Gene targeted mutagenesis and replacement can be used to modify gene sequence in genomic background without position effect or insertion inactivation in transgenic plants. Targeted mutagenesis organism has little biosafety concerns free of transgenes or marker genes. Gene targeted mutagenesis and replacement in high plants now appears to be a potential tool for gene functional analysis in situ, crops genetic improvement and molecular design. Zinc finger nuclease(ZFN)is most important and would be widely used in gene targeted mutagenesis and replacement through introducing double-strand breaks in genome. Strategies for gene targeted mutagenesis and replacement in plants is discussed. ZFN is described in detail from its structure, operation model and application in plants. Developmental prospect of ZFN in plant gene targeted mutagenesis and replacement is also discussed.

Background The incidence of dry eye is increasing among young adults because of wide usage of video display terminal.But the early diagnosis of dry eye still presents challenge to medical practitioners.The accurate diagnosis and treatment of the dry eye,therefore,is a topic of high interest to researchers.Previous examination outcome of dry eye is interferred primarily due to invasive procedure.It is very important to search an examination approach.Objective This study was to use Keratograph 5M,a non-invasive ocular surface analyzer to evaluate the influence of watching video display terminal on ocular surface and tear film.Methods Eighty-one eyes of 81 health volunteers among 18-30 years were enrolled in Affiliated Eye Hospital of Nanchang University from March 1,2015 to November 10,2015 under the informed consent,including 39 males and 42 females.The subjects watched the computer for continuously 3 hours under the nature light,and ocular surface related examinations were performed and compared before and after video display terminal exposure,including non-invasive tear film break-up time (NITBUT),tear meniscus height,conjunctival hyperemia scoring,limbal congestion scoring,corneal fluorescein staining scoring,meibomian gland imaging and lipid layer analysis.Results The number of eyes with visual fatigue,dryness,pain,blurring and conjunctival congestion was significantly increased after 3-hour video display terminal exposure in comparison with before (all at P＜0.01).The initial NITBUT and mean NITBUT were (6.086± 3.701) s and (9.103 ± 4.680) s,and tear meniscus height was (0.190 ± 0.032) mm after trail,which were significantly lower than (11.445 ±4.964) s,(14.626 ±4.467) s and (0.212 ±0.040) mm of before trail,respectively;The conjunctical hyperemia scoring and limbal congestion scoring were 0.869 ±0.311 and 0.572 ±0.276 after trial,which were significantly higher than 0.780 ± 0.306 and 0.509 ± 0.266 before trail,showing significant differences before and after exposure of video display terminal (all at P＜0.01).The intraocular pressure and the eye number of different scores of corneal fluorescence staining,abnormal meibomian gland and different morphological lipid layer of tear were unchanged before and after exposure of video display terminal.Conclusions Long-term exposure of video display terminal results in significant and temporary adverse influence on tear film and ocular surface.Keratograph 5M non-invasive ocular sudace analyzer can objectively assess overall ocular surface conditions.

Objective To explore the mechanisms by which hydrogen sulfide (H2S) regulates collagen metabolism during hypoxic pulmonary vascular structural remodeling. Methods Wistar rats were randomly divided into control group (n=6), hypoxia group (n = 6) and hypoxia+NaHS group(n= 6) H2S content in plasma was measured with spectrophotometry. The expression of collagen I was detected by immunohistochemistry. The expression of tissue inhibitor of metalloproteinase-1(TIMP-1) mRNA was detected with situ hybridization Results Compared with hypoxia group, H2S content in plasma increased significantly and the expressions of collagen I and TIMP-1 mRNA in pulmonary arteries were down- regulated in hypoxia + NaHS group. The H2S content in plasma negatively correlated with TIMP-I mRNA expression. Conclusions H2S increases the degradation of collagen in small and media pulmonary arteries of rats under hypoxia by decreasing the synthesis of TIMP-1, and therefore, attenuats the collagen remodeling in pulmonary arteries of hypoxic rats.

