AIM:This study aims to investigate of perodic expression,distribution and interaction between es-trogen receptor α(ERα)and ClC-3 chloride channel,and their relevance to the cell cycle specificity of tamoxifen(TAM)in anti-breast cancer treatment.METHODS:We utilized a web database to analyze the correlation between ERα and ClC-3 expression.Three-dimentional molecular simulation software and co-immunoprecipitation were employed to detect and analyze the interactions between these two proteins.To assess cell cycle dynamics,we performed thymidine(TdR)double-blocking release assay and used nocodazole to block the cell cycle,with subsequent analysis via flow cytometry.Cell viability was measured by MTT assay.Western blot was conducted to evaluate the protein expression levels of ERα and ClC-3,while immunofluorescence staining was utilized to assess their subcellular distribution.RESULTS:(1)Anal-ysis from the web database revealed a significant correlation between ERα and ClC-3 expression,and co-immunoprecipita-tion confirmed their interaction.(2)We successfully obtained human breast cancer T47D cells at different cycle stages us-ing the TdR double-blocking release method and nocodazole treatment.(3)Treatment with TAM primarily inhibited T47D cell viability during G2/M phase.(4)Both ERα and ClC-3 exhibited cyclic variations in protein expression,with their sub-cellular distributions showing periodicity and co-localization.(5)Protein interactions between ERα and ClC-3 were ob-served across all cell cycle phases;(6)After TAM treatment,ERα expression peaked in G2/M phase,while ClC-3 expres-sion remained unaffected.CONCLUSION:Our findings demonstrate cyclic differences in the expression and distribution of ERα and ClC-3 in human breast cancer T47D cells,along with confirmed interactions between these two proteins.The cyclic properties of ERα may play a role in mediating the cell cycle specificity of TAM's anti-breast cancer effect.

AIM:This study aims to investigate of perodic expression,distribution and interaction between es-trogen receptor α(ERα)and ClC-3 chloride channel,and their relevance to the cell cycle specificity of tamoxifen(TAM)in anti-breast cancer treatment.METHODS:We utilized a web database to analyze the correlation between ERα and ClC-3 expression.Three-dimentional molecular simulation software and co-immunoprecipitation were employed to detect and analyze the interactions between these two proteins.To assess cell cycle dynamics,we performed thymidine(TdR)double-blocking release assay and used nocodazole to block the cell cycle,with subsequent analysis via flow cytometry.Cell viability was measured by MTT assay.Western blot was conducted to evaluate the protein expression levels of ERα and ClC-3,while immunofluorescence staining was utilized to assess their subcellular distribution.RESULTS:(1)Anal-ysis from the web database revealed a significant correlation between ERα and ClC-3 expression,and co-immunoprecipita-tion confirmed their interaction.(2)We successfully obtained human breast cancer T47D cells at different cycle stages us-ing the TdR double-blocking release method and nocodazole treatment.(3)Treatment with TAM primarily inhibited T47D cell viability during G2/M phase.(4)Both ERα and ClC-3 exhibited cyclic variations in protein expression,with their sub-cellular distributions showing periodicity and co-localization.(5)Protein interactions between ERα and ClC-3 were ob-served across all cell cycle phases;(6)After TAM treatment,ERα expression peaked in G2/M phase,while ClC-3 expres-sion remained unaffected.CONCLUSION:Our findings demonstrate cyclic differences in the expression and distribution of ERα and ClC-3 in human breast cancer T47D cells,along with confirmed interactions between these two proteins.The cyclic properties of ERα may play a role in mediating the cell cycle specificity of TAM's anti-breast cancer effect.

