Objective: To explore the effects of Cldn14 gene knockout on renal metabolism and stone formation in rats，so as to provide reference for research in the field of urinary calium metabolism and stone formation. Methods: Cldn14 gene knockout homozygous rats and wild-type rats of the same age were randomly divided into 4 groups：wild-type control (WC) group，wild-type ethylene glycol (WE) group，gene knockout control (KC) group and gene knockout ethylene glycol (KE) group，with 10 rats in each group.The WE and KE groups were induced with ethylene glycol + ammonium chloride to form kidney stones，while the WC and KC groups received normal saline gavage.After 4 weeks of standard maintenance feeding，the urine samples were collected to detect the venous blood.The kidneys were collected for HE，Pizzolatto's staining and transmission electron microscopy.The protein in renal tissues was extracted to detect the expressions of Claudin16 and Claudin19. Results: Crystal deposition was observed in the renal tubular lumen of the WE and the KE groups，and more crystals were detected in the KE group.The WE group had a large number of intracytoplasmic black crystalline inclusions observed in renal tubular epithelial cells under transmission electron microscope，followed by the KE and KC groups.Compared with WC and WE groups，KC and KE groups had significantly decreased serum calcium and magnesium levels but significantly increased urinary calcium level.In addition，the urinary calcium level was higher in the WE group than in the WC group and higher in the KE group than in the KC group.The KE group had lower level of Claudin16，but there was no significant difference in the level of Claudin19 among the 4 groups(P>0.05). Conclusion: Knockout of Cldn14 gene alone cannot effectively reduce urinary calcium excretion or reduce the risk of stone formation in rats，which may be related to the decrease of Claudin16 level.

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) functions as a key regulator in inflammation and cell death and is involved in mediating a variety of inflammatory or degenerative diseases. A number of allosteric RIPK1 inhibitors (RIPK1i) have been developed, and some of them have already advanced into clinical evaluation. Recently, selective RIPK1i that interact with both the allosteric pocket and the ATP-binding site of RIPK1 have started to emerge. Here, we report the rational development of a new series of type-II RIPK1i based on the rediscovery of a reported but mechanistically atypical RIPK3i. We also describe the structure-guided lead optimization of a potent, selective, and orally bioavailable RIPK1i, 62, which exhibits extraordinary efficacies in mouse models of acute or chronic inflammatory diseases. Collectively, 62 provides a useful tool for evaluating RIPK1 in animal disease models and a promising lead for further drug development.

BACKGROUND:C2 ceramide reduces the formation of Alpha-Synuclein(α-Syn)oligomers as the protein phosphatase 2A agonist,which has an important regulatory effect on cell aging in the central nervous system. OBJECTIVE:To investigate the protective mechanism of C2 ceramide on dopaminergic neurons. METHODS:Twenty-five C57BL/6 mice were randomly divided into control group,model group,C2 ceramide low-,medium-and high-dose groups(n=5 per group).Except for the control group,a mouse model of Parkinson's disease was established by injecting mutant A53T α-Syn oligomers into the left striatum in the other groups.On the 30th day after the striatal injection,three C2 ceramide groups were intragastrically administered with C2 ceramide(1,5,10 μg/g)dissolved in saline at one time,while the control and model groups were administered with the same amount of saline within 30-90 days after modeling,for a total of 60 days.Behavioral changes in each group of mice were observed during this period.On the 90th day after striatal injection,mouse brain tissue was extracted by perfusion under anesthesia,and the changes of dopaminergic neurons in the midbrain substantia nigra were analyzed by immunohistochemical staining.The levels of α-Syn oligomerization and phosphorylation in the midbrain of mice were detected by ELISA,and the changes of enzyme activities related to α-Syn phosphorylation were analyzed. RESULTS AND CONCLUSION:C2 ceramide had an ameliorating effect on Parkinson's disease-like dyskinesia in mice caused by the striatal injection of mutant A53T α-Syn oligomers.High-dose C2 ceramide showed better effects on dyskinesia in mice with Parkinson's disease(P<0.01).The mutant A53T α-Syn oligomers significantly reduced the number of dopaminergic neurons in the substantia nigra of mice(P<0.01),while the number of dopaminergic neurons in the substantia nigra increased significantly in the C2 ceramide high-dose group(P<0.01).The levels of α-Syn oligomers and phosphorylated α-Syn in the brain were significantly reduced in the C2 ceramide high-dose group compared with the model group(P<0.01),while the level of ceramide was increased(P<0.05)and the activity of protein phosphatase 2A was significantly upregulated(P<0.01).To conclude,C2 ceramide can attenuate the neurotoxic effects induced by oligomerized α-Syn by the phosphorylation modification environment of α-Syn in mouse midbrain tissue and protect against the reduction in the number of nigrostriatal dopaminergic neurons in mice,thereby reducing the degree of dyskinesia in Parkinson's disease.

