To analyze the distribution of serotypes and antimicrobial resistance of clinical Salmonella isolates from multiple centers across China.

This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.
Animals ; Smad3 Protein/genetics* ; CRISPR-Cas Systems/genetics* ; Mice ; Podocytes/metabolism* ; Transforming Growth Factor beta1/pharmacology* ; Cell Line ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics*

Objective:To explore the predictive value of uterine artery ultrasound parameters combined with four coagulation indicators test for placental abruption in late pregnancy.Methods:A total of 160 pregnant women with placental abruption during the late pregnancy who were diagnosed and treated in our hospital from Jan. 2021 to Dec. 2022 were collected as the observation group, and 160 pregnant women with normal delivery were regarded as the control group. According to different grading of placental abruption, 65 cases were classified as grade I, 54 cases as grade II, and 41 cases as grade III. Ultrasound parameters of uterine artery [uterine artery pulsatility index (PI), resistance index (RI), ratio of maximum systolic blood flow velocity (S) to maximum diastolic blood flow velocity (D) (S/D) ], and four coagulation parameters [prothrombin time (PT), fibrinogen (FIB), thrombin time (TT), and activated partial thromboplastin time (APTT) ] were compared; Logistic regression analysis was conducted to analyze the factors affecting placental abruption in late pregnancy; receiver operating characteristic (ROC) curve was applied to analyze the predictive value of uterine artery ultrasound parameters combined with four coagulation indicators test for placental abruption in late pregnancy.Results:Compared with the control group, PI, RI, and S/D ratios of pregnant women with placental abruption in the late pregnancy in the observation group were obviously increased [ (1.26 ± 0.22) vs. (0.95 ± 0.14), (0.65 ± 0.12) vs. (0.48 ± 0.06), (3.46 ± 0.63) vs. (2.57 ± 0.45) ] (P<0.05) ; compared with the control group, PT, TT, and APTT in pregnant women with placental abruption in the late pregnancy in the observation group were obviously increased [ (12.90 ± 1.42) vs. (10.24 ± 1.14), (15.06 ± 1.24) vs. (12.67 ± 1.08), (30.32 ± 2.55) vs. (25.48 ± 2.10) ] ( P<0.05), the FIB level was obviously decreased [ (3.09 ± 0.37) g/L vs. (3.96 ± 0.58) g/L] (P<0.05) ; compared with the grade I group, as the grading of placental abruption increased, PI, RI, S/D ratio, PT, TT, and APTT in grade II group and grade III group increased in turn ( P<0.05) and the level of FIB decreased in turn ( P<0.05) ; Logistic regression analysis showed that mechanical injury, polyhydramnios, premature rupture of membranes, pregnancy diabetes, pregnancy induced hypertension, PI, RI, S/D ratio, PT, TT, APTT were the risk factors for placental abruption in late pregnancy ( P<0.05), and FIB was the protective factor for placental abruption in late pregnancy ( P<0.05) ; the area under the ROC curve (AUC) the combined detection of PI, RI, S/D ratio, PT, FIB, TT, and APTT for placental abruption in late pregnancy was 0.982, which was better than their individual predictions (Z combined test-PI=6.118, P<0.001; Z combined test-RI=6.080, P<0.001; Z combined test-S/D ratio=6.690, P<0.001; Z combined test-PT=5.837, P<0.001; Z combined test-FIB=6.500, P<0.001; Z combined test-TT=6.439, P<0.001; Z combined test- PTT=6.112, P<0.001) . Conclusions:The PI, RI, S/D ratio, PT, TT, APTT increase, and FIB level decreases in pregnant women with placental abruption in the late pregnancy, they are related to different grades of placental abruption. Combined detection has good predictive value for placental abruption in pregnant women in the late pregnancy.

