Screening for novel enzymes from metagenome and SIGEX,as a way to improve it

Metagenomics has been successfully applied to isolate novel biocatalysts from the uncultured microbiota in the environment. Two types of screening have been used to identify clones carrying desired traits from metagenomic libraries: function-based screening, and sequence-based screening. Both function- and sequence- based screening have individual advantages and disadvantages, and they have been applied successfully to discover biocatalysts from metagenome. However, both strategies are laborious and tedious because of the low frequency of screening hits. A recent paper introduced a high throughput screening strategy, termed substrate-induced gene-expression screening (SIGEX). SIGEX is designed to select the clones harboring catabolic genes induced by various substrates in concert with fluorescence activated cell sorting (FACS). This method was applied successfully to isolate aromatic hydrocarbon-induced genes from a metagenomic library. Although SIGEX has many limitations, it is expected to provide economic advantages, especially to industry.     

Practical reactor systems for yeast cell immobilization using biomass support particles

The technique of cell immobilization using porous support particles (biomass support particles) has been successfully applied to yeast cells. Two reactor configurations exploiting the use of these particles have been developed and assessed for use in aseptic yeast fermentations. A liquid-fluidized bed fermenter has been devised for use with particles denser than the fermentation liquor whilst a gas-stirred circulating bed fermenter proved suitable for particles of essentially neutral buoyancy. Both systems have been operated successfully for extended periods of continuous operation. The utilization of biomass support particle technology in such reactors provides a practical and robust system for immobilized cell reactors. This technology offers significant opportunities for further development.     

An on-chip magnetic bead separator using spiral electromagnets with semi-encapsulated permalloy.

A new planar bio-magnetic bead separator on a glass chip has been designed, fabricated and tested. The separator is composed of micromachined semi-encapsulated spiral electromagnets and fluid channels, which have been separately fabricated and then bonded. The device was tested with super-paramagnetic beads of mean diameter 1 microm which were suspended in a buffered solution. When a DC current of 300 mA was applied to the inductor, the bio-magnetic beads were successfully separated on the electromagnets, showing a functional capability as a magnetic bead separator. To evaluate separation rate and capability, the inductance measurement method has been introduced and the inductance variation according to the separation rate has been characterized. Using this separator, cells or cell fragments and magnetic beads bonded with protein or enzyme suspended in bio-buffer solutions can be successfully separated from their suspensions, envisaging a filterless bio-separator.     

佐剂诱导关节炎大鼠模型的建立及成纤维样滑膜细胞的分离

目的建立类风湿性关节炎（RA）动物模型;从炎性关节滑膜中分离成纤维样滑膜细胞（FLS）。方法应用热灭活结核杆菌H37Ra菌株与矿物油混合制备改良的佐剂,尾根部皮内注射Lewis大鼠诱导关节炎;剪取成功诱导关节炎大鼠的病变踝关节,从中剥离滑膜组织,充分剪碎后采用胶原酶消化法分离成纤维样滑膜细胞。结果成功诱导了大鼠关节炎,发病率为100%,发病时间有规律,组织学表现与RA相似;成功在体外培养了FLS并对其进行了鉴定,掌握了其生长形态和特征。结论成功制备RA动物模型并获得FLS,为今后RA发病机制探索和药物评价提供了良好的体内动物模型和体外细胞模型。     

Concise synthesis of clarhamnoside, a novel glycosphingolipid isolated from the marine sponge Agela clathrodes

The first total synthesis of a novel alpha-galactoglycosphingolipid clarhamnoside has been achieved through a straightforward strategy. A thiogalactosyl donor with a benzylidene group at C-4 and C-6 and nonparticipating p-methoxybenzyl group at C-2 was successfully employed in the stereocontrolled syntheses of alpha-GalGSLs. The N-Phth-protected trifluoroacetimidate donor for terminal disaccharide was successfully applied in constructing the [GalNAc beta-(1-->6)-Gal] glycosidic linkage.     

