Generation and Selection of Specific Aptamers Targeting Brucella Species through an Enhanced Cell-SELEX Methodology

Brucellae are Gram-negative, aerobic, non-motile coccobacilli causing brucellosis in man and animals. The disease is one of the most significant yet neglected global zoonoses. Especially in developing countries, brucellosis is causing public health problems and economic losses to private animal owners and national revenues. Composed of oligonucleotides, aptamers are chemical analogues of antibodies that are promising components for developing aptamer-based rapid, sensitive, and specific tests to identify the Brucella group of bacteria. For this purpose, aptamers were generated and selected by an enhanced protocol of cell systematic evolution of ligands by exponential enrichment (cell-SELEX). This enhanced cell-SELEX procedure involved the combination of both conventional and toggle cell-SELEX to boost the specificity and binding affinity to whole Brucella cells. This procedure, combined with high-throughput sequencing of the resulting aptamer pools, comprehensive bioinformatics analysis, and wet lab validation assays, led to the selection of a highly sensitive and specific aptamer for those Brucella species known to circulate in Egypt. The isolated candidate aptamer showed dissociation constant (K_D) values of 43.5 ± 11, 61.5 ± 8, and 56 ± 10.8 nM for B. melitensis, B. abortus, and B. suis, respectively. This is the first development of a Brucella-specific aptamer using an enhanced combination of conventional and toggle cell-SELEX to the authors’ best knowledge.     

高通量筛选研究茯砖茶对FXR模型的作用

将茯砖茶水提液分别用三氯甲烷、乙酸乙酯、正丁醇萃取,制得三氯甲烷层、乙酸乙酯层、正丁醇层及水层样品,茶渣用95%的乙醇提取,制得乙醇提取物,通过高通量药物筛选测定各样品对法尼酯衍生物X受体(FXR)模型的激活和抑制作用,研究茯砖茶的降脂减肥功效,结果显示茯砖茶各部份对FXR都有抑制作用,乙酸乙酯层样品激活作用较明显,从中分离制得儿茶素EGCG及ECG单体对FXR模型有很好的激活作用,证明茯砖茶有较强的降脂功能,有望开发成天然的降脂药物。茯砖茶中其他活性成分的分离有待进一步进行。     

基于高通量测序的浓香型白酒窖池古菌群落结构分析

本试验利用罗氏GS junior高通量测序平台分析了30年窖龄的浓香型白酒窖池窖泥古菌群落结构,结果发现:3口窖池样品共获得8706条有效序列,282个OTUs分类;样品中古菌的Shannon曲线值在4~5之间就趋于稳定,Chao曲线的值随着序列数的增加而增长。窖泥中古菌类群主要集中在Euryarchaeota(广古菌门)下的Thermoplasmata class(热原体纲)中的Thermoplasmataceae(热原体科),Methanobacteria class(甲烷杆菌纲)中的Methanobacteriaceae(甲烷杆菌科),Methanococci class(甲烷球菌纲)中的Methanomicrobiaceae(甲烷微菌科)、Methanocorpusculaceae(甲烷八叠球菌科)和Methanosarcinaceae(甲烷粒菌科);Saccharococcus sp.和WCHD3-02也有检测到。其中Thermoplasmataceae和Methanobacteriaceae占绝对优势,分别为39%和27%,其它菌类Methanomicrobiaceae占9%、Methanosarcinaceae占7%和Methanocorpusculaceae占4%、Saccharococcus sp.(糖球菌属)占1%和WCHD3-02占1%,unclassified序列占12%;对甲烷菌类进行深入分析,发现了Methanoculleus marisnigri strain、Methanoculleus bourgensis strain、Methanosarcina siciliae strain、Methanobacterium、Methanocorpusculum和Methanomicrobia等种属的菌种。     

酱香型白酒窖底泥微生物组成分析

对酱香型白酒发酵窖池中不同时间的窖底泥及环境土样中的微生物区系构成进行了测序分析。扩增样本中细菌16S r DNA的V6区并采用Illumina高通量测序平台进行深度测序。结果表明,酱香型白酒厂周边土壤中的微生物多样性极其丰富,相比窖底泥中的微生物组成更加复杂。在土壤驯化成窖底泥的过程中,微生物的复杂度逐渐降低,随着窖底泥的不断驯化,Lactobacillaceae(乳杆菌科)、Clostridiaceae(梭菌科)和Ruminococcaceae(瘤胃菌科)等厌氧微生物逐渐成为主要优势种群。土壤微生物为窖底泥驯化提供了重要的微生物来源,酱香型白酒生产地域周边土壤对于窖底泥的形成具有一定的影响。     

