Objective To investigate the anti-intrahepatic cholangiocarcinoma （ICC） activity and mechanism of 2-heptyl-3-hydroxy-4（1H）-quinolone （PQS）, a secondary metabolite of polar marine microorganisms. Methods Patient-derived organoid （PDO） models were established using intrahepatic cholangiocarcinoma （ICC） tumor tissues obtained from Renji Hospital, Shanghai Jiao Tong University School of Medicine. Hematoxylin and eosin （HE） staining was performed to assess the histomorphological characteristics of both patient ICC tissues and corresponding PDOs. Immunohistochemistry （IHC） was employed to evaluate CYP3a expression in patient ICC tissues and PDOs. The antiproliferative activity of PQS against stably passaged PDOs was determined using an adenosine triphosphate （ATP）-based bioluminescence assay, and dose-response curves were fitted to calculate the half-maximal inhibitory concentration （IC₅₀） for assessing the anti-ICC efficacy of PQS. In the human intrahepatic cholangiocarcinoma RBE cell model, the effects of PQS on RBE cell proliferation were evaluated by the cell counting kit-8 （CCK-8） assay; colony formation capacity was assessed by the plate colony formation assay; cell cycle distribution and apoptosis were analyzed by flow cytometry; and the protein expression levels of cyclin-dependent kinase 2（CDK2）, CDK4, RelA（p65）, and nuclear factor-κB1（p50） were detected by Western blotting. Results Two ICC PDO models were successfully established. Histomorphological observation revealed that the PDO tissues after serial passaging exhibited morphological features essentially consistent with the corresponding patient ICC tissues, both presenting as cystic vesicle-like structures. Immunohistochemical analysis demonstrated that CYP3a was expressed in both PDO tissues and patient ICC tissues. ATP-based bioluminescence assay results indicated that PQS effectively suppressed ATP content in PDO tissues, with a fitted dose-response curve yielding an IC₅₀ value of 2.49 µmol/L. In the RBE cell model, PQS inhibited RBE cell viability in a concentration-dependent manner, and the fitted dose-response curve yielded an IC₅₀ value of 1.05 µmol/L. Furthermore, PQS at concentrations of 1, 2, and 4 µmol/L significantly suppressed colony formation of RBE cells, arrested the cell cycle at the S phase, induced apoptosis, and downregulated the expression of proteins associated with the NF-κB signaling pathway. Conclusion ICC PDO models were successfully established, confirming the anti-ICC activity of PQS. PQS inhibited RBE cell proliferation, potentially via the NF-κB signaling pathway, by arresting the cell cycle at the S phase and inducing apoptosis of RBE cells

Objective To investigate the anti-intrahepatic cholangiocarcinoma （ICC） activity and mechanism of 2-heptyl-3-hydroxy-4（1H）-quinolone （PQS）, a secondary metabolite of polar marine microorganisms. Methods Patient-derived organoid （PDO） models were established using intrahepatic cholangiocarcinoma （ICC） tumor tissues obtained from Renji Hospital, Shanghai Jiao Tong University School of Medicine. Hematoxylin and eosin （HE） staining was performed to assess the histomorphological characteristics of both patient ICC tissues and corresponding PDOs. Immunohistochemistry （IHC） was employed to evaluate CYP3a expression in patient ICC tissues and PDOs. The antiproliferative activity of PQS against stably passaged PDOs was determined using an adenosine triphosphate （ATP）-based bioluminescence assay, and dose-response curves were fitted to calculate the half-maximal inhibitory concentration （IC₅₀） for assessing the anti-ICC efficacy of PQS. In the human intrahepatic cholangiocarcinoma RBE cell model, the effects of PQS on RBE cell proliferation were evaluated by the cell counting kit-8 （CCK-8） assay; colony formation capacity was assessed by the plate colony formation assay; cell cycle distribution and apoptosis were analyzed by flow cytometry; and the protein expression levels of cyclin-dependent kinase 2（CDK2）, CDK4, RelA（p65）, and nuclear factor-κB1（p50） were detected by Western blotting. Results Two ICC PDO models were successfully established. Histomorphological observation revealed that the PDO tissues after serial passaging exhibited morphological features essentially consistent with the corresponding patient ICC tissues, both presenting as cystic vesicle-like structures. Immunohistochemical analysis demonstrated that CYP3a was expressed in both PDO tissues and patient ICC tissues. ATP-based bioluminescence assay results indicated that PQS effectively suppressed ATP content in PDO tissues, with a fitted dose-response curve yielding an IC₅₀ value of 2.49 µmol/L. In the RBE cell model, PQS inhibited RBE cell viability in a concentration-dependent manner, and the fitted dose-response curve yielded an IC₅₀ value of 1.05 µmol/L. Furthermore, PQS at concentrations of 1, 2, and 4 µmol/L significantly suppressed colony formation of RBE cells, arrested the cell cycle at the S phase, induced apoptosis, and downregulated the expression of proteins associated with the NF-κB signaling pathway. Conclusion ICC PDO models were successfully established, confirming the anti-ICC activity of PQS. PQS inhibited RBE cell proliferation, potentially via the NF-κB signaling pathway, by arresting the cell cycle at the S phase and inducing apoptosis of RBE cells

