[摘 要] 目的：构建人乳头瘤病毒16型（HPV16） L1蛋白纳米抗体初级文库，通过筛选鉴定获得一株HPV16 L1特异性纳米抗体。方法：以HPV 16 L1蛋白为抗原对羊驼进行免疫，采用噬菌体展示技术构建初级抗体文库。经3轮淘选，采用ELISA法鉴定阳性克隆，将阳性反应最强克隆的VHH序列进行真核表达。经亲和纯化、凝胶过滤层析纯化、SDS‑PAGE和WB法鉴定，获得目的纳米抗体；采用表面等离子共振（SPR）技术检测纳米抗体与HPV 16 L1蛋白之间的亲和力，CCK-8法检测纳米抗体对人永生化角质细胞HaCat的毒性，荧光素酶报告基因实验检测纳米抗体对HPV 16假病毒的中和活性。结果：初级文库库容为1.304 × 1010，丰度为6.5 × 109个/mL，ELISA法鉴定获得36个阳性克隆。表达、纯化获得蛋白单体与二聚体，经鉴定为目的纳米抗体（命名为Nb）。Nb与HPV 16 L1蛋白结合的亲和力为35.41 nmol/L。Nb实验组HaCat细胞增殖活力与空白组没有显著差异（P > 0.05）。与阴性组比较，0.1和1 μmol/L Nb均能抑制假病毒感染293FT细胞（均P < 0.01）。结论：成功获得一株纯度较好、亲和力较高，对上皮细胞没有明显毒性作用、有效抑制HPV 16假病毒感染293FT细胞的纳米抗体Nb，为防治HPV 16感染提供了有效的候选抗体类药物。

The CRISPR/Cas systems comprising the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas protein is an acquired immune system unique to archaea or bacteria. Since its development as a gene editing tool, it has rapidly become a popular research direction in the field of synthetic biology due to its advantages of high efficiency, precision, and versatility. This technique has since revolutionized the research of many fields including life sciences, bioengineering technology, food science, and crop breeding. Currently, the single gene editing and regulation techniques based on CRISPR/Cas systems have been increasingly improved, but challenges still exist in the multiplex gene editing and regulation. This review focuses on the development and application of multiplex gene editing and regulation techniques based on the CRISPR/Cas systems, and summarizes the techniques for multiplex gene editing or regulation within a single cell or within a cell population. This includes the multiplex gene editing techniques developed based on the CRISPR/Cas systems with double-strand breaks; or with single-strand breaks; or with multiple gene regulation techniques, etc. These works have enriched the tools for the multiplex gene editing and regulation and contributed to the application of CRISPR/Cas systems in the multiple fields.
Gene Editing ; CRISPR-Cas Systems/genetics* ; Bacteria/genetics* ; Archaea ; Bioengineering

ObjectiveTo determine the influence of COVID-19 prevention and control on the epidemic characteristics and dynamics of notifiable infectious diseases in the first quarters， Zhejiang Province， and to explore more effective countermeasures against infectious diseases. MethodsDescriptive epidemiology was conducted to determine the change in notifiable infectious diseases during the prevention and control of COVID-19 in Zhejiang Province by retrieving the data of notifiable infectious diseases from 2017 to 2022 in the Chinese information system for disease control and prevention. Cumulative reported new cases of notifiable infectious diseases in the first quarters of 2017‒2019 were compared with that of 2020‒2022. ResultsA total of 546 753 cases of notifiable infectious diseases were newly reported in the first quarters of 2017‒2019， with an average incidence of 321.92/10⁵. In contrast， a total of 509 908 cases of notifiable infectious diseases were newly reported in the first quarters of 2020‒2022， during which the COVID-19 epidemic occurred， with an average incidence of 270.39/10⁵. The incidence in 2020‒2022 significantly declined by 51.53/10⁵， compared with that in 2017‒2019 （χ²=8 072.06， P<0.001）. In the first quarters of 2020‒2022， the average incidence of zoonotic diseases and vector-borne diseases decreased by more than 50%. In addition， the incidence of respiratory， enteric， blood-borne， and sexually transmitted diseases declined to certain degree. ConclusionThe decline in the newly reported cases of non-COVID-19 notifiable infectious diseases in the first quarters of 2020‒2022 indicates that the countermeasures against COVID-19 epidemic， such as multi-disease co-prevention， multi-sectoral collaboration， societal mobilization and personal hygiene and protection， may also decrease the incidence of multiple infectious diseases. It suggests the countermeasures are effective， which would provide evidence for routine prevention and control of infectious diseases in future.