基于高通量测序探究东北粳稻储藏期间真菌群落的演替

为探究不同初始水分的东北粳稻在模拟实仓环境储藏的条件下,稻谷中真菌群落随储藏时间的演替变化。选取水分在14.50%~15.50%的稻谷HW和13.50%~14.50%稻谷LW,进入模拟储藏实验。结果表明,在第一和第二储藏期高水分稻谷HW水分含量由15.50%降低至15.09%,低水分稻谷LW水分含量由14.21%降低至13.59%,直至第三储藏期保持平稳。基于可操作性分类单元（Operational Taxonmic Units,OTUs）的物种分类分析,两种水分的稻谷真菌群落经历了不同的演替变化。高水分稻谷在每个储藏期,真菌群落都发生了明显演替,稻瘟病菌（Magnaporthe_grisea）、金黄色蝶形担孢酵母（Papiliotrema_aurea）、弯孢菌（Curvularia_inaequalis）、黑孢霉（Neosetophoma_samararum）、汉纳酵母菌（Hannaella_sinensis）在储存结束时成为了最终的优势菌种。低水分稻谷在第二储藏期真菌群落发生明显演替,优势菌种为弯孢菌（Curvularia_inaequalis）、汉纳酵母菌（Hannaella_sinensis）、Hannaella_zeae。     

数字化高温大曲发酵过程中微生物群落结构的变化

采用Illumina MiSeq高通量测序技术对数字化高温大曲和传统高温大曲进行微生物群落多样性解析;同时根据微生物物种信息和理化特性进行相关性分析和生物信息学分析。结果表明,两种高温大曲在发酵过程中微生物群落结构均有显著变化。发酵结束时,两种高温大曲的细菌都以克罗彭斯特菌属（Kroppenstedtia）、糖多孢菌属（Saccharopolyspora）、芽孢杆菌属（Bacillus）、魏斯氏菌属（Weissella）和乳酸杆菌属（Lactobacillus）为主要优势菌属,真菌以嗜热子囊菌属（Thermoascus）、嗜热真菌属（Thermomyces）和曲霉菌属（Aspergillus）为主。非度量多维尺度分析和层级聚类分析结果表明,相同时间时两种大曲的微生物组成大体相似。     

Quantitative Assessment of Affinity Selection Performance by Using DNA-Encoded Chemical Libraries

DNA-encoded chemical libraries are often used for the discovery of ligands against protein targets of interest. These large collections of DNA-barcoded chemical compounds are typically screened by using affinity capture methodologies followed by PCR amplification and DNA sequencing procedures. However, the performance of individual steps in the selection procedures has been scarcely investigated, so far. Herein, the quantitative analysis of selection experiments, by using three ligands with different affinity to carbonic anhydrase IX as model compounds, is described. In the first set of experiments, quantitative PCR (qPCR) procedures are used to evaluate the recovery and selectivity for affinity capture procedures performed on different solid-phase supports, which are commonly used for library screening. In the second step, both qPCR and analysis of DNA sequencing results are used to assess the recovery and selectivity of individual carbonic anhydrase IX ligands in a library, containing 360 000 compounds. Collectively, this study reveals that selection procedures can be efficient for ligands with sub-micromolar dissociation constants to the target protein of interest, but also that selection performance dramatically drops if 10⁴ copies per library member are used as the input.     

不同时期排泥对培养SBBR生物膜的影响

序批式生物膜反应器（sequencing biofilm batch reactor, SBBR）是应用广泛的污水处理方法。为探究不同时期排泥对SBBR污染物去除效果与微生物群落结构的影响，本研究设置了挂膜初期、中期和后期进行排泥的反应器处理生活污水，同时结合16S rDNA高通量测序技术对微生物群落结构进行分析，并采用机器学习（machine learning, ML）的方法，在传统的微生物优势种分析基础上，对测序数据进行深度挖掘，寻找造成组间差异的关键物种。水质测定结果显示，COD去除效果在不同时期排泥的SBBR间没有明显差异，出水COD均低于30mg/L。挂膜中期排泥的SBBR的NH₃-N去除率先达到稳定且高于前期和后期排泥的系统。高通量测序结果显示，各SBBR中微生物优势种均以降解有机物的物种为主。挂膜中期排泥的SBBR中，ML筛选得到的NH₃-N去除相关物种（Hydrogenophaga、Gemmata和Nitrospira）与差异关键物种丰度更高，微生物群落结构稳定性更强，可从微生物层面解释分析SBBR污染物去除效果的差异。     

游离氨对活性污泥系统中硝化性能和硝化群落结构的影响

建立了4个平行的SBR处理合成废水,游离氨(FA)浓度分别为0.5、5、10、15 mg/L,命名为S0.5、S5、S10和S15,4个系统的脱氮性能在整个实验过程中均很好(平均值为98.7％),利用FA对亚硝酸氧化细菌(NOB)的抑制作用,结合过程控制,成功在S10和S15系统中实现短程硝化.在建立短程硝化途径的过程...     

High-Throughput Experimentation in Oxidation Catalysis

High-throughput experimentation in catalysis comprises the following components: (i) automated high-throughput synthesis, (ii) testing in Stage I and Stage II, for which to some extent novel assays are necessary, (iii) data handling and experimental design tools, and (iv) robotics. This contribution covers these topics, using examples from the research of the authors, but also from the literature, in order to illustrate the problems and opportunities associated with high-throughput experimentation in catalysis, focusing particularly on heterogeneous catalysis.