To explore the effect of Mlk3 (mixed lineage kinase 3) deficiency on blood pressure, Mlk3 gene knockout (Mlk3KO) mice were generated. Activities of sgRNAs targeted Mlk3 gene were evaluated by T7 endonuclease I (T7E1) assay. CRISPR/Cas9 mRNA and sgRNA were obtained by in vitro transcription, microinjected into zygote, followed by transferring into a foster mother. Genotyping and DNA sequencing confirmed the deletion of Mlk3 gene. Real- time PCR (RT-PCR), Western blotting or immunofluorescence analysis showed that Mlk3KO mice had an undetectable expression of Mlk3 mRNA or Mlk3 protein. Mlk3KO mice exhibited an elevated systolic blood pressure compared with wild-type mice as measured by tail-cuff system. Immunohistochemistry and Western blotting analysis showed that the phosphorylation of MLC (myosin light chain) was significantly increased in aorta isolated from Mlk3KO mice. Together, Mlk3KO mice was successfully generated by CRISPR/Cas9 system. MLK3 functions in maintaining blood pressure homeostasis by regulating MLC phosphorylation. This study provides an animal model for exploring the mechanism by which Mlk3 protects against the development of hypertension and hypertensive cardiovascular remodeling.
Animals ; Mice ; Mice, Knockout ; CRISPR-Cas Systems ; Blood Pressure ; Gene Knockout Techniques ; Zygote

Bladder cancer is the most common malignancy of the urinary system. Compound Kushen Injection (CKI) is a Chinese medicinal preparation that has been widely used in the treatment of various types of cancers in the past two decades. However, the pharmacological effect of CKI on bladder cancer is not still completely understood. In the current study, network pharmacology combined with bioinformatics was used to elucidate the therapeutic mechanism and potential targets of CKI in bladder cancer. The mechanism by which CKI was effective against bladder cancer was further verified in vitro using human bladder cancer cell line T24. Network pharmacology analysis identified 35 active compounds and 268 target genes of CKI. Bioinformatics data indicated 5500 differentially expressed genes associated with bladder cancer. Common genes of CKI and bladder cancer suggested that CKI exerted anti-bladder cancer effects by regulating genes such as MMP-9, JUN, EGFR, and ERK1. Functional enrichment analysis indicated that CKI exerted therapeutic effects on bladder cancer by regulating certain biological processes, including cell proliferation, cell migration, and cell apoptosis. In addition, Kyoto Encyclopedia of Genes and Genomes enrichment analysis implicated pathways related to cancer, bladder cancer, and the PI3K-Akt signaling pathway. Consistently, cell experiments indicated that CKI inhibited the proliferation and migration of T24 cells, and induced their apoptosis. Moreover, RT-qPCR and Western blot results demonstrated that CKI was likely to treat bladder cancer by down-regulating the gene and protein expression of MMP-9, JUN, EGFR, and ERK1. CKI inhibited the proliferation and migration, and induced the apoptosis of T24 bladder cancer cells through multiple biological pathways and targets. CKI also exhibited significant effects on the regulation of key genes and proteins associated with bladder cancer. Overall, our findings provide solid evidence and deepen current understanding of the therapeutic effects of CKI for bladder cancer, and further support its clinical use.
Computational Biology ; Drugs, Chinese Herbal ; Humans ; Network Pharmacology ; Phosphatidylinositol 3-Kinases ; Urinary Bladder Neoplasms/genetics*

Objective:To explore the changes of brain activity in drug-resistant or drug-controlled medial temporal lobe epilepsy patients by the method of functional connectivity density (FCD), and to analyze their correlation with the course of the disease.Methods:According to the definition of drug-resistant epilepsy by the International League Against Epilepsy in 2010, 146 patients with medial temporal lobe epilepsy who were clearly diagnosed as unilateral hippocampal sclerosis in Jinling Hospital, Nanjing University School of Medicine from July 2009 to February 2019 were divided into drug control group ( n=73) and drug-resistant group ( n=73). The 3.0 T resting state functional magnetic resonance scan was performed on all subjects to compare the difference in FCD between the two groups, and calculate the correlation between the FCD value of the brain area and the course of the disease between the two groups of patients. Results:There was significant difference between the two groups in FCD. Compared with the drug control group, the drug-resistant group had significantly lower FCD values in the insula, lenticular nucleus, thalamus, hippocampus and precentral gyrus on the side of the epileptogenic focus. The FCD value of the precuneus on the side of the epileptogenic focus in the drug-resistant group was negatively correlated with the duration ( r=-0.30, P=0.01). Conclusions:The FCD of patients with drug-resistant medial temporal lobe epilepsy was lower than that of the drug control group. In addition, there may be progressive damage to the brain. The difference is helpful for exploring the pathophysiological mechanisms related to drug resistance in patients with medial temporal lobe epilepsy, and finding reliable neuroimaging markers related to drug resistance.

