Subject recruitment is an important part of clinical researches. It should follow the ethical principles of fairness and representativeness. The recruitment process should pay attention to protecting the privacy of sub-jects. Various recruitment materials must be approved by the ethics committee before use. Whether could recruit el-igible subjects during Phase I clinical trials will have a significant impact on the results, which should attract the relevant persons′attention including the ethics committee, researchers, clinical research associate and the sponsor.

Objective To detect the content of plasma ctDNA and the mutation rate of BRAF V600E in plasma of patients with thyroid carcinoma ,and to explore its clinical significance. Methods Plasma ctDNA was extracted from 16 patients with thyroid carcinoma and 59 patients with benign thyroid nodules by using the blood genomic DNA Extraction Kit. The ctDNA content was detected by fluorescence quantitative PCR ,and the mutation of circulating BRAF V600E was detected by PCR and sequencing. Then the clinical significance was analyzed by combined detection analysis. Results The content of ctDNA in thyroid cancer group was significantly higher than that in benign nodule group (P < 0.01). BRAF V600E mutation detection showed that the mutation rate was 43.75%,but benign nodules had no mutation. Parallel combined detection improved the sensitivity and the specific-ity of the combined detection was higher. Conclusion Combined detection of ctDNA and BRAF V600E in plasma is helpful for differential diagnosis of benign and malignant thyroid nodules.

Objective To investigate the methods of screening specific aptamers for (EpCAM)Gpositive prostate cancer (PCa)cells by cellGSELEX technique.Methods A random DNA library was designed to screen EpCAMGspecific DNA aptamers from human prostate cancer cells expressing EpCAM molecule by cellGSELEX technique.After 12 rounds of in vitro screening,DNA products were cloned and sequenced.Flow cytometry and cellular immunofluorescence were used to detect the specific binding ability of aptamers to target cells.Results Two aptamers of Ep1 and Ep2 were selected.Both of them could specifically bind to EpCAMGpositive cancer cells LNCap,PCG3 ,DU1 45 , and HEK293T cells transfected with target molecule.The binding rates of Ep1 were 61.0%,74.3%,5 9.1% and 60.3%.The binding rates of Ep2 were 65.1%,77.8%,54.2% and 58.3%.Neither of them could bind to HEK293T cells transfected with empty vector with the binding rate of 5.4% in Ep1 and 3.3% in Ep2,respectively.Flow cytometry analysis and confocal images indicated that the EpCAM aptamers could specifically recognize human PCa cells expressing EpCAM,but could not bind to EpCAMGnegative cells.Conclusion EpCAM aptamers derived from cellGSELEX technology can recognize and bind to EpCAMGpositive PCa cells specifically,which may provide new ideas for the specific diagnosis and targeted therapy of prostate cancer,and lay an experimental basis for the other specific diagnosis and treatment schemes of malignant tumors.

Objective:To analyze the viral nucleic acid and cytokines in 12 children with 2019-nCoV infection.Methods:Clinical and laboratory data of the children diagnosed with 2019-nCoV infection in Guangzhou Women and Children′s Medical Center from January to April 2020 were retrospectively analyzed. Throat and anal swabs were collected on alternate days for the detection of 2019-nCoV nucleic acid by fluorescence quantitative PCR. Flow cytometry was used to detect serum cytokines including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-17F, IL-22, TNF-α and TNF-β during the early (both throat and anal swab tests were positive), the intermediate (throat swab test was negative, while anal swab test remained positive), and the convalescence (both throat and anal swab tests were negative) stages of infection.Results:A total of 12 children were enrolled in this study. The male-to-female ratio was 5∶1. The average age was (7.0±4.3) years. There were two asymptomatic, five mild and five common cases. No severe or critical cases were involved. Initially, throat and anal swab nucleic acid tests were simultaneously positive in nine children newly diagnosed in our hospital and the median time of viral shedding in throat swab was longer than that in throat swab [32 (4.5, 45.0) d vs 3 (2, 9) d, Z=11.0, P=0.010]. The median difference of viral shedding time between anal swab and pharyngeal swab was 25.5 (1.5, 42.8) d. The overall levels of serum cytokines IL-17A, IL -4 and IL-5 in different stages of the disease (early, intermediate and convalescence stage) were statistically different ( Z or F, P values were 8.33, 0.016; 5.36, 0.010 and 6.56, 0.004, respectively), and a significant increase was observed in the intermediate stage of infection. IL-17F, IL-2 and IL-22 were all increased during the infection, but there was no significant statistical difference among the three stages ( P>0.05). Conclusions:It was noted that intestinal viral shedding needed a longer time. Although the infectivity has not been determined, higher requirements have been put forward for disease prevention and control. Cytokines secreted by Th2 and Th17 cells were involved in the immune response in children with non-severe 2019-nCoV infection. Monitoring viral shedding and cytokine changes in pediatric patients would be conducive to disease assessment.

