OBJECTIVEExpress and purify four single-stranded DNA-binding (SSB) proteins, and evaluate the binding of SSB proteins on HCV RNA.

METHODSThe expression plasmids of four SSB proteins were conducted, termed TTH, SSOB, KOD and BL21, respectively. The BL21 (DE3) was transformed by the expression plasmid of TTH, Transetta (DE3) were transformed by the expression plasmid of SSOB, KOD and BL21, then protein expression was induced with IPTG, the expression products were analysised by SDS-PAGE. To evaluate the binding of SSB on HCV RNA, RNA-SSB protein complexes were applied to a 1.2% TAE agarose gel.

RESULTSSuitable competent cells were transformed with the expression plasmids, induced by IPTG. SSB proteins were purified by affinity chromatography, to visualize their purity all SSB proteins were applied to SDS-PAGE analysis. All four proteins showed single clear bands. We have successfully obtained the SSB protein expression plasmid, expressed and purified SSB protein. TAE agarose gel electrophoresis was used to confirm SSB protein-RNA binding activity. The each of SSB-RNA complex migrated more slowly than the sole RNA, which suggested SSB protein could specifically bind to RNA.

CONCLUSIONSWe have expressed and purified four SSB proteins, and for the first time found that SSB protein can bind HCV RNA. Our results may provide a basis for future studies of the novel functions of SSB proteins on RNA.

DNA, Single-Stranded ; genetics ; metabolism ; DNA-Binding Proteins ; chemistry ; isolation & purification ; metabolism ; Hepacivirus ; Hepatitis C ; metabolism ; virology ; Humans ; Molecular Weight ; Protein Binding ; RNA, Viral ; genetics ; metabolism

Biotechnology supremacy is a newly-advanced power theory. It is a superior dominance of military biotechnological application based on the microcosm of life structure within a certain period of time. The advancement of biotechnology supremacy and modern biotechnology has created the concept of bio-micro-frontier, which involves information and defense resources of all living ultra-micro-organisms with national and regional characteristics. Being feasible both in theory and practice, the implementation of bio-micro-frontier system is strategically important. This article explores the implementation of bio-micro-frontier in terms of strategy and tactics, which will add a unique dimension to future military transformation and active defense.

Objective To investigate the relationship between the apolipoprotein B (ApoB) gene Xba Ⅰ and EcoR Ⅰ polymorphisms and cholelithiasis in Han and Mongolian population in the Midwest Area of Inner Mongolia.Methods The clinical data of 100 patients with cholelithiasis and 115 healthy individuals at the First Affiliated Hospital of Medical College of Baotou from April to October in 2010 were collected.A case-control study which detected ApoB alleles of patients with cholelithiasis (cholelithiasis group) and healthy individuals (control group) in Han nationality and Mongolian nationality in the Midwest Area of Inner Mongolia was conducted by polymerase chain reaction-restriction fragment length polymorphism,which included Xba Ⅰ (X + X +,X + X-,X-X-,X +,X-) and EcoR Ⅰ (E + E +,E-E-,E + E-,E +,E-).The serum lipid (including triglyceride,total cholesterol,high density lipoprotein and low density lipoprotein) levels in different groups were detected.The count data and the measurement data were analyzed using the chi-square test and t test,respectively.Results Genotype X + X + was not found in the Han and Mongolian population,and Xba Ⅰ (X +) or EcoR Ⅰ (E-) alleles was not found in the Mongolian population.The levels of low density lipoprotein were (2.8 ± 0.9)mmol/L in the cholelithiasis group,which was significantly higher than (1.9 ± 0.8) mmol/L of the control group in the Han population (t =2.800,P ＜ 0.05).The levels of high density lipoprotein and low density lipoprotein were (1.7 ± 0.3) mmol/L and (3.5 ± 0.8) mmol/L of the cholelithiasis group,which were significantly higher than (1.2 ± 0.3) mmol/L and (2.8 ± 0.9) mmol/L of the control group in the Mongolian population (t =7.596,2.549,P ＜ 0.05).The levels of triglyceride,total cholesterol,high density lipoprotein and low density lipoprotein of the cholelithiasis group in the Mongolian population were (3.1 ± 1.6) mmol/L,(5.6 ± 1.0) mmol/L,(1.7 ± 0.3) mmol/L and (3.5 ± 0.8) mmol/L,which were significantly higher than (1.2 ± 0.6) mmol/L,(4.4 ± 1.2) mmol/L,(1.3 ± 0.3) mmol/L and (2.8 ± 0.9) mmol/L of the cholelithiasis group in the Han population (t =5.501,3.667,4.448,3.430,P ＜ 0.05).The levels of triglyceride,total cholesterol,low density lipoprotein were (2.6 ± 1.7) mmol/L,(5.1 ± 1.1) mmol/L and (2.8 ± 0.9) mmol/L of the control group in the Mongolian population,which were significantly higher than (1.3 ±0.7)mmol/L,(3.9 ±0.9) mmol/L and (1.9 ±0.8) mmol/L of the control group in the Han population (t =4.298,4.772,3.888,P ＜ 0.05),while the level of high density lipoprotein was significantly higher of the control group in the Han nationality than the control group in the Mongolian population (t =1.997,P ＜ 0.05).The levels of low density lipoprotein in patients with genotypes X + X-,X-X-of the cholelithiasis group in the Han population were (2.7 ± 0.1) mmol/L and (2.6 ± 1.0) mmol/L,and the levels of low density lipoprotein in patients with genoeypes E + E ±,E + E-/E-E-were (2.6 ± 1.0) mmol/L and (2.5±0.4)mmol/L,with no significant difference (t=0.225,0.124,P＞0.05).Conclusion In the Midwest area of Inner Mongolia,the Mongolian population might be more susceptible to cholelithiasis than the Han population.No relationship between the rare alleles X +,E-and the increase of blood lipids,which indicates that X + and E-of ApoB may not be a risk factor of cholelithiasis.