Background Qiang minority is minority groups of China with the special habits and customs and living condition. So whether the spectrum of disease and bacteria spectrum in conjunctiva are similar with Han nationality is worth paying attention. Objective Present survey was to obtain the data about bacterial species in conjunctival sac in Qiang minority population with the age 40 years old and more and the compare with matched Han nationality population. Methods This survey study was performed as the standardized training and protocol. A total of 212 eyes of 106 individuals from Qiang minority in Beichuan county and 640 eyes of 320 subjects from Han nationality in Mianyang city received questionnaire survey and ophthalmological examination. The secretion of the inferior palpebral conjunctival sac was embrocated and inoculated on blood plate for 48-72 hours. The bacteria was separated and identified. This study was approved by the Ethic Committee of Sichuan Provicial People' s Hospital. Orally informed consent was obtained before the medical procedure. Results All the examinee finished the survey and examination with a good compliance. No significant difference was found in the demography between these two groups of population. The multiple bacterial positive rate in conjunctival sac was 59. 4% in Qiang minority and that of Han people was 66. 3% with a considerably difference between them (χ2 = 2. 27,P = 0. 13). The multiple bacterial species were simultaneously detected in 26.2% in Qiang minority population and 11.88% Han people, showing evidently difference (χ2 = 106. 40, P = 0. 00 ) . The positive rate of corynbaccterium in conjunctival sac of Qiang minority was statistically lower than that of Han people (20. 7% versus 45. 0% ,χ2 =31. 75 ,P = 0. 00) ,but there was no statistical difference in the positive rate of staphylococcus epidemics between two groups (χ2 = 1. 89 ,P = 0. 17). Conclusion The bacteria positive rate in conjunctiva sac is resemble in the population over 40 years in both the Qiang minority and Han nationality. The simple bacterial species is found in majority people in two groups of subjects. The positive rate of multiple bacterial strains coexistence is more in the Qiang minority. The bacterial strains is different between Qiang minority and Han nationality.

Objective To establish high-performance liquid chromatography with electrochemical detector(HPLC-ECD) method for the determination for metanephrine and normetanephrine in 24 h urine, and provide a superior test for the diagnostic of pheochromocytomas over plasma/urine catecholamine.Methods MCX solid-phase cartridge was used for extraction of metanephrine and normetanephrine,HPLC-ECD was used for their measurements.The intra-assay CVs,interassay CVs and recoveries of metanephrine and normetanephrine were also calculated.104 hypertensive patients without pheochromocytomas and 5 pheochromocytomas patients were selected in this study.The concentrations of metanephrine and normetanephrine were compared with the plasma and 24h urinary catecholamines concentrations.Results The intra-assay CV,inter-assay CV and recovery of metanephrine were 5.9%, 7.5%,91.1% respectively;the intra-assay CV,inter-assay CV and recovery of normetanephrine were 6.3%,6.6%,88.5%,respectively.The MN,NMN,plasma CA and urine CA of all pheochromocytomas patients were positive.MN and NMN were negative in controls,while plasma CA and urine CA are false positive in 15 patients and 14 patients in controls,respectively.Conclusions The study establish a fast and accurate method for quantification of metanephrine and normetanephrine in 24 h urine by HPLC-ECD.These findings also prove that it is the best biochemical assays for pheochromocytomas at present.

Objective To study the genotype distribution of insulin receptor substrate-2(IRS-2)gene 1057G/A polymorphism in Han population from Southwest China,and to explore its association with the metabolism of glucose and lipids,insulin resistance and islet?-cell function in type 2 diabetic patients and subjects with impaired glucose tolerance(IGT).Methods A total of 929 Hans[462 patients with type 2 diabetes(DM group) and 164 subjects with IGT(IGT group)and 303 normal controls(NC group)]from Chongqing and nearby regions were screened for 1057G/A polymorphism of IRS-2 gene by PCR-RFLP assay.Body mass index(BMI),plasma glucose,serum insulin and lipid profile,high-sensitive C-reactive protein(hsCRP)and non-esterified fatty acid were measured.Homeostasis model assessment of insulin resistance(HOMA-IR)and disposition index(DI)were used to estimate insulin resistance and?-cell function respectively.Results In DM group,A allele frequency was significantly lower than that in NC group(0.326 vs 0.388,X~2=6.19,P=0.01).Compared with NC group,AA genotype frequeney was lower and GG genotype frequeney was higher in DM group(0.104 vs 0.135 and 0.452 vs 0.360 respectively,X~2=6.80,P