As a hot spot in clinical research today, immune checkpoint inhibitor has been recommended by guidelines in the first- and second-line treatments of advanced cervical cancer as immune monotherapy or combination therapy. It has also achieved good efficacy in clinical practice. In locally advanced cervical cancer, immune checkpoint inhibitors have been included in the guidelines for adjuvant therapy, and good tumor regression effects have been achieved in clinical practice. Based on the results of existing trials, immune checkpoint inhibitors have also shown good clinical potential as neoadjuvant therapy. Furthermore, the issue of immunotherapy rechallenge has increasingly captured clinicians’ attention, offering a potential new therapeutic strategy for cervical cancer patients with prior immunotherapy exposure. In this article, the clinical application and research progress of immune checkpoint inhibitors in the treatment of cervical cancer in recent years are summarized to provide valuable ideas and directions for clinical treatment.

Bacitracin is a broad-spectrum cyclic peptide antibiotic, and mainly produced by Bacillus. Energy metabolism plays as a critical role in high-level production of target metabolites. In this study, Bacillus licheniformis DW2, an industrial strain for bacitracin production, was served as the original strain. First, our results confirmed that elimination of cytochrome bd oxidase branch via deleting gene cydB benefited bacitracin synthesis. Bacitracin titer and ATP content were increased by 10.97% and 22.96%, compared with those of original strain, respectively. Then, strengthening cytochrome aa3 oxidase branch via overexpressing gene qoxA was conducive to bacitracin production. Bacitracin titer and ATP content were increased by 18.97% and 34.00%, respectively. In addition, strengthening ADP synthesis supply is also proven as an effective strategy to promote intracellular ATP accumulation, overexpression of adenosine kinase DcK and adenylate kinase AdK could all improve bacitracin titers, among which, dck overexpression strain showed the better performance, and bacitracin titer was increased by 16.78%. Based on the above individual methods, a method of combining the deletion of gene cydB and overexpression of genes qoxA, dck were used to enhance ATP content of cells to 39.54 nmol/L, increased by 49.32% compared to original strain, and bacitracin titer produced by the final strain DW2-CQD (DW2ΔcydB::qoxA::dck) was 954.25 U/mL, increased by 21.66%. The bacitracin titer produced per cell was 2.11 U/CFU, increased by 11.05%. Collectively, this study demonstrates that improving ATP content was an efficient strategy to improve bacitracin production, and a promising strain B. licheniformis DW2-CQD was attained for industrial production of bacitracin.
Bacillus licheniformis ; metabolism ; Bacitracin ; biosynthesis ; Energy Metabolism ; genetics ; Industrial Microbiology ; methods

