To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus (IBV) Beaudette strain, the ectodomain region of the Spike gene (1,302 bp) of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA. This recombinant was designated as BeauR-H120(S1). BeauR-H120(S1) was directly used as the DNA template for the transcription of viral genomic RNA in vitro. Then, the transcription product was transfected into Vero cells by electroporation. At 48 h post-transfection, the transfected Vero cells were harvested, and passaging continued. A syncytium was not observed until the recombinant virus had passed through four passages. The presence of rBeau-H120(S1) was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR. Western blot analysis of rBeau-H120 (S1)-infected Vero cell lysates demonstrated that the nucleocapsid (N) protein was expressed, which implied that rBeau-H120(S1) could propagate in Vero cells. The TCIDs0 and EIDs0 data demonstrated that the titer levels of rBeau-H120(S1) reached 10(590+/-0.22)TCID50/mL and 10(6.13+/-0.23)EID50/mL in Vero cells and 9-day-old SPF chicken embryos, respectively. Protection studies showed that the percentage of antibody-positive chickens, which were vaccinated with rBeau-H120(S1) at 7 days after hatching, rose to 90% at 21 days post-inoculation. Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41 strain (103EID50/chicken). This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis.
Animals ; Cercopithecus aethiops ; Chick Embryo ; Chickens ; Coronavirus Infections ; veterinary ; virology ; Infectious bronchitis virus ; chemistry ; genetics ; growth & development ; metabolism ; Poultry Diseases ; virology ; Protein Structure, Tertiary ; Spike Glycoprotein, Coronavirus ; chemistry ; genetics ; metabolism ; Transfection ; Vero Cells

AIM:To investigate the clinical effect of anti-vascular endothelial growth factor (VEGF) drugs combined with laser photocoagulation for diabetic macular edema (DME).METHODS: Totally 94 patients (141 eyes) with DME from June to December 2015 in our hospital were selected and randomly divided into combined group of 47 cases (68 eyes, ranibizumab combined with laser therapy) and the control group of 47 cases (73 eyes, laser treatment).The levels of best corrected visual acuity (BCVA), macular central retinal thickness (CRT), total macular volume (TMV) and macular edema level were compared between the two groups at different time after treatment.RESULTS: The mean values of BCVA in the combined group were higher than those in the control group at 2, 6 and 12wk, and the difference was statistically significant (P<0.05).At 2, 6 and 12wk after treatment, the CRT and TMV values of the combined group were lower than those of the control group, the difference was statistically significant (P<0.05).After treated for 12wk, patients with macular edema of combined group was 80.9% in mild level, 17.7% in moderate level, 1.5% in severe level, those of the control group was 60.0%, 31.5%, 5.5%, the difference between the two groups was statistically significant (P<0.05).CONCLUSION: The effect of anti-VEGF drugs combined with laser therapy for DME patients is better than that of single laser therapy alone.

The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.
Animals ; Chromatography, High Pressure Liquid ; Diterpenes ; metabolism ; Glucosides ; metabolism ; Hydrolysis ; Mass Spectrometry ; Metabolic Networks and Pathways ; Pinaceae ; chemistry ; Rats

China ; epidemiology ; Epidemiologic Methods ; Humans ; Wounds and Injuries ; epidemiology

Child, Preschool ; Chondromatosis, Synovial ; diagnosis ; diagnostic imaging ; surgery ; Diagnostic Errors ; Female ; Humans ; Tibial Fractures ; diagnosis ; Tomography, X-Ray Computed

Objective To observe the therapeutic action of choline on As2O3 induced electrecardiogram (ECG)QT interval prolongation and to further explore molecular biological mechanisms.Methods 40 guinea pigs were divided into 5 groups randomly with 8 rats in each group:control group intravenously injected with saline, experimental group with 0.4,0.8,1.6 ms/kg of choline and As2O3 group with 8 mg/kg choline plus 1.6 mg/kg As2O3.After a series of concentration of As2O3 was intravenously given,ECG was monitored at different time(0,10, 30,60,90,120 min),and corrected QT interval(QTc)was studied.Total RNA Wa$abstracted from the guinea pigs hearts after 6 h,and the effects of choline on altered L-type calcium channel α1c and potassium channel GPERG mRNA expression caused by As2O3 in cardiomyocytes of guinea pig were studied by the method of reverse transcription polyme,chain reaction(RT-PCR).Results In 0.8 mg/kg As2O3 group,the value of QTc at 60, 90 and 120 min respectively being 354 ±22.366± 31 and 368 ±29 waa significantly higher than that of control groups[(325±26,336 ±26 and 324 ±20)at same time] with a significant difference(P<0.05 or<0.01);the value of QTc at 90 and 120 rain was significantly higher than that of O min group(334 ±12),the difference being statistically significant(P<0.05).In 1.6 mg/kg As2O3 group,the value of QTc at 10,30,60,90 and 120 min respectively being 362±33,380±21,382±35,388±39 and 388 ±31 was significantly higher than that of control groups[(328 ±20.324 ±25,325 ±26,336±26 and 324 ±20)at same time]with a significant difference (P<0.05 or<0.01);the value of QTc at 10,30,60,90 and 120 min Was significantly higher than that of O min group(329 ±31),the difference being statistically significant(P<0.05).In choline+As2O3group,the value of qrc at 30,60,90 and 120 min was 337 ±17,341±15,344 ±22 and 343 ±19,significantly lower than that of 1.6 mg/kg As2O3 group,the difference being statistically significant(P<0.05 or<0.01).The RT-PCR results indicated that the value of L-type calcium channel α1c mRNA expression at the concentration of 1.6 mg/kg As2O3 was 1.27±0.14 vs 1.02±0.12 of control group(P<0.01),and it was 1.10 ±0.13 in choline+As2O3 group(P<0.05 Vs 1.6 mg/kg As2O3 group).The value of potassium channel GPERG mRNA expression were 1.29 ±0.11,1.22±0.12 and 1.27±0.16 at the dose of 0.4,0.8,1.6 mg/kg As2O3(P>0.05 VS 1.23±0.08 of control group).In choline+ As2O3 group,the value was 1.30±0.14(compared with 1.6 mg/kg As2O3 group,P>0.05).Conclusions Choline normalizes QTc abnormality during As2O3 application,and modulating changed calcium channel mRNA expression induced by As2O3 may be one of the mechanisms.