Objective To investigate the expression of human leucocyte antigen G (HLA-G) in urothelial carcinoma after renal transplantation,and to analyse the relationship between HLA-G expression and the various clinical and pathological parameters.Methods 29 patients with urothelium carcinoma after renal transplantation for the first time from January 2005 to June 2016 were selected as the experimental group,the age range was 32-70 years,with an average of (55.5 ± 8.1) years.29 non-transplanted patients with urothelial carcinoma as the control group 1,the age range was 36-74 years,with an average of (57.9 ± 8.2) years.15 cases of normal urinary tract epithelial were from cystoscopy biopsy as the control group 2.Immunohistochemical method was used to detect the difference of HLA-G expression between the three groups.The clinical and pathological data of patients with urothelial carcinoma after renal transplantation were analyzed.Results The expression rate of HLA-G was 79.3％ (23/32) in patients with urothelial carcinoma after renal transplantation,37.9％ (11/32) in non-transplanted group and 0 (0/15) in normal urinary tract epithelium group.The expression rate of HLA-G in non-transplanted group was significantly higher than that in normal urinary tract epithelium group (P ＜ 0.05).The expression rate of HLA-G in patients with urothelial carcinoma after renal transplantation was significantly higher than that in nontransplanted group and normal urinary tract epithelium group (P ＜ 0.05).Conclusions HLA-G is associated with the occurrence of urothelial carcinoma after renal transplantation.It may provide a new idea for the prevention and treatment of urinary tract epithelium after renal transplantation.

Objective To investigate the diagnostic value of calcification and cystic lesion of CT findings in differentiating pancreatic head ductal carcinoma (PHDA)from mass-forming chronic pancreatitis (MFCP)of the pancreatic head.Methods The clinic data and CT findings of 30 cases with PHDA and 24 cases with MFCP of the pancreatic head,which were confirmed by surgery and pathology were analyzed retrospectively.The images were reviewed independently by two expert radiologists with a double-blind method.An independent sample t test and chi-square test were used to compare the data of imaging findings between two groups.Results ① Calcification was found in 14 cases (58.33%)with MFCP and in 3 cases (10%)with PHDA (P＜0.001).The percentage of patchy,punctate and mixed calcification were 28.57% (n=4),14.29% (n=2)and 57.14% (n=8)in MFCP,0% (n=0),66.67% (n=2)and 33.33% (n=1) in PHDA,respectively.② Necrotic cyst was founded in 7 cases (29.17%)with MFCP and 18 cases (60%)with PHDA(P＜0.05). Pseudocysts were demonstrated in 14 cases (58.33%)with MFCP and in 3 cases (10%)with PHDA (P＜0.001).Honeycombed change with tension within or around the lesion were demonstrated only in patients with MFCP.In addition,normal tissue of the pancreas was found within the lesion in 11 cases (45.83%)of MFCP and none in PHDA,which showed significant difference between two groups.Conclusion Mixed calcification and honeycomb with tension of CT findings are of significant value in differentiating PHDA from MFCP of the pancreatic head.

