BackgroundMouse has been used in laboratory studies as the model of ocular diseases.Electroretinogram (ERG)is a non-invasive method for primary examination to evaluate retinal function.Though flash ERG has been widely applied in the mouse ocular disease model for the functional assessment of the retinal outer layer,pattern ERG(PERG) is seldom used for inner retinal evaluation.ObjeetiveThe present experimental study was to investigate the recording parameters and method,wave characteristics of PERG and influencing factors in mouse,and to build the foundation for further research.Methods Thirty C57BL/6 mice aged 6 weeks old were included in this research.RETLport ( Roland Consult,Germany) was adopted for the recording of PERG.The positive needle electrode was placed in the cornea,and the reference and earth electrodes were placed under the derma in the cheek and tail.The PERG under different temporal frequencies (0.5,1.0,2.0 and 4.0 Hz),and special frequencies (0.05,0.10,0.20 cpd) were recorded in a photopic environment,and different contrast ratio (95％ and 99％ ) of stimulator or different transmission bands ( 1-100 Hz,5-30 Hz) in the same temporal frequency and spatial frequency were regulated to analyze the influence on mouse PERG.The use of animals was in compliance to the Regulations for the Administration of Affairs Concerning Experimental Animals by the State Science and Technology Commission.ResultsThe latency of N1 PERG showed a negative N1wave at around 37 ms and positive P wave at about 86 ms in adult mice.The amplitude of N1-P was 2-6 μV.Different spatial frequency,temporal frequency and contrast can affect the final results,and the different temporal frequencies were statistically significant.The wave was stable and the amplitude was unaffected at 5-30 Hz transmission bands with pronounced interference (mean amplitude of N1-P waves were(3.40±0.71),(5.08±0.88),(3.21±1.54),(3.85±1.96)μV in 0.5,1.0,2.0,4.0 Hz,F=7.43,P=0.00).ConclusionsPERG wave from adult mouse is similar to that from human.It is a useful method in evaluating the inner retinal function.Appropriate stimulating parameters are critical for recording.

Background Acanthamoeba keratitis is a sort of serious infectious eye disease with high causing-blindness rate.Acanthamoeba keratitis often is misdiagnosed as fungal keratitis or viral keratitis in the early stage.Because conventional clinical diagnosis methods show a low specificity and take a long time,timely treatment often is delayed.Conventional PCR does not apply well because the lesion sample is not enough to extract DNA.However,direct PCR can amplify 18S rRNA conserved sequence of acanthamoeba keratitis without the extraction of DNA.Objective This study was to discuss the feasibility for rapid diagnosis of acanthamoeba keratitis using direct PCR to amplify the gene 18S rRNA fragment.Methods Ten acanthamoeba strains were isolated from 10 eyes with acanthamoeba keratitis in Qingdao Eye Hospital.The sensitivity of the direct PCR assay was tested using different numbers of amoebas.The specificity of the assay was tested using DNA extracted from acanthamoeba,candida albicans,pseudomonas aeruginosa,herpes simplex virus-1 (HSV-1) and normal human corneal epithelial cell.Acanthamoeba keratitis models were established using infected method in clean 6-week-old female BALB/c mice.Corneal lesion samples were obtained 1 day,3,5,7,10,15 days after modeled.The effectivity and feasibility of the direct PCR assay for rapid diagnosis of acanthamoeba keratitis were evaluated and compared with culture method,corneal smear examination and real-time PCR.Results Direct PCR primers could only amplify DNA of acanthamoeba rather than other pathogens,and 10 stains of acanthamoeba were detected at least in each sample.During the development of acanthamoeba keratitis in the mice,the diagnosis positive rate of direct PCR was 80.0％,90.0％,80.0％,70.0％,70.0％ and 50.0％ in 1 day,3,5,7,10,15 days after modeled with the total positive rate 73.3％,which was higher than 31.7％ of culture method,56.7％ of corneal smear examination and 61.7％ of realtime PCR,with a significant difference between the direct PCR and culture method (P =0.005),but no significant difference was seen in the total positive rate between the direct PCR and real-time PC R (P =0.172) or corneal smear examination (P =0.056).Conclusions The direct PCR assay is a simple,rapid,highly specific and sensitive method for the rapid diagnosis of acanthamoeba keratitis,especially for the limited lesion sample.

