Objective: To establish a method for detecting the duration of the synthesis-phase(Ts) and the total cell-cycle(Tc) based on the dual labeling of 5-ethynyl-2′deoxyuridine(EdU) and 5-Bromodeoxyuridinc(BrdU). Methods: HEK293T cells were double labeled with EdU and BrdU and then subjected to immunofluorescence staining. The fluorescence signals were collected using a High-Content Imaging System. The numbers of EdU-positive and BrdU-positive cells in the entire cell population were counted. Based on the cell cycle calculation formula in a previously published article, it was improved to accurately calculate the duration of Ts and Tc of cells. The cell cycle time of human embryonic stem cells was calculated using the established method. Results: Based on the kinetic parameters of the EdU/BrdU dual labeling, the Ts of human embryonic stem cells was 10.9 h, and the Tc was 15.3 h. Conclusion The EdU/BrdU dual labeling method can be used to detect the duration of the cell cycle, providing an accurate and efficient method for cell cycle analysis.

Objective To monitor the susceptibility of clinical isolates to antimicrobial agents in tertiary hospitals in major regions of China in 2022.Methods Clinical isolates from 58 hospitals in China were tested for antimicrobial susceptibility using a unified protocol based on disc diffusion method or automated testing systems.Results were interpreted using the 2022 Clinical &Laboratory Standards Institute(CLSI)breakpoints.Results A total of 318 013 clinical isolates were collected from January 1,2022 to December 31,2022,of which 29.5％were gram-positive and 70.5％were gram-negative.The prevalence of methicillin-resistant strains in Staphylococcus aureus,Staphylococcus epidermidis and other coagulase-negative Staphylococcus species(excluding Staphylococcus pseudintermedius and Staphylococcus schleiferi)was 28.3％,76.7％and 77.9％,respectively.Overall,94.0％of MRSA strains were susceptible to trimethoprim-sulfamethoxazole and 90.8％of MRSE strains were susceptible to rifampicin.No vancomycin-resistant strains were found.Enterococcus faecalis showed significantly lower resistance rates to most antimicrobial agents tested than Enterococcus faecium.A few vancomycin-resistant strains were identified in both E.faecalis and E.faecium.The prevalence of penicillin-susceptible Streptococcus pneumoniae was 94.2％in the isolates from children and 95.7％in the isolates from adults.The resistance rate to carbapenems was lower than 13.1％in most Enterobacterales species except for Klebsiella,21.7％-23.1％of which were resistant to carbapenems.Most Enterobacterales isolates were highly susceptible to tigecycline,colistin and polymyxin B,with resistance rates ranging from 0.1％to 13.3％.The prevalence of meropenem-resistant strains decreased from 23.5％in 2019 to 18.0％in 2022 in Pseudomonas aeruginosa,and decreased from 79.0％in 2019 to 72.5％in 2022 in Acinetobacter baumannii.Conclusions The resistance of clinical isolates to the commonly used antimicrobial agents is still increasing in tertiary hospitals.However,the prevalence of important carbapenem-resistant organisms such as carbapenem-resistant K.pneumoniae,P.aeruginosa,and A.baumannii showed a downward trend in recent years.This finding suggests that the strategy of combining antimicrobial resistance surveillance with multidisciplinary concerted action works well in curbing the spread of resistant bacteria.

Objective:To investigate the safety and efficacy of endoscopic ultrasound-guided coil embolization for gastric varices.Methods:Patients with portal hypertension who received endoscopic ultrasound-guided coil embolization for the prophylaxis of gastric variceal bleeding between November 2016 and August 2020 at Zhongshan Hospital, Fudan University were included in the study. The primary evaluation index was the post-operative loss of blood flow in gastric varices, and the secondary indices included the safety of coil embolization, rebleeding rate, portal vein thrombosis changes and patient survival.Results:A total of 13 patients with isolated gastric varices and portal systemic shunt were enrolled, including 6 males and 7 females with the median age of 58 years. The median maximum diameter of the target vessel was 40 mm and the median number of coils used was 2.7. All puncture sites were in the lower part of the esophagus near the cardia, and no active bleeding occurred after deployment of coils. Flow in the gastric varices were confirmed absent by Doppler imaging. In a median follow-up period of 403 days, 2 patients had rebleeding, with the one-year rebleeding rate of 9.1%.Two patients developed portal venous thrombosis after the operation. Two patients died, and the one-year survival rate was 90.0%.Conclusion:Endoscopic ultrasound-guided coil embolization might be an effective option for the treatment of isolated gastric varices with portal systemic shunts.

