Reduced sister chromatid cohesion acts as a tumor penetrance modifier

Sister chromatid cohesion (SCC) is an important process in chromosome segregation. ESCO2 is essential for establishment of SCC and is often deleted/altered in human cancers. We demonstrate that esco2 haploinsufficiency results in reduced SCC and accelerates the timing of tumor onset in both zebrafish and mouse p53 heterozygous null models, but not in p53 homozygous mutant or wild-type animals. These data indicate that esco2 haploinsufficiency accelerates tumor onset in a loss of heterozygosity (LOH) sensitive background. Analysis of The Cancer Genome Atlas (TCGA) confirmed ESCO2 deficient tumors have elevated number of LOH events throughout the genome. Further, we demonstrated heterozygous loss of sgo1, important in maintaining SCC, also results in reduced SCC and accelerated tumor formation in a p53 heterozygous background. Surprisingly, while we did observe elevated levels of chromosome missegregation and micronuclei formation in esco2 heterozygous mutant animals, this chromosomal instability did not contribute to the accelerated tumor onset in a p53 heterozygous background. Interestingly, SCC also plays a role in homologous recombination, and we did observe elevated levels of mitotic recombination derived p53 LOH in tumors from esco2 haploinsufficient animals; as well as elevated levels of mitotic recombination throughout the genome of human ESCO2 deficient tumors. Together these data suggest that reduced SCC contributes to accelerated tumor penetrance through elevated mitotic recombination.     

Fast identification of thermostable beta‐glucosidase mutants on cellobiose by a novel combinatorial selection/screening approach

Engineering costly cellulases on natural cellulosic substrates is of importance for emerging biomass‐based biorefineries. Directed enzyme evolution is becoming a popular tool, but identification of desired mutants from a large mutant library remains challenging sometimes. In this work, we demonstrated a novel combinatorial selection/screening strategy for finding thermostable beta‐glucosidase on its natural substrate—cellobiose. First, selection was conducted through complementation of beta‐glucosidase for non‐cellobiose‐utilizing Escherichia coli so that only the cells expressing active beta‐glucosidase can grow on a M9 synthetic medium with cellobiose as the sole carbon source (selection plate). Second, the clones on the selection plates were duplicated by using nylon membranes. After heat treatment, the nylon membranes were overlaid on M9/cellobiose screening plates so that remaining activities of thermostable beta‐glucosidase mutants hydrolyzed cellobiose on the screening plates to glucose. Third, the growth of an indicator E. coli strain that can utilize glucose but not cellobiose on the screening plates helped detect the thermostable beta‐glucosidase mutants on the selection plates. Several thermostable mutants were identified from a random mutant library of the Paenibacillus polymyxa beta‐glucosidase. The most thermostable mutant A17S had an 11‐fold increase in the half‐life of thermoinactivation at 50°C. Biotechnol. Bioeng. 2009;103: 1087–1094. © 2009 Wiley Periodicals, Inc.     

Structure-based design of COX-2 selectivity into flurbiprofen

Comparative computer modeling of the X-ray crystal structures of cyclooxygenase isoforms COX-1 and COX-2 has led to the design of COX-2 selectivity into the nonselective inhibitor flurbiprofen. The COX-2 modeling was based on a postulated binding mode for flurbiprofen and took advantage of a small alcove in the COX-2 active site created by different positions of the Leu384 sidechain between COX-1 and COX-2. The design hypothesis was tested by synthesis and biological assay of a series of flurbiprofen analogs, culminating in the discovery of several inhibitors having up to 78-fold selectivity for COX-2 over COX-1.     

Production of biocommodities and bioelectricity by cell‐free synthetic enzymatic pathway biotransformations: Challenges and opportunities

Cell‐free synthetic (enzymatic) pathway biotransformation (SyPaB) is the assembly of a number of purified enzymes (usually more than 10) and coenzymes for the production of desired products through complicated biochemical reaction networks that a single enzyme cannot do. Cell‐free SyPaB, as compared to microbial fermentation, has several distinctive advantages, such as high product yield, great engineering flexibility, high product titer, and fast reaction rate. Biocommodities (e.g., ethanol, hydrogen, and butanol) are low‐value products where costs of feedstock carbohydrates often account for ～30–70% of the prices of the products. Therefore, yield of biocommodities is the most important cost factor, and the lowest yields of profitable biofuels are estimated to be ca. 70% of the theoretical yields of sugar‐to‐biofuels based on sugar prices of ca. US$ 0.18 per kg. The opinion that SyPaB is too costly for producing low‐value biocommodities are mainly attributed to the lack of stable standardized building blocks (e.g., enzymes or their complexes), costly labile coenzymes, and replenishment of enzymes and coenzymes. In this perspective, I propose design principles for SyPaB, present several SyPaB examples for generating hydrogen, alcohols, and electricity, and analyze the advantages and limitations of SyPaB. The economical analyses clearly suggest that developments in stable enzymes or their complexes as standardized parts, efficient coenzyme recycling, and use of low‐cost and more stable biomimetic coenzyme analogs, would result in much lower production costs than do microbial fermentations because the stabilized enzymes have more than 3 orders of magnitude higher weight‐based total turn‐over numbers than microbial biocatalysts, although extra costs for enzyme purification and stabilization are spent. Biotechnol. Bioeng. 2010. 105: 663–677. © 2009 Wiley Periodicals, Inc.     

Structural investigations of the extracellular polysaccharides elaborated by Beijerinckia mobilis

The extracellular mucilage from Beijerinckia mobilis, a member of the Azotobacteriaceae, after removal of contaminating protein, was separated into a neutral polysaccharide (N-2, 10%); a neutral, dialysable fraction (N-1, 5%), consisting of glucose and oligosaccharides containing glucose, arabinose, and rhamnose; and an acidic polysaccharide (85%). N-2 (mol. wt, 1900) was highly branched and comprised glucopyranose, mannopyranose, and arabinofuranose residues (1:1:1). The various linkages were determined. The acid fraction was a polymer of high molecular weight composed of L-guluronic acid (65%), D-glucose (15%), and D-glycero-D-mannoheptose (20%), together with acetic and pyruvic acids. From the results of methylation, periodate oxidation, and partial hydrolysis, a branched molecule with a backbone of guluronic acid and heptose, and side chains of glucose and guluronic acid is proposed. Pyruvic acid was found to be acetal-linked to 2?5% of the heptose residues. The similarities between this polysaccharide and that from the related species Azotobacter indicum are discussed.     

每搏量变异度在围手术期容量治疗监测中的应用

每搏量变异度是动态的容量监测指标.机械通气患者心肺的相互作用是每搏量变异度的产生基础,通过动脉压力波形分析技术可以进行连续监测.每搏量变异度能够准确预测容量治疗反应,与静态的血流动力学参数相比,对于优化心输出量和组织氧供更有优势,但也存在一定的局限性.每搏量变异度受多种因素影响且不能用于自主呼吸和心律失常的患者.临床应用时应该综合考虑其影响因素,结合其他的指标和方法指导容量治疗.     

Characterisation of sucrose lactate and other oligosaccharides found in the cladophorales

Oligosaccharides not previously recorded in the Cladophorales (green algae) have been separated from the 80% ethanolic extract and characterised. 4-O-Lactyl-β- -fructofuranosyl - -glucopyranoside (sucrose lactate) was found only in Cladophora laetevirens and a fresh-water Rhizoclonium species. Panose, maltotriose, maltotetraose, and 6-O- -glucosylmaltotriose appear to be common to all the species examined¹.