Objective To verify the feasibility of Cas9 microinjection in frozen-thawed pronuclear embryos, based on the model of pronuclear embryos of C57BL/6J mice by in vitro fertilization.Methods After fertilized mouse pronuclear embryos cultured in vitro, one-cell and 2-cell embryos were frozen using EFS20/40 cryopreservation tube.The next day recovered and then cultured.The recovery rate and survival rate of the one-cell and 2-cell embryos were compared.The frozen-thawed and fresh pronuclear embryos were injected with Cas9 mixture and injection buffer into the cytoplasm, and then cultivated to 2-cell embryos,and the survival rate and development rate of the 2-cell embryos were compared.Results The recovery rate of frozen-thawed one-cell embryos was 92.5%, the survival rate was 92.8%, the recovery rate of 2-cell embryos was 90.5% and the survival rate was 95.8%, showing no significant difference.Furthermore, the survival rate of fresh one-cell embryos after Cas9 injection was 92.7%, the survival rate of one-cell embryos of the blank group was 97.5%.While the survival rate of Cas9 injected frozen-thawed one-cell embryos was 82.6%, and that of the blank group was 92%,showing a significant difference between the frozen-thawed injected group and other groups(P < 0.05).The development rate of 2-cell embryos after Cas9 injection was not significantly different.Conclusions Frozen-thawed pronuclear embryos can be used for Cas9 microinjection.

Objective To understand the influencing factors of the post competency of health technical personnel in township hospitals in a prov-ince,so as to provide scientific reference for improving health professionals'post competency. Methods Using stratified cluster random sam-pling,1242 health professionals from township health centers and village clinics of 14 cities were extracted and then surveyed with questionnaire. Results The average score of the respondents'post competency was 3.43,which meant they could afford most of the work. Multiple linear re-gression analysis showed that the main influencing factors of the professionals'post competency included gender,age,specialty,and training. The female,senior,nursing and health-care professionals with training exhibited a better post competency. Conclusion To realize the further develop-ment of professionals'post competency,medical students should not only be early clinical and more clinical,but also actively participate in the training and continue the education after entering the clinic. On the other hand ,the government should define the function orientation of the town-ship healthy centers and explore human resource management mode based on the post competency.

Bronchial asthma is a common respiratory disease characterized by chronic airway inflammation.In recent years,fractional exhaled nitric oxide as a non-invasive assessment of airway inflammation detection methods is widely used to determine the nature of airway inflammation,judge the responsiveness of steroids therapy,help to clinical diagnosis and differential diagnosis,adjust hormone therapy,evaluate prognosis.This paper discusses about fractional exhaled nitric oxide's research progress,production and role,the normal standard,clinical application in children with wheezing and related diseases.

Objective To study the toxicokinetics of Cymermethrin and its metabolites in dog bile and provide evidence for forensic cases of identification of Cymermethrin poisoning. Methods 1/4LD50 doses of Cymermethrin were given to 6 male dogs by oral perfusion after the gallbladder fistula surgery on them,and their bile were collected at different time, in which Cymermethrin and its metabolites were extracted by Liquid-liquid extraction with dichloromethane and detected by HPLC-MS-MS. The qualitative analysis was based on retention time and MRM ions. The quantitative analysis was based on an internal standard method and calibration curve. Toxicokinetics equations of Cymermethrin and its metabolites in the bile were established from the c-t curves which were fitted by the WinNonlin toxicokinetics software meanwhie toxicokinetics parameters were obtained. Results The toxicokinetics of Cymermethrin met first-order dynamic equation. The Tmax of Cymermethrin(CYM), 3-phenoxybenzoic acid (3-PBA), 3-(2,2-Dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate (DCVA) respectively were 1.52±0.30,1.29±0.04,0.93±0.41 h ; The Cmax of CYM, 3-PBA, DCVA respectively were 0.38±0.03,7.9±1.32,30.9±16.24 μg/mL ; The T1/2 of CYM, 3-PBA, DCVA respectively were3.93±0.71,1.36±0.11,4.49±2.81 h; Conclusion The toxicokinetics of Cymermethrin in dog bile met first-order dynamic equation ; The toxicokinetics model and parameters of Cymermethrin can provide evidence for forensic identification of Cymermethrin poisoning cases.