Lung cancer is the leading cause of cancer-related deaths, with high morbidity and mortality, as well as poor prognosis in China and worldwide. Despite the recent advances in surgery, irradiation, chemotherapy, and targeted therapy, the curative effect of non-small cell lung cancer (NSCLC) in advanced cancer patients is unsatisfactory, and the five-year survival rate of patients remains low. Immunotherapeutic approaches, such as checkpoint inhibitors, active vaccination, and adoptive vaccination, have been given in-creasing attention for the treatment of patients with NSCLC. Results of phase I clinical trials show a higher remission rate, and the out-comes of phases II and III clinical trials are under exploration. This review provides an overview of the latest advances and challenges in immunotherapy for NSCLC.

OBJECTIVEUsing the health economics methodology to assess the screening program on gastric cancer in Zhuanghe high risk area for gastric cancer, from 2001 to 2003 and to assess the feasibility on cost of the screening program and to provide a basis for the popularization of the two-time gastric cancer screening methodology.

METHODSThree major techniques of medical economics namely cost-effective analysis (CEA), cost-benefit analysis (CBA) and cost-utility analysis (CUA) were used to assess the screening program. The screening program was composed of two steps: (1) epidemiological survey and detection of blood pepsinogen; (2) gastroscopy and biopsy of membrane. 'Number of deaths reduced' was used to evaluate the effect during cost-effective analysis while cost-benefit analysis would include the evaluation on the direct cost and indirect cost, direct benefit and indirect benefit as well as the cost-benefit ratio (CBR). During CUA, a questionnaire of WHOQOL-BREF was used to assess the value of the utility while the number of quality adjusted life year (QALY) saved by the screening program was also computed. The direct cost of per saved QALY was also calculated.

RESULTSDada from CEA showed that: investing every 8448 Yuan on screening program and treatment in Zhuanghe high risk area of gastric cancer, one gastric cancer patient could be avoided. Results from CBA showed that: direct cost was 1,260,000 Yuan while indirect cost was 40 621 Yuan with direct benefit as 101 500 Yuan and indirect benefit as 1 540 979 Yuan. The total cost however, was 1,300,621 Yuan with total benefit as 2,555,979 Yuan and CBR was 1:1.97. Data from CUA showed that: a total number of 331.44 QALY was saved, 11.43 QALY was saved by reducing one death, 3802 Yuan per QALY was saved in high risk area of gastric cancer, through this screening program.

CONCLUSIONThe screening program of gastric cancer appeared to be an economic and society-beneficial measure regarding primary prevention in high risk area of gastric cancer. We also suggested that in the future, evaluations through health economics methodologies on different screening programs be carried out in the same population to solve the problem of comparability.

Adult ; China ; Cost-Benefit Analysis ; Feasibility Studies ; Female ; Health ; Humans ; Male ; Quality-Adjusted Life Years ; Risk ; Stomach Neoplasms ; diagnosis ; economics

