In order to develop a positive quality control products for the detection of porcine repro-ductive and respiratory syndrome virus(PRRSV)nucleic acid by real-time fluorescent quantitative PCR(RT-qPCR),the positive quality control products of PRRSV-1 and PRRSV-2 M genes were prepared using armored RNA technology of MS2 phage.PRRSV-1 and PRRSV-2 M genes were amplified,purified and recovered,and ligated into pET28b vector containing MS2 mature enzyme protein gene and capsid protein.After transformed into BL21(DE3),the gene products were in-duced by IPTG and purified by PEG6000 precipitation method to prepare the armored RNA virus-like particles(AR-PRRSV)containing PRRSV M gene.Following the performance evaluation,as the positive quality control products of PRRSV-1 and PRRSV-2 M genes,AR-PRRSV1M and AR-PRRSV2M were calculated using YY/T 1652-2019 standard.Results showed that it had a good u-niformity,stable storage for the armored virus-like particles at-20,4,25 ℃ for 60 d,and 37 ℃ for 30 d.The prepared armored virus-like particles AR-PRRSV1M and AR-PRRSV2M were deter-mined by digital quantitative PCR(ddPCR)after preliminary quantification by RT-qPCR.The 104 copies/μL of AR-PRRSV1M and AR-PRRSV2M ddPCR fixation was(1.33+0.50)× 104 cop-ies/μL.The above results indicates that the AR-PRRSVM can be used as the quality control of the whole detection process(nucleic acid extraction,reverse transcription and RT-qPCR).

Objective:To investigate the distribution of intraocular pressure (IOP) in high-altitude population aged 18 years and over in Xining, Qinghai and establish the reference interval (RI) of IOP.Methods:A cross-sectional study was conducted in Xining, Qinghai Province at 2.271 km above sea level from September 2019 to May 2020.Ophthalmic examinations and IOP measurement were conducted among subjects from Physical Examination Center of Qinghai Provincial People's Hospital.The subjects who had been living in Xining without leaving for three months were enrolled.Ophthalmic examinations included vision examination, IOP measurement, slit-lamp microscopy, fundus photography, anterior and posterior segment optical coherence tomography.IOP was measured using Goldmann applanation tonometry under local anesthesia.Subjects with factors that could cause significant changes in IOP and affect the accuracy of IOP measurement, and those who were unable to receive IOP measurement were excluded.Subjects were grouped according to sex, age and ethnicity, and the distribution and RI of IOP were compared among all groups.This study adhered to the Declaration of Helsinki.The study protocol was approved by the Ethics Committee of Beijing Tongren Hospital, Capital Medical University (No.TRECKY2017-024). Written informed consent was obtained from each subject.Results:A total of 6 120 subjects (6 120 eyes) aged 18-90 years old were enrolled, including 2 850 males and 3 270 females with average age of (45.54±13.85) years.The average IOP of high-altitude population in Xining, Qinghai Province was (14.32±1.93) mmHg (1 mmHg=0.133 kPa), with the RI of 10.54-18.10 mmHg.The average IOP was (14.42±1.98) mmHg in male with the RI of 10.54-18.30 mmHg, (14.23±1.88) mmHg in female with the RI of 10.55-17.91 mmHg.The IOP of male was higher than that of female ( t=3.71, P<0.001). The IOP of Han, Tibetan, Hui and other nationalities were (14.38±1.91), (13.93±2.06), (14.21±1.87), (13.94±1.95) mmHg, respectively, with a statistically significant overall difference ( F=6.73, P<0.001). The IOP of Han nationality was significantly higher than that of Tibetan, Hui and other nationalities, and the differences were statistically significant (all at P<0.05). Conclusions:RI of IOP in high-altitude population from Xining, Qinghai is lower compared with normal altitude area.

