Randomized Controlled Trial Design Based on Patient Cohorts： Methods and Applications of Trials Within Cohorts / 中国实验方剂学杂志

Trials within cohorts （TwiCs） are design methods derived from randomized controlled trials （RCTS）. They have been widely used in chronic disease areas such as tumors and cardiovascular diseases. The basis of the TwiCs design is a prospective cohort of specific diseases. When RCTS need to be implemented， some patients meeting the inclusion and exclusion criteria are randomly sampled from the cohort to receive "trial interventions"， while the remaining patients in the cohort who meet the inclusion and exclusion criteria continue to receive conventional treatment as control groups. By comparing the efficacy differences between the intervention measures of the trial group and the control group， the efficacy of intervention measures was evaluated. Within the cohort， the same process could be repeated to carry out multiple RCTS， so as to evaluate different intervention measures or compare the efficacy of different doses or timing of interventions. Compared with classical RCTS， TwiCs make it easier to recruit patients from the cohort and have higher external validity， providing a new research paradigm for improving the efficiency and applicability of RCTS in clinical practice. However， TwiCs may also face the challenge of poor compliance of patients in the cohort. Researchers need to take effective measures to control these patients in the design and operation of TwiCs. This article focused on the methodological key points during the implementation of TwiCs， including multi-stage informed consent （patients are informed of consent at three stages： entering the cohort， entering the trial group， and after the trial）， randomization procedures （only random sampling of patients from the cohort to receive "trial interventions"）， sample size calculation， and statistical analysis methods. The article also compared the differences between TwiCs and traditional RCTS and illustrated TwiCs research design and analysis with examples， so as to provide new research ideas and methods for clinical researchers.

Progress of circulating-tumor DNA in the precise medicine for advanced-stage cancer / 中华检验医学杂志

Circulating tumor DNA (ctDNA) detection is one of "liquid biopsy"analysis. It has shown promising clinical application in cancer precise medicine due to its noninvasive sampling and real-time observation. Nowadays, ctDNA analysis has been clinically validated and successfully applied in advanced cancer, including selecting personalized treatment, monitoring treatment responses, minimal residual disease and recurrence. Although there has been rapidly increasing interests in ctDNA, its clinical application in cancer precise medicine is still facing few challenges, especially in screening and diagnosing cancer at early stages. Here, the latest progress in the applications and challenges of ctDNA in advancedstage tumor therapy were reviewed to provide references for future studies on ctDNA.

Analysis on the main virulence genes and molecular typing of non-O1/non-O139 Vibio cholerae in ;bloodstream infection / 中华传染病杂志

Objective To analyze the virulence genes and molecular typing of non-O1/non-O139 Vibio cholerae in bloodstream infection,and to provide the scientific basis for its diagnosis,treatment, prevention and controls.Methods Five Vibio cholerae strains were obtained from blood samples of five inpatients with sepsis in Ruian People ’s Hospital from 2012 to 2015 . Morphological examination, biochemical identification,drug sensitivity test and multilocus sequence typing (MLST)classification analysis of strains were conducted.Totally 17 virulence genes were detected by PCR amplification.Results These five suspected Vibrio cholerae isolates were confirmed as non-O1/non-O139 Vibrio cholerae . Drug susceptibility test showed that all the strains were sensitive to tetracycline,ciprofloxacin,piperacillin and tazobactam, meropenem, amikacin and gentamicin; one strain was resistant to trimethoprim/sulfamethoxazole;all were resistant to ampicillin.MLST analysis showed that all strains were new sequence types (ST),belonging to ST268,ST269,ST267,ST270 and ST271 ,and two novel alleles of RY03(mdh:60 and pyrC:86)were discovered.Virulence genes testing showed that the five strains were divided into 4 virulence genotypes:RY02 and RY04 (hlyA + toxR + hap + rtxA + nanH + vasH + vasA +vasK + ),RY01 (hlyA +toxR +hap +rtxA +nanH +vasH -vasA +vasK - ),RY03 (hlyA +toxR +hap +rtxA +nanH - vasH + vasA + vasK + ) and RY05 (hlyA + toxR + hap + rtxA + nanH + vasH - vasA - vasK - ). Conclusions Non-O1/non-O139 Vibrio cholerae can cause human bloodstream infection in immunocompromised patients.The pathogenic factors may be related to the virulence genes of hlyA, toxR,hap ,rtxA and T6SS.

