ObjectiveTo identify the presence of side population (SP) cells in human ovarian cancer cell line OVCAR-3 and to investigate whether SP cells have the characteristics of cancer stem cells.MethodsSP and non-SP (NSP) cells from OVCAR-3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342.Limiting dilution transplantation assay,realtime PCR,and drug sensitivity assay were performed to compare the tumorigenic ability,differentiation ability in vivo,the mRNA expressiou of stemness marker (Oct-4,Klf4,and Nanog) and ATP-binding cassette (ABC) transporter (ABCG2,ABCB1,and ABCC2),and response to multiple drugs (cisplatin,paclitaxel,doxorubicin,and mitoxantrone )between SP and NSP cells.ResultsA few of SP cells [ ( 1.13 ±0.39) ％ ] which were sensitive to reserpine were identified in OVCAR-3 cells.The injection of as few as 102 SP cells initiated tumors in two of five mice.Tumor latency was 52 -61 days.However,the NSP cells did not generate any tumors in mice until 104 NSP cells were injected (two of five mice).Tumor latency was 64 - 98 days.Tumorigenicity of SP cells was enhanced by at least 100-fold than that of NSP cells.The SP cells regenerated both SP [ ( 2.09 ± 0.73 ) ％ ] and NSP populations in vivo with a fraction size that was comparable to the original population.The mRNA expression ofstemness genes Oct-4,Klf4 and ABC transporters ABCG2,ABCC2 genes were elevated in SP cells compared to NSP cells,the fold changes were 1.95±0.41 (P＜0.05),4.26 ±0.63 (P＜0.01),3.22±0.36 (P＜0.01),and 1.76±0.26 (P＜0.01 ),respectively.The relative activity of SP and NSP cells were 0.757 ± 0.105 versus 0.474 ± 0.035 (P＜0.01),0.521 ±0.092 versus 0.384 ±0.073 (P＜0.05),0.742 ±0.051 versus 0.526 ±0.088 (P ＜0.01 ),and 0.690 ± 0.096 versus 0.466 ± 0.112 ( P ＜ 0.01 ) when they exposed to 0.25 μg/ml cisplatin, 0.01μmol/Lpaclitaxel, 0.25μmol/Ldoxorubicin, and0.05μg/mlmitoxantrone,respectively.ConclusionsSP cells from OVCAR-3 have enhanced self-renewal,differentiation,and tumorinitiating capacity compared to NSP cells.The mRNA expression of stemness genes and ABC transporters are markedly elevated in SP cells,which showed resistance to multiple chemotherapeutic drugs and have characteristics of cancer stem-like cells.Therefore,SP phenotype could be used as a marker to isolate the cancer stem-like cells in ovarian cancer.

Objective To identify differentially expression of microRNAs associated with expression of estrogen receptor α(ERα)and progesterone receptor(PR)between type Ⅰ and type Ⅱ endometrial adenocarcinoma.Methods Two kinds of endometrial adenocarcinoma cell lines,Ishikawa and KLE,was transplanted into node mice and biopsied to identify the expression of ERα,PR and p53,and test their response to estrogen and progesterone.Cultured the two cell lines under the estrogen-free and progesteronefree circumstance,total RNA was isolated to identify the differentially expressed microRNAs by microarray for prediction the microRNAs which target ESR1 and PGR by software miRANDA and TargetScan,and then was validated by real-time PCR in two cell lines cultured both in vivo and in vitro and ten specimens from patients.Results Ishikawa cell line was confirmed from type Ⅰ endometrial adenocarcinoma.KLE cell line was confirmed from typeⅡendometrial adenocarcinoma.One hundred and twenty-six differentially expressed microRNAs between the two cell lines were identified by mieroRNA microarray,among of which may target ESR1 inchded hsa-miR-100,99a,and may tgrget PGR included hsa-miR-378,768-3p-The differential expression of hsa-miR-100,99a,378,768-3p identified by microarray between Ishikawa and KLE in vivo and in vitro was equal to that by real-time PCR,while Hsa-miR-100 was significantly down expressed in type Ⅰ group specimens compared to type Ⅱ group(P＜0.01).Conclusion Hsa-miR-100 is significantly down-expressed in type Ⅰ endometrial adenocarcinoma compared to type Ⅱ,which may be a great potential to target ESR1.

Objectives To explore the relationship between illness perceptions and health behavior in the subjects. Methods Revised illness perception questionnaire and health promoting lifestyle profileⅡ (HPLPⅡ) were used to measure 101 patients′illness perception and health behavior between the groups. Results The HPLPⅡscore of LEAD was(2.17 ± 0.29) with the score of dimension. The course of disease, prognosis and perception of emotion were negatively related to health behaviour; personal control and primaty congtion of drsease were postively related to health behaviour (all P<0.01). Conclusion Medical personnel should pay attention to the relationship between illness perceptions and health behavior in patients with LEAD and effective measures should be taken to improve the patients′illness perceptions, promote their choices and persistence in health behavior and improve the quality of life.

