Developments,applications, and prospects of cryo‐electron microscopy

Cryo‐electron microscopy (cryo‐EM) is a structural biological method that is used to determine the 3D structures of biomacromolecules. After years of development, cryo‐EM has made great achievements, which has led to a revolution in structural biology. In this article, the principle, characteristics, history, current situation, workflow, and common problems of cryo‐EM are systematically reviewed. In addition, the new development direction of cryo‐EM—cryo‐electron tomography (cryo‐ET), is discussed in detail. Also, cryo‐EM is prospected from the following aspects: the structural analysis of small proteins, the improvement of resolution and efficiency, and the relationship between cryo‐EM and drug development. This review is dedicated to giving readers a comprehensive understanding of the development and application of cryo‐EM, and to bringing them new insights.     

新冠病毒刺突糖蛋白结构与功能的生物信息学分析及原核表达

SARS-CoV-2是一种高致病性且传播迅速的病原体,通过刺突糖蛋白(Spike glycoprotein,S蛋白)识别宿主细胞表面的受体来实现入侵和感染。对S蛋白进行系统的生物信息学分析和原核表达,有助于深入理解S蛋白的功能和阐明该蛋白介导病毒感染的分子机制。本文采用Protparam、Pfam、TMHMM、ExPASy-ProtScale、PSORTⅡ、SignalP、UniProt、NetPhos 3.1、NetNGlyc 1.0、NetOGlyc 4.0和BLAST等生物信息学软件和数据库对S蛋白的理化性质、亚细胞定位、翻译后修饰及相互作用网络等生物学特性进行了系统分析。利用Clustal X2和MEGA7.0软件对该蛋白进行了基于氨基酸序列的同源性分析和系统进化分析。最后,通过分子克隆技术构建重组表达载体pET-22b-S并进行原核表达。结果显示,S蛋白由1273个氨基酸组成,分子量141.2 kD,等电点6.24,有两个卷曲螺旋结构,一个跨膜螺旋区,疏水性较强。S蛋白包含刺突受体结合结构域和S2糖蛋白结构域,主要分布于宿主细胞的内质网膜和细胞膜,含有136个潜在的磷酸化位点和20个可能的糖基化位点。与SARS-CoV-2 S蛋白序列一致性最高的是SARS冠状病毒、SARS冠状病毒WH20和蝙蝠冠状病毒HKU3,均为76%。SARS-CoV-2与SARS冠状病毒和蝙蝠冠状病毒聚为一大支,提示它们可能具有共同的祖先。S蛋白主要在细菌裂解液离心之后的沉淀中表达,这为后续的结构分析和疫苗研发奠定了基础。S蛋白在SARS冠状病毒和蝙蝠冠状病毒之间保守性较高,提示其在病毒入侵过程中具有重要功能。SARS-CoV-2与SARS冠状病毒和蝙蝠冠状病毒可能具有共同的祖先。本研究为SARS-CoV-2 S蛋白的表达纯化、结构与功能研究提供了重要的数据基础,有助于全面揭示S蛋白的生物学功能,同时为设计和筛选靶向S蛋白的新型抗病毒药物提供了科学依据。     

应用底栖动物完整性指数B-IBI评价溪流健康

B-IBI指数是溪流生态系统健康评价常用指标之一。据安徽黄山地区溪流的33个底栖动物样点数据(11个参照点,22个受损点),对21个生物参数进行分布范围、Pearson相关性和判别能力分析,确定B-IBI指数由总分类单元数、EPT分类单元数、前3位优势分类单元%、粘附者%、敏感类群%和BI指数构成。分别用3分制、4分制和比值法统一各参数量纲,B-IBI指数值即为累加各构成指数的分值或比值。B-IBI箱线图分析表明,上述3种方法计算出的B-IBI值有较高判别能力(IQ=3),能很好地判别参照水体和受损水体,且3者之间具高相关性(r>0.90)。分别依据参照样点和所有样点B-IBI值建立健康评价标准,比较3分制、4分制和比值法对评价结果的准确性,表明用比值法统一各参数量纲并依据所有样点的B-IBI值建立的健康评价标准的准确性优于3分制和4分制法。建立了适合祁门县溪流生态系统健康评价B-IBI标准B-IBI>3.59健康,2.7～3.59亚健康,1.8～2.69一般,0.9～1.79差,B-IBI<0.9极差。祁门县21个样点的水体,14个健康,6个亚健康,1个一般。B-IBI与电导率(r=-0.62,p<0.01)和生境质量(r=0.65,p<0.01)显著相关。     

晚疫病菌基因组SSR扩增产物两种检测方法的比较与改进

以32份马铃薯或番茄晚疫病菌基因组DNA为模板,分析了琼脂糖凝胶电泳法和聚丙烯酰胺凝胶电泳法对10对SSR引物多态性检测的影响,同时比较了聚丙烯酰胺凝胶电泳两种银染法(常规银染法和快速银染法)的差异。结果显示,聚丙烯酰胺凝胶电泳较琼脂糖凝胶电泳分离效果好,具有更高的多态性检出率,其中快速银染法与常规银染法的检出结果相同,但常规银染步骤繁琐,整个过程要用1-2h,而快速银染只需约15min,且染色背景较浅,条带清晰。聚类分析显示,聚丙烯酰胺凝胶电泳快速银染法可将供试晚疫病菌株分布于更多的聚类组,显示出更高的遗传变异度。可见,聚丙烯酰胺凝胶电泳快速银染法结合SSR分子标记技术,可有效地用于马铃薯或番茄晚疫病菌群体遗传多样性分析。     

