地表甲虫多样性指数显著高于其他生境。不同大小甲虫对沙漠化的响应模式不同,大中型甲虫对沙漠化的响应较小型甲虫敏感,这在5月表现尤为明显。地表甲虫与环境因子的RDA分析结果表明,12个植被和土壤环境因子解释了49.8%的地表甲虫群落变异,其中植被环境解释了甲虫群落变异的16.3%,土壤环境解释了甲虫群落变异的4.2%,植被和土壤环境相互作用解释了甲虫群落变异的29.3%。pRDA分析结果表明,草本物种丰富度、灌木盖度、土壤有机碳含量和粗砂含量是影响地表甲虫分布的主要环境因子,它们解释了43.7%的地表甲虫群落变异。Pearson相关分析表明,草本物种丰富度与地表甲虫活动密度呈显著正相关,而与地表甲虫均匀度呈显著负相关;灌木盖度与地表甲虫多样性呈显著正相关;地表甲虫物种丰富度与灌木盖度和草本物种丰富度均呈显著正相关。此外,研究还发现戈壁琵甲、克氏扁漠甲、中华砚甲和甘肃齿足象可以用于指示FSD生境,东鳖甲属昆虫可以用于指数SFSD生境,谢氏宽漠王可以用于指示IL及ASD生境。     

肠内营养对乳腺癌术后化疗患者肠道菌群及生活质量的影响

目的研究肠内营养对乳腺癌术后化疗患者肠道菌群、生活质量、营养指标水平的影响。方法选取2012年7月至2018年1月于我院进行乳腺癌术后化疗的91例女性乳腺癌患者为研究对象,根据是否进行肠内营养将患者分为肠内营养组和对照组,观察化疗前后2组患者肠道菌群、生活质量、营养指标水平的变化及不良反应发生情况。结果治疗前2组患者肠道菌群、生活质量、营养指标水平差异无统计学意义(均P0.05)。治疗后,肠内营养组患者肠道双歧杆菌数量为(6.7±0.5)lg CFU/g、乳杆菌数量为(8.5±0.4)lg CFU/g,均显著高于治疗前,同时显著高于对照组(均P0.05)。治疗后肠内营养组患者出现反酸、腹胀、便秘等胃肠道不良反应的比例与对照组比较差异无统计学意义(均P0.05)。治疗后两组患者总体健康、认知功能、情绪功能、角色功能、社会功能、活力、躯体疼痛、躯体功能评分显著高于治疗前(均P0.05),且肠内营养组患者生活质量各指标的评分显著高于对照组(均P0.05)。治疗后肠内营养组患者血清总蛋白(TB)、血清清蛋白(ALB)、血清前清蛋白(PA)、转铁蛋白(TF)水平均显著高于对照组(均P0.05)。结论肠内营养可预防乳腺癌术后化疗患者肠道菌群的紊乱及营养不良的发生,安全有效,有助于提高乳腺癌患者的生活质量。     

Found again: the extremely rare <Emphasis Type="Italic">Codonorchis canisioi</Emphasis> (Orchidaceae: Codonorchideae) reappears after being missing for 78 years

To date, the endangered terrestrial orchid Codonorchis canisioi was only known from two collections at its type locality. This plant has not been collected since 1935 and is currently considered as Critically Endangered in the Brazilian list of threatened flora. We recently found two small populations about 174 km from the type locality. We thus seized the opportunity to make a complete record of morphological traits, aiming to fill the gaps of the short and non-illustrated protologue. Plant, flower (including details of column and pollinarium) and fruit features are here illustrated and discussed in detail. Overall, plant and flower features are similar to those of the widespread Patagonian C. lessonii. Yet, both species can be separated by a number of vegetative and floral features. Female Augochlorine Halictidae bees were recorded as pollinators. Fruits mature quickly (15–19 days after pollination). Plants wither by middle October, resting underground as very small (3–5 mm diameter) roundish tubers. The two newly found populations are in privately owned areas and prone to both fire and cattle grazing. Due to the small size of each population (estimated in less than 200 individuals), it is likely that this species would still be considered as CR (Critically Endangered) when formally and properly surveyed under the IUCN criteria. Since plants are small and strongly seasonal, more field surveys are necessary to locate other potentially overlooked populations.     

研究报告：CRBGP通过抑制FAK-AKT通路和白介素-6的分泌抑制胶质瘤U251细胞的活性

目的电压门控钠通道(voltage-gated sodium channels,VGSCs)表达于胶质瘤U251细胞,并影响U251细胞的增殖、侵袭和凋亡。富含半胱氨酸的颊腺蛋白(cysteine-rich buccal gland protein,CRBGP)是一种从日本七鳃鳗颊腺中分离出来的VGSCs阻断剂。本文旨在探究CRBGP是否因具有VGSCs阻断功能从而能够抑制U251细胞的活性。方法首先采用MTT法检测了CRBGP对U251细胞增殖的作用,并分别利用莱特-吉姆萨、FITC-phalloidin和Hoechst 33258染色法观察CRBGP处理后U251细胞的形态、细胞骨架和细胞核。细胞外基质蛋白如IV型胶原蛋白、纤连蛋白和层黏连蛋白用于检测CRBGP对U251细胞黏附的影响。此外,本文采用transwell法检测CRBGP处理后U251细胞的迁移和侵袭能力,并通过Western blot方法探讨CRBGP诱导U251细胞凋亡并抑制其运动的内在机理。最后,通过酶联免疫吸附测定法检测CRBGP对U251细胞的抑炎效果。结果 CRBGP通过线粒体依赖途径诱导U251细胞凋亡,...