ObjectiveTo construct lentiviral vectors that interferes with the expression of sphingosine-1-phosphate receptor 1(S1 PR1) and stably transfect the lentiviral vectors into THP-1 cells, in order to study the effect of S1 PR1 on macrophage function and the mechanism of S1 PR1 in tumor development at the cellular level.MethodsAccording to the sequence of S1 PR1 gene(1901) registered in NCBI database, three pairs of primers were designed for the shRNA sequence of this gene. The target gene was amplified by PCR, inserted into vector pLKO.1-puro, and the corresponding lentiviral plasmids were constructed,which were identified by enzymatic digestion electrophoresis and sequencing. The correct lentiviral vector plasmids were named pLKO.1-S1 PR1-shRNA1, pLKO.1-S1 PR1-shRNA2, and pLKO.1-S1 PR1-shRNA3, and the vector pLKO.1-puro containing stuffer sequence was used as control(named pLKO.1-S1 PR1-shNC). The lentiviral vector plasmids and lentivirus packaging plasmids were co-transfected into 293T cells for virus packaging, and the titer of virus solution was determined after concentration. The screening concentration of puromycin(0, 0. 5, 1. 0, 1. 5, 2. 0, 2. 5 μg/mL), culture time(0, 24, 48, 72 h)and MOI(10, 20, 30, 40, 50) of THP-1 cells were determined. THP-1 cells were infected with the lentivirus under the optimum conditions and screened by puromycin. The relative mRNA and protein expression of S1 PR1 in THP-1 cells of each group were detected by RT-qPCR and Western blot respectively.ResultsEnzymatic digestion electrophoresis identification and sequencing indicated that pLKO.1-S1 PR1-shRNA1, pLKO.1-S1 PR1-shRNA2, and pLKO.1-S1 PR1-shRNA3 lentiviral vectors were correctly constructed. The lentivirus titers of shNC, shRNA1, shRNA2 and shRNA3 groups were 9. 5 × 10~9, 4. 25 × 10~9,2 × 10~9 and 4. 4 × 10~9 TU/mL, respectively. THP-1 cells were infected with the lentivirus at the optimum MOI of 50 for 72 h.After screening with 1. 5 μg/mL puromycin, the relative expression levels of S1 PR1 mRNA in shRNA1, shRNA2 and shRNA3 groups were significantly lower than that in shNC group(F = 44. 916, P < 0. 001); the relative expression level of S1 PR1 protein in shRNA1 group decreased with no significant difference(t = 1. 921, P > 0. 05), while the relative expression levels of S1 PR1 protein in shRNA2 and shRNA3 groups decreased significantly(t = 8. 730 and 6. 957, respectively, each P < 0. 05).ConclusionThe lentiviral vectors interfering with S1 PR1 expression and THP-1 cell lines stably expressing the vectors were successfully constructed, which can be used for further related research.

Objective:To investigate the mechanism by which histone deacetylase 6 (HDAC6) exerts immune protective effects in Chlamydia trachomatis respiratory tract infection. Methods:Wild-type (WT) C57BL/6 mice and HDAC6 gene knockout (HDAC6 -/-) mice were used to establish mouse models of Chlamydia muridarum ( Cm) respiratory infection by nasal inhalation of Cm. qPCR and Western blot were used to detect the relative expression of HDAC6 in lung tissues of WT mice after Cm infection. Intracellular factor staining was used to detect the percentages and absolute numbers of CD4 + T cell subsets (Th1, Th17 and Th2 cells)in mouse lung tissues after infection. The levels of IL-4 in spleen cell culture supernatants were measured by ELISA. One-way or two-way analysis of variance (ANOVA) was used for statistical analysis. Results:Cm respiratory tract infection significantly promoted the expression of HDAC6 at both mRNA and protein levels in lung tissues of WT mice ( P<0.001). HDAC6 -/- mice lost more weight than WT mice and took longer to recover to the normal level. Chlamydia load ( P<0.001 and P<0.05) and pathological damage ( P<0.05 and P<0.000 1) in lung tissues were more serious in HDAC6 -/- mice than in WT mice at 7 and 14 d after infection. Neither the percentage nor the absolute number of Th1 (CD4 + IFN-γ + T) and Th17 (CD4 + IL-17 + T) cells showed significant differences between WT and HDAC6 -/- mice, while the percentage and absolute number of Th2 (CD4 + IL-4 + T) cells increased in HDAC6 -/- mice ( P<0.05 and P<0.01). Moreover, in HDAC6 -/- mice, the expression of IL-4 mRNA increased ( P<0.000 1) and the level of IL-4 in the splenic cell culture supernatants increased ( P<0.01). Conclusions:HDAC6 plays an immune protective role in Cm infection. It can reduce the susceptibility of host to Cm respiratory tract infection and alleviates the pathological damage in lung tissues by inhibiting the immune response of Th2 cells.