BACKGROUND: The application of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) antibodies has greatly improved the clinical outcomes of lung cancer patients. Here, we retrospectively analyzed the efficacy of PD-1 antibody therapy in locally advanced non-surgical or metastatic lung cancer patients, and preliminarily explored the correlation between peripheral blood biomarkers and clinical responses. METHODS: We conducted a single center study that included 61 IIIA-IV lung cancer patients who received PD-1 antibody treatment from March 2020 to December 2021, and collected the medical record data on PD-1 antibody first-line or second-line treatment. The levels of multiple Th1 and Th2 cytokines in the patient's peripheral blood serum, as well as the phenotype of peripheral blood T cells, were detected and analyzed. RESULTS: All the patients completed at least 2 cycles of PD-1 monoclonal antibody treatment. Among them, 42 patients (68.9%) achieved partial response (PR); 7 patients (11.5%) had stable disease (SD); and 12 patients (19.7%) had progressive disease (PD). The levels of peripheral blood interferon gamma (IFN-γ) (P=0.023), tumor necrosis factor α (TNF-α) (P=0.007) and interleukin 5 (IL-5) (P=0.002) before treatment were higher in patients of the disease control rate (DCR) (PR+SD) group than in the PD group. In addition, the decrease in absolute peripheral blood lymphocyte count after PD-1 antibody treatment was associated with disease progression (P=0.023). Moreover, the levels of IL-5 (P=0.0027) and IL-10 (P=0.0208) in the blood serum after immunotherapy were significantly increased compared to baseline. CONCLUSIONS Peripheral blood serum IFN-γ, TNF-α and IL-5 in lung cancer patients have certain roles in predicting the clinical efficacy of anti-PD-1 therapy. The decrease in absolute peripheral blood lymphocyte count in lung cancer patients is related to disease progression, but large-scale prospective studies are needed to further elucidate the value of these biomarkers.
Humans ; Lung Neoplasms/metabolism* ; Interleukin-5/therapeutic use* ; Tumor Necrosis Factor-alpha/therapeutic use* ; Retrospective Studies ; Programmed Cell Death 1 Receptor ; Biomarkers ; Immunotherapy ; Disease Progression ; B7-H1 Antigen

OBJECTIVE To evaluate the quality and existing problems of Compound Paracetamol and Chlorphenamine Tablets for Infant. METHODS Using legal standards to inspect sampled samples, and several analytical methods were subsequently established or improved for the exploratory research on related substances, assay, dissolution, subdivision characteristics of scored tablets, and genotoxic impurities. RESULTS All samples met the regulatory specification, and the pass rate was 100%. However, the control for related substances was lacking, and the method for assay and content uniformity was defective. The exploratory studies showed that the assay and content uniformity met the requirements, and the risk of related substances and genotoxic impurities was acceptable. As a divisible tablet, the subdivision characteristics could not meet the requirements of the scored tablet, and the dissolution performance of some enterprise samples could not meet the requirements of similar products in foreign pharmacopoeias. CONCLUSION The overall quality of this product is adequate. However, due to the early time on the market, some quality attributes no longer meet the requirements of current regulations or relevant guidelines. The manufacturers should further optimize the prescription and production process, referring to foreign similar products. The current specification needs to be revised and improved.

