DNA ligation using a disposable microfluidic device combined with a micromixer and microchannel reactor

A novel PDMS and glass-based microfluidic device consisting of a micromixer and microreactor for DNA ligation is described in this article. The new passive type planar micromixer is 10.33 mm long and composed of a straight channel integrated with nozzles and pillars, and the microreactor is composed of a serpentine channel. Mixing was enhanced by convective diffusion facilitated by the nozzles and pillars. The performance of the micromixer was analytically simulated and experimentally evaluated. The micromixer showed a good mixing efficiency of 87.7% at a 500 μL/min flow rate (Re = 66.5). DNA ligation was successfully performed using the new microfluidic device, and ligation time was shortened from 4 h to 5 min. When used for on-chip ligation, this new micromixer offers advantages of disposability and portability.     

SNP genotyping by DNA photoligation: application to SNP detection of genes from food crops

We describe a simple and inexpensive single-nucleotide polymorphism (SNP) typing method, using DNA photoligation with 5-carboxyvinyl-2′-deoxyuridine and two fluorophores. This SNP-typing method facilitates qualitative determination of genes from indica and japonica rice, and showed a high degree of single nucleotide specificity up to 10 000. This method can be used in the SNP typing of actual genomic DNA samples from food crops.     

氯胺酮对腹腔感染脓毒症小鼠生存率的影响

目的: 探讨早期使用氯胺酮是否延迟盲肠结扎穿孔(CLP)所致脓毒症小鼠的死亡时间。方法: 60只CLP小鼠,分别于术后1、6 h 腹腔注射氯胺酮0、3、6、12 mg·100 g^-1,观察小鼠死亡情况并比较累计生存率。结果: 氯胺酮延长CLP小鼠死亡时间(P<0.05);氯胺酮6 mg·100 g^-1组小鼠生存时间明显长于3 mg·100 g^-1组和12 mg·100 g^-1组(P<0.05)。结论: 氯胺酮延迟CLP小鼠死亡时间,并呈现剂量依赖性。     

A FRET Sensor to Monitor Bivalent SUMO–SIM Interactions in SUMO Chain Binding

The small ubiquitin‐like modifier (SUMO) can be assembled into polymeric chains as part of its diverse biochemical signal pattern upon conjugation to substrate proteins. SUMO chain recognition is facilitated by receptor proteins that contain at least two SUMO‐interacting motifs (SIMs). Little is known about the structure of SUMO chains, both in an unliganded form and upon complexation with multi‐SIM protein partners. A FRET sensor has been developed based on a linear dimer of human SUMO‐2 as a minimal SUMO chain analogue. The synthetic acceptor and donor dyes were conjugated by maleimide and copper‐catalyzed click chemistry to each of the two SUMO subunits. FRET changes were only observed in the presence of di‐ or multi‐SIM ligands. Alteration of the short linker sequence between SIMs 2 and 3 of RNF4 showed a great tolerance, and hence, structural flexibility, of the SUMO dimer for bivalent binding of adjacent SIMs. The di‐SUMO FRET sensor reports on the binding of SIM clusters of the proteins C5orf25 and SOBP; this suggest that these can bind to adjacent subunits of a SUMO chain. The developed FRET sensor will be a useful tool to study the importance of SIM and linker sequences, as well as biochemical and structural properties of SUMO chains and multi‐SIM proteins.     

Specifically immobilised aldo/keto reductase AKR1A1 shows a dramatic increase in activity relative to the randomly immobilised enzyme

The difference between site-specific and random immobilisation of the aldo/keto reductase AKR1A1 was explored. AKR1A1 was recombinantly expressed as a thioester by the intein strategy. The thioester was selectively modified with a biotin label by the expressed protein ligation method, and subsequent immobilisation on streptavidin templates was performed. Adsorption of wild-type AKR1A1 to streptavidin templates and of biotinylated AKR1A1 to uncoated templates was used to study randomly immobilised enzymes. Investigation of the kinetic parameters revealed remarkably improved activity for the site-specifically immobilised enzyme, which was comparable to that of the wild-type enzyme in solution and 60-300-fold greater than that of the randomly immobilized enzymes. Furthermore, the enzyme was surprisingly stable. No loss of activity was observed for over a week, and even after 50 days more than 35% of activity was maintained.