Upper respiratory tract is directly connected with the external environment， and its natural immune system is the first line of defense against pathogens. In antiviral infection， interferon （IFN） is the main component of the antiviral natural immune system and IFN-λ is a newly discovered immune effector molecule that is mainly produced in the mucosal barrier. IFN-λ exerts a biological role through Janus kinase （JAK） and signal transducer and activator of transcription （STAT） signaling pathway， and plays an important part in regulating innate and acquired immunity of respiratory mucosa. IFN-λ principally expresses on the mucosal barrier with a long-lasting antiviral impact and controls immune-inflammatory damage， which is becoming a new focus of antiviral immunity research in the upper respiratory tract， especially in fighting against 2019 novel coronavirus diseases （COVID-19）. Thus， we summarize the research progress of IFN-λ antiviral immunity in the upper respiratory tract to provide new insight in the prevention and treatment of viral infection in the upper respiratory tract.

Corona virus disease 2019 （COVID-19） has led to more than 500 million cases， including 6.19 million deaths. Mutated strains of SARS-CoV-2 （alpha， beta， gamma， delta and omicron） have successively emerged. Currently the omicron variant is gradually replacing the delta variant， driving a surge of cases in most countries including China， posing a great challenge to the prevention and treatment of COVID-19. In the face of the severe pandemic situation， a large number of non-medical volunteers are fighting on the front lines of the war against the pandemic and are vulnerable to exposure due to lack of professional knowledge. This paper introduces the routine skills training of volunteers and emergency response， aiming to provide behavioral guidance for non-medical volunteers in designated isolation facilities.

Objective:To investigate the relationship between serum vascular endothelial growth factor (VEGF) levels and white matter high signal and non-dementia vascular cognitive dysfunction in patients with cerebral small vascular disease (CSVD).Methods:Total 106 patients with CSVD who were admitted to the Department of Neurology of the First Affiliated Hospital of Xinxiang Medical College from April 2019 to December 2020 were enrolled.They were divided into vascular cognitive impairment no dementia group (VCIND group, n=47) and no vascular cognitive impairment group (N-VCI group, n=59)according to mini-mental assessment scale (MMSE), Montreal cognitive assessment (MoCA) scale and activity of daily living scale (ADL). Serum VEGF levels were detected by enzyme-linked immunosorbent assay (ELISA). The baseline data, serum VEGF levels, MoCA score and Fazekas score were compared between the two groups.The correlation between serum VEGF level and white matter high signal and cognitive function was analyzed.SPSS 19.0 software was used for data processing.The statistical methods were t-test, Chi square test, nonparametric test, Logistic regression analysis, Pearson correlation analysis and Spearman correlation analysis. Results:There were significant differences in serum VEGF level((464.18±114.58)pg/mL, (414.17±45.80)pg/mL, F=22.880), MoCA score((13.07±6.48), (20.17±4.06), F=17.920) and Fazekas score (4(3, 5), 3(1, 3), Z=-4.189)between the two groups (all P<0.05). The level of VEGF( β=0.008, OR=1.008, 95% CI=1.001-1.015, P<0.05) was the influencing factor of cognitive function in patients with CSVD .The level of VEGF was negatively correlated with the total score of MoCA, attention and calculation power, and orientation ability ( r=-0.345, -0.373, -0.445, all P<0.05) and it was positively correlated with the total Fazekas score and the Fazekas score of paraventricular and deep white matter ( r=0.392, 0.495, 0.302, all P<0.05). There was a linear trend between the high signal grade of paraventricular and deep white matter and VCIND (both P<0.05). Conclusion:Serum VEGF level is correlated with cognitive function and white matter hyperintensity in patients with CSVD.The increase of VEGF level may be a factor reflecting cognitive dysfunction.In addition, with the increase of white matter hyperintensity level, the risk of VCIND in CSVD is increased.