BACKGROUND:Macrophage migration inhibitory factor(MIF)is a pleiotropic cytokine,which is secreted in different types of stem cells and can regulate the proliferation,differentiation and migration of various types of stem cells.Our previous research has confirmed that human embryonic stem cells secrete MIF and that its concentration in the culture medium is relatively stable.However,whether MIF is involved in the survival,proliferation and differentiation of human embryonic stem cells remains unclear. OBJECTIVE:To investigate the effects of MIF on survival,proliferation,and differentiation of human embryonic stem cells. METHODS:(1)Human embryonic stem cells H9 were cultured.The growth curve of cells was detected and plotted by CCK-8 assay.Enzyme-linked immunosorbent assay was used to determine the level of MIF in the medium.(2)To determine the effects of exogenous MIF on the survival and proliferation of human embryonic stem cells,different groups were established:the control group,which was cultured in stem cell medium without any modifications;the exogenous MIF group,which was treated with different concentrations(30,100,300 ng/mL)of MIF in the stem cell medium;the MIF inhibitor ISO-1 group,which was treated with different concentrations(2,7,21 μmol/L)of ISO-1 in the stem cell medium;and the MIF+ISO-1 group,which was treated with different concentrations of ISO-1 along with 100 ng/mL of MIF.Cell viability was assessed using the CCK-8 assay.(3)To further elucidate the effect of MIF gene on survival and proliferation of human embryonic stem cell,the MIF knockout H9 cell line was constructed by CRISPR-Cas 9 technology to observe the lineage establishment.(4)To determine the effect of high concentrations of MIF on human embryonic stem cell differentiation,100 ng/mL MIF and 100 ng/mL of CXCR4 neutralizing antibody were separately added to the normal stem cell culture medium.The expression levels of self-renewal factors(KLF4,c-MYC,NANOG,OCT4,and SOX2)and differentiation transcription factors(FOXA2,OTX2)were measured using real-time quantitative polymerase chain reaction,immunofluorescence staining,and western blot analysis. RESULTS AND CONCLUSION:(1)The logarithmic growth phase of H9 cells was between 3-6 days.Under normal growth conditions,human embryonic stem cells secreted MIF at a concentration of approximately 20 ng/mL,independent of cell quantity.(2)Compared to the control group,the addition of different concentrations of MIF had no effect on the proliferation of human embryonic stem cells(P>0.05).ISO-1 significantly inhibited the proliferation of human embryonic stem cells,with a stronger inhibition observed at higher concentrations of ISO-1(P<0.05).The addition of MIF in the presence of ISO-1 reduced the inhibitory effect of ISO-1(P<0.05).(3)Real-time quantitative polymerase chain reaction showed that knocking out 50%of the MIF gene resulted in a significant decrease in the growth vitality of human embryonic stem cells and failure to establish cell lines.(4)Adding 100 ng/mL exogenous MIF to the culture medium resulted in a decrease in the mRNA,protein,and fluorescence expression levels of the self-renewal transcription factor KLF4,while the mRNA,protein,and fluorescence expression levels of the differentiation factor FOXA2 increased.(5)When 100 ng/mL CXCR4 neutralizing antibody was added to the culture medium,the mRNA and protein expression levels of KLF4 increased,while the mRNA and protein expression levels of FOXA2 decreased,contrary to the expression trend observed in the MIF group.In conclusion,the endogenous secretion of MIF by human embryonic stem cells is essential for their survival.The addition of MIF to the culture medium does not promote the proliferation of human embryonic stem cells.However,it can lead to a decrease in the expression of the self-renewal factor KLF4 and an increase in the expression of the transcription factor FOXA2.This provides a clue for further investigation into the effects and mechanisms of MIF on the differentiation of human embryonic stem cells.The MIF-CXCR4 axis plays a regulatory role in this process.