To knock out β-lactoglobulin (BLG) gene and insert human lactoferrin (hLF) coding sequence at BLG locus of goat, the transcription activator-like effector nucleases (TALEN) mediated recombination was used to edit the BLG gene of goat fetal fibroblast, then as donor cells for somatic cell nuclear transfer. We designed a pair of specific plasmid TALEN-3-L/R for goat BLG exon III recognition sites, and BLC14-TK vector containing a negative selection gene HSV-TK, was used for the knock in of hLF gene. TALENs plasmids were transfected into the goat fetal fibroblast cells, and the cells were screened three days by 2 μg/mL puromycin. DNA cleavage activities of cells were verified by PCR amplification and DNA production sequencing. Then, targeting vector BLC14-TK and plasmids TALEN-3-L/R were co-transfected into goat fetal fibroblasts, both 700 μg/mL G418 and 2 μg/mL GCV were simultaneously used to screen G418-resistant cells. Detections of integration and recombination were implemented to obtain cells with hLF gene site-specific integration. We chose targeting cells as donor cells for somatic cell nuclear transfer. The mutagenicity of TALEN-3-L/R was between 25% and 30%. A total of 335 reconstructed embryos with 6 BLG-/hLF+ targeting cell lines were transferred into 16 recipient goats. There were 9 pregnancies confirmed by ultrasound on day 30 to 35 (pregnancy rate of 39.1%), and one of 50-day-old fetus with BLG-/hLF+ was achieved. These results provide the basis for hLF gene knock-in at BLG locus of goat and cultivating transgenic goat of low allergens and rich hLF in the milk.
Animals ; Animals, Genetically Modified ; genetics ; Female ; Fibroblasts ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Goats ; genetics ; Humans ; Lactoferrin ; genetics ; Lactoglobulins ; genetics ; Milk ; chemistry ; Nuclear Transfer Techniques ; Plasmids ; Pregnancy ; Transfection

The paper is aimed to study the difference in yield and quality at different harvest time and determine the optimum harvest of planting Gentiana in Ludian traditional harvest period. The authors analyzed the variation in fresh weight, dry weight, dry discount rate, length, diameter, volume and the content of gentiopicroside, loganin acid, alcohol-soluble extract and total ash and made a comprehensive appraisal of yield, appearance quality and intrinsic quality by gray relational distance ideal Comprehensive Evaluation method. The results showed that there is a big difference in yield and quality both 2-year-old and 3-year-old Gentiana harvested in traditional harvest period and the comprehensive evaluation more better when harvested more later. It can be seen, Gentiana harvested the later had a better yield and quality in Ludian traditional harvest period. The harvest of Gentiana can be appropriate delayed depending on the particular circumstances of production.
Agriculture ; methods ; China ; Gentiana ; anatomy & histology ; growth & development ; metabolism ; Iridoid Glucosides ; metabolism ; Organ Size ; Quality Control ; Time Factors

Gene knockout by ZFNs (zinc-finger nucleases) is efficient and specific, and successfully applied in more than 10 organisms. Currently, it is unclear whether this technology can be used for knocking-out enhanced green fluorescent protein (EGFP) gene in transgenic goats. Here we constructed and used ZFN-coding plasmids to produce genetic knockouts in the cells of cloned fetus produced from donor cells by microinjection of EGFP gene. Following introduced plasmids into caprine primary cultured fetus fibroblasts by electroporation, targeting of a transgene resulted in sequence mutation. Using the flow cytometric analysis, we confirmed the disappearance of EGFP expression in treated cells. Sequence from PCR products corresponding to targeted site showed that insertion of a G into the exon of EGFP resulted in frame shift mutation. These results suggest that ZFN-mediated gene targeting can apply to caprine fetus fibroblasts, which may open a unique avenue toward the creation of gene knockout goats combining with somatic cell nuclear transfer.
Animals ; Base Sequence ; Cloning, Organism ; Electrophoresis ; Endonucleases ; genetics ; metabolism ; Fetus ; Fibroblasts ; metabolism ; Gene Knockout Techniques ; Gene Targeting ; methods ; Goats ; Green Fluorescent Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Zinc Fingers

