Characterization of N-phosphoryl oligopeptide libraries by ESI-MS and HPLC-MS

N-diisopropyloxyphosphoryl oligopeptide libraries were constructed through the transformation from the homo-oligopeptide libraries, which were synthesized with the assistance of phosphorus oxychloride. The molecular mass distributions of N-phosphoryl oligopeptide libraries were monitored by ESI-MS, and the components identification and quantitative analysis were carried out by HPLC-MS.     

A chitin-oligomer binding peptide obtained by screening of a phage display random peptide library and its affinity modulation corresponding to oxidation–reduction state

Screening of a phage display random 9-mer peptide library, in which cysteine residues were at the both terminals of the 7-mer random region, was performed to obtain an oligopeptide that recognizes a chitin-oligomer. Affinity of the obtained peptide (Cys-Ser-Arg-Thr-Thr-Arg-Thr-Arg-Cys) to chitotriose was modulated by its oxidation–reduction state. Only the oxidized form exhibited specific binding to the target molecule, chitotriose. This is the first report of reversible affinity modulation of a synthetic oligopeptide which can recognize a neutral saccharide.     

Diversity of the Clostridium coccoides group in human fecal microbiota as determined by 16S rRNA gene library

Fecal microbiota were analyzed in seven healthy individuals by 16S rRNA gene libraries (universal library) using universal primers, and the Clostridium coccoides group libraries using the universal primer 27F and the C. coccoides group-specific primer Erec482. The universal libraries were used in our previous studies. The 972 clones obtained from two different primer set libraries belonged to the C. coccoides group and were classified into 139 operational taxonomic units (OTU) (at least 98% sequence similarity). Of these, 41 OTU were detected commonly from universal libraries and C. coccoides group libraries. One hundred and ten OTU were detected from the C. coccoides group libraries. Fifteen new OTU were isolated from the C. coccoides group libraries in human gut. Most of the OTU did not correspond to known species, thus representing as-yet-uncultured bacteria. We also detected OTU that related to the butyrate-producing bacteria. The C. coccoides group consisted of an average of 35 OTU, although there were differences in the number and the type of species in each individual. When fecal microbiota were analyzed using universal libraries, the OTU belonging to the C. coccoides group detected in elderly individuals were fewer than those detected in adult individuals. When C. coccoides group libraries were used, the numbers of OTU in elderly and adult individuals were not different. Interindividual differences in the composition of OTU belonging to the C. coccoides group were also observed in fecal microbiota.     

ESI-MS Studies of Hetro-peptide Libraries by Phosphorus Oxychloride Activation

Peptide libraries by phosphorus oxychloride activation were developed and several hetro-peptide libraries were synthesized and analyzed by Bio-Tools^TM software with ESI-MS/MS technique. The products of the libraries were studied and the diversity of the peptide libraries was discussed. It was found that the reaction of phosphorus oxychloride with l-Val/l-Leu produced the most abundant hetro-peptide libraries with 68 kinds of peptides sub-libraries based on molecular weight difference.     

ESI-MS studies on novel liquid homo-peptide libraries and their conjugate libraries directed by phosphorus oxychloride

Summary The reactions of natural non-polar amino acids with phosphorus oxychloride were investigated. Some non-polar amino acids, such as L-Phe, L-Leu and L-Val were treated with phosphorus oxychloride, then quenched with water or some bioactive compounds (L-menthol, cinchonidine and benzylamine), and yielded the corresponding peptide and peptide conjugate libraries. ESI-MS/MS was used to study the products of the reaction and confirm the structure of the library. This paper reports a simple method to synthesize the homo-oligo-peptide libraries and peptide conjugated libraries.     

Protein structure prediction using mutually orthogonal Latin squares and a genetic algorithm

We combine a new, extremely fast technique to generate a library of low energy structures of an oligopeptide (by using mutually orthogonal Latin squares to sample its conformational space) with a genetic algorithm to predict protein structures. The protein sequence is divided into oligopeptides, and a structure library is generated for each. These libraries are used in a newly defined mutation operator that, together with variation, crossover, and diversity operators, is used in a modified genetic algorithm to make the prediction. Application to five small proteins has yielded near native structures.     

cDNA文库的构建策略及其应用

cDNA文库在基因分离和克隆中具有重要的作用。从cDNA文库中能筛选出所需要的目的基因,并直接用于该目的基因的表达。cDNA文库是发现新基因和研究基因功能的基础工具。随着分子生物学技术的发展。cDNA文库构建方法有了很大改进和提高,就cDNA文库的构建方法及其应用进行综述。     

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel.     