Vibrio parahaemolyticus, the main pathogen causing seafood related food poisoning worldwide, has strong biofilm formation ability. ToxR is a membrane binding regulatory protein, which has regulatory effect on biofilm formation of V. parahaemolyticus, but the specific mechanism has not been reported. c-di-GMP is an important second messenger in bacteria and is involved in regulating a variety of bacterial behaviors including biofilm formation. In this study, we investigated the regulation of ToxR on c-di-GMP metabolism in V. parahaemolyticus. Intracellular c-di-GMP in the wild type (WT) and toxR mutant (ΔtoxR) strains were extracted by ultrasonication, and the concentrations of c-di-GMP were then determined by enzyme linked immunosorbent assay (ELISA). Three c-di-GMP metabolism-related genes scrA, scrG and vpa0198 were selected as the target genes. Quantitative real-time PCR (q-PCR) was employed to calculate the transcriptional variation of each target gene between WT and ΔtoxR strains. The regulatory DNA region of each target gene was cloned into the pHR309 plasmid harboring a promoterless lacZ gene. The recombinant plasmid was subsequently transferred into WT and ΔtoxR strains to detect the β-galactosidase activity in the cellular extracts. The recombinant lacZ plasmid containing each of the target gene was also transferred into E. coli 100λpir strain harboring the pBAD33 plasmid or the recombinant pBAD33-toxR to test whether ToxR could regulate the expression of the target gene in a heterologous host. The regulatory DNA region of each target gene was amplified by PCR, and the over-expressed His-ToxR was purified. The electrophoretic mobility shift assay (EMSA) was applied to verify whether His-ToxR directly bound to the target promoter region. ELISA results showed that the intracellular c-di-GMP level significantly enhanced in ΔtoxR strain relative to that in WT strain, suggesting that ToxR inhibited the production of c-di-GMP in V. parahaemolyticus. qPCR results showed that the mRNA levels of scrA, scrG and vpa0198 significantly increased in ΔtoxR strain relative to those in WT strain, suggesting that ToxR repressed the transcription of scrA, scrG and vpa0198. lacZ fusion assay showed that ToxR was able to repress the promoter activities of scrA, scrG and vpa0198 in both V. parahaemolyticus and E. coli 100λpir. EMSA results showed that His-ToxR was able to bind to the regulatory DNA regions of scrA and scrG, but not to the regulatory DNA region of vpa0198. In conclusion, ToxR inhibited the production of c-di-GMP in V. parahaemolyticus via directly regulating the transcription of enzyme genes associated with c-di-GMP metabolism, which would be beneficial for V. parahaemolyticus to precisely control bacterial behaviors including biofilm formation.
Vibrio parahaemolyticus/metabolism* ; Escherichia coli/metabolism* ; Bacterial Proteins/metabolism* ; Transcription Factors/genetics* ; Gene Expression Regulation, Bacterial

Objective:To study the transcriptional regulation of pilABCD by the master quorum sensing (QS) regulator OpaR in Vibrio parahaemolyticus. Methods:Total RNAs were extracted from the wild type (WT) and opaR mutant (Δ opaR) strain. Quantitative real-time PCR (qPCR) was employed to calculate the transcriptional variation of pilA (the first gene of pilABCD operon) between WT and Δ opaR. The regulatory DNA region of pilABCD was cloned into the corresponding restriction endonuclease sites of pHRP309 harboring a promoterless lacZ reporter gene. The recombinant pHRP309 plasmid was then transferred into WT and Δ opaR, respectively, to detect the β-galactosidase activity in cellular extracts using a β-Galactosidase Enzyme Assay System (Promega). The primer extension assay was applied to map the transcription start site of pilABCD using the total RNAs extracted from the WT strain as the template. The regulatory DNA region of pilABCD was amplified by PCR, and the over-expressed His-OpaR was purified under native conditions with nickel loaded HiTrap Chelating Sepharose columns (Amersham). Thereafter, the electrophoretic mobility shift assay (EMSA) was applied to analyze the DNA-binding activity of His-OpaR to the target DNA in vitro, and the DNase I footprinting assay was further employed to detect the DNA-binding sites of His-OpaR within the target DNA. Results:The results of qPCR and LacZ fusion assays showed that OpaR activated the transcription of pilABCD, leading to a gradual increase in the expression level of pilA with the extension of culture time. The primer extension assay detected only one transcription start site located at 155 bp upstream of pilA. The results of EMSA and DNase Ⅰ footprinting assays showed that His-OpaR protected two DNA regions located from -246 to -197 bp and -181 to -131 bp upstream of pilA. Conclusions:Vibrio parahaemolyticus OpaR activated the transcription of pilABCD in a direct manner.