Objective To investigate the alterations of serological endothelin(ET) in patients with kawasaki disease(KD) and its relation with coronary dilatation(CAD).Methods Serological ET were measured in 50 cases of patients with KD in acute phase as well as subacute phase; 30 cases of patients with acute febrile infection(IC) in infective phases and 30 healthy children(HC).Results 1.ET in subacute phase significantly increased than that in acute phase.2.In both acute and subacute phases of KD, ET significantly elevated higher than that in HC; and ET in subacute phase significantly elevated than that of IC. 3. Fifty-four percentage patients with KD were complicated with CAD. For CAD subgroup, ET had no difference with CAD subgroup in subacute phase.Conclusion ET has still increases in subacute of KD,which indicates the relation with coronary artery lesion.

Objective: To study the dynamic changes of type Th22 cell immunological response during atherosclerosis process in experimental mice in order to provide a new theoretical basis for atherosclerosis therapy. Methods: 8 weeks C57BL/6J mice were divided into 2 groups: Experiment group,n=24 ApoE-/- mice and Control group, n=24 normal mice. All animals received high fat diet and the following indexes were compared between 2 groups at 0, 4, 8, 12 weeks after treatment: aortic atherosclerotic lesions were deifned by Oil red O staining, dynamic changes of Th22 cells in spleen were measured by lfow cytometry, mRNA expressions of interleukin-22 (IL-22), IL-22R1, AhR and T-bet in aorta were examined by RT-PCR, blood levels of IL-22 was detected by ELISA. Results: Compared with Control group, Experiment group had the increased area of aortic atherosclerosis (the ratio of plaque area/lumen area) and Th22 cell (CD4+ IL-22+/CD4+T cell) amount, elevated mRNA expressions of IL-22, IL-22R1, AHR, T-bet in aorta and higher blood levels of IL-22 at all time points, the differences between each time point (except 0 week) had the statistic meaning,P<0.05. In Experiment group, the differences between 2 adjacent time points, for the area of aortic atherosclerosis and mRNA expressions of AHR, T-bet: 4 weeks vs 0 week, 8 weeks vs 4 weeks, 12 weeks vs 8 weeks all had statistic meaning; for Th22 cell amount: 4 weeks vs 0 week, 8 weeks vs 4 weeks had statistic meaning and 12 weeks vs 8 weeks had no real distinction; for mRNA expressions of IL-22, IL-22R1 and blood levels of IL-22: 4 weeks vs 0 week had statistic meaning and 8 weeks vs 4 weeks, 12 weeks vs 8 weeks had no real distinctions. Conclusion: Hyperactive immunological response of Th22 cells might be involved in atherosclerosis process, the relevant mechanism should be further studied.

Objective To study the regulation of microRNA 199a (miR-199a) on adhesion,migration and invasion ability of human eutopic endometrial stromal cells (ESC) from patients with endometriosis.Methods ESC were transfected with miR-199a mimics or negative control (NC) RNA by lipofectamine 2000.The adhesion,migration and invasion ability of ESC were detected by cell adhesion assay,scratch assay,cell migration assay and matrigel invasion assay,respectively.Luciferase reporter assay was used to evaluate whether IKKβ was the target gene of miR-199a.The expression of ikappa B kinase beta (IKKβ),inhibitory kappa B alpha (IκB-α),phospho-IκB-α (p-IκB-α) and nuclear factor-kappa B (NF κB) protein were measured by western blot.Results ( 1 ) Adhesion potential:the adhesion inhibitory rates were ( 14 ± 4 )％ in miR-199a group and 0 in control group,which showed significant difference (P＜0.01 ).(2) Migration and invasion:in the scratch assay,ESC transfected with miR-199a exhibited a lower scratch closure rate than that of controls.In migration and invasion assays,the migration and invasion ability of miR-199a group were significantly decreased compared with those of NC group [ 130 ± 31 vs.247±36 (P＜0.01); 63 ± 15 vs.133 ± 17 (P＜0.01),respectively].(3) The luciferase activity of miR-199a group was significantly lowered than that of control group [ 0.160 ± 0.006 vs.0.383 ± 0.083 ( P ＜0.01 ) ].The protein levels of IKKβ,p-IκB-α,IκB-α and NF-κB of 0.350 ±0.195,0.443 ±0.076,1.970 ±0.486 and 0.454 ± 0.147 in miR-199a group were significantly different compared with the NC group in which the protein levels were set at 1.000 ( P ＜ 0.01 ).Conclusions miR-199a can inhibit the adhesion,migration and invasion of the ESC.IKKβ is the target gene of miR-199a in ESC.One of the mechanisms of the inhibition effect is probably that miR-199a inhibits the activation of NF-κB signaling pathway by targeting IKKβ gene.