Objective To investigate the clinical features of pneumocystis pneumonia (PCP) in patients with chronic kidney disease. Methods Clinial data of 21 cases of the primary and secondary kidney diseases complicated with PCP,excluding renal transplantation,were analyzed retrospectively. Results Twenty-one cases consisted of 6 cases of primary renal diseases and 15 eases of secondary renal diseases.Twenty patients (95.2%) were receiving immunesuppressive therapy at the PCP onset.Main manifestations were fever,progressive dyspnea,cough with no or seldom sputum.Twenty patients presented obvious hypoxemia and 12 of them were type I respiratory failure.X-ray and CT imaging of 20 patients revealed diffuse pulmonary interstitial shadows or ground glass opacities in both lungs.All the patients were treaed with trimethoprim-sulfamethoxazole.Eleven patients died accounting for 52.3%.Compared with the survivors,elder age (60.91±15.08 vs 44.50±14.83,P＜0.05),lower blood oxygen pressure at onset [(48.11±19.05)mm Hg vs (65.91±13.13)mm Hg,P＜0.01],higher percentage of respirator application and other secondary lung infection were found in dead patients.No PCP relapsed after average 16-month follow-up in the survival patients. Conclusions PCP is a severe complication with high mortality during immunosuppressive therapy in patients with chronic renal disease.Early diagnosis and proper treatment are important to improve prognosis.

OBJECTIVETo analyze the proportion and associated factors of taking subsequent confirmation test among men who have sex with men (MSM) after being tested positive in oral fluid HIV antibody test.

METHODSBy using successive sampling, 1 003 MSM, who were tested positive in oral fluid HIV antibody test in China-Bill & Melinda Gates Foundation AIDS prevention Program (Extension program) in Beijing during May 1 to December 31, 2013, were recruited. The inclusion criteria included: the objects were men who reported having sex with men; the objects aged more than 18 years old; the objects were tested positive in oral fluid HIV antibody test; the objects had not been reported as HIV positives in China Information System for Disease Control and Prevention previously. According to the program strategy, MSM grassroots organizations transferred the respondents to seek subsequent confirmation tests in specific Center for Disease Control and Prevention (CDCs) or hospitals. The subsequent confirmation tests included: fingertip blood HIV antibody rapid test, venous blood Enzyme Linked Immunosorbent Assay (ELISA) HIV antibody test and venous blood Western Blot (WB) HIV antibody test. Chi-square test was adopted to compare the proportion of taking subsequent confirmation tests in different groups. Nonconditional multivaritae binarylogistic regression analysis was taken to identify the associated factors with whether taking subsequent confirmation tests and to calculate the OR (95% CI) values.

RESULTSThe 1 003 respondents were (30.9 ± 9.1) years old. Among all objects, 87.8% (881/1 003) of them took fingertip blood HIV antibody rapid tests and the positive rate was 85.4% (752/881). 98.0% (737/752) of those who were identified as positive in fingertip blood HIV rapid tests took ELISA and WB tests, and the positive rate was 94.4% (696/737). Comparing with those who were expected to seek subsequent confirmation tests in CDCs, the OR (95% CI) value of those who were expected to seek tests in hospitals was 5.10 (1.69-15.36). The OR (95% CI) values of those who used condom sometimes and those who never used condom in anal sex were 5.81 (2.14-15.77) and 3.45 (2.00-5.97) respectively, in comparison with those who reported not having anal sex or using condom consistently in anal sex during the past 6 months. Comparing with the respondents recruited from the internet, the OR (95% CI) values of those recruited in bathrooms, parks/toilets and bars were 0.17 (0.05-0.53), 0.10 (0.04-0.29) and 0.22 (0.06-0.79) respectively. The likelihood of taking subsequent confirmation test decreased with the increase of number of male sexual partners in the past 3 months, and the OR (95% CI) value was 0.92 (0.86-0.99).

CONCLUSIONThe potential HIV positive MSM in the bathroom, park/toilet and bars are less likely to take subsequent confirmation test. Those who do not use condom consistently during anal sex are more likely to seek subsequent confirmation test. Medical organization conducting subsequent confirmation tests is more likely to increase the confirmation test rate of potential HIV positive MSM. The number of male sexual partners has negative correlation with whether to accept the subsequent confirmation test.