Objective To investigate the function of sorafenib on the growth of hepatocellular carcinoma by establishing subcutaneous transplantation tumor model with nude mice.To explore the effect of sorafenib on circadian clock gene expression in hepatoma cells.Methods Mouse tumor model was established by implanting hepatocarcinoma cell (HepG2) subcutaneously in Balb/C nude mice.Sixteen experimental mice were randomly divided into two groups:sorafenib treatment group (n =8) and solvent control group (n =8).The nude mice were treated with sorafenib (100 mg/kg) and DMSO daily by intragastric administration,respectively.The volume of tumors was recorded every 3 days.The expressions level of circadian clock genes (Per1,Per2,Per3,CLOCK,Cry1,Cry2,BMAL1 and CKIε) were detected by real-time polymerase chain reaction (Real-time PCR).The correlations between the size of the xenografts and the expressions of the circadian clock genes were further analyzed.Results Compared with the control group,the tumor size in the sorafenib treatment group were significantly smaller comparing with the control group.Results of Real-time PCR showed that the expression level of Per1,Cry1 and BMAL1 mRNA was remarkably decreased in the treatment group (Per1,P =0.02;Cry1,P =0.002;BMAL1,P =0.035),the differences were statistically significant.Correlation analysis showed that the size of subcutaneous transplantation tumorsin nude mice was negatively correlated with the expressions of Per1,Per2,Cry1 and Cry2 mRNA in control group.While,the size of subcutaneous transplantation tumors was negatively correlated with the expressions of Per2,Per3 and BMAL1 levels in the sorafenib treatment group.Conclusions There is a negative correlation between the expression levels of some biological clock genes and the size of transplantation tumor in nude mice.Sorafenib treatment significantly inhibited the growth of hepatocellular carcinoma in nude mice and down-regulation the expressions of Per1,Cry1 and BMAL1 mRNA in hepatoma cells.

Objective To investigate the influence of high-fat diet in the intestinal flora,fecal weight and its water content in rats, and to clarify the effect and significance of high-fat diet in the occurrence of obesity forming. Methods Twenty Sprague-Dawley (SD)rats were randomly divided into normal diet (ND)group and high-fat diet (HF)group (n=10).The rats in ND group were fed with normal diet,and the rats in HF group were fed with diet rich in oil and fat.The fresh feces were collected separately on days 1,15,30,and 49 for analysis of weight, water content,and intestinal flora.Results On the 49th day,the wet weight and water content of feces of the rats in ND group were (6.61 ± 0.17)g and (37.07 ± 3.04)%,respectively,while those in HF group were (4.46±0.30)g and (18.04±2.23)% (P<0.05).Compared with ND group ,the fecal pellets in HF group were increased obviously from the 7th day (P<0.05).There were obvious changes in intestinal microbial populations of HF group. The counts of enterococci, bifidobacteria, Lactobacillus, Bacteroides bacteria were significantly decreased on the day 49, but the count of Escherichia coli was increased significantly (P < 0.05 ). Conclusion High-fat diet can result in decrease for weight,water content,and pellets of feces;it can change the structure of intestinal flora. As result, there is a possibility that all of changes above can promote obesity in the future.

Objective To investigate the effect of knocking-down Yes-associated protein (YAP)on the biologi-cal characteristics of SNB19 glioblastoma cell.Methods The expression of YAB in SNB19 was knockdown by YAB small interfering RNA (YABsiRNA).The downregulation of YAP expression was identified by Western blot analysis. The proliferative ability of cell was determined by methyl thiazoyl terazolium (MTT).The invasive ability of cell was examined by Transwell assay.Flow cytometry and Annexin V staining were used to detect the cell cycle and apoptosis respectively.The results were analyzed by the statistical software SPSS18.0.Results The expression of YAP in the cells transfected with YAPsiRNA was significantly reduced.The cell proliferation activity of SNB19 cells was inhibited, which decreased from (100.00 ±0.00)%to (52.32 ±3.10)%(F=33.00,P<0.01).The cell cycle was arrested in G0-G1 phase (F=8.76,P<0.01).The cell invasive ability was attenuated apparently,which decreased from (163.20 ±10.10)to (37.71 ±2.52)(F=282.05,P<0.01).The apoptosis ratio of the tumor cell which transfected with YAPsi-RNA was increased from (3.56 ±0.35)%to (18.99 ±0.66)%,(F=931.99,P<0.01).Conclusion Knocking-down YAP expression in glioma cells could inhibit the proliferative activity and invasive ability of SNB19 cell and could induce cell apoptosis.YAP could be served as a potential target for the gene therapy of glioma.