Objective:To construct the sphingosine kinase 1(SPHK1)overexpression lentiviral vector,and to establish the SKOV3 lentiviral stable transfection cell line.Methods:According to the SPHK1 data information provided by the National Center for Biotechnology Information(NCBI)database,the primers were designed and synthesized,the target gene was amplified,and connected to the GV492 plasmid treated with Bam HⅠ and AgeⅠ restriction enzymes to construct the SPHK1 overexpression lentiviral vector;the positive clones were selected for PCR and sequencing identification;the lentiviral plasmid and the lentiviral packaging auxiliary plasmid were co-transfected into the HEK-293T cells for packaging and titer determination;according to the measured optimal multiplicity of infection(MOI)of 10,the corresponding lentiviral amounts in various groups were transfected into the SKOV3 cells,and the SKOV3 cells were divided into blank group(without treatment),GV492 control group(GV492 control lentivirus infected SKOV3 cells),and GV492-SPHK1 overexpression group(GV492-SPHK1 overexpression lentivirus infected SKOV3 cells,ov-SPHK1 group);the optimal concentration of 2 mg·L-1 puromycin was used to screen the stably transfected SKOV3 cell line;after 48 h,the medium was changed and replaced with 1 mg·L-1 puromycin for screening for 14 d;the morphology and fluorescence expression of the cells were observed under fluorescence microscope;real-time fluorescence quantitative PCR(RT-qPCR)method was used to detect the expression levels of SPHK1 mRNA in the SKOV3 cells in various groups;Western blotting method was used to detect the expression level of SPHK1 protein in the SKOV3 cells in various groups.Results:The PCR sequencing results showed that the gene sequence of the SPHK1 overexpression lentiviral vector was completely consistent with the target sequence,and the SPHK1 overexpression lentiviral vector was successfully constructed;the titer determination results showed that the lentiviral titers in GV492 control group and ov-SPHK1 group were 5×1011 and 8×1011 TU·L?1,respectively;the SKOV3 cells in GV492 control group and ov-SPHK1 group were in good state and showed strong fluorescence expression,suggesting that the SKOV3 stable transfection cell line overexpressing SPHK1 was successfully established;the RT-qPCR results showed that compared with blank group and GV492 control group,the expression level of SPHK1 mRNA in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01);the Western blotting results showed that compared with blank group and GV492 control group,the expression level of SPHK1 protein in the SKOV3 cells in ov-SPHK1 group was significantly increased(P<0.01).Conclusion:The SPHK1 overexpression lentiviral vector is successfully constructed,and the SKOV3 stable transfection cell line is established.