Synthesis and biological evaluation of new opioid agonist and neurokinin-1 antagonist bivalent ligands

Newly designed bivalent ligands-opioid agonist/NK1-antagonists have been synthesized. The synthesis of new starting materials-carboxy-derivatives of Fentanyl (1a-1c) was developed. These products have been transformed to 'isoimidium perchlorates' (2a-c). The new isoimidium perchlorates have been successfully implemented in nucleophilic addition reactions, with l-tryptophan 3,5-bis(trifluoromethyl)benzyl ester to give the target compounds-amides (3a-c). Perchlorates (2a-c) successfully undergo reactions with other nucleophiles such as alcohols, amines or hydrazines. The obtained compound 3b exhibited μ-opioid agonist activity and NK1-antagonist activity and may serve as a useful lead compound for the further design of a new series of opioid agonist/NK1-antagonist compounds.     

利用Red同源重组系统进行牛β酪蛋白基因敲除

利用EL350基因工程菌进行同源重组, 成功进行基因敲除已有报道, 但利用该系统进行乳腺生物反应器质粒构建的研究却未见报道。实验采用含有完整的牛b 酪蛋白基因的CSN2质粒作为基因打靶的载体, 设计不同的同源臂, 成功地敲除了b 酪蛋白基因的编码区。并且同时利用同源重组技术对敲除不同大小的DNA片段的效率进行了研究。为进一步利用CSN2质粒两端的调控序列, 插入新的基因, 研究其表达功能, 或者进行乳腺生物反应器的研究奠定了基础。     

借助慢病毒将EGFP基因导入西藏小型猪胎儿成纤维细胞

目的借助慢病毒将EGFP基因导入西藏小型猪胎儿成纤维细胞（porcine embryonic fibroblasts,PEFs）,以基于PEFs建立慢病毒介导的外源基因体外投递系统。方法取35d西藏小型猪胚胎,酶消化法分离培养西藏小型猪的PEFs;按Invitrogen公司推荐的标准程序进行慢病毒（携带EGFP基因）包装（脂质体介导的瞬时转染）,随后用病毒上清感染PEFs,24～48h后荧光显微镜下观察是否见绿色荧光以证实慢病毒是否成功生产和成功感染PEFs。结果成功分离培养西藏小型猪的PEFs,按标准程序生产的携带EGFP基因慢病毒高效率感染西藏小型猪的PEFs。结论针对西藏小型猪的PEFs建立了相应的慢病毒介导的外源基因体外投递系统,为相关后续研究打下了良好基础。     

Structural proteomics by NMR spectroscopy

Structural proteomics is one of the powerful research areas in the postgenomic era, elucidating structure-function relationships of uncharacterized gene products based on the 3D protein structure. It proposes biochemical and cellular functions of unannotated proteins and thereby identifies potential drug design and protein engineering targets. Recently, a number of pioneering groups in structural proteomics research have achieved proof of structural proteomic theory by predicting the 3D structures of hypothetical proteins that successfully identified the biological functions of those proteins. The pioneering groups made use of a number of techniques, including NMR spectroscopy, which has been applied successfully to structural proteomics studies over the past 10 years. In addition, advances in hardware design, data acquisition methods, sample preparation and automation of data analysis have been developed and successfully applied to high-throughput structure determination techniques. These efforts ensure that NMR spectroscopy will become an important methodology for performing structural proteomics research on a genomic scale. NMR-based structural proteomics together with x-ray crystallography will provide a comprehensive structural database to predict the basic biological functions of hypothetical proteins identified by the genome projects.     

Cryoballoon ablation of focal atrial tachycardia originating from right atrial appendage: Case report and review of the literature

The right atrial appendage (RAA) is a rare site of focal atrial tachycardia (AT). Sometimes, catheter ablation cannot successfully be accomplished at this location due to the difficulty in reaching the exact ablation site as well as the associated possible life-threatening complications like pericardial tamponade or perforation. Although radiofrequency (RF) ablation is preferred for the treatment of RAA tachycardias, alternative tools may be required in rare instances. This report presents a case of RAA tachycardia that was not terminated by RF ablation, instead, has been successfully ablated using cryoballoon. In addition, an overview of the literature and therapeutic options for the AT originating from RAA have also been included.     

Recent developments in mass spectrometry-based quantitative phosphoproteomics.

Protein phosphorylation is a reversible post-translational modification that is involved in virtually all eukaryotic cellular processes and has been studied in great detail in recent years. Many developments in mass spectrometry (MS)-based proteomics have been successfully applied to study protein phosphorylation in highly complicated samples. Furthermore, the emergence of a variety of enrichment strategies has allowed some of the challenges associated with low phosphorylation stoichiometry and phosphopeptide copy number to be overcome. The dynamic nature of protein phosphorylation complicates its analysis; however, a number of methods have been developed to successfully quantitate phosphorylation changes in a variety of cellular systems. The following review details some of the most recent breakthroughs in the study of protein phosphorylation, or phosphoproteomics, using MS-based approaches. The majority of the focus is placed on detailing strategies that are currently used to conduct MS-based quantitative phosphoproteomics.     