Objective:This study aimed to explore the effect of Yes-associated protein (YAP) on the growth of the human glioma cell line LN229. Methods:YAPsiRNA was transfected into LN229 cells to knock down the YAP expression. The downregulation of the YAP expression was identified through Western Blot analysis. Colorimetric assay using methyl-thiazolyl-tetrazolium was applied to evaluate cell proliferation ability. Cell invasive activity was examined using Transwell assay. Flow cytometry and AnnexinV were used to detect cell cycle and apoptosis, respectively. The relevant molecules regulating proliferation, invasion, cell cycle progression, and apoptosis were examined through Western Blot analysis. Results:The YAP expression was downregulated after YAPsiRNA was trans-fected into LN229 glioma cells. Reduced YAP expression could arrest the cell cycle at G0/G1 phase, inhibit cell proliferation and inva-sion, and promote apoptosis. The expression of the proliferating cell nuclear antigen (Ki-67), matrix metallopeptidase-9 (MMP-9), cy-clin D1, and Bcl-2 were downregulated. Conclusion:The downregulation of YAP in LN229 cells suppresses cell proliferation and inva-sion, as well as promotes cell apoptosis. This study provides a novel evidence for further study on Hippo-YAP signal pathway in molec-ular pathology of glioma.

Objective To study the inhibitory effect of knocking down miR-30a-5p on the U87 human glioma xenograft growth and its possible mechanism.Methods Nude mice bearing subcutaneous U87 human glioblastoma were established and separated into three groups (eight for each group) by randomized digital table method,including control group,scr-ODN treated group and AS-miR-30a-5p treated group.After relevant subcutaneous injection treatment,tumor size was measured every other day until the observation period ended.Researchers executed the animals after the treatment,stripped tumor tissues and extracted RNA and protein.Real-time PCR was conducted to detect the expression of miR-30a-5p.The histopathological characteristics and proliferation and apoptosis biological characters (including SEPT7,PCNA,cyclin D1,MMP-2,apoptosis related factor P53,bcl-2 and caspase3) were evaluated by HE and immunohistochemical staining,Westem blot analysis respectively,and the cell apoptosis was detected by TUNEL method.Results In AS-miR-30a-5p treated group,the tumor growth was delayed and the final tumor volume was smaller than that in the control and scr-ODN treated group (F =7.167,P ＜0.05),and the expression of miR-30a-5p was knocked down.The expression of PCNA,cyclin D1 were significantly downregulated while P53,SEPT7 and caspase3 up-regulated.Apoptotic index was increased significantly.Conclusion As-miR-30a-5p suppresses the growth of U87 human gliomas xenografts significantly.Malignant phenotype of tumors are reversed to a considerable degree.Therefore,miR-30a-5p can be a candidate for targeted therapy of human glioma.

The miR-17-92 cluster,consisting of six individual microRNAs,including miR-17,miR-20a,miR-18a,miR-19a,miR-19b and miR-92a-1,is a kind of typical oncogenic polycistron microRNA.MiR-19 (containing miR-19a and miR-19b),as the key oncogenic component in miR-17-92 cluster,is overexpressed in human cancers including lymphomas,leukaemia,lung cancer,breast cancer and multiple myeloma,and has been one of research focus in recent years.It is found that miR-19 promotes tumor growth,invasion and metastasis through negative regulation of target genes such as PTEN,PP2A,Bim,SOCS1,and is correlated strongly with PI3K-AKT-mTOR pathway.MiR-19 plays an important role in tumor genesis and development.