Objective To construct and validate a prediction model for urosepsis in patients after upper urinary tract stone surgery using various machine learning algorithms.Methods A total of 7 464 upper urinary tract stone patients who underwent surgery at the Sixth Affiliated Hospital of Guangxi Medical University from Jun.2018 to Jun.2023 were enrolled and randomly assigned to training(5 224 cases)or validation sets(2 240 cases)at a ratio of 7∶3.Among them,622(8.33%)cases developed urosepsis postoperatively.Six machine learning algorithms,including extreme gradient boosting(XGBoost),logistic regression,light gradient boosting machine(LightGBM),random forest(RF),adaptive boosting(AdaBoost),and gradient boosting decision tree(GBDT),were used to construct prediction models for postoperative urosepsis.The model's predictive ability and clinical benefits were evaluated using receiver operating characteristic(ROC)curves,Shapley additive explanation(SHAP)analysis,calibration curves,and decision curve analysis(DCA).Results The clinical features included body mass index(BMI),number of surgeries,heart rate,Barthel index,venous thrombo embolism(VTE)risk assessment,gender,American Society of Anesthesiologists(ASA)grade,urinary nitrite,and urinary leukocyte in the models.In the training set,the XGBoost,LightGBM,and RF models performed excellently,with area under curve(AUC)values of ROC curves reaching 1.00.In the validation set,the logistic regression model performed the best,with an AUC value of ROC curve of 0.76,showing good predictive stability and calibration.The AdaBoost and GBDT models followed with AUC values of 0.74 and 0.75,respectively,while the AUC values of the LightGBM,XGBoost,and RF models were 0.71,0.70,and 0.68.In terms of model interpretability,SHAP analysis showed the contribution of variables in a descending order as:heart rate,urinary leukocytes,gender,BMI,Barthel index,VTE risk assessment,urinary nitrite,number of surgeries,and ASA grade.Conclusion A logistic regression model for early risk prediction of postoperative urosepsis in upper urinary tract stone patients has been successfully constructed.This model has good predictive performance and calibration,and can effectively assist clinical diagnosis.

Objective:To evaluate the efficacy of angiogenesis inhibitors and poly ADP-ribose polymerase (PARP) inhibitors in the treatment of recurrent ovarian cancer using a mesh meta-system.Methods:Subject terms were used to search Pubmed, Embase, the Cochrane Library and web of science databases to collect randomized controlled trials related to angiogenesis inhibitors and PARP inhibitors in the treatment of recurrent ovarian cancer. The search time was established until January 1, 2024. Outcome measures included progression-free survival (PFS) and overall survival (OS). Bias risk assessment was performed using Revman 5.4 software and mesh meta-analysis was performed using gemtc package in R 4.3.1 software.Results:34 randomized controlled trials were included in the PFS and 26 in the OS. Olaparib ( HR=0.63, 95% CI: 0.40-0.99), rucaparib ( HR=0.48, 95% CI: 0.24-0.99), niraparib ( HR=0.49, 95% CI: 0.26-0.93), niraparib+ bevacizumab ( HR=0.17, 95% CI: 0.05-0.61), chemotherapy+ bevacizumab+ maintenance bevacizumab ( HR=0.54, 95% CI: 0.3-0.97) and chemotherapy+ bevacizumab ( HR=0.50, 95% CI: 0.31-0.81) had longer PFS than conventional platinum-based chemotherapy/chemotherapy+ placebo. Niraparib+ bevacizumab had the longest PFS of all pharmacological interventions. Chemotherapy plus bevacizumab ( HR=0.72, 95% CI: 0.57 -0.88) had a longer OS than conventional platinum-based chemotherapy/chemotherapy plus placebo. Conclusions:There is limited evidence that angiogenesis inhibitors alone (bevacizumab) or PARP inhibitors alone (niraparib, olaparib, and rucaparib) can improve PFS or OS in recurrent ovarian cancer, and that the combination of angiogenesis inhibitors and PARP inhibitors may be more beneficial in prolonging PFS or OS in recurrent ovarian cancer.

Objective:To investigate the mechanism by which histone deacetylase 6 (HDAC6) exerts immune protective effects in Chlamydia trachomatis respiratory tract infection. Methods:Wild-type (WT) C57BL/6 mice and HDAC6 gene knockout (HDAC6 -/-) mice were used to establish mouse models of Chlamydia muridarum ( Cm) respiratory infection by nasal inhalation of Cm. qPCR and Western blot were used to detect the relative expression of HDAC6 in lung tissues of WT mice after Cm infection. Intracellular factor staining was used to detect the percentages and absolute numbers of CD4 + T cell subsets (Th1, Th17 and Th2 cells)in mouse lung tissues after infection. The levels of IL-4 in spleen cell culture supernatants were measured by ELISA. One-way or two-way analysis of variance (ANOVA) was used for statistical analysis. Results:Cm respiratory tract infection significantly promoted the expression of HDAC6 at both mRNA and protein levels in lung tissues of WT mice ( P<0.001). HDAC6 -/- mice lost more weight than WT mice and took longer to recover to the normal level. Chlamydia load ( P<0.001 and P<0.05) and pathological damage ( P<0.05 and P<0.000 1) in lung tissues were more serious in HDAC6 -/- mice than in WT mice at 7 and 14 d after infection. Neither the percentage nor the absolute number of Th1 (CD4 + IFN-γ + T) and Th17 (CD4 + IL-17 + T) cells showed significant differences between WT and HDAC6 -/- mice, while the percentage and absolute number of Th2 (CD4 + IL-4 + T) cells increased in HDAC6 -/- mice ( P<0.05 and P<0.01). Moreover, in HDAC6 -/- mice, the expression of IL-4 mRNA increased ( P<0.000 1) and the level of IL-4 in the splenic cell culture supernatants increased ( P<0.01). Conclusions:HDAC6 plays an immune protective role in Cm infection. It can reduce the susceptibility of host to Cm respiratory tract infection and alleviates the pathological damage in lung tissues by inhibiting the immune response of Th2 cells.