[摘 要] 目的：探讨表皮生长因子受体（epidermal growth factor receptor，EGFR）基因突变与非小细胞肺癌（non-small cell lung cancer，NSCLC）伴脑转移患者预后的相关性，为改善NSCLC合并脑转移患者预后、指导个体化治疗提供临床依据。方法：回顾性分析福建省立医院2013年1月1日至2018年9月30日期间收治的88例NSCLC合并脑转移患者的临床资料，随访取得患者的死亡时间，随访截止日期为2019年10月31日。收集和分析的临床资料包括性别、年龄、吸烟史、病理类型、基因检测、治疗情况、无进展生存期（progression free survival，PFS）、总生存期（overall survival，OS）等。运用生存分析（Kaplan-Meier生存时间曲线）评价EGFR突变型患者的预后，以单因素分析（log-rank检验）预测影响EGFR-TKI治疗效果的因素。结果：88例NSCLC脑转移患者有57例为EGFR突变型，其中位PFS（MPFS）为13.0个月（95%CI：11.951~14.049），明显高于EGFR野生型患者（P=0.003），患者中位生存期（median survival time，MST）为29.0个月（95%CI：20.531~37.468），明显高于EGFR野生型（P=0.001）。EGFR突变型中，Exon19-del突变组患者较Exon21 L858R突变组患者OS有延长趋势（P=0.05），Exon19-del+Exon20T790M突变组患者OS较Exon21 L858R突变组有延长趋势（P=0.077）。结论：EGFR突变组较野生型组NSCLC脑转移患者预后相对好些，且携带19外显子单一缺失突变的患者预后最好。

Objective To generate various types of diabetic retinopathy ( DR) fundus images automatically by computer vision algorithm. Methods A method based on deep learning to generate fundus images was proposed,which used the vascular vein of the fundus image and the text description of lesions as the constraint conditions to generate fundus image. The text description was encoded by using a long short-term memory ( LSTM) , and the vascular vein image was encoded by a convolutional neural network (CNN). Then the encoded information was combined and used to generate a fundus image by generative adversarial networks ( GAN ) . Results The results showed that the algorithm can generate realistic fundus images. However, the image detail features were not obvious because the text-encoded recurrent neural network ( RNN ) loss function did not converge well. Conclusions Using the GAN can generate realistic DR fundus images, which has certain application value in expanding medical data. However,the generation of detail features in small areas still needs improvement.

Objective To compare the clinical effects of HC video laryngoscope and fiberoptic bronchoscope (FOB)in guidance of endotracheal intubation for patients undergoing cervical surgery.Meth-ods A total of 50 patients (ASA I or II)with cervical vertebra injury,nerve root cervical spondylopathy or cervical spondylotic myelopathy in the first affiliated hospital of Wenzhou medical university were selected, all of whom were undergone selective cervical operations between March 2014 and June 2015.The patients were randomly divided into two groups (n =25):HC video laryngoscope group (group H)and FOB group (group F).After induction of anesthesia,HC video laryngoscope and FOB were used for tracheal intuba-tion.⑴ Intubation time and success rates of intubation were recorded,and visual analogue scale (VAS) was used to evaluate the difficulty of intubation;⑵ Mean arterial pressure (MAP),heart rate (HR),and rate-pressure product (RPP)before incubation (Tb),immediate after intubation (T0),and 1min after in-cubation (T1)were recorded;(3)Intubation-related complications were recorded.Results ⑴ The dura-tion of intubation (19.7 ±7.1 )s in the group H was significantly shorter than that of group F (51.9 ± 19.2)s (P <0.05).The one-time success rate of intubation in group H (92%)was higher than that of group F (64%),with statistically significant differences (P <0.05).Intubation was easier in group H (P <0.05).⑵ MAP,HR and RPP of group H at T0 were higher than those of group F.And at T1,there were no statistical differences between groups in MAP,HR and RPP (P >0.05).⑶ The incidence of complications (sore throat)was lower in group H (P <0.05).Conclusions Compared to FOB,HC vid-eo laryngoscope-guided selective cervical operation has the characteristics of higher success rate of intubat-ion,shorter operation time,and easier operation,more stable hemodynamics and lower incidence of intuba-tion complications.Therefore,HC video laryngoscope is a safe and effective method in the intubation for pa-tients undergoing cervical surgery.