Beijing ; Condoms ; HIV Antibodies ; analysis ; HIV Seropositivity ; diagnosis ; Homosexuality, Male ; Humans ; Male ; Mass Screening ; Patient Acceptance of Health Care ; Risk-Taking ; Sexual Behavior ; Sexual Partners ; Surveys and Questionnaires

With high accuracy and precision,next generation sequencing (NGS) has provided a powerful tool for clinical testing of genetic diseases.To follow a standardized experimental procedure is the prerequisite to obtain stable,reliable,and effective NGS data for the assistance of diagnosis and/or screening of genetic diseases.At a conference of genetic testing industry held in Shanghai,May 2019,physicians engaged in the diagnosis and treatment of genetic diseases,experts engaged in clinical laboratory testing of genetic diseases and experts from third-party genetic testing companies have fully discussed the standardization of NGS procedures for the testing of genetic diseases.Experts from different backgrounds have provided opinions for the operation and implementation of NGS testing procedures including sample collection,reception,preservation,library construction,sequencing and data quality control.Based on the discussion,a consensus on the standardization of the testing procedures in NGS laboratories is developed with the aim to standardize NGS testing and accelerate implementation of NGS in clinical settings across China.

[Objective]To investigate the association between screen content and the frequency of screen exposure at the age of one and a half years and hyperactive behavior in preschool,and to explore how the association is affected by the interaction between outdoor activities and screen behaviors,which could provide theoretical basis and feasible solutions for the prevention and intervention of behavioral problems in childhoood.[Methods]The survey was conducted from June 2022 to June 2023 in Huicheng District,Huizhou (China) stratified by whole cluster sampling methods. Parents and teachers of 5648 children in 61 kindergartens were sampled for questionnaire surveys. The Conners Teacher Rating Scale (TRS) was used to investigate hyperactive behavior. A self-administered questionnaire was used to investigate basic demographic information of children,screen content,frequency of screen exposure and outdoor activities at the age of one and a half years. Multivariate logistic regression was used to explore the association between video screen behavior and hyperactive behavior and its interaction with outdoor activities by controlling for covariates such as children's age,gender,and parental education.[Results]Result showed the overall prevalence of 3.2％ for hyperactive behavior,2.1％ for conduct problems,2.1％ for hyperactivity problems,1.3％ for inattention-passivity problems,and 0.9％ for hyperactivity index. After adjusting for confounding factors,multiple logistic regression analysis showed that screen exposure of "two to four times a week" at one and a half years old was associated with an increased detection rate of hyperactive behaviors in preschool children,with an estimated ORs (95％ CI) of 1.682 (1.141,2.480). Daily screen exposure was associated with increased detection rates of hyperactive behavior,conduct problems,hyperactivity issues,inattention-passivity problems,and hyperactivity index in pre-school age. The estimated ORs (95％ CI) were 2.136 (1.218,3.746),2.321 (1.185,4.546),2.300 (1.208,4.380),2.776 (1.267,6.085) and 3.640 (1.525,8.687),respectively. But the above associations were not found in children who were engaged in daily outdoor activities at the age of one and a half years (P value for interaction<0.001). No association was found between screen content and hyperactive behavior (P>0.05).[Conclusions]Frequency of screen exposure in early childhood is significantly associated with hyperactive behavior problems in preschool,and outdoor activities could weaken the correlation between high-frequency screen exposure and hyperactive behavior,suggesting that parents and schools should prioritize scientifically guiding children's video viewing behavior and outdoor activities,ensuring a well-arranged daily life,to lay a good foundation for the healthy development of children's behavior.

Pseudorabies virus(PRV)is the etiological agent of pseudorabies in pigs,which is char-acterized by dyspnea,reproductive disorders,and neurological diseases,and it spreads widely a-round the world.Since 2011,the newly emerged PRV variants have resulted in poor immunity pro-tection of traditional vaccine strains,and the original method of vaccine strain preparation is time-consuming and labor-intensive.Therefore,it is urgently needed to develop an efficient screening method of the vaccine strain at present.Using CRISPR/Cas9 gene editing technology in this study,two single guide RNAs(sgRNA)were designed targeting the virulence gene TK of PRV variant strain CH/JX/2016,and then the enhanced green fluorescent protein the reporter(EGFP)gene was inserted at the TK locus by a homologous repair plasmid.After multiple rounds of plaque puri-fication,the rPRV-ΔTK/EGFP strain was obtained.The results showed the cleavage efficiency of the two sgRNAs was extremely high.The preparation of rPRV-ΔTK/EGFP strain was succeed af-ter only three rounds of purification,and the EGFP expressed normally.The CRISPR/Cas9 system can edit the PRV gene simply,rapidly,and efficiently,and exhibits great potential in the construction of vaccine candidate strains.Meanwhile,the rescued rPRV-ΔTK/EGFP strain not only could be used as a tracer strain in PRV variant infection progresses,but also for subsequent antivi-ral drug screening.