基于高通量测序技术的三叉苦幼苗转录组数据分析

目的获得三叉苦Melicope pteleifolia转录组信息特征。方法以三叉苦幼苗根、茎、叶混合样品为对象,采用二代高通量测序平台Illumina HiSeq~(TM) 2000进行转录组测序并进行系统的生物信息学分析。结果转录组测序分析共获得47 045 040条高质量序列(clean reads),Trinity de novo组装获得67 956条unigenes,平均长度787 nt。BLAST分析显示分别有42 749(61.92%)、31 152(45.84%)、26 563(39.0 9%)、17 481(25.72%)条unigenes在NR、Swiss-port、KOG、KEGG数据库得到注释信息,参与生物过程、细胞组分和分子功能3个GO类别的47个小组,共9807条unigenes注释到130个KEGG代谢通路中,筛选到19条次生代谢通路,KOG功能分类分析获得25个不同的KOG功能类群。预测共有高等植物转录因子56个家族;借助MISA软件发现7 748个SSRs,三碱基重复SSRs数量最丰富,有4 117个,出现频率为53.1%,五碱基重复SSRs相对较少,占2.2%。结论利用高通量测序技术和生物信息分析获得三叉苦转录组信息特征,为后续三叉苦功能基因的挖掘、次生代谢途径解析及其调控机制研究奠定基础。     

Recent progress on the diagnosis of 2019 Novel Coronavirus

Coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has become a global pandemic. Therefore, convenient, timely and accurate detection of SARS‐CoV‐2 is urgently needed. Here, we review the types, characteristics and shortcomings of various detection methods, as well as perspectives for the SARS‐CoV‐2 diagnosis. Clinically, nucleic acid‐based methods are sensitive but prone to false‐positive. The antibody‐based method has slightly lower sensitivity but higher accuracy. Therefore, it is suggested to combine the two methods to improve the detection accuracy of COVID‐19.     

Membrane-associated mucins of the ocular surface: New genes,new protein functions and new biological roles in human and mouse

The mucosal glycocalyx of the ocular surface constitutes the point of interaction between the tear film and the apical epithelial cells. Membrane-associated mucins (MAMs) are the defining molecules of the glycocalyx in all mucosal epithelia. Long recognized for their biophysical properties of hydration, lubrication, anti-adhesion and repulsion, MAMs maintain the wet ocular surface, lubricate the blink, stabilize the tear film and create a physical barrier to the outside world. However, it is increasingly appreciated that MAMs also function as cell surface receptors that transduce information from the outside to the inside of the cell. A number of excellent review articles have provided perspective on the field as it has progressed since 1987, when molecular cloning of the first MAM was reported. The current article provides an update for the ocular surface, placing it into the broad context of findings made in other organ systems, and including new genes, new protein functions and new biological roles. We discuss the epithelial tissue-equivalent with mucosal differentiation, the key model system making these advances possible. In addition, we make the first systematic comparison of MAMs in human and mouse, establishing the basis for using knockout mice for investigations with the complexity of an in vivo system. Lastly, we discuss findings from human genetics/genomics, which are providing clues to new MAM roles previously unimagined. Taken together, this information allows us to generate hypotheses for the next stage of investigation to expand our knowledge of MAM function in intracellular signaling and roles unique to the ocular surface.     

Consensus summary report for CEPI/BC March 12–13, 2020 meeting: Assessment of risk of disease enhancement with COVID-19 vaccines

A novel coronavirus (CoV), Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in late 2019 in Wuhan, China and has since spread as a global pandemic. Safe and effective vaccines are thus urgently needed to reduce the significant morbidity and mortality of Coronavirus Disease 2019 (COVID-19) disease and ease the major economic impact. There has been an unprecedented rapid response by vaccine developers with now over one hundred vaccine candidates in development and at least six having reached clinical trials. However, a major challenge during rapid development is to avoid safety issues both by thoughtful vaccine design and by thorough evaluation in a timely manner. A syndrome of “disease enhancement” has been reported in the past for a few viral vaccines where those immunized suffered increased severity or death when they later encountered the virus or were found to have an increased frequency of infection. Animal models allowed scientists to determine the underlying mechanism for the former in the case of Respiratory syncytial virus (RSV) vaccine and have been utilized to design and screen new RSV vaccine candidates. Because some Middle East respiratory syndrome (MERS) and SARS-CoV-1 vaccines have shown evidence of disease enhancement in some animal models, this is a particular concern for SARS-CoV-2 vaccines. To address this challenge, the Coalition for Epidemic Preparedness Innovations (CEPI) and the Brighton Collaboration (BC) Safety Platform for Emergency vACcines (SPEAC) convened a scientific working meeting on March 12 and 13, 2020 of experts in the field of vaccine immunology and coronaviruses to consider what vaccine designs could reduce safety concerns and how animal models and immunological assessments in early clinical trials can help to assess the risk. This report summarizes the evidence presented and provides considerations for safety assessment of COVID-19 vaccine candidates in accelerated vaccine development.     

