The risk of stroke increases significantly after transient ischemic attack (TIA). TIA is an independent risk factor for cerebral infarction. This article review s the advances in prediction models of stroke risk after TIA in order to conduct rapid and accurate risk assessment and stratification in patients w ith TIA and develop timely reasonable treatment strategies, thereby reducing the risk of stroke.

Objective To observe the influences of VEGF transfection on the proliferation and ultrastructure of hBMSCs. Methods Three groups were enrolled in the experiment: group of Ad-VEGF transfection, group of empty Ad transfection, and the control group. Cells were observed continually by inverted phase contrast microscope, and stained by HE. Proliferation of cells was tested by MTT and by flow cytometry analysis. Results Histological observation and observation through inverted phase contrast microscope showed that the cells were of the similar morphology in the 3 groups. As time elapsed, the amount of cells increased, but still no obvious differences were found in optical density (OD) value of hBMSCs.Groups B, C, A had a similar percentage of DNA G1 period and a similar proliferation index (PrI) ( P >0. 05). Conclusion Transfeetion of VEGF has no obvious influence on the prohferation and morphologyof hBMSCs in vitro.

Restriction endonucleases are important molecular biology tools for DNA recombination.Because of the cleavage of DNA,their recombinant expression is difficult with low yields and complicated purification processes.In commercial productions,the technology that uses specific methylases to protect host DNA from digestion of the expressed restriction enzymes was cumbersome and practically limited.For solving this problem the expression of restriction enzyme Not Ⅰ was performed by using the DNA methylase M.Sss Ⅰ derived from Spiroplasma sp.MQ1 which specifically kept CpG sequence methylated.The methylated DNA was protected from the cutting of Not Ⅰ whose recognition sequence contained CpG.The gene of methylase M.Sss Ⅰ was introduced into Escherichia coli ER2566 and constitutively expressed,resulting in the CpG methylation pattern of the host DNA.Restriction enzyme Not Ⅰ was successfully expressed in this E.coli strain.Furthermore,by adding a purification tag to one terminus of the enzyme,recombinant Not Ⅰ was prepared as a highly purified and active product through two simple Ni-affinity and anion exchange chromatography steps.This expression system can be applied for the preparation of a series of restriction enzymes with CpG in their recognition sequences.

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.
Encephalitis Virus, Japanese ; enzymology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; Protease Inhibitors ; chemistry ; RNA Helicases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Serine Endopeptidases ; metabolism ; Viral Nonstructural Proteins ; metabolism

To obtain active hFKBP52 protein for screening novel neu rotrophic drugs. Semi-nested and overlap PCR and affinity chromatography were u sed. hFKBP52 gene was cloned successfully from human fetal brain cDNA library, a nd then highly expressed (about 30%) as fusion protein in pET28a(+) vector syste m. The recombinant protein was purified as one band on SDS-PAGE. The purified h FKBP52 showed peptidyl-prolyl cis-trans isomerase (PPIase) activity, simil ar to the wild type.

Rhesus retinal vascular endothelial cell line RF/6A cells were treated with human insulin. Cell proliferation, migration, and lumen formation, as well as the expressions of vascular endothelial growth factor-A ( VEGF-A ), VEGF-A receptors, and phosphorylated receptors were measured. Insulin promoted RF/6A cell proliferation, migration, and lumen formation ( all P＜0. 01 ). Insulin increased the expression of VEGF-A mRNA and improved its protein activity ( all P＜0. 05 ), and promoted the expression of VEGFR2 mRNA and its phosphorylation ( both P＜0. 01 ). There was no significant difference in the expression of VEGFR1 mRNA among the groups ( P＞0. 05 ).

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.
Chloroplasts ; DNA, Chloroplast ; genetics ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Dendrobium ; classification ; genetics ; Plants, Medicinal ; classification ; genetics

OBJECTIVETo identify the Cynomorii Herba and its analogues species using DNA barcoding technique.

METHODTotal genomic DNA extracted from all materials using the DNA extraction kit. The internal transcribed spacer 2 (ITS2) regions were amplified using polymerase chain reaction (PCR), and purified PCR products were sequenced bi-directionally. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner 3.7.1. The Kimura 2-Parameter (K2P) distances and GC content were computed using MEGA 5. 0. Species identification analyses were conducted through the species identification system for traditional Chinese medicine and neighbor-joining (NJ) trees.

RESULTThe ITS2 sequence lengths of Cynomorii Herba were 229 bp. The average intra-specific genetic distances of Cynomorii Herba were 0.003. The average inter-specific genetic distances between Cynomorii Herba and its adulterants species were 0.760. The results showed that the minimum inter-specific divergence is larger than the maximum intra-specific divergence. The species identification system for traditional Chinese medicine and NJ trees results indicated that Cynomorii Herba and its adulterants species can be easily identification.

CONCLUSIONThe ITS2 region is an efficient barcode for identification of Cynomorii Herba, which provide a new technique to ensure clinical safety in utilization of traditional Chinese medicine.

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.

Objective To explore the methods of Y-V vascular pedicle lengthening technique for cover-age of the defect of great toe after wrap-around flap transfer, and decrease the morbidity of donor site in great toes. Methods Fifteen patients received three kinds of flap for immediate resurfacing of donor defect of the great toes during wrap-around flap transfer, all flaps were harvested with Y-V vascular pedicle lengthening technique. Among them, 9 cases received the free lateral tarsal flaps transplantation for coverage of defect in donor great toes, 5 cases was repaired by medial tarsal flaps, and only one was treated by the medial plantar flap. Results All the flaps survived postoperatively. The medial plantar flap encountered the venous crisis in postoperative 3 days, and regained the normal blood supply after continuous bleeding for 2 days. All patients were satisfied with cosmetic and functional outcome in average 10- month follow-up. The appearance and sensory function of donor toe repaired by medial tarsal flaps was best among three ones. Conclusion Depending on the Y-V vascular pedicle lengthening technique, the local pedical flaps of foots are enlarged scope for coverage of great toe after wrap-around flap harvest, which can decrease the complication of donor site at the most.