Objective To study the initial pumping speed of sodium citrate in single plasma exchange with regional citrate anticoagulation(RAC).Methods From January to December 2021,15 patients and 67 times of treatment with local sodium citrate anticoagulation single plasma exchange in the hospital were in-cluded in the study.According to the initial pumping speed of sodium citrate,they were included in the low-speed group(n=33)and the high-speed group(n=34).The transmembrane pressure,filter pressure drop and venous pressure were compared between the two groups at 30 minutes,one hour and two hours after treatment.The free calcium concentration after plasma separator at 15 minutes and one hour after treatment,and the coagulation of plasma separator and extracorporeal circulation pipeline at the end of treatment were compared between the two groups.The concentration of free calcium,blood gas analysis and electrolyte were compared at the beginning of treatment,one hour after treatment and at the end of treatment.Results The free calcium concentration after the filter was monitored at 15 minutes and one hour of treatment in both groups was within the effective range of anticoagulation recommended by the guidelines.There were no lips,fingertip numbness and hand-foot convulsions in the two groups during the treatment,and no bleeding oc-curred after the treatment.There were four cases of hypocalcemia and two cases of alkalosis in the low-speed group,and 13 cases of hypocalcemia and eight cases of alkalosis in the high-speed group.The difference be-tween the two groups was statistically significant(P＜0.05).There were 15 cases of grade Ⅰ coagulation and five cases of grade Ⅱ coagulation in plasma separator and pipeline in the low-speed group,while there were 14 cases of grade Ⅰ coagulation and four cases of grade Ⅱ coagulation in plasma separator and pipeline in the high-speed group.There was no significant difference between the two groups(P＞0.05).Conclusion In plasma exchange treatment,according to the low initial pumping speed,RAC can not only ensure the anticoagulant effect,but also reduce the incidence of complications such as hypocalcemia and alkalosis.

Objective:To analyze the fail mode of neoadjuvant therapy combined with surgery for locally advanced esophageal squamous cell carcinoma (ESCC) after long-term follow-up.Methods:Clinical data of consecutive 238 patients with locally advanced resectable ESCC who underwent neoadjuvant therapy combined with surgery in Zhejiang Cancer Hospital from September 2012 to October 2019 were retrospectively analyzed. The failure mode in the whole cohort was analyzed after long-term follow-up. The overall survival (OS) and disease free survival (DFS) rates were analyzed by Kaplan-Meier method. Survival differences were determined by log-rank test.Results:The pathological complete response (pCR) rate was 42.0% in 238 patients. After a median follow-up of 46.1 months, tumor progression occurred in 96 patients (40.3%), including 25 patients (10.5%) with local recurrence, 61 patients (25.6%) with distant metastases, and 10 patients (4.2%) with simultaneous local recurrence and distant metastases. The median OS and DFS were 64.7 months and 49.9 months. And the 3-, 5-, and 7-year OS and DFS rates were 70.0%, 52.8%, 36.4% and 63.5%, 42.5%, and 30.0%, respectively. The 3-, 5-, and 7-year locoregional recurrence-free survival rates and distant metastasis-free survival rates were 86.0%, 71.4%, 61.2% and 70.6%, 55.9%, 43.0%. Compared with non-pCR patients, the overall progression rate and distant metastasis rate of pCR patients were lower (26.0% vs. 50.7%, 16.0% vs. 32.6%, both P<0.05). And the 3-, 5-, and 7-year OS (83.0% vs. 60.2%, 69.7% vs. 41.7%, 50.4% vs. 27.7%, all P<0.001) and DFS rates (80.4% vs. 51.4%, 63.9% vs. 31.2%, 45.9% vs. 20.3%, all P<0.001) were significantly better in pCR patients. Conclusions:Distant metastasis is the main failure mode of patients with locally advanced ESCC after neoadjuvant therapy. Patients with postoperative pCR can achieve better long-term survival.

Objective:To analyze the failure patterns and survival after stereotactic body radiotherapy (SBRT) in patients with T 1-2N 0M 0 non-small cell lung carcinoma (NSCLC). Methods:Clinical data of early-stage NSCLC patients who received SBRT at Zhejiang Cancer Hospital from January 2012 to September 2018 were retrospectively analyzed. The primary observed endpoint was the pattern of disease progression, which was divided into intra-field recurrence, regional lymph node recurrence and distant metastasis. Overall survival (OS) and progression-free survival (PFS) were calculated by Kaplan-Meier method. Univariate analysis was conducted by log-rank test, and multivariate analysis was performed by Cox's model.Results:A total of 147 patients with 156 lesions were included. The median follow-up time was 44.0 months (16.5-95.5 months). A total of 57 patients (38.8%) progressed: 14 patients (24.5%) had recurrence with the 1-, 3-, and 5-year local recurrence rates of 2.0%, 10.9%, and 14.3%, respectively; 36 patients (63.2%) had Distant metastasis with the 1-, 3- and 5-year distant metastasis rates of 12.2%, 22.4% and 28.6%, respectively; and 7 patients (12.3%) had recurrence complicated with distant metastasis. The 3-, 5- and 7-year OS rates were 80.5%, 64.2% and 49.9% for all patients, respectively. The median OS was 78.4 months. The 3-, 5- and 7-year PFS rates were 64.8%,49.5% and 41.5%, with a median PFS of 57.9 months (95% CI: 42.3-73.5 months). Univariate and multivariate analyses showed that biologically equivalent dose and age were the factors affecting the efficacy of SBRT (both P<0.05). Conclusion:Distant metastasis is the main failure pattern in patients with T 1-2N 0M 0 NSCLC after SBRT. High-risk population should be selected for further systematic treatment to improve the efficacy.