Objective To study the value of carotid plaque magnetic resonance imaging (MRI) in pre-operation assessment in the elderly patients with carotid atherosclerosis and explore the possibility of minimizing the contrast weightings to gain sweeptime. Methods Totally 70 elderly patients with cerebral ischemia (average age of 68.8 years) underwent carotid MRI and digital subtraction angiography (DSA) due to the appearance of carotid plaque detected by ultrasound. Carotid plaque MRI was acquired with 3.0T MR scanner and 8 channel surface coil. The standard carotid plague MRI program included pre-and post-contrast T1 weighted imaging (T1WI), T2 weighted imaging, proton density weighted imaging and 3D time of flight MR angiography (3D TOF MRA). All these program were divided into two combinations: the 5-sequence MRI (all the sequences) and 2-sequence MRI (T1WI and TOF MRA). Digital subtraction angiography (DSA) in coronal and lateral views of carotid artery was performed with GE Advantx LCN+. The software SPSS 13.0 was used to statistically analyze the difference between MRI and DSA, and that of two sequence combinations was used in the detection of luminal stenosis and fibrous cap (FC) rupture. Results Totally 135 arteries were analyzed while 3 arteries in one patient were excluded due to the poor quality image and stent placement. The degree of luminal stenosis were (38.3±31.0)% and (38.5±30.9)%, respectively, detected by the two MRI sequence-combination with no significant difference (t=2.447, P＞0.05) and was (35.1±31.8)% by DSA. There was a good concordance between MRI and DSA in luminal stenosis detection (Kappa value: 0.773). No statistical difference was found between two MR sequence combinations in detecting FC rupture (both in 36 vessels). DSA detected FC rupture of 16 vessels, showing remarkably difference contrast to MRI(χ2=12.0, P＜0.01). Conclusions MRI can accurately detect the luminal stenosis and FC rupture. The short time scanning resulting from sequence optimization could make MRI much more suitable than DSA to do the pre-operation assessment for senile carotid atherosclerotic patients.

Objective To research ischemic postconditioning on heart function after myocardial ischemia-reperfusion(I/R),and the protective mechanisms. Methods Thirty-two rats were divided into four groups:I/R group ( n = 8 ) , ischemic postconditioning group (n=8),myocardial ischemic postconditioning+ NSC-74859 (STAT3 inhibitor) group(n=8),and control group(n=8). Establish a model of rat to observe changes of the heart rate,LVSP,+dp/dtmax,-dp/dtmax,coronary flow and myocardial enzyme spectrum in each group un-der different conditions. Results Compared with ischemia-reperfusion group,heart rate of the reperfusion period,CK and LDH of coronary ef-fluent in the ischemic postconditioning group were obviously lower,while left ventricular systolic pressure,change of intraventricular pressure, and coronary effluent volume increased obviously. And after inhibition of STAT3 expression,this protective effect decreased significantly. Con-clusion Ischemic postconditioning can provide potent cardioprotective effect in which STAT3 mediates the cardioprotective effects.

Objective To compare the efficacy of MRI and DSA in assessment of carotid artery stenosis and atherosclerotic plaque.Methods Forty-six patients with carotid plaque detected by ultrasound were enrolled in this study,and 89 carotid arteries were evaluated by MRI and DSA.MRI examination was acquired with 3.0 T MR scanner and 8 channel phase-array surface coil.The MRI sequences consisted of pre-and post-contrast T1WI,T2WI,PDWI,TOF.Anterior-posterior and lateral views of carotid artery were performed on DSA.The degree of carotid artery stenosis was evaluated by the NASCET standard.Fibrous cap rupture,intraplaque hemorrhage,and calcification were also evaluated on MRI and DSA.Statistical comparison was performed with the Kappa value and paired Chi-square test.Results The degree of carotid artery stenosis was 50％ (16％-78％) on MRI and 47％ (7％-73％) on DSA.Two imaging modalities were in good consistency in evaluation of the degree of stenosis ( Kappa =0.882,P ＜ 0.01 ).There was statistical difference in detecting fibrous cap rupture by MRI and DSA (34 vessels vs 10 vessels,respectively,x2 =20.346,P ＜ 0.01 ).Furthermore,thirty-seven vessels with intraplaque hemorrhage and 71 vessels with calcification in the plaque were found on MRI but none on DSA.Conclusion MRI is a reliable tool in assessment of the degree of the carotid stenosis and it is superior to DSA in detecting fibrous cap rupture,intraplaque hemorrhage,and calcification.