Objective:To evaluate the efficacy of angiogenesis inhibitors and poly ADP-ribose polymerase (PARP) inhibitors in the treatment of recurrent ovarian cancer using a mesh meta-system.Methods:Subject terms were used to search Pubmed, Embase, the Cochrane Library and web of science databases to collect randomized controlled trials related to angiogenesis inhibitors and PARP inhibitors in the treatment of recurrent ovarian cancer. The search time was established until January 1, 2024. Outcome measures included progression-free survival (PFS) and overall survival (OS). Bias risk assessment was performed using Revman 5.4 software and mesh meta-analysis was performed using gemtc package in R 4.3.1 software.Results:34 randomized controlled trials were included in the PFS and 26 in the OS. Olaparib ( HR=0.63, 95% CI: 0.40-0.99), rucaparib ( HR=0.48, 95% CI: 0.24-0.99), niraparib ( HR=0.49, 95% CI: 0.26-0.93), niraparib+ bevacizumab ( HR=0.17, 95% CI: 0.05-0.61), chemotherapy+ bevacizumab+ maintenance bevacizumab ( HR=0.54, 95% CI: 0.3-0.97) and chemotherapy+ bevacizumab ( HR=0.50, 95% CI: 0.31-0.81) had longer PFS than conventional platinum-based chemotherapy/chemotherapy+ placebo. Niraparib+ bevacizumab had the longest PFS of all pharmacological interventions. Chemotherapy plus bevacizumab ( HR=0.72, 95% CI: 0.57 -0.88) had a longer OS than conventional platinum-based chemotherapy/chemotherapy plus placebo. Conclusions:There is limited evidence that angiogenesis inhibitors alone (bevacizumab) or PARP inhibitors alone (niraparib, olaparib, and rucaparib) can improve PFS or OS in recurrent ovarian cancer, and that the combination of angiogenesis inhibitors and PARP inhibitors may be more beneficial in prolonging PFS or OS in recurrent ovarian cancer.

Objective:To analyze the clinical features of MDA5 antibody positive dermatomyositis (MDA5-DM) and to provide evidence for early diagnosis and treatment.Methods:From March 2019 to June 2021, 272 patients with anti-MDA5-DM from the Nanjing Medical University myositis-associated interstitial lung disease cohort were enrolled, with 76 patients with anti-synthetase syndrome (ASS) as the control group. The clinical characteristics and the occurrence of interstitial lung disease were analyzed. T-test was used for normally distributed and variance-homogeneous independent samples, Mann-Whitney U test for non-normally distributed data, and chi-square test or Fisher′s exact test for dichotomous variables. Results:Among the 272 anti-MDA5-DM patients, 88.6% (241/272) developed interstitial lung disease (ILD), and 33.8% (92/272) developed rapidly progressive ILD (RP-ILD). The six-month all-cause mortality rate of anti-MDA5-DM patients was 16.9% (46/272), and it was as high as 47.8% (44/92) for those with RP-ILD. Compared with ASS patients, anti-MDA5-DM patients had a significantly higher proportion of males, arthritis, Gottron's sign, heliotrope rash, V-sign, periungual erythema, and skin ulcers ( P<0.05). The levels of ALT, AST, and ferritin were significantly increased ( P<0.05). Compared with non-RP-ILD patients, RP-ILD patients had a significantly higher proportion of males [35.9%(33/92) vs. 23.3%(42/180), χ2=4.79, P=0.029], higher levels of LDH [387 (276, 547) U/L vs. 310 (245, 400) U/L, Z=-3.67, P<0.001], ESR [45.5 (29.25, 63.25) mm/1 h vs. 31.2 (20, 51) mm/1 h, Z=-3.71, P<0.001], CRP [10.9 (4.1, 25.2) mg/L vs. 4.54 (2.58, 9.08) mg/L, Z=-4.97, P<0.001], ferritin [1 340 (650, 2 000) ng/ml vs. 556 (203, 1 186) ng/ml, Z=-4.40, P<0.001], and a higher proportion of anti-Ro52 antibody and anti-MDA5 antibody co-positivity [87.0%(80/92) vs. 52.2%(94/180), χ2=31.87, P<0.001]. Conclusion:Anti-MDA5-DM patients are prone to develop RP-ILD and have poor prognosis.