Objective:To investigate the characteristics of distribution and drug resistance of urinary bacteria in the mid-stream urine of patients with infectious stones.Methods:The retrospective study analyzed the clinical data of 254 patients with infectious stones in the First Affiliated Hospital of Guangzhou Medical University from September 2016 to September 2018. All patients were treated with PCNL. Overall, there were 101 male and 153 female patients, with the mean age of(51.5±12.3) years, and the mean stone burden of 1443.5(660.8, 2837.5) mm2. There were 58 (22.8%) patients with hypertension, 17(6.7%) patients with diabetes and 195(76.8%)with hydronephrosis. The mid-stream urine samples were obtained for bacterial culture and susceptibility test, and the results of urine culture and antimicrobial susceptibility were recorded and analyzed.Results:Of 254 patients involved in this study, 89(35.0%) were positive and 165 (65.0%) were negative for urinary bacterial culture of the mid-stream. The proportion of patients with positive urine bacterial culture of the mid-stream who had positive urine leucocytes, positive urine nitrite and postoperative pyrexia were 86.5%(77/89), 64.0%(57/89), 25.8%(23/89), respectively, which was higher than that of patients with negative urine bacterial culture of the mid-stream [50.3%(83/165), 14.5%(24/165), 14.5%(24/165), P<0.05]. Four teen kinds of bacteria were detected from the mid-stream urine, and the three bacteria with the highest detection rate in turn were Escherichia coli of 38.2%(34/89), Proteus mirabilis of 15.7%(14/89), and Pseudomonas aeruginosa of 11.2%(10/89). The results of this study showed that three common bacteria had high resistance to drug including Cefazolin, Cefuroxime, Cefuroxime ester, Ampicillin and Co-trimoxazole (all resistance rate>40%). The resistance rates of Escherichia coli and Pseudomonas aeruginosa to Ciprofloxacin and Levofloxacin were higher than or equal to 40%. The resistance rates of Escherichia coli and Proteus mirabilis to meropenem, imipenem, ertapenem, piperacillin/tazobactam and amikacin were all lower than 10%. In addition, the resistance rates of Escherichia coli to nitrofurantoin and tigecycline and Proteus mirabilis to tobramycin, aztreonam and cefoxitin were all less than 10%. The resistance rates of Pseudomonas aeruginosa to ceftazidime, cefepime, gentamicin and aztreonam were less than 10%. Conclusions:The highest detection rate of urinary bacteria in culture of the mid-stream with infectious stones are Escherichia coli, Proteus mirabilis and Pseudomonas aeruginosa, all of which showed high resistance to Ampicillin, Cotrimoxazole, and some cephalosporins. Escherichia coli and Pseudomonas aeruginosa showe high resistance to Ciprofloxacin and Levofloxacin, and all of the three bacteria have low resistance rates to some β-Lactamase inhibitor complex and carbapenems, suggesting a reference for clinical empirical medical treatment.

Objective:To explore the use of internet-based continuous visual recognition task (MemTrax test, MTX) as a rapid screening tool for amnestic mild cognitive impairment (aMCI).Methods:Sixty-four patients with aMCI and 64 individuals with normal cognition as healthy controls were enrolled respectively from Department of Neurology and Health Examination Center of the First Affiliated Hospital of Kunming Medical University from August 2018 to December 2019. Montreal Cognitive Assessment (MoCA) scale and MTX were adopted to assess the cognitive function of all subjects. The total adjusted MoCA scale score, correct rate of MTX, reaction time of MTX and MTX score were obtained and statistically analyzed.Results:The adjusted MoCA scale scores of aMCI patients and healthy controls were 19 (14, 24) and 26 (24, 27; Z=6.795), the correct rate of MTX of aMCI patients and healthy controls were 74% (60%, 80%) and 88% (84%, 94%; Z=8.359), and the MTX score of aMCI patients and healthy controls were 51.11±14.07 and 70.56±14.91 ( t=7.590), respectively, all with statistically significant difference ( P<0.001). Reaction time of MTX of aMCI patients and healthy controls was 1.401 (1.253, 1.590) s and 1.277 (1.163, 1.410) s, respectively ( Z=3.083, P<0.01). After adjustment for age, physical or mental occupation, exercise, hypertension, hyperlipidemia, stroke, sleep time, as well as smoke, the linear regression showed that the aMCI patients had a significant decrease of adjusted MoCA score, correct rate of MTX and MTX score ( P<0.001), and an extension of reaction time of MTX ( P=0.071), compared with the controls. By MTX and MoCA scale assessment, the best cutoff value was 81% for correct rate of MTX and 23 for adjusted MoCA scale score respectively for the prediction of aMCI (with sensitivity of 79.7%, 93.8% respectively, and specificity of 68.8%, 82.8% respectively). The area under the curve (AUC) of correct rate of MTX was 0.93 (95% CI 0.89-0.97, P<0.001), and the AUC of adjusted MoCA score was 0.85 (95% CI 0.78-0.91, P<0.001). There was a statistically significant difference in paired comparison of the two AUCs (χ2=4.620, P<0.05). Conclusion:MTX acts better for the detection of aMCI than MoCA scale, and correct rate of MTX<81% can be considered as the existence of MCI.