Objective:To investigate the proliferation kinetics of T cells in patients with B-cell hematologic malignancies who received CAR19 T cell therapy.Methods:Observational study. Flow cytometry was used to monitor the levels of CAR19+and CAR19-T cell expansion and the dynamic changes of T lymphocyte subsets before and after CAR19 T cell therapy. The 52 patients with B-cell hematologic malignancies (including 12 B-ALL and 40 NHL) who received CAR19 T cell therapy in the First Affiliated Hospital of Soochow University from November 2021 to December 2023 were recruited in this study. Patients were divided into complete response group and incomplete response group according to the efficacy evaluation criteria in the treatment guidelines for B-cell hematologic malignancies. T test or non-parametric rank sum test were used to compare the differences of CAR19+and CAR19-T cell subsets between the two groups.Results:At the peak of CAR19+T cell expansion, there was no statistic difference of CAR19+T cell subsets between the complete response group and the incomplete response group. After 6 months, the percentage of CD4+T cells (CD3+CD4+CD8-) in CAR19-T cells in patients was lower than the pre-treatment level(48.0+27.2,63.1+19.7,<0.01), and the percentages of CD197+CD45RA+and CD197-CD45RA-subsets recovered to the pre-treatment level, while the percentage of CD197-CD45RA+subset(4.2+3.0,21.1+15.6,<0.01) was lower than the pre-treatment level. The percentage of CD8+T cells (CD3+CD4-CD8+) returned to pre-treatment level after 6 months, CD197-CD45RA-subset in CD8+T cells returned to pre-treatment level, while CD197+CD45RA+subset(16.6+8.7,35.1+30.1,<0.01),CD197+CD45RA-subset(18.7+9.1,25.8+19.1,<0.01) were still lower than pre-treatment level.Conclusion:After CAR19 T cell treatment, there was no significant differences in the proportions of CAR19+T cell subsets in patients with different therapeutic effects. After treatment, the proportion of CAR19-CD3+CD4-CD8+cells recovered earlier than CD3+CD4+CD8-cells, and the dynamic changes of each subgroup were different. This therapeutic regimen has a great impact on the subpopulation of CAR19-T cells in vivo, and the reconstruction of such T cells takes a long time.

AIM:To investigate the role of ClC-3 chloride channel in the promotion of radio sensitization of na-sopharyngeal carcinoma CNE-2Z cells by dihydroartemisinin(DHA).METHODS:MTT was used to detect the inhibito-ry effect of DHA on the viability of CNE-2Z cells and normal nasopharyngeal epithelial NP69-SV40T cells,the radio sensi-tization effect of DHA on CNE-2Z cells was detected by cloning assay,the expression of ClC-3 protein was detected by Western blot,the expression of ClC-3 protein was down-regulated by siRNA technology,and the chlorine current of cells was recorded by whole cell patch-clamp technology.RESULTS:(1)Compared with NP69-SV40T cells,DHA selective-ly inhibited the proliferation of CNE-2Z cells,with IC10 values of(13.020±4.831)μmol/L and(5.244±1.050)μmol/L,respectively(P＜0.01).(2)The results of clonal formation experiments showed that DHA had a radio sensitizing effect on CNE-2Z cells,with a radio sensitization ratio of 1.9.(3)DHA could activate the chlorine channel of CNE-2Z cells and produce an outward chlorine current,but had no effect on the chlorine channel of NP69-SV40T cells.(4)DHA promoted the expression of ClC-3 chloric channel protein in CNE-2Z cells(P＜0.01).(5)Chlorine channel blocker NPPB could in-hibit the radio sensitizing effect of DHA on CNE-2Z cells by 1.84 times,and also inhibited the chlorine current activated by DHA.(6)the down-regulation of CNE-2Z ClC-3 protein could inhibit the radio sensitization effect of DHA on CNE-2Z cells by 4.19 times,and the activation of chlorine current by DHA on CNE-2Z cells was no longer produced.CONCLU-SION:DHA has a radio sensitizing effect on nasopharyngeal carcinoma CNE-2Z cells,which is likely to be related to the activation of ClC-3 chloride channel.

Objective:To investigate the proliferation kinetics of T cells in patients with B-cell hematologic malignancies who received CAR19 T cell therapy.Methods:Observational study. Flow cytometry was used to monitor the levels of CAR19+and CAR19-T cell expansion and the dynamic changes of T lymphocyte subsets before and after CAR19 T cell therapy. The 52 patients with B-cell hematologic malignancies (including 12 B-ALL and 40 NHL) who received CAR19 T cell therapy in the First Affiliated Hospital of Soochow University from November 2021 to December 2023 were recruited in this study. Patients were divided into complete response group and incomplete response group according to the efficacy evaluation criteria in the treatment guidelines for B-cell hematologic malignancies. T test or non-parametric rank sum test were used to compare the differences of CAR19+and CAR19-T cell subsets between the two groups.Results:At the peak of CAR19+T cell expansion, there was no statistic difference of CAR19+T cell subsets between the complete response group and the incomplete response group. After 6 months, the percentage of CD4+T cells (CD3+CD4+CD8-) in CAR19-T cells in patients was lower than the pre-treatment level(48.0+27.2,63.1+19.7,<0.01), and the percentages of CD197+CD45RA+and CD197-CD45RA-subsets recovered to the pre-treatment level, while the percentage of CD197-CD45RA+subset(4.2+3.0,21.1+15.6,<0.01) was lower than the pre-treatment level. The percentage of CD8+T cells (CD3+CD4-CD8+) returned to pre-treatment level after 6 months, CD197-CD45RA-subset in CD8+T cells returned to pre-treatment level, while CD197+CD45RA+subset(16.6+8.7,35.1+30.1,<0.01),CD197+CD45RA-subset(18.7+9.1,25.8+19.1,<0.01) were still lower than pre-treatment level.Conclusion:After CAR19 T cell treatment, there was no significant differences in the proportions of CAR19+T cell subsets in patients with different therapeutic effects. After treatment, the proportion of CAR19-CD3+CD4-CD8+cells recovered earlier than CD3+CD4+CD8-cells, and the dynamic changes of each subgroup were different. This therapeutic regimen has a great impact on the subpopulation of CAR19-T cells in vivo, and the reconstruction of such T cells takes a long time.