Objective:To analyze the survival efficacy, prognostic factors and failure patterns of patients with esophageal squamous cell carcinoma (ESCC) underwent postoperative radiotherapy (PORT) using modified clinical target volume (CTV) based on postoperative high-frequency recurrence regions, so as to provide reference for the further optimization of CTV of PORT.Methods:The patients with ESCC underwent radical operation in Comprehensive Cancer Center of Nanjing Drum Tower Hospital, Affiliated Hospital of Nanjing University Medical School from April 28, 2014 to November 29, 2018 were retrospectively analyzed. Patients with stage pT 3-4aN 0 or N +, who underwent PORT with modified CTV based on postoperative high-frequency recurrence regions, were included in the study. Kaplan-Meier method was used to calculate overall survival (OS) and locoregional recurrence free survival (LRFS) , adverse events of patients were evaluated, Cox proportional hazards model was used for univariate and multivariate survival analysis, and the failure patterns of patients after PORT were analyzed. Results:A total of 85 patients were included in this study, and the median follow-up time was 52.6 months. The median OS of the whole group was 74.1 months. The 1-year, 2-year and 3-year OS rates were 97.6%, 84.7% and 71.7% respectively. The median LRFS was not reached, and the 1-year, 2-year and 3-year LRFS rates were 92.9%, 78.6% and 71.5% respectively. The incidence of grade 3-4 adverse events was 17.6% (15/85) , mainly including lymphopenia, bone marrow suppression, gastrointestinal reaction and skin reaction. Univariate analysis of OS after PORT showed that the degree of differentiation (set G1+G1-2+G2 group as the control group, G2-3+G3 group HR=4.19, 95% CI: 1.91-9.17, P<0.001; NA+basal-like group HR=4.16, 95% CI: 1.29-13.44, P=0.017) and postoperative stage ( HR=2.19, 95% CI: 1.09-4.39, P=0.030) were the influencing factors of OS. Cox multivariate analysis showed that the degree of differentiation was an independent prognostic factor for OS after PORT (set G1+G1-2+G2 group as the control group, G2-3+G3 group HR=5.24, 95% CI: 2.30-11.93, P<0.001; NA+basal-like group HR=4.83, 95% CI: 1.33-17.62, P=0.017) . The first failure patterns analysis showed that 39 cases (45.9%) had recurrence, among which, 22 cases (25.9%) had locoregional recurrence with the median onset time of 15.2 months after operation, 19 cases (22.4%) had distant metastasis with the median onset time was 14.1 months after operation, and 2 cases (2.4%) were mixed failure mode. Among the locoregional recurrence, 16 cases (72.7%) recurred in the radiation field. Among all the local recurrence sites, the lymph node drainage regions in the supraclavicular, upper middle mediastinum and upper abdominal perigastric/celiac artery trunk areas were the most common sites. Among the distant metastatic organs, lung, bone and liver metastases were the most common. Conclusion:Patients of ESCC with high risk of recurrence after radical esophagectomy have long survival time and high safety after PORT with modified CTV according to the high-frequency recurrence regions. It is worthy of further confirmation by multicenter, large sample and prospective clinical trials.

Objective To retrospectively evaluate the influence of prostate volume on prostate cancer detection using elastography targeted transperineal biopsy. Methods A total of 573 consecutive patients suspicious for prostate cancer were enrolled in this study. Patients underwent combined elastography-targeted biopsy and 10 core-systematic transperineal biopsy.In correlation with prostate biopsy pathology, the sensitivity of elastography-targeted biopsy and systematic biopsy were compared among four subgroups with different prostate volume.Results The overall prostate cancer detection rate was 42.9% (246/573). The increase in cancer detection rate by elastography-targeted biopsies was 9.1% (52/573).In patients with prostate volume of ≤30 ml,30-50 ml,50 -80 ml and >80 ml,the sensitivity of elastography targeted biopsy were 91.1% (72/79),81.3% (87/107),70.5% (31/44) and 50.0% (8/16),respectively ( P =0.000).The sensitivity of systematic biopsy were 77.2% (61/79),77.6% (83/107),86.4% (38/44) and 75.0% (12/16),respectively,in comparison among these four groups ( P = 0.601). For patients with prostate volume ≤30 ml,the sensitivity of elastography targeted biopsy was significantly higher than that of systematic biopsy (P= 0.028). Conclusions Prostate cancer detection rate can be improved by elastography targeted biopsy. Prostate volume is correlated with the accuracy of elastography. The sensitivity of elastography targeted biopsy is higher in patients with a smaller prostate gland.

Objective To establish a novel method using a nano microfluidic chip to detect circulating tumor cells (CTCs) in peripheral blood in gallbladder carcinoma,and to study the relationship between CTCs with clinicopathology and prognosis in these patients.Methods The peripheral blood samples of 51 patients with gallbladder carcinoma were collected from June 2014 to January 2017.The CTCs from peripheral blood samples were detected by a novel nano microfluidic chip.This study aimed to study the correlation between CTCs with the clinical and pathological features.The significance of CTCs on prognosis in patients with gallbladder carcinoma was also analyzed.Results The positive rate of CTCs in the peripheral blood of gallbladder carcinoma patients was 43.1％ (22/51).There were significant correlations between CTCs with liver metastasis (P ＜ 0.05) and Nevin staging (P ＜ 0.05).The 1-and 2-year overall survival (OS) in patients with CTCs were 70.7％ and 35.3％,and the 1-and 2-year OS in patients without CTCs were 92.0％ and 56.1％.There was a significant difference in the 2-year OS (P ＜ 0.05) but no significant difference in the 1-year OS between the 2 groups of patients (P ＞ 0.05).Conclusions Peripheral blood CTCs in patients with gallbladder carcinoma were efficaciously detected by a novel nano microfluidic chip.Peripheral blood CTCs was closely related to the Nevin staging and liver metastasis.CTCs could serve as a potential prognostic indicator in patients with gallbladder carcinoma.