Objective:To acquire more scientific, accurate and efficient unplanned extubation (UEX) quality management data by using information technology to directly take the quality index data of unplanned extubation from the nursing medical record system.Methods:From June 2019 to August 2019, a knowledge base of pipeline management including the name of pipeline, pipeline evaluation, pipeline care measures, and reasons for unplanned extubation of pipeline was established in Sir Run Run Shaw Hospital Affiliated to Zhejiang University School of Medicine. Through the analysis of each node of pipeline management, pipeline management system was established. And the non-planned pipeline management data set was directly collected to the nursing quality management system for statistics and analysis, which was verified.Results:In clinical application from September 2019 to september 2020, the direct collection of unplanned extubation quality index data saved 496 hours of nursing labor costs per month, and the accuracy of direct data collection increased from 95.8% (161/168) in trial operation to 100.0% (494/494). The underreporting rate of adverse events decreased to 0. Satisfaction with the application of the quality index management system for unplanned extubation was (4.35 ± 0.73) points.Conclusions:The direct collection of quality index data for unplanned extubation improves the accuracy of the data. Nurses no longer need to report unplanned extubation incidents. The clinical nursing information system and the nursing management system are interconnected to improve the management efficiency of unplanned extubation. It is worthy of application and promotion in clinic.

Background Studies show that retinal neurodegeneration may precede retinal microvascular changes in diabetes mellitus.The apoptosis of retinal ganglion cells (RGCs) is an early finding in retinal neurodegeneration.Toll-like receptor 4 (TLR4) is proved to be up-regulated in diabetic rats retina.However,the impact of TLR4 on RGCs damage in retinal neurodegeneration is poorly understood.Objective The aim of this study was to investigate the expressing change of TLR4 induced by high glucose in RGCs in order to offer a basis for the prevention diabetic retinal neurodegeneration and the study on targeting drugs.Methods RGCs were isolated and purified from the retinas of SPF SD rats aged postnatal 1-3 days by using papain digestion method and then were identified by immunofluorescence technology to detect the expression of Brn3a,a specific marker of RGCs.The cells were divided into normal control group and 10,20,30 mmol/L glucose groups.The expressions of TLR4 mRNA and protein in the ceils were detected by real-time fluorescence quantitative PCR and Western blot analysis in 24 and 48 hours after addtion of glucose.All procedures performed in studies were in accordance with the Association for National Institutes of Health (NIH) Statement for the Care and Use of Laboratory Animals recommendations.The protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University.Every effort was made to minimize animal discomfort and stress.Results The normal cells grew well with the shape of near roundness after inoculaton.The cells were gradually enlarged and clustered with obvious axons and dendrites 24 hours after purifying.Brn3a showed the positive expression in cultured cells.At 24 hours and 48 hours after glucose culture,the cell structures were gradually invisible in most cells.The expressions of TLR4 mRNA in the cells were 0.945 ±0.237,1.180±0.193 and 0.827±0.213 at 24 hours and 1.509±0.422,2.433±0.617 and 1.435±0.410 at 48 hours after culture in the 10,20 and 30 mmol/L glucose groups,respectively,which were significantly higher than 0.600±0.099 and 0.724±0.302 in the normal control group (all at P＜0.01).The expressions of TLR4 protein in the cells were 0.442±0.147,0.626±0.128 and 0.330±0.153 at 24 hours and 0.464±0.121,0.930±0.441 and 0.394±0.158 at 48 hours after culture in the 10,20 and 30 mmol/L glucose groups,respectively,which were significantly higher than 0.090±0.050 and 0.094±0.070 in the normal control group (all at P＜0.01).Conclusions A large number of RGCs die in a high-glucose environment in vitro,meanwhile,the expression of TLR4 up-regulates in the cells,indicating that TLR4 maybe participate in the damage of RGCs induced by high glucose.

OBJECTIVETo understand epidemiological characteristics of Haemophilus influenzae (Hi) infection in hospitalized children with lower respiratory tract infection (LRTI) in west Sichuan China.

METHODThe multicenter prospective cross-sectional design was used; four hospitals in west Sichuan China were chosen as research field, sputum bacterial culture was done and biological typing, PCR identification and drug sensitivity test of Hi epidemic strains were carried out among 0-17y hospitalized patients with LRTI in four hospitals located in west Sichuan China.