Aggregatibacter actinomycetemcomitans leukotoxin: From mechanism to targeted anti‐toxin therapeutics

Aggregatibacter actinomycetemcomitans is a Gram‐negative bacterium associated with localized aggressive periodontitis, as well as other systemic diseases. This organism produces a number of virulence factors, all of which provide some advantage to the bacterium. Several studies have demonstrated that clinical isolates from diseased patients, particularly those of African descent, frequently belong to specific clones of A. actinomycetemcomitans that produce significantly higher amounts of a protein exotoxin belonging to the repeats‐in‐toxin (RTX) family, leukotoxin (LtxA), whereas isolates from healthy patients harbor minimally leukotoxic strains. This finding suggests that LtxA might play a key role in A. actinomycetemcomitans pathogenicity. Because of this correlation, much work over the past 30 years has been focused on understanding the mechanisms by which LtxA interacts with and kills host cells. In this article, we review those findings, highlight the remaining open questions, and demonstrate how knowledge of these mechanisms, particularly the toxin's interactions with lymphocyte function‐associated antigen‐1 (LFA‐1) and cholesterol, enables the design of targeted anti‐LtxA strategies to prevent/treat disease.     

利用规律间隔成簇短回文重复序列/相关蛋白9系统敲减α2δ1对人肝癌细胞系Hep-12干性的影响

目的 通过规律间隔成簇短回文重复序列/相关蛋白9(CRISPR/Cas9)系统敲减人肝癌细胞系Hep12中电压依赖性钙离子通道 α2δ1的表达，观察α2δ1敲减后对肝癌细胞干性的影响。方法 设计3对靶向α2δ1的向导 (sgRNA)，长度为20 bp，构建到lenti CRISPRv2-puro载体上，然后在体外检测sgRNA切割活性，利用慢病毒包装系统包装含有sgRNA的重组质粒，将包装好的病毒感染Hep-12细胞，2 d后加入嘌呤霉素筛选。利用Western blotting验证α2δ1的敲减效果和干性相关基因的表达。通过成球实验检测其体外自我更新能力的变化。结果 测序结果显示，sgRNA成功插入载体质粒；体外切割实验显示，3条sgRNA均有切割活性；Western blotting结果显示，α2δ1基因的表达显著降低，干性相关基因B细胞特异性莫洛尼白血病病毒插入位点1(BMI1)和Nanog的表达显著被抑制；无血清培养基成球实验结果表明，敲减α2δ1导致Hep-12细胞的体外自我更新能力减弱。结论 利用CRISPR/Cas9技术成功构建敲除α2δ1基因的Hep-12细胞系；敲除α2δ1基因后能抑制Hep-12细胞肿瘤干细胞样特性。     

基于规律成簇的间隔短回文重复序列及其相关蛋白技术检测乙型肝炎病毒共价闭合环状DNA方法的建立

目的建立一种基于规律成簇的间隔短回文重复序列及其相关蛋白(CRISPR/Cas13a)的乙型肝炎病毒(HBV)共价闭合环状DNA(HBV cccDNA)检测方法。方法提取2017年6月至2020年10月于首都医科大学附属北京佑安医院就诊的4例乙型肝炎患者肝脏总DNA后,使用HindⅢ内切酶和质粒安全性ATP依赖DNA酶(PSAD)分别进行酶切;根据松弛环状DNA(rcDNA)和cccDNA的结构差异,设计特异性扩增HBV cccDNA的引物,对酶切后的产物进行滚环扩增(RCA)和PCR扩增;并筛选crRNA,建立基于CRISPR/Cas13a技术的HBV cccDNA检测新方法。结果利用α-1-抗胰蛋白酶(A1AT)和HBV表面抗原(HBsAg)引物对双重酶切后的产物进行扩增,验证产物中HBV基因组的存在;利用HBV cccDNA和HBV rcDNA引物对PSDA酶切后的产物扩增,验证了产物中只存在HBV cccDNA;利用RCA后的阳性样本作为模板梯度稀释,然后进行PCR扩增转录后使用CRISPR/Cas13a检测,计算出检测下限为10拷贝/μl。结论本研究建立了RCA-PCR-CRISPR-Cas13a的新型检测方法,可对HBV cccDNA进行高灵敏度和高特异性检测,为乙型肝炎患者抗病毒治疗评估、治疗终点的确定以及调整治疗方案提供了有效的监测手段。     

曲霉菌对唑类抗真菌药物的耐药机制研究进展

侵袭性曲霉病是由致病曲霉菌引起的危及生命的真菌感染,主要致病菌为烟曲霉,而唑类抗真菌药物是临床治疗的首选。目前已上市的唑类抗真菌药物包括伊曲康唑,伏立康唑,泊沙康唑和伊沙康唑。然而,流行病学研究发现曲霉菌对唑类抗真菌药物的耐药率呈逐年上升趋势,给临床治疗造成了威胁。其中,Cyp51A蛋白突变、外排系统高表达以及环境耐药机制等多种因素都参与唑类抗真菌药物的耐药。因此,本文综述近年来有关曲霉菌对唑类抗真菌药物的耐药机制,以期为耐药监测、耐药菌控制和新药研发提供理论依据。