Objective:To investigate the relationship between serum matrix metalloproteinase-9 (MMP-9) level and the location and severity of bleeding in patients with cerebral microbleeds(CMBs).Methods:A total of 60 CMBs patients admitted to the Department of Neurology of the First Affiliated Hospital of the Xinxiang Medical University from January 2019 to August 2020 were selected as subjects as the CMBs group, and 60 healthy controls without nervous system diseases in outpatient physical examination during the same period were selected as the control group. The clinical data and biochemical indicators of the two groups were collected. Serum MMP-9 levels were measured by enzyme linked immunosorbent assay (ELISA). According to susceptibility weighted imaging (SWI), CMBs patients were divided into grade 1 group ( n=24), grade 2 group ( n=19) and grade 3 group ( n=17), and according to the micro analytical rating scale (MARS), the CMBs patients were divided into the lobar group ( n=19), the deep or infratentorial group ( n=17) and the mixed group ( n=24).The relationship between serum MMP-9 level and the location and severity of CMBs was analyzed. SPSS 19.0 software was used for data statistical analysis.One-way ANOVA, t-test and rank sum test were used for comparison. Logistic regression analysis was used to analyze the influencing factors. Pearson correlation analysis and Spearman correlation analysis were used for correlation analysis. Results:The level of MMP-9 in CMBs group was significantly higher than that in control group (208.13(142.25, 285.88) μg/L, 149.50(93.40, 186.51)μg/L), and the difference was statistically significant ( P<0.05). Serum MMP-9 level was a risk factor of CMBs ( β=1.322, OR=3.750, 95% CI=2.038-7.997, P=0.002). The difference of level of MMP-9 in different severity of CMBs was statistically significant (147.55(109.25, 266.47)μg/L, 242.12(147.55, 288.80)μg/L, 270.42(203.43, 364.27)μg/L, P=0.017). Serum MMP-9 level was positively correlated with the number of CMBs ( r=0.371, P=0.003). The difference of MMP-9 level of CMBs in different locations were statistically significant (249.77(158.43, 338.46)μg/L, 188.83(138.52, 243.15)μg/L, 210.65(144.25, 255.78)μg/L, P=0.013). The increased serum MMP-9 level was a risk factor for CMBs( β=0.401, OR=1.122, 95% CI=1.004-1.204, P=0.036). Conclusion:The increased level of serum MMP-9 may be a risk factor of CMBs, especially for CMBs in cerebral lobesand, and the level of MMP-9 is positively correlated with the severity of CMBs.

OBJECTIVE: To explore the feasibility and safety of robotic surgical system for radical gastrectomy after liver transplantation. METHODS: A 65-year-old male patient with gastric cancer after liver transplantation underwent radical distal subtotal gastrectomy using Da Vinci surgical system at the General Surgery Department of Southwest Hospital Affiliated to the Army Military Medical University on October 23,2018. The placement of Trocars was arranged using five-hole method. No metastatic tumors were found during intraperitoneal exploration and the first hepatic hilum was found to be wrapped with omentum majus. The tumor located at gastric antrum near the lesser curvature. Then the first and the second station lymph nodes were dissected successively. Distal gastrectomy, Billroth II and Brown anastomosis were performed. The anatomical changes of upper abdomen and gastric lymph reflux after liver transplantation were analyzed. RESULTS: Radical distal gastrectomy with D2 lymphadenectomy was successfully performed under the whole robotic surgical system. The operative time was 315 minutes,and blood loss was 145 ml. A total of 19 lymph nodes were dissected, of which 11 were metastatic lymph nodes. The operative difficulty was to separate the adhesion around the hepatic hilum precisely so as to avoid the damage of hepatic surface, as well as the colon hepatic flexure and duodenum which were closely adhered to hepatic hilum. Meanwhile,it was necessary to pay attention to protetion for the common bile duct and portal vein. The endoscopic wrist joint of the robot surgical system was flexible and delicate, which had obvious advantages in the process of anatomical separation of the adhesions among organs and adhesions around denuded common hepatic artery without normal vascular sheath. Semi-liquid diet was provided on the third day after operation. The immunosuppressants were resumed on the third day after operation. The patient was discharged on the 7th day postoperatively without any complications. There were no abdominal bleeding, incision infection,anastomotic leakage, anastomotic stenosis and other complications. Two months after operation, the patients diet and daily life is normal. CONCLUSION The robotic surgical system is safe and feasible for gastric cancer surgery after liver transplantation.
Aged ; Feasibility Studies ; Gastrectomy ; methods ; Humans ; Laparoscopy ; Liver Failure ; surgery ; Liver Transplantation ; Lymph Node Excision ; Male ; Robotic Surgical Procedures ; Stomach Neoplasms ; surgery