In this study, the buffering capacity of amphiphilic pH-sensitivity copolymer poly(2-ethyl-2-oxazoline)-cholesteryl methyl carbonate (PEOZ-CHMC) was evaluated. The ammonium sulfate gradient method was used to prepare doxorubicin hydrochloride (DOX x HCl)-loaded liposomes (DOX-L), and then the post-insertion method was used to prepare PEOZ-CHMC and polyethylene glycol-distearoyl phosphatidyl ethanolamine (PEG-DSPE) modified DOX x HCl-loaded liposomes (PEOZ-DOX-L and PEG-DOX-L). The physico-chemical properties, in vitro drugs release behavior, cellular toxicity and intracellular delivery of liposomes were evaluated, separately. The results showed that PEOZ-CHMC has a satisfactory buffering capacity. The sephadex G-50 column centrifugation method and dynamic light scattering were used to determine the encapsulation efficiency (EE) and particle size of liposomes. The EE and particle size of DOX-L were (97.3 ± 1.4) % and 120 nm, respectively, and the addition of PEOZ-CHMC or PEG-DSPE had no influence on EE and particle size. The zeta potentials of three kinds of liposomes were negative. The release behavior of various DOX liposomes in vitro was investigated by dialysis method. In phosphate buffer solution (PBS) at pH 7.4, DOX x HCl was released from PEOZ-DOX-L in a sustained manner. While in PBS at pH 5.0, the release rate of DOX x HCl from PEOZ-DOX-L increased significantly, which suggested DOX x HCl was released from PEOZ-DOX-L in a pH-dependent manner. The intracellular delivery of liposomes was investigated by confocal laser scanning microscopy (CLSM). The CLSM images indicated that PEOZ-DOX-L showed efficient intracellular trafficking including endosomal escape and release DOX x HCl into nucleus, as well as the DOX-L and PEG-DOX-L had no this effect. The cytotoxicity of liposomes against MCF-7 cells was detected by using MTT assay. The results showed that antiproliferative effects of PEOZ-DOX-L enhanced with pH value decreased, whereas DOX-L and PEG-DOX-L did not have any significant difference in inhibitions at different pH conditions. Therefore, the problems of the inhibition of cellular uptake of liposomes and the failed endosomal escape of pH-sensitive liposomes by PEG chain can be overcome by the pH-sensitive liposomes constructed by PEOZ-CHMC.
Cell Nucleus ; Doxorubicin ; analogs & derivatives ; chemistry ; Endosomes ; Formates ; chemistry ; Humans ; Liposomes ; chemistry ; MCF-7 Cells ; Microscopy, Confocal ; Particle Size ; Phosphatidylethanolamines ; Polyamines ; chemistry ; Polyethylene Glycols ; chemistry

Objective To investigate the prevalence and molecular epidemiology of clinical isolates of plasmid-mediated 16S rRNA methylases-producing Klebsiella pneumoniae. Methods From January 2006 and September 2007, 337 non-replicate clinical isolates of Klebsiella pneumoniae were consecutively collected from inpatients in a teaching hospital in Wenzhou, China. All of the isolates were identified by the automated microbiology systems. MICs of amikacin, gentamicin and tobramycin were determined by agar dilution method. The isolates were investigated for the presence of ESBLs by the CLSI-recommended confirmatory tests. PCR was used to detect 16S rRNA methylase genes, ESBL genes and class Ⅰ integrase gene. The homology of these isolates was analyzed by pulse-field gel electrophoresis (PFGE). Results Sixty-four ( 19. 0% ), 28 ( 8. 3% ) and 55 ( 16. 3% ) of 337 isolates were resistant to gentamicin, amikacin and tobramycin, respectively. Twenty-one (6. 2% ) isolates carried 16S rRNA methylase genes (3 for armA, 13 for rmtB, 5 for both armA and rmtB) and high-level resistant to gentamicin, amikacin and tobramycin ( MICs ≥256 mg/L). Nineteen of 21 isolates with 16S rRNA methylase genes were ESBL producers, blaCTX-M-14-like, blaCTX-M-like and blaSHV-12-like were predominant genotypes of ESBLs. The plasmids of 13 isolates were transferred into the recipients E. co/iJ53. PCR and sequence analysis revealed that blaCTX-M-14-like,blaCTX-M-15-like and blaSHV-12-like were co-transferred with the armA or the rmtB to the recipients. All transconjugants harbored intll and blaTEN-1. Of the 21 isolates, 14 patterns were obtained by PFGE. Conclusion Both horizontal gene transfer and clonal spread were responsible for the dissemination of the rmtB or the armA gene in Klebsiella pneumoniae.