Objective To search for new biomarkers to predict prognosis in colorectal cancer (CRC) patients.Methods A prognostic model was developed for colorectal cancer with immune-related genes from the cancer ge-nome atlas (TCGA) database using one-way Cox regression analysis and least absolute shrinkage and selection op-erator (LASSO) regression analysis.Moreover, the immune infiltration characteristics of patients in high and low risk groups was compared by sstimation of stromal and immune cells in malignant tumor tissues using expression da-ta (ESTIMATE) and cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT) .In addition, the expression levels of immune checkpoints were analyzed in patients from different risk groups.The sen-sitivity of patients in the two risk groups to chemotherapeutic agents was also compared based on genomics of drug sensitivity in cancer (GDSC).Results It was found that the prognostic model constructed based on immune genes could better predict the overall survival (OS) of CRC patients,and the results showed area under curve (AUC) values of 0.764 (95％ CI:0.751-0.793), 0.773 (95％ CI:0.761 -0.779), and 0.760 (95％ CI:0.742 -0.774) for 1-, 3-, and 5-year OS, respectively.Patients in the low-risk group had higher expression levels of im-mune checkpoints and more abundant immune cells such as T cells (P<0.001) , dendritic cells (P<0.001) , macrophages (P<0.001) , neutrophils (P<0.001) .Patients in the high-risk group might be more sensitive to some chemotherapeutic agents such as axitinib, imatinib, methotrexate, pazopanib, rapamycin, sunitinib and tasig-arnib.Conclusion A prognostic model based on 19 immune genes was effective in predicting the prognosis of CRC patients.The number and activity of immune cells in the immune microenvironment in different patients may be an important factor influencing their response to immunocheck inhibitors and chemotherapeutic agents.

Objective:To investigate the role of dendritic cells (DC) in Chlamydia muridarum ( Cm) respiratory infection and their effect on adaptive immune response. Methods:C57BL/6 mice were exposed to 1×10 3 inclusion-forming units (IFU) of Cm through inhalation to establish the mouse model of Cm respiratory infection. The proportion of CD11c + MHCⅡ + DC and the expression of costimulatory molecules (CD40, CD80 and CD86) in spleen tissues were detected by flow cytometry on 0, 3 and 7 d after infection. The expression of IL-12p40, IL-10 and IL-6 at mRNA level in spleen tissues was detected by qPCR. Mouse splenic DC isolated on 7 d after Cm infection were sorted by magnetic beads and then transferred to recipient mice. Th1 response in the recipient mice was measured using intracellular cytokine staining 14 d after infection. Results:Cm respiratory infection induced massive infiltration of DC and promoted the expression of costimulatory molecules on splenic DC. The expression of IL-12 and IL-10 at mRNA level in splenic DC reached the peak on 3 d after infection. Transferring the splenic DC of Cm-infected mice into the recipient mice could alleviate the disease condition in the recipient mice after Cm infection with reduced Cm inclusion-forming units in lung tissues and significantly increased proportion of Th1 cells in lung and spleen tissues. Conclusions:Cm respiratory infection could induce the maturation and activation of DC, which promoted Th1 immune response. DC played an important role in Cm infection.