Specific and Sensitive Detection of Phytophthora nicotianae by Nested PCR and Loop‐mediated Isothermal Amplification Assays

Phytophthora nicotianae is an important soilborne plant pathogen. It causes black shank in tobacco and other commercially important crop diseases. Early and accurate detection of P. nicotianae is essential for controlling these diseases. In this study, primers based on the Ras‐related protein gene (Ypt1) of P. nicotianae were tested for their specific detection of the pathogen using nested PCR and LAMP assays. For specificity testing, DNA extracts from 47 P. nicotianae isolates, 45 isolates of 16 different oomycetes and 25 isolates of other fungal species were used; no cross‐reaction with other pathogens was observed. The sensitivity assay showed that the nested PCR and LAMP assays had detection limits of 100 fg and 10 fg genomic DNA per 25‐μl reaction, respectively. Furthermore, the nested PCR and LAMP assays were used for the detection of DNA from naturally P. nicotianae‐infected tobacco tissues and soil. Our results suggest that the LAMP assay has the greatest potential for the specific detection of P. nicotianae in regions that are at risk of contracting tobacco black shank disease and that the Ypt1 gene is a novel and effective target of P. nicotianae LAMP visual detection.     

Phenotypic and Genotypic Characterization of Phytophthora infestans Isolates from China

A total of 288 (202 from potato and 86 from tomato) isolates of Phytophthora infestans were collected from 1998 to 2007 in China. The isolates were characterized based on mating type, in vitro metalaxyl sensitivity, virulence on potato differentials, allozymes of glucose-6-phosphate isomerase ( Gpi ), peptidase ( Pep ), and mitochondrial DNA (mtDNA) haplotype and examined by DNA-based simple sequence repeat (SSR) and random amplified polymorphic DNA (RAPD) fingerprinting. The majority (283 of 288) of the isolates were of the A1 mating type, the other three were the A2 mating type and two were the A1A2 mating type. Resistance to metalaxyl was frequently observed, with 248 (86.1%) resistant, 21 (7.3%) intermediate and 19 (6.6%) sensitive isolates identified. Virulence was assessed for 125 isolates on a set of 11 potato differentials and 61 races were detected. Most isolates were virulent on the differential genotype with gene R3, and all known virulence genes were found, with race 3.4.7.11 being the most common. This pattern did not appear to be associated with geographic origin, sample type, mating type or metalaxyl sensitivity. The dominant banding patterns for Gpi were 100/100/111 (176 isolates) and 100/100 (109 isolates), but genotypes 86/100 and 100/111 were also identified. All isolates tested were homozygous (100/100) at the Pep locus. The majority (205 of 288) of isolates tested was of mtDNA haplotype IIb, 76 were haplotype IIa and seven were the rare Ib haplotype. The genetic diversity of 60 representative isolates from China was assayed by two types of molecular markers, RAPD and SSR. A high level of polymorphism was found. The results demonstrated the diverse phenotypic and genotypic structure of the current populations of P. infestans in China.     

果蝇共生菌降低有害真菌的毒性

【背景】目前对于如何解决有害真菌对黑腹果蝇的致死性病理研究较少,对共生菌抑制有害真菌的研究引起普遍关注。【目的】检测黑腹果蝇共生菌对病原性真菌的拮抗作用,揭示共生菌提高果蝇的适合度。【方法】利用PDA培养基分离黑腹果蝇食物中真菌;利用形态和rDNAITS基因序列比对进行真菌的鉴定;通过测量菌落直径、孢子数量以及菌丝分枝数量以评定真菌的生长;利用存活率评估病原真菌的毒性;建立无菌和悉生模型,通过发育历期验证其共生菌与病原性真菌的竞争作用;利用双向选择食物装置检测共生菌抑制病原真菌的效果。【结果】从果蝇食物中分离出的真菌经鉴定为拟茎点霉(Phomopsis),可显著地降低成年果蝇的存活率和延缓果蝇发育。东方醋酸杆菌在体外可明显抑制拟茎点霉的生长,有效地减轻拟茎点霉对果蝇的致死作用,挽救了拟茎点霉导致的果蝇发育延滞,改善了果蝇产卵对拟茎点霉的趋避作用。【结论】拟茎点霉是果蝇的一株条件性病原真菌,而东方醋酸杆菌可以有效地减轻拟茎点霉对果蝇生长发育和存活率的损害,从而提高果蝇适合度。     

Age-dependent changes in the gut microbiota and serum metabolome correlate with renal function and human aging

Human aging is invariably accompanied by a decline in renal function, a process potentially exacerbated by uremic toxins originating from gut microbes. Based on a registered household Chinese Guangxi longevity cohort (n = 151), we conducted comprehensive profiling of the gut microbiota and serum metabolome of individuals from 22 to 111 years of age and validated the findings in two independent East Asian aging cohorts (Japan aging cohort n = 330, Yunnan aging cohort n = 80), identifying unique age-dependent differences in the microbiota and serum metabolome. We discovered that the influence of the gut microbiota on serum metabolites intensifies with advancing age. Furthermore, mediation analyses unveiled putative causal relationships between the gut microbiota (Escherichia coli, Odoribacter splanchnicus, and Desulfovibrio piger) and serum metabolite markers related to impaired renal function (p-cresol, N-phenylacetylglutamine, 2-oxindole, and 4-aminohippuric acid) and aging. The fecal microbiota transplantation experiment demonstrated that the feces of elderly individuals could influence markers related to impaired renal function in the serum. Our findings reveal novel links between age-dependent alterations in the gut microbiota and serum metabolite markers of impaired renal function, providing novel insights into the effects of microbiota-metabolite interplay on renal function and healthy aging.