Background Epidemiological surveys showed that the types of bacteria are different in the conjunvical sac from different nationalities,which possibly is associated with living environment.To characterize the types of conjunctival bacteria involved is important for the prevention and treatment of infectious eye diseases.Objective The present survey was to obtain data about bacterial species in the conjunctival sac in the Tibetan minority population aged over 40 years old and compared with the matched Han nationality population.Methods The standardized training and protocol were performed before this survey.A total of 290 eyes of 145 individuals from the Tibetan minority and 346 eyes of 173 subjects from the Han nationality were enrolled in this study in Ganzi Autonomous Prefecture,who had received questionnaire surveys and ophthalmological examinations.The secretion of the inferior palpebral conjunctival sac was embrocated and inoculated and grown on blood plates for 48-72 hours.The bacteria were isolated and identified.This study was approved by the Medical Ethic Committee of the Sichuan People Hospital.Oral informed consent was obtained from the subjects.Results No significant differences were seen in the constituent ratio of the gender as well as the age between the Tibetan minority and Han nationality in this study (x2 =0.987,P=0.3202;t=1.142,P=0.254).There was a significant difference in the proportions of farmers and herdsmen between the two groups(x2 =8.557,P =0.000).The positive rate of bacterial cultivation in Tibetan individuals was 50.74％,showing a statistically significant decrease in comparison with the Han people(60.4％)(x2=6.042,P=0.014).There was no statistical difference in the multiple bacterial species between the two groups (11.0％ in Tibetan,11.6％ in Han people)(x2 =0.0271,P =0.869).The rate of staphylococcus epidemics was 26.6％ in the Tibetan minority and that of Han population was 33.2％,without a significant difference between them (x2 =3.350,P=0.060).No significant difference was seen in the ratio of corynbacterium infection between the two population(15.9％ vs.17.3％)(x2 =0.248,P =0.618).Conclusions The ratio of bacterial cultivation in Tibetans is statistically lower than that of the Han people.The types and distribution of bacteria are similar in the Tibetan and Han nationality.

Objective To explore the biomechanical function of PCL and its different bundles and examine the biomechanical impact of posterior cruciate ligament (PCL) integrity on the medial femoral condyle. Methods Twelve fresh human cadaveric knee specimens were subjected to different axial load (0-800 N) at 0°, 30°,60°, and 90°of knee flexion. Four surgical treatments were carried out for biomechanical testing: PCL intact, anterolateral bundle (ALB) rupture, posteromedial bundle (PMB) rupture and PCL rupure. During the test, strains of middle part of the medial femoral condyle were calculated. Results At O°knee flexion, increasing strain of the medial femoral condyle was detected in PMB rupture and PCL rupture under all loading conditions. No significant difference of strain of the medial femoral condyle was noted between PCL intact and ALB rupture under any loading conditions. Compared to PMB rupture, PCL rupture had not higher strain of the medial femoral condyle under all loading conditions. At 30°, 60° and 90° knee flexion, increasing strain of the medial femoral condyle was noted in ALB rupture under higher loading conditions and PCL rupture under all loading conditions. ALB rupture under lower loading conditions and PMB rupture under all loading conditions did not significantly increased strain of the medial femoral condyle. PCL rupture had higher strain of the medial femoral condyle than ALB rupture under most of loading conditions.Conclusion The data suggest that PMB is the major stabilizing bundle of PCL in full extension, ALB is the major stabilizing bundle of PCL in knee flexion, and both bundles function through the ROM in a codominant fashion. Partial and complete ruptures of PCL may have hazardous biomechanical impacts on the medial femoral condyle during normal movement.