RESULTTotally 5 748 cases with LRTI in four hospitals were investigated in west Sichuan from Nov. 2013 to April 2014 and the rate of sputum culture was 46.96% (2,699/5 748). The total pathogenic bacteria positive rate of sputum culture was 43.53% (1,175/2 699), and 279 Haemophilus influenzae (Hi) strain in 272 cases were isolated, the Hi positive rate was 10.08% (272/2 699). All the strains (100%) were non-typeable Haemophilus influenzae (NTHi ) indentified by PCR. The main biotype of 279 strains was type Ⅰ with 39.07% (109/279) and type Ⅳ with 50.90% (142/279) ; 272 cases were enrolled in this survey, 12.50% (34/272) had broncheolitis, the rest of lower respiratory infection was 87.50 % (238/272), and 2.57% (7/272) was neonatal pneumonia, 2.21%(6/272)was pneumonia complicated with sepsis; in four hospitals the overall positive rate of Hi in inpatients with lower respiratory infection was 10.21%, 28.96%, 4.80%, 10.21% (χ(2) = 112.561, P = 0.000) and the positive rate of Hi inpatients with broncheolitis was 11.92%, 20.93%, 4.76%, and 66.67% (Fisher exact probability P = 0.001), with the rest lower respiratory infection was 9.96%, 30.90%, 4.81%, 9.85% (χ(2) =108.876, P = 0.000); 2.87% (8/279) bacterial strains of β-lactamase-nonproducing-ampicillin-intermediary (BLNAI) distributed in four hospitals, and 1.79% (5/279) bacterial strains of β-lactamase-nonproducing-ampicillin-resistant (BLNAR), 0.72% (2/279) bacterial strains of β-lactamase-positive amoxicillin-clavulanate-resistance (BLPACR) were found in two hospitals respectively.

CONCLUSIONAll the Hi isolated from sputum were non-typeable among 0-17y inpatients with LRTI and the main biotype were type Ⅰ and type Ⅳ in west Sichuan China. Much attention should be paid to BLNAR and BLPACR strains found in the west Sichuan region.

Ampicillin ; Child ; Child, Hospitalized ; China ; epidemiology ; Cross-Sectional Studies ; Drug Resistance, Bacterial ; Epidemiologic Studies ; Haemophilus Infections ; epidemiology ; Haemophilus influenzae ; Humans ; Microbial Sensitivity Tests ; Pneumonia, Bacterial ; epidemiology ; Polymerase Chain Reaction ; Prospective Studies ; Respiratory Tract Infections ; epidemiology ; microbiology ; Sepsis ; epidemiology ; beta-Lactamases

Objective To investigate the distribution of breast invasive ductal carcinoma with different molecular subtypes ,and to study the relationship between molecular subtypes and clinical pathological features in Xinjiang area .Methods A total of 621 patients with IDC from January 2013 to January 2014 was classified into Luminal A type,Luminal B type,HER-2 overexpression type and Basal -like type,different molecular subtypes according to the estrogen receptor(ER),progesterone receptor(PR),epidermal growth factor receptor -2(HER-2) and Ki-67,and compared the statistics combining with the clinical pathological characteristics of IDC .Re-sults The result showed that Luminal B type accounted for 53.1%,Luminal A type,HER-2 overexpression type and Basal-like type accounted for 14.5%,15.9%,16.4%,respectively.There were significant differences between the molecular subtypes and tumor size ,histological grade ,tumor pathological stage ,the expression of ER , PR and HER-2(P<0.05 for all).There were no correlations between the molecular subtypes and age ,as well as lymph node metastasis(P>0.05).Conclusion There is a close relationship between invasive ductal carcino-ma of different molecular subtypes and clinical pathological characteristics .The molecular subtypes are beneficial to treatment guidelines for clinical individual .

Objective To explore a method to separate and purify apolipoprotein Ⅰ from human serum conveniently and efficiently. Methods Apolipoprotein Ⅰ was separated and pufified by ultracentrifugation and affinity chromatography. Then the purified apolipoprotein Ⅰ was analyzed by SDS-polyacrylamide gel electrophoresis and agar gel double immunodiffusion test. Results The purified apolipoprotein Ⅰ was satisfactory. Conclusion This method had good reliability and was convenient and economical.

Objective To investigate the effect and s ig nificance of GYLZ-RCC18 (Genebank accession number:BE825133) on RCC cell line GRC-1. Methods To detect the expression of GYLZ-RCC18 by means of RT-PCR in both the renal cell carcinoma cell line G RC-1 and the normal kidney cell line HK-2. 20bp GYLZ-RCC18 antisense oligon u clotide packed with liposome was transfected into GRC-1 cell.The change in gro wth speed,proliferation activity,apoptosis,mortality and morphology of GRC-1 w ere observed. Results The expression of GYLZ-RCC18 in R CC was much higher than in the normal kidney.After transfection of GYLZ-RCC18 a ntisense oligonuclotide,the mortality of GRC-1 increased significantly.At the s ame time the proliferation activity and growth speed were retarded remarkably.Th e antisense oligonuclotide induced apoptosis of GRC-1 throughout the observati on time. Conclusions GYLZ-RCC18,a RCC relative novel ge ne,overexpression would stimulate the growth and proliferation activity of RCC.A poptosis and mortality of the RCC cell were also retarded.So,transfection of ant isense oligonuclotide could inhibit the generation and development of RCC provid ing a new approach to the research of RCC.