Objective:To identify and validate secreted effector proteins of Chlamydia psittaci ( C. psittaci) through bioinformatic prediction and experimental verification, and to characterize their subcellular localization in host cells. Methods:Potential effector proteins were predicted using bioinformatics tools. Candidate effectors were fused to β-lactamase through the constructed expression vectors, and these vectors were transformed into C. psittaci. The secretion of these candidate effectors was evaluated by β-lactamase translocation assays. Eukaryotic expression vectors of confirmed effectors were transfected into host cells to determine their intracellular localization patterns. Results:Bioinformatic analysis identified 29 candidate effector proteins. Experimental validation confirmed the secretion of five effectors, with four exhibiting cytoplasmic localization and one displaying nuclear localization in host cells.Conclusion:This study characterizes five novel C. psittaci secreted effector proteins, providing critical insights for investigating the molecular pathogenesis of psittacosis.

Objective : To explore the therapeutic effect of extracellular vesicles(EVs) of Aspergillus flavus(A. flavus) on A. flavus infection. Methods: The EVs of A. flavus were isolated using ultracentrifugation and detected/identified by nanoparticle tracking analysis(NTA). Quantitative real-time PCR(qRT-PCR) and enzyme-linked immunosorbent assay(ELISA) kits were used to assess the effect of A. flavus EVs on the polarization of bone marrowderived macrophages(BMDMs) and the expression levels of several cytokines,including tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),interleukin-6(IL-6),and interleukin-10(IL-10). The Galleria mellonella(G.mellonella) infection model was constructed. Three concentration groups of A. flavus EVs(0. 2,2,and 20 μg/larvae) were set as the experimental groups,and PBS was used as the control group for the infection model.Meanwhile,three forms of negative control groups were established,including the PBS group,the pierced controlgroup, and the negative control group. The therapeutic effect of A. flavus EVs on A. flavus infection was evaluated by the survival rate of the G. mellonella infection models. Results: The particle size of A. flavus EVs ranged from 20 to550 nm. A. flavus EVs could polarize BMDMs into both M1 and M2 phenotypes and induce the production of cytokines,including TNF-α,IFN-γ,IL-6,and IL-10. The results of the G. mellonella infection model showed that A.flavus EVs could improve the survival rate of G. mellonella after A. flavus infection. Conclusion The EVs produced by A. flavus can promote the expression of both pro-inflammatory and anti-inflammatory cytokines in BMDMs,induce M1 polarization and M2 polarization of BMDMs,and increase the survival rate of G. mellonella after A. flavus infection.

Objective:To identify and validate secreted effector proteins of Chlamydia psittaci ( C. psittaci) through bioinformatic prediction and experimental verification, and to characterize their subcellular localization in host cells. Methods:Potential effector proteins were predicted using bioinformatics tools. Candidate effectors were fused to β-lactamase through the constructed expression vectors, and these vectors were transformed into C. psittaci. The secretion of these candidate effectors was evaluated by β-lactamase translocation assays. Eukaryotic expression vectors of confirmed effectors were transfected into host cells to determine their intracellular localization patterns. Results:Bioinformatic analysis identified 29 candidate effector proteins. Experimental validation confirmed the secretion of five effectors, with four exhibiting cytoplasmic localization and one displaying nuclear localization in host cells.Conclusion:This study characterizes five novel C. psittaci secreted effector proteins, providing critical insights for investigating the molecular pathogenesis of psittacosis.

Objective To examine the mediating role of empathy and emotional competence in the association between family functioning and internalizing and externalizing problem behaviors among adolescents in China.Methods In this study,we used the data from the June-July 2022 survey of Chengdu Positive Child Development(CPCD)cohort.All respondents were 5th-9th graders from six primary or secondary schools in Chengdu.The Achenbach Child Behavior Checklist(CBCL),the Chinese Family Assessment Instrument(C-FAI),the empathy subscale of the Chinese version of the Interpersonal Reactivity Index(C-IRI),and the emotional competence(EC)subscale of the Chinese Positive Youth Development Scale(CPYD)were used to evaluate the respondents'internalizing and externalizing problem behaviors,family functioning,empathy,and emotional competence,respectively.The average score derived from the total score of a scale divided by the number of entries in each dimension was used as the final score of the scale.Independent samples t-tests or one-way analysis of variance(ANOVA)were performed to examine the differences in family functioning,empathy,emotional competence,and internalizing and externalizing problem behaviors across student groups with different demographic characteristics(sex,grade,and region).Pearson correlation analysis was conducted to examine the relationship between family functioning,empathy,emotional competence,and internalizing and externalizing problem behaviors.AMOS 24.0 was used to validate the hypothesized model and structural equation modeling was used to analyze the mediating effects of empathy and emotional competence between family functioning and internalizing and externalizing problem behaviors among adolescents.Results A total of 3026 eligible participants were included,including 1548(51.16%)male students and 1478(48.84%)female students.Among the respondents,798(26.37%)were 5th graders,738(24.39%)were 6th graders,567(18.74%)were 7th graders,614(20.29%)were 8th graders,and 309(10.21%)were 9th graders.In addition,2064(68.21%)of all respondents were from urban areas and 962(31.79%)were from rural areas.The results of the difference analysis showed that the differences in adolescents'internalizing and externalizing problem behaviors were statistically significant between students of different grades(P=0.004),and that the differences in family functioning and empathy scores were also statistically significant between students of different grades(all P<0.001),whereas the differences in adolescents'internalizing and externalizing problem behaviors were not statistically significant between sexes and regions(P=0.919,0.959).The results of correlation analysis showed that family functioning scores(the higher the score,the worse the family functioning)were significantly negatively correlated with empathy and emotional competence(r=-0.482,-0.432,P<0.01),and significantly positively correlated with internalizing and externalizing problem behaviors(r=0.220,P<0.01).Empathy was significantly positively correlated with emotional competence(r=0.402,P<0.01).Empathy and emotional competence were significantly negatively correlated with all the dimensions of internalizing and externalizing problem behaviors(r=-0.115,-0.305,P<0.01).Emotional competence partially mediated the relationship between family functioning and adolescents'internalizing and externalizing problem behaviors,with a mediation effect value being 0.042(95%[confidence interval]CI:0.031-0.057).Empathy and emotional competence had chain mediation effect between family functioning and adolescents'internalizing and externalizing problem behaviors,with the value of the mediation effect being 0.010(95%CI:0.007-0.014).Conclusion Family functioning influences adolescents'internalizing and externalizing problem behaviors in a direct way and through the chain-mediating roles of empathy and emotional competence.

