可转化人工染色体文库的构建与鉴定

随着人类和植物基因组计划的实施,能容纳大片段的人工染色体载体发展迅速。而用YAC、BAC和PAC等基因组文库进行目的基因的筛选,在获得候选克隆后,通常要进行亚克隆,然后对每个亚克隆逐一进行基因功能互补试验,不仅工作量大,而且有遗漏目的基因的危险。TAC载体含P1质粒和Ri质粒的复制子,可直接转化植物,大大加速了工作进程。概括性的叙述了TAC载体的发展,TAC文库构建的程序及文库鉴定的问题。     

Generation of EST and Microarray Resources for Functional Genomic Studies on Chicken Intestinal Health

Abstract

Expressed sequenced tags (ESTs) and microarray resources have a great impact on the ability to study host response in mice and humans. Unfortunately, these resources are not yet available for domestic farm animals. The aim of this study was to provide genomic resources to study chicken intestinal health, in particular malabsorption syndrome (MAS), which affects mainly the intestine. Therefore a normalized and subtracted cDNA library containing more than 7000 clones was prepared. Randomly chosen clones were sequenced for control purposes. New ESTs were found and multiple ESTs not identified in the chicken intestine before were observed. The number of non‐specific ESTs in this cDNA library was low. Based on this normalized and subtracted library a cDNA microarray was made. In a preliminary hybridization experiment with the microarray, genes were identified to be up‐ or downregulated in MAS infected chickens. This indicates that the generated resources are valuable tools to investigate chicken intestinal health by whole genome expression analysis approaches.     

Synthesis of a small library of bivalent α-d-mannopyranosides for lectin cross-linking

A small library of bivalent α-d-mannopyranosides having rigid linkers was constructed in order to evaluate the effects of inter-saccharide distances upon multivalent binding interactions with plant and bacterial lectins. To this end, iodoaryl and propargyl α-d-mannopyranosides were synthesized and the former treated with TMS-acetylene under palladium chemistry to provide their corresponding ethynylaryl derivatives. A library of 15 dimeric members was then obtained using Lewis acid catalyzed glycosidation, aryl–aryl homocoupling, transition metal catalyzed Sonogashira cross-coupling reactions, and oxidative Glaser homocoupling.     

An on-bead assay for the identification of non-natural peptides targeting the androgen receptor-cofactor interaction

An efficient and rapid on-bead screening method was established to identify non-natural peptides that target the Androgen Receptor-cofactor interaction. Binding of the Androgen Receptor ligand binding domain to peptide sequences displayed on beads in a One-Bead-One-Compound format could be screened using fluorescence microscopy. The method was applied to generate and screen both a focussed and a random peptide library. Resynthesis of the peptide hits allowed for the verification of the affinity of the selected peptides for the Androgen Receptor in a competitive fluorescence polarization assay. For both libraries strong Androgen Receptor binding peptides were found, both with non-natural and natural amino acids. The peptides identified with natural amino acids showed great similarity in terms of preferred amino acid sequence with peptides previously isolated from biological screens, thus validating the screening approach. The non-natural peptides featured important novel chemical transformations on the relevant hydrophobic amino acid positions interacting with the Androgen Receptor. This screening approach expands the molecular diversity of peptide inhibitors for nuclear receptors.     

微创经皮肾镜钬激光联合气压弹道碎石术治疗马蹄肾结石

目的:探讨微创经皮肾镜钬激光联合气压弹道碎石术治疗马蹄肾结石的可行性与效果,以期指导临床诊治。方法:回顾性分析在2010年8月~2013年8月期间接受微创经皮肾镜钬激光联合气压弹道碎石术治疗的12例马蹄肾结石患者的疗效及并发症。结果:在12例患者中,一期结石清除率为83.3%(10/12),2例需要二期取石。单通道取石11例,双通道取石1例。1例患者为上盏通道,其余患者均为中盏通道。平均手术时间105 min,术中平均出血量50 m L,平均住院天数14d。未出现严重并发症,无患者需要输血。结论:微创经皮肾镜钬激光联合气压弹道碎石术是治疗马蹄肾结石的安全、有效的方法,具有损伤小、出血少、恢复快等优点。     

灌洗法配合透明质酸钠治疗膝骨关节炎性滑膜炎

目的：探讨双套管针穿刺膝关节腔内闭式灌洗,配合透明质酸钠治疗膝骨关节炎性滑膜炎的临床疗效。方法：回顾性分析52例膝关节积液明显的膝骨关节炎性滑膜炎患者的临床效疗。结果：无药物反应发生,有3例出现关节红、肿、痛,但2～3天后症状缓解。术后随访3～26月,治愈好转率达96％,平均治愈好转时间为18、4天。2例于治疗半年后复发,经再次治疗后康复。结论：此方法对膝关节呈Ⅰ～Ⅲ期改变的骨关节炎,特别是合并滑膜炎患者有较好疗效。     

Application of the concept of oxime library screening by mass spectrometry (MS) binding assays to pyrrolidine-3-carboxylic acid derivatives as potential inhibitors of γ-aminobutyric acid transporter 1 (GAT1)

In the present study, the concept of oxime library screening by MS Binding Assays was successfully extended to N-substituted lipophilic pyrrolidine-3-carboxylic acid derivatives in the pursuit of varying the amino acid motif in order to identify new inhibitors for GAT1 and to broaden structure–activity-relationships for this target, the most abundant GABA transporter in the central nervous system. For the screening, 28 different oxime sub-libraries were employed that were generated by simple condensation reaction of an excess of pyrrolidine-3-carboxylic acid derivatives carrying a hydroxylamine functionality with various sub-libraries each assembled of eight aldehydes with broadly varying chemical structures and functionalities. The compounds responsible for the activity of an oxime sub-library were identified by deconvolution experiments performed by employing single oximes. Binding affinities of the oxime hits were confirmed in full-scale competitive MS Binding Assays. Thereby, oxime derivatives with a 1,1′-biphenyl moiety were found as the first inhibitors of mGAT1 comprising a pyrrolidine-3-carboxylic acid motif with affinities in the submicromolar range.     

Advances in Lead Generation

Lead Generation represents a critical drug discovery phase where chemical starting points and their respective mechanism of action, quality, and potential liabilities are largely predefined. Recent advances such as DNA-encoded libraries or fragment-, chemical biology-, and virtual screening-based approaches are today as common as traditional High Throughput Screening. Innovations in characterizing lead quality have allowed more informed decision-making by discovery teams. The key challenge today is to individually tailor the right mix of methods for each project to facilitate data integration with the purpose of creating multiple high-quality lead series, ultimately translating to reduced chemistry-related pipeline attrition.