Comparison of library screening techniques used in the development of dsDNA ligands

The gel retardation and FID (fluorescent intercalator displacement) techniques have been compared for the selection of dsDNA binding ligands out of library mixtures. The selection procedure involves the synthesis and screening of unnatural oligopeptide libraries based on an iterative deconvolution procedure. Both methods yield comparable selection results and binding constants for the selected compounds, meaning that they can be considered as complementary in the discovery process of new antigene compounds. Furthermore, a quinazolin-2,4-dione amino acid has been identified as possessing interesting properties for interaction with dsDNA.     

Effects of signal sequence on phage-displayed disulfide-stabilized single chain antibody variable fragment (sc-dsFv) libraries

Phage-displayed single chain variable fragment (scFv) libraries are powerful tools in antibody engineering. Disulfide-stabilized scFv (sc-dsFv) with an interface disulfide bond is structure-wise more stable than the corresponding scFv. A set of recently discovered signal sequences replacing the wild type (pelB) signal peptidase cleavage site in the c-region has been shown to be effective in rescuing the expression of sc-dsFv libraries on the phage surface. However, the effects of the other regions of the signal sequence on the expression of the sc-dsFv libraries and on the formation of the interface disulfide bond in the phage-displayed sc-dsFv have not been clear. In this work, selected novel signal sequence variants in the h-region were shown to be equally effective in promoting sc-dsFv library expression on the phage surface; the expression level and complexity of the sc-dsFv libraries were comparable to the corresponding scFv libraries produced with the wild-type (pelB) signal sequence. The interface disulfide bond in the phage-displayed sc-dsFv was proven to form to a large extent in the library variant ensemble generated with signal sequence variants in both the h-region and the c-region. The sc-dsFv engineering platform established in this work can be applied to many of the known scFv molecules which are in need of a more stable version for the applications under harsh conditions or for longer shelf-life.     

医院图书馆建立学科馆员制度的制约因素探讨

目的：探讨在医院图书馆建立学科馆员制度和开展学科化服务的必要性与可行性。方法：通过上海市三级医院图书馆的人员、信息服务基础、数字化资源建设及学科馆员设置比例等的调研数据,分析其影响医院图书馆开展学科化服务的制约因素。结果：目前医院图书馆开展学科化服务存在：大环境、制度、评估指标、人员素质、和数字化资源建设六方面的主要制约因素。结论：学科馆员制度设置和学科化服务的开展是医院图书馆发展的方向和主流。通过对制约因素的揭示,在现有的条件和状态下,可通过制度保证、完善评估机制和标准、人员的继续教育和资源共建共享等措施,逐步提高医院图书馆学科馆员设置比例,以提升医院图书馆服务水平。     

肺癌消减杂交文库中共同基因的生物信息学分析及验证

抑制性消减杂交(suppression subtractive hybridization,SSH)技术因操作简单、筛选效率高、假阳性率低等优点,被广泛用于生物与医学领域．该文拟找出用SSH所建立的10个肺癌差异表达基因文库中重复出现的基因,结果显示,其中6个正向杂交文库中,出现2次的基因有41个,出现3次的基因有4个;其中4个反向杂交文库中,出现2次的基因有6个．进一步用生物信息学分析发现,这些重复出现的差异表达基因参与了多种细胞功能,如基因转录、蛋白翻译、细胞粘附、DNA修复、氧化还原反应等,并涉及多条信号通路．值得注意的是,在这些差异表达基因中,核糖体蛋白相关基因占比最多．然后,用实时定量PCR检测文库中的TIMP3、GPX3、HSP90B1、CD9等基因的表达,结果显示,SSH文库中差异表达基因的上调和下调趋势具有较好的特异性．该研究为发现肺癌相关基因提供有价值的线索．     

噬菌体肽库技术的应用

噬菌体肽库是由大量带有不同肽段的单个噬菌体组成的重组噬菌体库,通过分析筛选到的多肽的结构和序列,可以了解蛋白质分子之间的相互作用。随着生物技术的发展,噬菌体肽库技术在基因治疗、抗原表位定位、确定核酸结合蛋白、基因疫苗研究和药物筛选等方面得到广泛应用并取得了很大进展。     