Objective To detect circulating tumor cells (CTCs) in the peripheral blood of patients with pancreatic cancer using a new nano microfluidic chip and to explore the relationship between CTCs and clinicopathological feature,postoperative recurrence and prognosis of pancreatic cancer.Methods The peripheral blood samples of 53 patients with pancreatic cancer who underwent curative resection in the second affiliated hospital of Jiaxing college of medicine were collected from January 2015 to January 2017.The CTCs from peripheral blood were detected by novel nano microfluidic chip.The cut-off value for CTCs-positive and negative groups was 1 CTC.The relationship between CTCs positive and postoperative recurrence and prognosis of pancreatic cancer were evaluated.Results The number of CTCs for 23 pancreatic cancer of 53 patients ranged from 5 to 196 per ml,and the mean number was 78.5 ± 44.7 per ml;the rate of CTCs-positive patients was 43.4％ (23/53).There were significant correlation between CTCs with vascular invasion (P =0.001),but but CTCs was not correlated with the gender,age,the presence of clinical symptoms,tumor size,pathological type,lymph metastasis and TNM stage.31 patients had tumor recurrence,and the rate of tumor recurrence was 58.5％.Among them,there were 13 cases with tumor local recurrence,10 cases with tumor distant metastasis (including liver,lung,kidney,etc.) and 8 cases with both tumor local recurrence and distant metastasis.The median recurrence free survival time of all patients was 14.0 months (13.0-17.0) and the median overall survival time was 19.0 months (15.5-24.0).The cumulative one-year and two-year recurrence free survival rate were 66.9％,12.2％ for patients with CTCs-positive and 88.3％,42.2％ for CTCs-negative patients,and the differences were statistically significant (both P ＜ 0.05).The cumulative one-year and two-year overall survival rate were 85.4％,33.6％ for patients with CTCs-positive and 96.3％,62.2％ for CTCs-negative.There was no difference in statistics in cumulative one-year overall survival rate and with a statistically significant difference in cumulative two-year overall survival rate (P =0.028).Conclusions Peripheral blood CTCs of pancreatic cancer can be effectively detected by a novel nano microfluidic chip.There were significant correlation between CTCs with vascular invasion and survival time after surgery.CTCs may be a potential prognostic indicator of the postoperative recurrence and prognosis of pancreatic cancer.

Objective To explore the diagnostic value of k ras mutation detection of specimen obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA)for pancreatic cancer.Methods Seventy-eight patients with pancreatic carcinoma and 49 patients with pancreatic benign diseases were collected from January 2013 to December 2015.All the patients underwent EUS-FNA and cell or tissue samples were collected.DNA was extracted from the samples,and the codon 12 and 13 mutation of K-ras gene was detected by specific nano capture probe.Liquid cytology was also conducted.The sensitivity and specificity of the two methods were compared.Results The K-ras mutation rate was 92.3％ (72/78) in 78 patients with pancreatic carcinoma,which was obviously higher than the mutation rate of 20.4％ (10/49) in 49 patients with pancreatic benign tumors,and the difference was statistically significant (x2 =68.002,P =0.000).The sensitivity of the cytology examination of EUS-FNA specimens in the diagnosis of pancreatic carcinoma was 75.6％,the specificity was 87.8％,Youden index was 0.634,the positive and negative predictive value was 6.196 and 0.227.The detection of K-ras mutations had a sensitivity of 92.3％ and a specificity of 79.6％ in the diagnosis of pancreatic carcinoma,the Youden index was 0.719,the positive and negative predictive value was 4.524 and 0.096.K-ras mutation detection had a higher sensitivity compared with cytology,and the difference was statistically significant (x2 =8.47,P =0.004).The sensitivity was 94.9％ and specificity of 95.9％ using the combination of K-ras mutations and cytology for diagnosing pancreatic cancer,and the specificity was obviously increased compared with only k ras mutation detection and the difference was statistically significant (x2 =6.13,P =0.013).Conclusions K-ras mutation detection of EUS-FNA specimen using nano capture probe system can significantly improve the sensitivity of diagnosing pancreatic cancer,and the sensitivity and specificity could be further improved when combined with cytology.