Objective:To study the clinical phenotype and molecular genetic characteristics of a Chinese Han family with X-linked retinoschisis (XLRS), and to determine the associated gene variations.Methods:A pedigree investigation was performed.The clinical characteristics and pedigree analysis of a Han Chinese family line with XLRS was conducted in August 2021 at the Xiamen Eye Center Affiliated to Xiamen University.All patients and the carriers underwent comprehensive medical history collection and routine ophthalmological examinations, including visual acuity, non-contact tonometer, slit lamp microscope, direct ophthalmoscope, and optical coherence tomography.The proband and some patients underwent medical optometry, fundus photography or wide-angle fundus photography, and electroretinogram examination.Peripheral venous blood samples were collected from the family members, and whole exome sequencing (WES) analysis was performed on the proband samples.For variants screened by WES, the expanded verification in other patients and normal persons in the family was carried out by Sanger sequencing.Multiple bioinformatic tools were used to analyze the pathogenicity of variants.This study protocol was approved by the Ethics Committee of Xiamen Eye Center of Xiamen University (No.XMYKZX-KY-2021-012). Written informed consent forms were obtained from each subject or guardian of minors.CADD, FATHMM and other bioinformatics tools were used to analyze the pathogenicity of the variation sites.Results:The Han XLRS pedigree consisted of 8 individuals in 3 generations.Out of the 3 cases diagnosed with XLRS based on clinical evaluation, all were male.The mother of the proband was a carrier of related genes.There were 5 persons with normal phenotypes.There was no history of consanguineous marriages within the family, and the disease was shown to be intergenerational, which is consistent with the recessive inheritance of the X chromosome.None of the patients had a history of systemic disease or any other abnormal manifestations.The prevailing feature of ophthalmopathy was poor binocular vision since childhood.The proband and his younger brother had spoke split in the macula, and their grandfather showed atrophy of retinal nerve fibers.Genetic analysis revealed a hemizygous variation c. 214G>C: p.Glu72Gln in the RS1 gene in all the patients in this family.The proband's mother was heterozygous at this site, and all other phenotypically normal family members exhibited wild type at this site.This variant was predicted to be a deleterious variation and likely to cause disease based on bioinformatics analysis. Conclusions:The proband and patients in this Han Chinese family have the known c. 214G>C: p.Glu72Gln hemizygous variation of the RS1 gene and exhibit mild XLRS, which was consistent with the recessive inheritance of X chromosome.