BACKGROUND: Nowadays, biological tissue engineering is a growing field of research. Biocompatibility is a key indicator for measuring tissue engineering biomaterials, which is of great significance for the replacement and repair of damaged tissues. METHODS: In this study, using gelatin, carboxymethyl chitosan, and sodium alginate, a tissue engineering material scaffold that can carry cells was successfully prepared. The material was characterized by Fourier transforms infrared spectroscopy. In addition, the prepared scaffolds have physicochemical properties, such as swelling ratio, biodegradability.we observed the biocompatibility of the hydrogel to different adult stem cells (BMSCs and ADSCs) in vivo and in vitro. Adult stem cells were planted on gelatin-carboxymethyl chitosan-sodium alginate (Gel/SA/CMCS) hydrogels for 7 days in vitro, and the survival of stem cells in vitro was observed by live/died staining. Gel/SA/CMCS hydrogels loaded with stem cells were subcutaneously transplanted into nude mice for 14 days of in vivo culture observation. The survival of adult stem cells was observed by staining for stem cell surface markers (CD29, CD90) and Ki67. RESULTS: The scaffolds had a microporous structure with an appropriate pore size (about 80 lm). Live/died staining showed that adult stem cells could stably survive in Gel/SA/CMCS hydrogels for at least 7 days. After 14 days of culture in nude mice, Ki67 staining showed that the stem cells supported by Gel/SA/CMCS hydrogel still had high proliferation activity. CONCLUSION Gel/SA/CMCSs hydrogel has a stable interpenetrating porous structure, suitable swelling performance and degradation rate, can promote and support the survival of adult stem cells in vivo and in vitro, and has good biocompatibility. Therefore, Gel/SA/CMCS hydrogel is a strong candidate for biological tissue engineering materials.

Objective To investigate the diagnostic accuracy of FibroScan-AST (FAST) score in patients with high-risk nonalcoholic steatohepatitis (NASH) with a nonalcoholic fatty liver disease (NAFLD) activity score of ≥4 and significant liver fibrosis (F ≥2), along with comparison with other serological models. Methods A total of 84 consecutively admitted patients hospitalized in Ruijin Hospital from January 2015 to December 2020 and biopsy-confirmed NAFLD/NASH were included in this study, and FibroScan (liver stiffness measurement and controlled attenuation parameter) and blood biochemical tests were performed at one week before and after liver biopsy. A Kruskal-Wallis H analysis of variance was used for comparison between multiple groups, and Spearman's correlation coefficient was used to analyze the correlation between variables. The receiver operating characteristic (ROC) curve was plotted with pathological results as the "gold standard", and the area under the ROC curve (AUC) was calculated. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and classification accuracy were calculated based on the cut-off values determined by previous studies. In subgroup analysis, the patients were divided into subgroups based on different clinical indices to evaluate the diagnostic accuracy of each model, which was expressed as AUC (95% confidence interval [ CI ]). Results Among the 84 patients, 43 had high-risk NASH. The FAST score was 0.54(0.04-0.93) for all patients, and the FAST score for liver fibrosis stages F0-F4 was 0.26(0.06-0.73), 0.48(0.04-0.82), 0.61(0.13-0.75), 0.64(0.09-0.93), and 0.82(0.75-0.89), respectively, with a significant difference between stages ( H =23.360, P < 0.001). FAST score was positively correlated with liver fibrosis stage ( r =0.491, P < 0.001). NAFLD fibrosis score (NFS), fibrosis-4 (FIB-4), and aspartate aminotransferase-to-platelet ratio index were positively correlated with liver fibrosis stage ( r =0.230, 0.346, and 0.281, all P < 0.05), with a weaker correlation than FAST score. FAST score had an AUC of 0.725 (95% CI : 0.617-0.834, P < 0.001) in evaluating high-risk NASH. According to the low cut-off value determined by previous studies, FAST score ≤0.35 excluded high-risk NASH in 21 patients (25%) with an NPV of 71%; according to the high cut-off value, FAST score ≥0.67 helped to make a confirmed diagnosis of high-risk NASH in 19 patients (22.6%) with a PPV of 74%. NFS and FIB-4 had an AUC of 0.633(95% CI : 0.513-0.753) and 0.686(95% CI : 0.570-0.803), respectively, in the diagnosis of high-risk NASH ( P < 0.05). Conclusion FAST score can accurately determine the presence or absence of high-risk NASH in NAFLD patients with or without metabolic risk factors, and selection of appropriate cut-off values can help some patients avoid liver biopsy.