Live attenuated vaccines for human use.

Live attenuated vaccines have been successfully used for the prevention of a number of viral and bacterial diseases. Several vaccine strains have been utilized recently as expression vectors for cloned heterologous antigens. Through the use of recombinant DNA technology, candidate vaccine strains and vector systems have been developed and are undergoing clinical evaluation.     

溶解固氮蓝藻的细菌

固氮蓝藻作为水稻新肥源已获得良好增产效果。       

利用动态等位基因特异性杂交技术进行单核苷酸多态高通量分型

动态等位基因特异性杂交(dynamic allele-specific hybridization, DASH)是新发展起来的一种单核苷酸多态(single nucleotide polymorphisms, SNP)等位基因分型技术,具有快速、经济、准确、高通量、重复性好等优点.利用DASH技术,对96份正常人外周血DNA样品成功地进行了两个SNP位点的基因分型,并摸索实验条件,对该技术进行了优化.     

Functionalized polymeric liposomes. Efficient immobilization of alpha chymotrypsin

A polymerizable phospholipid has been synthesized having a latent aldehyde moiety in the head group (1). Photopolymerized vesicles which have been prepared from this phosphatidylcholine have been successfully conjugated to alpha chymotrypsin. A high degree of loading was achieved with significant retention of enzymatic function.     

Towards systems metabolic engineering of microorganisms for amino acid production

Microorganisms capable of efficient production of amino acids have traditionally been developed by random mutation and selection method, which might cause unwanted physiological changes in cellular metabolism. Rational genome-wide metabolic engineering based on systems and synthetic biology tools, which is termed 'systems metabolic engineering', is rising as an alternative to overcome these problems. Recently, several amino acid producers have been successfully developed by systems metabolic engineering, where the metabolic engineering procedures were performed within a systems biology framework, and entire metabolic networks, including complex regulatory circuits, were engineered in an integrated manner. Here we review the current status of systems metabolic engineering successfully applied for developing amino acid producing strains and discuss future prospects.     

Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics?

Morpholino phosphorodiamidate anti-sense oligonucleotides (MPOs) have recently emerged as a tool for gene-specific knockdown. MPOs have a great potential for both therapeutic applications and functional genomics. In particular, zebrafish are well suited for gene function studies using MPOs owing to their rapid external development, transparent embryos and the ease of delivery of the intervening MPOs. This paper describes principles of MPO action and the application of MPOs for gene function studies and therapeutics. In the field of functional genomics, the MPO strategy has been most successfully used to study gene function in zebrafish. Over 35 mutations have been successfully phenocopied and over 30 novel gene functions have been analysed in zebrafish using MPOs during the last two years. The essential controls that are required to avoid misinterpretation of experimental data when using MPOs for gene function analysis will also be described.     

微生物酶的分子改性和人工进化的研究进展

运用分子生物学技术对微生物来源的酶进行分子改性和人工进化在过去几年中取得了令人瞩目的进展。本文综述了用于酶分子改性和人工进化的主要分子生物学方法,如易错PCR技术、DNA体外随机拼接技术等及其在酶的分子进化和改性中应用成就。     

Cultivation of the Spirochætes Associated with Tropical Ulcer

Spiroch?tes from cases of tropical ulcer have been cultured in a modified Wenyon media and have now been successfully passed through thirty-two subcultures.-A pure culture has not yet been obtained.-Five inocultation experiments are described in which it has been shown that the spiroch?tes and fusiform bacilli, in mixed culture, are capable of proliferation and of producing ulcerated lesions in natives.     

Hydrophobic tag-assisted liquid-phase synthesis of a growth hormone-inhibiting peptide somatostatin

Hydrophobic tag-assisted liquid-phase peptide synthesis technique and disulfide bond formation have been well-combined, leading to the efficient and practical preparation of a growth hormone-inhibiting peptide somatostatin. Intramolecular disulfide bond formation has successfully been carried out even under relatively high concentrations, enabling the effective peptide modifications in preparative scale.