Objective At present, there is no study on effect of levetiracetam(LEV) on the gray matter structure remodeling in benign epilepsy children with central temporal spikes(BECTS).The purpose of this study was to study the influence of LEV on the gray matter structure in BECTS and to evaluate the mechanism of LEV on the brain structure of BECTS through using voxel-based MRI morphological(VBM) methods.Methods From January 2014 to September 2016, twenty-four BECTS treated with LEV(LEV group), twenty-four drug-na?ve BECTS(untreated group) and twenty-four normal children(normal group) consulted in department of Neurology, Nanjing Children′s Hospital and the Nanjing Military Region, Nanjing General Hospital were continuously included to receive three-dimensional T1-weighted imaging with 3T MRI and the gray matter volume was calculated by VBM.We compared the difference of grey matter volumes of the three groups and analyzed their correlation with epilepsy duration, age of onset and medication time and other clinical index.Results Compared with the normal group, the grey matter volume of bilateral thalamus were decreased, and the volume of bilateral Rolandic areas, anterior insula/frontal operculum/frontal triangle, left supplementary motor area, paracentral lobule, precentral gyrus, superior frontal gyrus and right middle frontal gyrus were increased in the untreated group, but the grey matter volume of the bilateral Rolandic areas, frontal operculum and left supplementary motor area were decreased in the LEV group.Compared with the untreated group, the grey matter volume of bilateral supplementary motor, left paracentral lobule, precentral gyrus, bilateral anterior insula/frontal operculum/frontal triangle, left superior frontal gyrus and right middle frontal gyrus in the LEV group were decreased.The grey matter volume of left anterior insula/frontal operculum areas was negatively correlated with the medication time in LEV group(r=-0.527, P<0.01).Conclusion T The mainly representations of BECTS are thalamic gray matter damage and epileptic-related cortical area irritation structural abnormalities, but the LEV could reshape the epilepsy-related cortical area and the gray matter in the brain area associated with clinical symptoms.

Background and objective Radiotherapy combined with chemotherapy or molecular targeted therapy remains the standard of treatment for brain metastases from non-small cell lung cancer (NSCLC). hTe aim of this study is to determine if the deferral of brain radiotherapy impacts patient outcomes.Methods Between May 2003 and December 2015, a total of 198 patients with brain metastases from NSCLC who received both brain radiotherapy and systemic therapy (chemo-therapy or targeted therapy) were identiifed. hTe rate of grade 3-4 adverse reactions related to chemotherapy and radiotherapy had no signiifcant difference between two groups. 127 patients received concurrent brain radiotherapy and systemic therapy, and 71 patients received deferred brain radiotherapy after at least two cycles of chemotherapy or targeted therapy. Disease speciifc-graded prognostic assessment was similar in early radiotherapy group and deferred radiotherapy group.Results Me-dian overall survival (OS) was longer in early radiotherapy group compared to deferred radiotherapy group (17.9 monthsvs 12.6 months;P=0.038). Progression free survival (PFS) was also improved in patients receiving early radiotherapy compared to those receiving deferred radiotherapy (4.0 monthsvs 3.0 months;P<0.01). Receiving tyrosine kinase inhibitor (TKI) therapy atfer the diagnosis of brain metastases as any line therapy improved the OS (20.0 monthsvs 10.7 months;P<0.01), whereas receiving TKI as ifrst line therapy did not (17.9 monthsvs 15.2 months;P=0.289).Conclusion Our study suggests that the use of deferred brain radiotherapy may resulted in inferior OS in patients with NSCLC who develop brain metastases. A prospec-tive multi-central randomized study is imminently needed.

Objective To explore the clinical efficacy of laparoscopic left hemihepatectomy for the treatment of intrahepatic bile duct stones.Methods The clinical data of 30 patients with left intrahepatic bile duct stones who were admitted to the Second Affiliated Hospital of Nancbang University from June 2013 to June 2014 were retrospectively analyzed.All the patients underwent laparoscopic left hemihepatectomy by the Glisson intra-and extra-pedicles vascular inflow occlusion techniques together with the removal of choledocholithiasis and right bile duct stones,and T tube placement or laparoscopic primary suture of common bile duct were selected according to the condition of bile duct.All the 30 patients were readmitted to hospital and detected by color Doppler ultrasound (CDUS),computed tomography (CT) and T tube cholangiography at postoperative month 1,and then received CDUS reexamination every 3 months.CT and MRI reexaminations were applied to patients with complication of residual stones if necessary.All the patients were followed up till July 2014.Results All the 30 patients were treated by laparoscopic hepatectomy with left hemihepatic vascular inflow occlusion,including 5 with conversion to open surgery and 25 with successful operation.The Glisson extra-and intra-pedicel vascular inflow occlusion techniques were used in 11 and 14 patients,respectively.The operation time and volume of blood loss were (158 ± 85) minutes and (405 ± 215) mL.Two patients received intraoperative blood transfusion.There were no residual stones in the 8 patients with choledocholithiasis by intraoperative choledochoscope,and primary suture of bile duct and T tube placement were done in 5 and 3 patients,respectively.No patients died.After operation,there were 2 patients with bile leakage and 1 with pleural effusion,and they were cured though drainage.One patient with subphrenic effusion was cured by B ultrasound-guided puncture and drainage.One patient had bleeding with the volume of blood loss of 500 mL,and was cured by conservative treatment.The duration of hospital stay in all the patients was (8.5 ± 2.3)days.No bile leakage and abdomen infection were detected by outpatient examination.The time of followup was 1-12 months,without recurrence of stones.Conclusion Laparoscopic left hemihepatectomy for the treatment of left intrabepatic bile duct stones is safe and feasible with satisfactory outcome.