Objective To investigate the effect of empathy on depressive symptoms in adolescents and to explore the potential mediating role of family functioning in the effect of empathy on depressive symptoms.Methods The 2022 cross-sectional data from the Chengdu Positive Child Development(CPCD)cohort were analyzed in the study.A survey was conducted in Chengdu in June 2022,involving 3 020 students in grades 5-8 from three randomly selected stratified schools.The Interpersonal Reactivity Index(IRI-C),the Chinese Family Assessment Instrument(C-FAI),and the Center for Epidemiologic Studies Depression Scale for Children(CES-DC)were used in the survey.Chi-square test or one-way analysis of variance was performed to examine the differences in various demographic characteristics(sex,grade,region,and total monthly household income)between groups of respondents,as well as the differences in family functioning,empathy,and depression.Pearson correlation coefficient was used to examine the correlation between family functioning,empathy,and depressive symptoms.Structural equation modeling and SPSS PROCESS component Model 4 were used to analyze whether family functioning played a mediating role in the effect of empathy on depressive symptoms in adolescents.Results The detection rate of depressive symptoms among survey respondents was 25.40%.The results of the difference analysis revealed significant differences in the detection rates of depressive symptoms among respondents of different grades,regions,and monthly household incomes(P<0.05).There was no significant difference in the detection rates of depressive symptoms between male and female students.There was a significant difference in the detection rate of depressive symptoms between respondents with different scores for family dysfunction and empathy ability(P<0.001).Correlation analysis results showed that empathy scores were negatively correlated with depression(r=-0.11,P<0.001),that family dysfunction was positively correlated with depression(r=0.29,P<0.001),and that empathy scores were negatively correlated with family functioning(r=-0.37,P<0.001).The mediating role of family dysfunction in the relationship between empathy and depressive symptoms was established,with the direct effect being 0.039(95% confidence interval[CI]:0.010-0.069,P<0.001)and the indirect effect value being-0.096(95% CI:-0.115--0.079,P<0.001).The direct effect value accounted for 28.89% of the total effect value,while the mediation effect value accounted for 71.11% of the total effect value.Conclusion The empathy ability of adolescents is correlated to depressive symptoms,and family functioning plays a mediating role between empathy and depressive symptoms in adolescents.It is suggested that adolescents'empathy ability and family functioning should be enhanced through multiple channels to reduce the occurrence of depressive symptoms.

Objective To establish high performance liquid chromatography(HPLC)characteristic chromatograms of Xinqingduyin Granules(composed of Taraxaci Herba,Lonicerae Japonicae Flos,Chrysanthemi Indici Flos,etc.)and content determination of chicory acid and glycyrrhizic acid,and to optimize the preparation process of Xinqingduyin Granules.Methods Using the characteristic chromatograms of Xinqingduyin and the retention rate of chicory acid and glycyrrhizic acid as indexes,we carried out orthogonal experiment to optimize the extraction process of Xinqingduyin,and studied the concentration process.The molding process of Xinqingduyin Granules was conducted by screening the types and dosage of auxiliary materials,then three batches of pilot experiments were carried out.Results HPLC characteristic chromatograms of Xinqingshuyin Granules and the determination methods of chicory acid and glycyrrhizic acid were established.The optimal preparation technology was as follows:8 times amount of water was added,the drug was decocted for 3 times,with 1 hour per time.After the extract was concentrated under reduced pressure at 80℃,the appropriate amount of steviol glycoside and lactose was added into the extract and mixed.One-step granulation and packaging were adopted.The retention rates of chicoric acid and glycyrrhizic acid in the 3 batches of Xinqingduyin Granules,which were prepared on the pilot scale,were(54.56±1.63)%and(54.96±1.08)%,and the rate of finished product was(87.47±0.49)%,respectively.The quality is uniform,and the characteristic map of Xinqingduyin Granules showed high similarity with that of decoction prepared from the same batch of slices.Conclusion The optimized preparation technology is reasonable,feasible and reproducible.This preparation can be used to obtain the granule with similar materials of Xinqingduyin decoction.