Short tandem repeats (STRs) have been widely used in forensic sciences such as stain analysis and paternity testing. Although most of STR typing could give the reliable and clear results, some unusual typing have been observed in forensic practice. The anomalous typing could result from a lot of causes, including DNA genetic variation, poor quality or quantity of DNA template, different typing system or method, nonspecific reaction in PCR or anomalous electrophoresis migration. The unusual results may disturb the right interpretation of STR typing.
DNA Fingerprinting/methods* ; Forensic Medicine/methods* ; Gene Frequency ; Genotype ; Humans ; Polymerase Chain Reaction/methods* ; Polymorphism, Genetic ; Tandem Repeat Sequences/genetics*

Adolescent ; Apolipoproteins E ; blood ; Humans ; Hyperlipoproteinemias ; complications ; Hypolipidemic Agents ; therapeutic use ; Kidney Diseases ; blood ; drug therapy ; pathology ; Kidney Glomerulus ; blood supply ; pathology ; Male ; Thrombosis ; pathology

Background The incidence of brain metastases in patients with breast cancer is approximately 10％-16％,and survival after diagnosis of brain metastases is usually short.This study was designed to evaluate the risk factors associated with brain metastases in advanced breast cancer patients,with a view to help predict patient groups with high risk of brain metastases.Methods In total,295 patients with advanced breast cancer were evaluated.All patients were pathologically confirmed and metastatic lesions were confirmed pathologically or by imaging.All patients were examined at least once every 6 months with head CT or MRI.Patients showing symptoms underwent immediate inspection,and brain metastatic lesions were confirmed by head CT and/or MRI.Results At a median follow-up of 12 months from the occurrence of metastases,brain metastases had occurred in 49 patients (16.6％).In our univariate analysis,variables significantly related to increased risk of brain metastases were hormone receptor-negative tumors,epidermal growth factor receptor 2 (HER2)-positive tumors,and multiple distant metastases.Patients with dominant tumor sites in soft tissue,or defined as Luminal A subtype,tended to have a lower risk of brain metastases than patients with visceral metastases,Luminal B subtype,triple-negative subtype or HER2-enriched subtype tumors.Conclusions Our results strongly suggest that factors such as Luminal B,triple-negative,and HER2-enriched subtypes are high risk factors for brain metastases.These data,therefore,provide pivotal clinical evidence towards a comprehensive understanding of the risk factors of brain metastases in advanced breast cancer patients.

OBJECTIVETo study the effect of hepatitis C virus nonstructural protein 3 N-terminal protein (HCV NS3-5') on hepatocyte transformation and tumor development.

METHODSQSG7701 cells were transfected with plasmid pRcHCNS3-5' (expressing HCV NS3 N-terminal protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological effect of transfected cells was observed through cell proliferation assay, anchor independent growth, and tumor development in nude mice. The expression of HCV NS3 and c-myc protein in the induced tumor was evaluated by immunohistochemistry.

RESULTSHCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5' and the positive signal was located in cytoplasm. The HCV NS3 expression and c-myc protein in the induced cytoplasm. Cell proliferation assay showed that the population doubling time in the pRcHCNS3-5' transfected cells was much shorter than that in the pRcCMV and non-transfected cells (24 h, 26 h, 28 h respectively). The cloning efficiencies of transfected cells with pRcHCNS3-5', pRcCMV and non-transfected cells were 33.0%, 1.5%, 1.1% respectively (P < 0.01). Tumor developed in nude mice inoculated with pRcHCNS3-5'transfected cells 15 days after the inoculation. HE staining showed hepatocarcinoma character and immunohistochemistry confirmed HCV NS3 and c-myc expression in the tumor tissue. The positive control group also showed tumor development, while no tumor mass obtained in the nude mice inoculated with pRcCMV and non-transfected cells even 40 days after the injection.

CONCLUSIONHCV NS3 N-terminal protein showed cell transformation and tumorigenic features.

Animals ; Cell Division ; Cell Transformation, Neoplastic ; Female ; Hepatocytes ; pathology ; Humans ; Liver Neoplasms, Experimental ; etiology ; Mice ; Mice, Nude ; Mitogen-Activated Protein Kinases ; metabolism ; Phosphorylation ; Transfection ; Viral Nonstructural Proteins ; toxicity