Background and objective Chemotherapy is a highly effcient primary treatment for extensive-stage small cell lung cancer (ES-SCLC). However, patients receiving such treatment are prone to develop drug resistance. Local treatment is palliative and thus can alleviate the local symptoms and improve quality of life, but limited evidence is available for prolonging survival. Hence, this study evaluated the role of local treatment in chemotherapy of patients with ES-SCLC. Methods A total of 302 ES-SCLC cases were enrolled in this retrospective study. Prognostic factors were analyzed by Kaplan-Meier and Cox multivariate proportional hazards model. Results Median progression-free survival (PFS) and median survival time (MST) of the patients were 4.4 and 10.4 months, respectively. 1-, 2-, and 3-year survival rates were 37.8%, 10.2%and 4.4%, correspondingly. hTe MST of the primary tumor radiotherapy plus chemotherapy group was 14.3 months, whereas that of the chemotherapy group was 8.2 months (P<0.01). hTe MSTs of multiple-site, single-site, and non-metastasis local treatments were 18.7, 12.3 and 8.9 months, respectively (P<0.01). hTe MSTs of initiative, passive, and non-metastasis local treatments were 16.0, 10.9 and 9.4 months, correspondingly (P<0.01). hTe MSTs of patients with prophylactic cranial irradiation (PCI) and those without PCI were 19.8 and 9.9 months, respectively (P<0.01). Primary tumor radiotherapy, metastasis local treat-ment, and PCI were independent prognostic factors for ES-SCLC. Conclusion Primary tumor radiotherapy, metastasis local treatment, and PCI can signiifcantly improve survival in patients with ES-SCLC.

Background and objective Lung cancer is currently the leading cause of cancer death, two thirds of patients are over the age of 65. Small cell lung cancer (SCLC) accounts for about15%-20%of all lung cancer. hTe objective of this study is to evaluate the survival of patients older than 65 with SCLC and analyze the independent prognostic factors in this group of patients. Methods A retrospective study has enrolled160 cases of lung cancer aged over 65. hTe prognostic factors were analyzed by Kaplan-Meier and Cox multivariate proportional hazards model. Results ①hTe median follow-up time was12 (2-109) months.1-, 3-, and 5-year survival rate was 47.1%,13.0%, 9.6%respectively, and 74.4%, 25.0%,19.7%for limited-stage (LD), and 36.8%, 8.7%, 5.8%for extensive-stage (ED). Median survival time (MST) of all the patients was12 months, 24 months for LD and11months for ED, respectively.②Multivariate analysis suggested that performance status (PS) pre-treatment, the change of PS atfer treatment, stage, liver metastases and thoracic radiotherapy were the independent prognostic factors in all patients.③For LD-SCLC patients, PS pre-treatment, thoracic radiotherapy were the independent prognostic fac-tors. hTe model of thoracic radiotherapy (concurrent chemoradiation vs sequential chemoradiation, early concurrent chemo-radiation vs late concurrent chemoradiation) and prophylactic cranial irradiation (PCI) did not show signiifcant difference.④For ED-SCLC patients, sex, the change of PS atfer treatment, chemotherapy, liver metastases, thoracic radiotherapy, PCI were the independent prognostic factors. Conclusion hTe survival time is related to PS and thoracic radiotherapy in eldly patients. Besides, it is also related to sex, chemotherapy, liver metastases and PCI for ED-SCLC.