Objective: To test the effectiveness of psychological flexibility training on career adaptability among middle school students who undertook psychological courses based on acceptance commitment therapy and the adolescent mental flexibility model(DNA-V), and to provide a reference plan to improve the mental health of middle school students. Methods: This study recruited 110 junior high school students (60 boys and 50 girls) from a middle-school in Beijing. The students were randomly divided by class into a DNA-V face-to-face course group(offline group n=33), a DNA-V online course group(online group n=40), and a regular school psychology course group(control group n=37). Louise Hayes DNA-V intervention program was condensed into a six-hour middle-school DNA-V psychology curriculum. Using the Avoidance and Fusion Questionnaire for Youth and the Career Adaptability Scale, changes in psychological flexibility and career adaptability were measured before(T1), one week after(T2), and two months after (T3) the intervention. Results: Linear mixed models were used for the analysis, while controlling for demographic variables. Psychological flexibility and career adaptability in the offline group were higher at T2 and T3 than at T1(psychological flexibility t=4.22, 3.11; career adaptablity t=3.05, 4.16, P<0.01), while the difference between T2 and T3 was not statistically significant. The psychological flexibility and career adaptability of the online group were not statistically significant at T1, T2, and T3. The psychological flexibility and career adaptability of the control group increased from T1 to T2(t=4.64, 2.47, P<0.05), but T3 decreased back to a level close to T1. Conclusion In terms of both psychological flexibility and career adaptability, the DNA-V face-to-face psychology course resulted in a retention period of at least two months.

Human bioequivalence testing is an important part of evaluating the quality of a formulation. Although these drugs have a large amount of safety data and clinical application data, they may still have ethical risks in healthy subjects. The definition of healthy volunteers, the general inclusion and exclusion criteria, auxiliary inclusion and exclusion criteria, and inclusion and exclusion criteria considering drug specificity are summarized. The basis for determining whether abnormal test values are clinically significant when screening healthy subjects and the considerations for improving the screening pass rate are discussed. It is expected to provide useful reference for the smooth implementation of human bioequivalence testing.

Objective To establish a one-step recombinase polymerase amplification (RPA) method for pathogen screening and rapid detection in the field targeting for five hemorrhagic fever related viruses (Zaire ebola virus, Sudan ebola virus, Marburg virus, Lassa virus and Yellow fever virus). Methods The specific nucleic acid (NA) fragments of each virus were selected as target genes by genome sequence analysis, and the primers and probes for RPA assays were designed according to the sequence. A series of diluted template genes were used for RPA detection to determine the sensitivity. The hemorrhagic fever-related viral nucleic acids were used for RPA detection to determine the specificity. The amplification experiments were carried out at different temperature ranging from 37℃ to 42℃ to validate the reaction temperature range. Results The RPA reaction systems of the five hemorrhagic fever viruses could effectively amplify the target genes, the sensitivities were between 1.5×102 and 1.5×103 copies. No cross reactions existed with the other hemorrhagic fever-related viral genes. Meanwhile, RPA assay could effectively amplify the target genes at 37-42℃. Conclusion The isothermal RPA assays of five hemorrhagic fever viruses are established, which may amply target genes fast and react at a wide temperature range, and be potentially useful for in field pathogens detection.

Objective To prepare quality control samples for St.Louis encephalitis virus(SLEV)molecular detection by constructing pseudovirus containing target sequences of SLEV.Methods According to the principles of armored RNA technique, the prM gene sequence of SLEV was cloned into the prokaryotic expression vector to generate recombinant plasmid pSE380-MS2-SLEV.Then, recombinant E.coli transformed with the corresponding plasmid was induced with IPTG to produce recombinant pseudovirus particles.The particles were purified by chloroform and further characterized by double enzyme digestion and transmission electron microscopy.The temperature sensitivity experiments and quantitative RT-PCR were performed to validate the potential of these pseudovirus particles as quality control samples.Results PCR amplification and sequencing analysis confirmed that the prM gene sequence of SLEV was cloned into vector pSE380-MS2.Transmission electron microscopy showed that homogenous spherical particles with a diameter of about 25 nm were produced upon IPTG induction.The SLEV genomic RNA within the pseudovirus particles was resistant to DNaseⅠand RNase A digestion, and remained stable for 20 days at 37℃.These samples were validated with quantitative RT-PCR for SLEV.Conclusion The RNase-resistant and stable pseudovirus particles containing prM fragment of SLEV are constructed successfully, which can be used as positive quality control samples for RNA extraction and molecular detection.