The reports on chemical constituents of Hyoscyamus niger were summarized. The compounds include alkaloids, saponins, lignans, coumarinolignans, flavonoids, and some other nonalkaloidal compounds. TLC, HPLC, and GC were used for the qualitative and quantitative analyses of some chemical constituents in H. niger. Modern pharmacological experiments showed that H. niger had the analgesic, anti-inflammatory, antipyretic, anticonvulsant, spasmolytic, antidiarrhoeal, antisecretory, bronchodilatory, urinary bladder relaxant, hypotensive, cardiosuppressant, vasodilator, antitumor, and feeding deterrent properties. In addition, the toxicities of this medicinal plant were also described.

Objective To evaluate the locating effects of a portable ultrasound for leg perforator flaps,and the clinical effects of leg perforator flaps for the wounds of the extremities.Methods Since December 2009 to March 2011,thirty-one cases of soft tissue defects of the extremities were treated with free vascularized leg perforator flaps in our center.A portable ultrasound were used for the locating of the perforator arteries of the leg before the operations.These flaps include 24 cases of the fibular artery perforator flaps (4 with simultaneous fibula transfer),three cases of the posterior tibia artery perforator flap,two cases of the medial gastrocnemius artery perforator flap,and 2 cases of the lateral gastrocnemius artery perforator flap.The flap size ranged from 15.0 cm × 8.0 cm to 1.0 cm × 1.5 cm.The caliber diameter of the perforator artery ranged from 0.2 mm to 1.8 mm.The wounds included 21 cases of the hand,five cases of the forearm,and 5 cases of the leg.Results The coincidence rate intraoperatively of the portable ultrasound was 96.8％.All the flaps survived and the wounds healed uneventfully.The donor site of the flaps were either closed directly or closed with partial split-thickness skin graft.Conclusion The portable ultrasound is an effective,reliable and accurate instrument for locating the leg perforator flaps.The leg perforator flaps have the advantages of convenient harvest,satisfactory thickness,less donor site morbidity,and high survival rate.

ObjectiveTo investigate the relationships between T and NK cells in peripheral blood and the relapse or metastasis of malignant tumors.MethodsFlow cytometry was used to test the percentages ofT and NK cells in peripheral blood of 48 malignant tumor patients with relapse or metastasis (relapse or metastasis group) and 48 malignant tumor patients without relapse or metastasis(control group).ResultsCompared with control group,the ratio of Th/Ts decreased,CD4+/CD25+ raised and the activities of NK cells decreased in relapse or metastasis group (0.95 ±0.52 vs.1.35 ±0.43,7.15 ±3.81 vs.6.01 ±2.67,0.2053 ±0.1135 vs.0.2501 ±0.0745)(P＜0.01 or ＜0.05).ConclusionsTesting the T and NK cells in peripheral blood of malignant tumor patients regularly can know their immune status and find the relapse or metastasis early.It is easy and useful in patients' follow-up.

Objective To assess the clinical application values of HC visual laryngoscope in emergency tracheal intubation for severe craniocerebral trauma patients with basilar fracture.Methods Retrospective analysis was used to review 60 patients of severe traumatic brain injury with basilar fracture in emergency resuscitation room who needed the hospital emergency tracheal intubation.There were 30 patients operated with visual HC laryngoscope (the observation group), and the other 30 patients underwent common direct laryngoscope (the control group).The index of the degree of exposure,intubation time,success rate of intubation were compared between the two groups.ResultsThere were 28 cases (93.33%) of Cormark-Lehane grading Ⅰ to Ⅱ in observation group,which was significantly higher than 20 cases (66.67%) in the control group(P< 0.05).The average intubation time in observation group was (34.3±6.7) seconds,and it was shorter than (44.5±5.7) seconds in the control group (P<0.05).In observation group,26 cases (86.67%) were of one-time successful intubation,which was higher than 19 cases (63.33%) in the control group,and the difference was statistically significant(P<0.05).ConclusionHC visual laryngoscope in emergency tracheal intubation for severe craniocerebral trauma patients with basilar fracture can improve the success rate of intubation,shorten the intubation time,reduce adverse reactions,and it is worthy of clinical application.