Objective To explore the correlations of intracranial pressure (ICP) with changes of neuron specific enolase (NSE),D-Dimer (D-D) and C-reactive protein (CRP) levels in patients with severe traumatic brain injury.Methods A serial of 35 patients with severe traumatic brain injury,admitted to our hospital from January 2012 to January 2014,were chosen as experimental group,and 20 healthy subjects performed physical examination in our Physical Examination Center at the same period were as controls.ICP monitoring was performed in these 35 patients.The patents were divided into two groups according to ICP:severely elevated ICP group (＞40 mmHg) and moderately elevated ICP group (20-40 mmHg).The NSE,D-D and CRP levels were measured,and these data were compared with those from the control group.The correlations of ICP with changes of NSE,D-D and CRP levels were analyzed.Results The levels of NSE,D-D and CRP in the severely elevated ICP group and moderately elevated ICP group were obviously higher than those in the control group ([12.11 ±2.35] lg/L,[0.39±0.61] mg/L,[3.72±0.69] mg/L) (P＜0.05).The levels ofNSE,D-D and CRP in the severely elevated ICP group ([104.08±7.90] μg/L,[1.55±0.26] mg/L,[47.66±8.60] mg/L) were also obviously higher than those in the moderately elevated ICP group ([61.89±30.35] μg/L,[0.93±0.32] mg/L,[30.87±9.84] mg/L)(P＜0.05).Significant positive correlations were noted between ICP and changes ofNSE,D-D and CRP levels in the patient group (regression equation:ICP=18.598+0.256 NSE [t=7.200,P=0.000],ICP=10.779+23.955D-D [t=10.29,P=0.000],ICP=9.932+0.771 CRP [t=8.423,P=0.000]).Multivariant stepwise regression analysis indicated the closest correlation between ICP and D-D (multiple correlation coefficient=0.873,coefficient of determination=0.762,F=105.917,P=0.000).Conclusions Significant positive correlations can be noted between ICP and changes of NSE,D-D and CRP levels,and the closest correlation is between ICP and D-D in patients with severe traumatic brain injury.The combined application of ICP and NSE,D-D and CRP levels can promote the diagnosis and treatment of severe traumatic brain injury patients.

Objective To evaluate the relationship between birth weight and nonalcoholic fatty liver diseases (NAFLD) in Chinese primary school students. Methods A cross-section study was conducted in ifve elementary schools in Gao Hang Town, Shanghai and 2163 students were enrolled in the study (1120 boys/1043 girls). Height, body weight, waist circumference and per-cent of body fat (bioelectrical impedance analysis) were measured by professional nutritionist after training. Birth weight, feeding pattern, height and body weight of parents were obtained by a self-completed questionnaire. NAFLD was diagnosed by ultra-sound. The risk factors of NAFLD were analyzed. Results The prevalence of NAFLD in the study population was 8.9%. The prevalence of NAFLD was signiifcantly higher in boys than that in girls (12.5%vs 5.0%, P<0.01). Logistic regression showed that sex (OR=1.97, 95%CI:1.21-3.21) and percent of body fat (OR=1.12, 95%CI:1.07-1.17) were the risk factors of NAFLD, and normal BMI was the protective factor of NAFLD (OR=0.09, 95%CI:0.04-0.19) in the study population. Conclusions The pre-valence of NAFLD is higher in boys than that in girls. Also overweight, and high percent of body fat are risk factors of NAFLD in children.

Objective:To investigate the regulation mechanisms of the bone marrow mesenchymal stem cells on lymphocyte pro -liferation of type I diabetic rats .Methods:The rat bone marrow mesenchymal stem cells were isolated , cultured and identified and the effect on lymphocyte proliferation of type Ⅰdiabetic rat was observed by MTT assay , and analyze the CD 4 +CD25 +regulatory T cell ra-tio, cell cycle and apoptosis of type I diabetes rat by flow cytometric .Results:B and C groups was significantly lower than the absor-bance values of group A,the differences between the data were statistically significant (P<0.05), C group was significantly lower than group B absorbance values, the difference was significant (P<0.05);the CD4 +CD25 +regulatory T cells of B and C groups were sig-nificantly higher than group A, the differences of the data were statistically significant (P<0.05), the CD4 +CD25 +regulatory T cell ratio of C group significantly higher than that group B , the differences were statistically significant (P<0.05);the apoptosis levels of B and C groups were significantly higher than group A , the differences were statistically significant (P<0.05), the apoptosis levels of C group were significantly higher in group B , the differences were statistically significant (P<0.05).Conclusion:Bone marrow mesen-chymal stem cells can significantly inhibit lymphocyte proliferation of type Ⅰdiabetic rats, and it may regulate CD4 +CD25 +regulatory T cells, promote apoptosis, thereby affecting the immune function of T lymphocytes , and play its rejection.