Objective Study on the stability of carbofuran and its metabolite carbofuran phenol in blood preserved at different conditions,in order to provide a scientific evidence for forensic identification of carbofuran poisoning death. Methods The dogs were given intragastric administration with 4LD50(13.5mg/kg) of carbofuran, the blood were collected and divided into five equally groups preserved at 20℃(NC2.5mg/mL), 20℃(1%NaF), 20℃, 4℃ and -20℃, respectively. The concentrations of carbofuran and carbofuran phenol in above samples were detected by GC-MS/MS with MRM at 0d、5d、7d、15d、40d、83d and 150d. Results The concentration of carbofuran in preserved blood were found to be significant decrease at 7d(P < 0.05), then a steady decline. In each condition, the concentration of carbofuran phenol in preserved blood showed an increasing trend firstly, then a declined tendency. The concentration of carbofuran and carbofuran phenol descending fast in blood at 20 ℃ (NC) and 20 ℃ (1%NaF).Conclusion Carbofuran and carbofuran phenol in preserved specimens are found to be decomposed. The decomposition is quick at 20℃ and slow at -20℃. Citrate sodium and sodium fluoride are not suit for anticoagulation and antiputrefactiva. Biological specimens used for forensic identification of the carbofuran poisoning should be stored at refriferated or freezed, and be analyzed as soon as possible.

Objective To develop a method of support liquid-liquid extraction (SLE) and simultaneous determination of 4 components in somedon and its 8 metabolites by GC–MS/MS. Methods Somedon and its metabolites were extracted by SLE and determined by GC-MS/MS in MRM mode. The qualitative analysis was based on retention time and ratio of ions. The quantitative analysis was based on internal standard method and calibration curve. Results After SLE and determination of 4 components in somedon and its 8 metabolites, the extraction rate were 37.57%~95.87%, the linear range were 0.12μg/mL~16.00μg/mL, the correlation coefficient(r)were 0.989 6~0.999 7, LOD were 0.08ng/mL~14.48ng/mL, the accuracy were 79.63%~122.90%, the interday and intraday precision were 0.99%~7.43% and 2.19%~10.60% respectively. Conclusion Simultaneous determination of somedon and its metabolites by GC–MS/MS in biological samples, which was rapid, simple, accurate and was high precision and recovery, can be used for qualitative and quantitative analysis in forensic cases.

Objective To investigate the expression and aberrant methylation of Ptchl gene in hedgehog signal pathway in carcinogenesis of human gastric cancer.Methods The total RNA and genomic DNA were extracted from 10 human gastric carcinoma tissues,adjacent tissues(＞3 cm from cancerous tissue)and gastric cancer eell line AGS.Ptchl mRNA expression was detected by real-time quantitative reverse transcription PCR.The pattern of CpG island in Ptchl gene 5'regulation sequence was analyzed by software and its methylation extent was tested by bisulfite sequencing PCR.Results The analysis of CpG island(starting-3950 bases upstream of the Ptchl mRNAla transcription start site and ending 2050 bases downstream)revealed that there were two CpG islands in Ptchl gene 5' regulation sequence(first CpG:-1139 bp～+860 bp;second CpG:+875 bp～+1692 bp).Bisulfite sequencing PCR analysis of 19 CpG sites included in the first CpG island(-870 bp～+229 bp)showed that there was methylation present in all cell lines and the average extent of the methylation of these CpG sites was significantly higher in cancerous tissues(64%±32%,ranged 16%～100%)than that in adjacent tissues(13%±14%,ranged 0%～42%,P＜0.05).There was a negative correlation of the Ptchl methylation with its expression.Conclusion The high methylation of Ptchl gene that involves in the carcinogenesis of human gastric carcer will be a new biomarker for gastric carcer.

With the rapid development of hospice care, more and more people pay attention to the quality of life of patients at the end of life. Due to the dialysis-related complications, heavy burden of comorbidities and cognitive impairment, elderly hemodialysis patients have poor quality of life, which brings heavy physiological, psychological and economic burdens to patients, families and society. Advance care planning can improve patients′ quality of life and save limited medical resources. By referring to relevant literature at home and abroad, this paper reviewed the overview of advance care planning, the necessity of discussing advance care planning with elderly hemodialysis patients, the implementation status and the obstacle factors. The purpose was to provide theoretical reference for better implementation of intervention treatment in elderly hemodialysis patients in China in the future.