Objective:In order to explore the impact of corona virus disease 2019(COVID-19)on the hospitalization of children with bronchiolitis and to improve clinicians′ understanding of the characteristics of bronchiolitis during the COVID-19 epidemic.Methods:This was a multicenter clinical study, and the data have been collected from 23 children′s medical centers in China.All the clinical data were retrospectively collected from children with bronchiolitis who were hospitalized at each study center from January 1, 2019 to December 31, 2021.The results included gender, age at hospitalization, length of stay, respiratory syncytial virus(RSV) test results, severity rating, ICU treatment, and the total number of children hospitalized with respiratory tract infection during the same period.The clinical data of children with bronchiolitis in 2019 before COVID-19 epidemic and in 2020、2021 during COVID-19 epidemic were statistically analyzed and compared.Results:According to a summary of data provided by 23 children′s medical centers, there were 4 909 cases of bronchiolitis in 2019, 2 654 cases in 2020, and 3 500 cases in 2021.Compared with 2019, the number of bronchiolitis cases decreased by 45.94% in 2020 and 28.70% in 2021.In 2019, 2020 and 2021, there were no significant differences in gender ratio, age, and duration of hospitalization.Compared with 2019, the ratio of bronchiolitis to the total number of hospitalizations for respiratory tract infection decreased significantly in 2020 and 2021( χ2=12.762, P<0.05; χ2=84.845, P<0.05).The proportion of moderate to severe bronchiolitis cases in both 2020 and 2021 was lower than that in 2019, and the difference was statistically significant ( χ2=4.054, P<0.05; χ2=8.109, P<0.05).There was no statistically significant difference in the proportion of bronchiolitis cases requiring ICU treatment between 2019, 2020, and 2021 ( χ2=1.914, P>0.05).In 2019, a total of 52.60%(2 582/4 909) of children with bronchiolitis underwent RSV pathogen testing, and among them, there were 708 cases with RSV positive, accounting for 28.00%.In 2020, 54.14%(1 437/2 654) of children with bronchiolitis underwent RSV pathogen testing, and there were 403 cases with RSV positive, accounting for 28.04%.In 2021, 66.80%(2 238/3 500) of children with bronchiolitis underwent RSV pathogen testing, and there were 935 cases with RSV positive, accounting for 41.78%.Compared with 2019 and 2020, the RSV positive rate in 2021 showed a significant increase( χ2=99.673, P<0.05; χ2=71.292, P<0.05). Conclusion:During the COVID-19 epidemic, the implementation of epidemic prevention and control measures reduced the hospitalization rate and severity of bronchiolitis, but did not reduce the positive rate of RSV detection.

Objective:To investigate the changes of various cytokines and coagulation function in B cell acute lymphoblastic leukemia(ALL) patients with different CRS scores during CD19-CAR-T cell immunotherapy.Methods:87 patients with B-ALL hospitalized in the First Affiliated Hospital of Soochow University and 30 normal controls were enrolled into this study from July 2018 to October 2020. The age of the patients was 32(20, 56) years old and 36(41.4%) were female. All these coagulation indicators, prothrombin time (PT), activated partial thromboplastin time (APTT), D-dimer, fibrinogen (Fg) were analyzed by automatic blood coagulation in B-ALL patients before and after treated with CAR-T cell. The ratio of CD19-CAR-T cells and the expression of IL-6, IL-10, IFN-γ, TFN-α, IL-2, IL-4, and IL-17A were analyzed using flow cytometry. The patients′ clinical parameters were detected, and the CRS classification of severity was made according to the standard of consensus.Results:Patients with CRS>3 had prolonged PT and APTT, increased D-dimer, and decreased fibrinogen ( P<0.05). The levels of cytokines of IFN-γ, IL-6, and IL-10 were significantly higher in patients with CRS>3 than that in controls ( P<0.05).The D-dimer level is positively correlated with IL-10. Conclusion:Patients with severe CRS grading have significant coagulation dysfunction in CD19-CAR-T cell immunotherapy. Cytokines IFN-γ, IL-6, and IL-10 may affect coagulation function and CRS grading during CD19-CAR-T cell immunotherapy.

Objective To explore the measurement of(1,3)-β-D glucan in plasma for the diagnosis of pulmonary fungal infections in pulmonary tuberculosis patients. Methods 40 pulmonary tuberculosis patients with pulmonary fungal infections in Guangzhou chest hospital from January 2015 to December 2015 were enrolled as a test group,among which 35 were confirmed and 5 were suspected pulmonary fungal infections. 52 pulmonary tuber-culosis patients without fungal infections were selected as a control group.(1,3)-β-D glucan content(G test)in this 92 patients plasma were detected. The results of G tests were compared with those from etiological diagnosis to assess the performance of G test. Results 13 strains of candida albicans,13 strains of aspergillus,2 strains of candida tropicalis,2 strains of candida glabrata and 6 strains of other yeast were obtained from patients of test group,but no fungal identified from those of control group. The median of G test in test group and in control group was 126.1 and 29.56 pg/mL,respectively,the level in test group was significantly higher than that in control group (P<0.001). 35 cases were identified as positive and 5 were negative in test group by G test ,while 41 cases were identified as negative and 11 were positive in control group. The sensitivity,specificity,positive predictive value, negative predictive value ,concordance and Youden index of G test were 87.5%,78.85%,76.09%,89.13%, 82.6%and 0.663,respectively. Conclusions Candida albicans and aspergillus are more common pathogens than the other fungi isolated from pulmonary tuberculosis patients with pulmonary fungal infection. G test ,used in pul-monary tuberculosis with pulmonary fungal infections diagnosis,is reliable and fast,and has a higher sensitivity, specificity and accuracy.