ObjectiveTo investigate the efficacy and safety of Fuzheng Huayu tablets (FZHY) combined with entecavir (ETV) in the treatment of chronic hepatitis B (CHB) liver fibrosis. MethodsA total of 52 patients with CHB liver fibrosis with an Ishak stage of ≥F3 who were treated in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine and Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine from April 2011 to January 2013 were enrolled and divided into FZHY combined with ETV group (combination group) and placebo combined with ETV group (control group), with 26 patients in each group, and the course of treatment was 48 weeks for both groups. Liver biopsy was performed before and after these treatment; clinical outcome was determined based on the reversal rate of Ishak stage for liver fibrosis and the improvement rate of histological activity index (HAI) for inflammation grade, and safety was evaluated based on electrocardiographic findings. Three datasets (full analysis set, per-protocol set, and safety dataset) were identified for analysis; the t-test or the Wilcoxon test was used for comparison of continuous data between two groups, and the CMH chi-square test, the chi-square test, or the Fisher’s exact test was used for comparison of categorical data between groups. ResultsOf all 52 patients, 46 underwent the two liver biopsies before and after treatment, with 22 in the combination group and 24 in the control group. At week 48 of treatment, there was a significant difference in the proportion of patients with Ishak stage reduced by ≥1 stage between the combination group and the control group (81.8% vs 54.2%, χ2=5.297, P=0.021). There was also a significant difference in the improvement rate of HAI grade between the combination group and the control group were (59.1% vs 25.0%, χ2=6.822, P=0.009). There were no significant differences between the two groups in the incidence rates of adverse events and serious adverse events, the safety analysis of vital signs, and laboratory safety indicators (all P＞0.05). ConclusionFZHY combined with ETV has significant advantages over ETV alone in improving liver fibrosis and inflammation, and antiviral therapy combined with anti-fibrosis therapy can bring better hepatic histological improvement for CHB patients. FZHY combined with ETV has good safety in the treatment of patients with CHB liver fibrosis.

Objective:To evaluate the 5-year survival outcome of patients with unresectable locally advanced non-small cell lung cancer (NSCLC) treated with Endostar in combination with platinum-based concurrent chemoradiotherapy.Methods:From March 2009 to June 2015, 115 patients with the unresectable locally advanced NSCLC from two prospective studies[Clinical trials 2009-2012(ClinicalTrials.gov NCT01894) and 2012-2015(ClinicalTrials.gov, NCT01733589)] were treated with Endostar in combination with platinum-based concurrent chemoradiotherapy. A total dose of 60-66 Gy was delivered in 30-33 fractions. Endostar was given 1 week prior to the beginning of radiotherapy, and repeated fortnightly during the concurrent chemoradiotherapy. After long-term follow up, survival outcome was evaluated in 104 patients treated with radiation dose of ≥60 Gy. Kaplan-Meier method was used for survival analysis. Univariate survival analysis was performed using the log-rank test.Results:Of 104 eligible patients, 60.6% of them had squamous carcinoma and 65.4% were classified in stage Ⅲ B. All the patients received ≥2 cycles of Endostar and 93.3% of them received 4 cycles of Endostar. The median follow-up time was 68.3 months. The median overall survival (OS) and median progression-free survival (PFS) were 31.3 and 13.9 months, respectively. The 3-year and 5-year OS were 45.6% and 35.7%, respectively. The 3-year and 5-year PFS were 27.1% and 24.9%, respectively. Univariate analysis indicated that sex, ECOG, pathological type, clinical stage, radiotherapy technique, chemotherapy regimen, chemotherapy cycle and cycle of Endostar use were not associated with OS. Late radiation injury occurred in 14.4% of patients, and no grade 4-5 late injury was observed. Conclusion:Patients with unresectable locally advanced NSCLC treated with Endostar fortnightly in combination with platinum-based concurrent chemoradiotherapy achieve better OS than historical data with tolerable toxicities.