Objective:To explore the risk factors associated with a three-month prognosis in patients with aortic acute cerebral infarction.Methods:A prospective study was conducted on 191 patients with aorthropathic acute cerebral infarction included in the Department of Neurology from June 2018 to December 2019, and the patients were divided into good prognosis group (153 cases) and poor prognosis group (38 cases) according to the MRS score of the patient's 3-month prognosis, and the general data, past medical history and blood pressure variability evaluation index (BPV) between the two groups were correlated analysis. The t-test was used to compare the measurement data with normal distribution, the χ 2 test was used to compare the counting data, and the Logistic regression analysis was used to analyze the risk factors. Results:The proportion of patients with diabetes history in the poor prognosis group (20.3% (31/153)), admission NIHSS score ((3.03±2.01) points), standard deviation (SD) ((12.06±4.46) mmHg) and coefficient of variation (CV) ((8.61±3.08)%) of systolic blood pressure at 24 h were lower than those in the good prognosis group (47.4% (18/38), (5.61±3.84) points, (14.75±3.46) mmHg, (10.41±2.18)%), the differences were statistically significant (the statistical values were χ 2=11.73, t=4.01, t=3.46, t=3.38; P values were 0.001, <0.001, 0.001, and 0.001, respectively). Because 24 h systolic blood pressure SD and 24 h systolic blood pressure CV had obvious collinearity, they were respectively included in the Logistic regression model. Taking diabetes history, NIHSS score and 24 h systolic blood pressure SD into the variables, the multivariate Logistic regression results of adverse prognostic risk factors in patients with acute cerebral infarction showed that the history of diabetes mellitus ( OR=3.649, 95% CI: 1.545-8.648, P=0.003), NIHSS score ( OR=1.472, 95% CI: 1.247-1.725, P<0.001) and 24 h systolic blood pressure SD ( OR=1.201, 95% CI: 1.085-1.336, P<0.001). Taking diabetes history, NIHSS score and 24 h systolic blood pressure CV into consideration, multivariate Logistic regression results of adverse prognostic risk factors in patients with acute cerebral infarction showed that the history of diabetes mellitus ( OR=4.695, 95% CI: 1.873-11.766, P=0.001), admission NIHSS score ( OR=1.922, 95% CI: 1.513-2.441, P<0.001) and 24 h systolic blood pressure CV ( OR=1.220, 95% CI: 1.045-1.425, P=0.012). All are independent risk factors influencing the prognosis of patients. Conclusion:The effect of 24 h systolic blood pressure SD and 24 h systolic blood pressure CV on patient prognosis was more valuable in clinical prediction, and the prognosis value of controlling blood glucose levels in patients with diabetes was higher in patients with cerebral infarction.

Objective:To explore prenatal ultrasound diagnostic clues of congenital dislocation of the knee(CDK).Methods:The prenatal ultrasonographic features of 13 CDK fetuses diagnosed from January 2013 to December 2018 in the Third Affiliated Hospital of Zhengzhou University were retrospectively analyzed, combined with the results of gene detections and pathological findings.Results:Thirteen fetuses were diagnosed by prenatal ultrasound during the second trimester. Two-dimensional and three-dimensional ultrasound showed dislocation of the knee(unilateral in 3 cases and bilateral in 10 cases). All cases were accompanied with multiple abnormalities, including talipes(5 cases), overlapping fingers(2 cases), short nasal bone(1 case), thicken nuchal fold(1 case), narrow thorax(1 case), abnormal vertebral development(1 case), etc. Ultrasound diagnosis included Larsen syndrome in 2 cases, arthrogryosis multiplex congenital in 3 cases, asphyxiating thoracic dysplasia in 1 case, and congenital dislocation of the knee in 7 cases. CDK was confirmed in all fetuses after termination of pregnancy. Three of them were known to undergo chromosome examinations with normal chromosomal microarray analysis (CMA). One case was confirmed to be Larsen syndrome by further second-generation sequencing combined with first-generation sequencing screening, suggesting there was FLNB gene mutation.Conclusions:CDK can exist isolated, and it can also be a common manifestation of various diseases. Therefore, attention should be paid to other associated abnormalities in the prenatal detection of knee flexion, and further detection of related genes can provide valuable information for genetic counseling.