藏酋猴微卫星富集文库的构建及微卫星分子标记的筛选

通过磁珠富集法构建了藏酋猴AC重复和AAAG重复的微卫星富集文库,分离微卫星序列并对其进行分析。将藏酋猴基因组DNA经Sau3AI酶切后纯化回收,连接特定接头。用生物素标记的探针与酶切片段杂交,捕获300～1000bp片段,随后将获得的片段连接到pMD-19T载体上,转化至JM109中,成功构建藏酋猴微卫星富集文库。(AC)n富集文库和(AAAG)n富集文库的阳性克隆率分别为50%和10%左右。根据测序得到的48个微卫星序列成功设计了24对引物,最终筛选出6个微卫星标记,这些标记将为藏酋猴的遗传多样性研究、圈养种群结构的分析和遗传图谱的构建等奠定一定的基础。     

Power and false-positive rate in QTL detection with near-isogenic line libraries

Libraries of near-isogenic lines (NILs) were used for quantitative trait locus (QTL) detection in model species and economically important crops. The experimental design and genetic architecture of the considered traits determine the statistical properties of QTL detection. The objectives of our simulation study were to (i) investigate the population sizes required to develop NIL libraries in barley and maize, (ii) compare NIL libraries with nonoverlapping and overlapping donor segments and (iii) study the number of QTLs and the size of their effects with respect to the power and the false-positive rate of QTL detection. In barley, the development of NIL libraries with target segment lengths of 10 c and marker distances of 5 c was possible using a BC₃S₂ backcrossing scheme and population sizes of 140. In maize, population sizes larger than 200 were required. Selection for the recipient parent genome at markers flanking the target segments with distances between 5 and 10 c was required for an efficient control of the false-positive rate. NIL libraries with nonoverlapping donor chromosome segments had a greater power of QTL detection and a smaller false-positive rate than libraries with overlapping segments. Major genes explaining 30% of the genotypic difference between the donor and recipient were successfully detected even with low heritabilities of 0.5, whereas for minor genes explaining 5 !or 10%, high heritabilities of 0.8 or 0.9 were required. The presented results can assist geneticists and breeders in the efficient development of NIL libraries for QTL detection.     

蝶兰胚珠的cDNA文库构建及其特异基因表达的研究

以蝶兰(Phalaenopsis“Mt.Kaala”cv SM9108)为材料,分别提取大孢子母细胞时期胚珠和成熟胚珠的PolyA RNA,反转录成cDNA,构建起两个cDNA文库。克隆筛选采用差异杂交法。从上述两个cDNA文库中,各选择一个筛选出的cDNA,对其在植物体不同器官和不同发育时期的胚珠内的表达进行了分析。结果表明该两个cDNA均为胚珠特异,并且分别在胚珠发育的特定时期表达。推测该两个cDNA的表达受胚珠内部的不同因子调控。     

各种RNA干扰库的构建及比较

不同种类的RNA干扰库已经被用于基因功能的研究之中,根据其构建方式和分子形式的不同,可将RNA干扰库分为4种类型,即化学合成的小干扰RNA（siRNA）库、化学合成的小发夹RNA（shRNA）表达库、酶切法构建的siRNA库和酶切法构建的shRNA表达库。简要介绍上述RNA干扰库的构建方法及其特点和局限。     

A facile route to dynamic glycopeptide libraries based on disulfide-linked sugar-peptide coupling

We report here that disulfide-linked dynamic glycopeptide libraries can be constructed from 1-thiosugar and cysteine-rich oligopeptide building blocks upon gentle air oxidation of a slightly basic (pH 7.8) aqueous solution thereof. A mixture of 1-thiogalactose and two oligopeptides H2N-CysGlyCysGly-CO2H and H2N-GlyCycCysGlyGly-CO2H, for example, affords a poorly HPLC-resolved disulfide library composed of various sugar-peptide conjugates and cyclic peptides, at least 10 of which can be identified by ESI mass spectrometry. The building components of disulfide members are exchangeable with each other in the presence of dithiothreitol as an initiator to allow dynamic equilibration. A preliminary SPR examination shows that the thiogalactose-derived library indeed contains active divalent galactoside species capable of cross-linking peanut lectin molecules.     

Comparison of large-insert,small-insert and pyrosequencing libraries for metagenomic analysis

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation'' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.