Retrospective analysis of distribution and antibiotic resistance of pathogenic bacteria in neonatal sepsis over 10 years / 临床儿科杂志

Objective To analyze distribution and antibiotic resistance pathogenic bacteria in neonatal sepsis in 10 years. Methods The distribution of pathogens and their antibiotic resistance were retrospectively analyzed in neonatal sepsis from January, 2004 to December, 2013. The results were compared between 2004-2008 and 2009-2013. Results The percentage of Streptococcus agalactiae rose from 1.0%to 4.2%and fungi rose from 1.9%to 7.9%in all pathogens in past 10 years. But the distribution of pathogenic bacteria was not significantly different. The ESBLs of Escherichia coli were increased from 28.6%to 36.0%(P>0.05). The Escherichia coli resistant to imipenem and meropenem had not been found. The Staphylococcus resistance to oxacallin and ampicillin/sulbactam was increased. The Staphylococcus resistant to vancomycin had not been found. Conclusions The main pathogens of neonatal sepsis are coagulase-negative staphylococci and E. coli. The fungi and Streptococcus agalactiae infections are signiifcantly increased.

Expression of specific marker molecules of long-term cultured human dermal papilla cells in vitro / 中华医学美学美容杂志

Objective To investigate the expression of specific marker molecules in hair-inducing activity of long-term cultured human dermal papilla cells (HDPCs) in vitro.Methods After dissected and cultured the HDPCs in vitro,the cells of passages 1 to 8 were used for experiments.The growth appearances of HDPCs in different passages were observed under inverted microscope.To detect the expression of specific marker molecules of long-term cultured HDPCs,the alkaline phosphatase (ALP) activity of the HDPCs was examined,and the specific genes ALP and insulin-like growth factor-1 (IGF-1) expression levels of HDPCs were determined by real-time quantitative PCR.Results After long-term cultured in vitro,the ALP and IGF-1 expression levels of HDPCs gradually decreased in different passages,as well as the display of the aggregated and cartouche growth.The ALP and IGF-1 expression levels of HDPCs in passage 1 was the highest,they were almost about 6.8-fold and 3.5-fold higher than the HDPCs in passage 8.The ALP staining of the HDPCs in passage 1 and passage 2 were evident,but the cells' ALP staining gradually became much weaker than the cells in the previous passages after the long-term cultured in vitro.Conclusions The expression levels of specific marker molecules ALP and IGF-1 of the HDPCs decrease gradually after long-term cultured in vitro,and the higher passage HDPCs lost the special aggregated and cartouche growth appearance,and hence lead to the loss of hair-inducing activity of HDPCs.

CD4 + CD25 + FoxP3 + T regulatory cells in subjects responsive or unresponsive to hepatitis B vaccination / 中南大学学报(医学版)

To determine CD4 + CD25 + T regulatory cells (Tregs),forkhead box P3 (FoxP3) mRNA expression and levels of cytokines secreted by peripheral blood mononuclear cells (PBMCs) in individuals responsive or unresponsive to hepatitis B (HB) vaccination,and to explore the relationships between immune response and immune regulatory cells or cytokines.Methods Based on the antibody against hepatitis B surface antigen (HBsAg) after HB vaccination,the CD4 + CD25 + Tregs frequencies in PBMCs from 18 responders,22 nonresponders and 10 non-immunized healthy controls were analyzed by flow cytometry.The expression of FoxP3 mRNA in PBMCs with or without stimulation of phytohemagglutinin ( PHA ) and HBsAg was analyzed by real-time quantitative PCR.Levels of IL-4,IL-12,IL-18,and IFN-γ secreted by PBMCs after PHA and HBsAg stimulation were analyzed by enzyme-linked immunosorbent assay.Results The ratio of CD4 + CD25 + Tregs to CD4 + T cells in the nonresponders was markedly higher than that in the responders (P ＜0.05),but lower than that in the controls (P ＜0.01 ).FoxP3 was differentially expressed among the responders,nonresponders,and controls in PBMCs before and after PHA and HBsAg stimulation,and nonresponders had the highest FoxP3 mRNA expression (P ＜ 0.05 or P ＜ 0.01 ).The content of IFN-γ by PBMCs after PHA and HBsAg stimulation was markedly lower in the nonresponders as compared with the controls and responders (P ＜ 0.05).However,there were no significant differences in the levels of IL-18,IL-4,and IL-12 from PBMCs after PHA and HBsAg stimulation between the responders and controls as well as the nonresponders (P ＞ 0.05 ).Conclusion CD4 + CD25 + FoxP3 + Tregs may be involved in the negative regulation of responses to hepatitis B vaccination.Immunologic non-responses to hepatitis B vaccination may be related to IFN-γhyposecretion in PBMCs.