Structural Insights into the Reaction between Hydrogen Peroxide and Di-iron Complexes at the Ferroxidase Center of Ferritin.

The Fe(II) oxidation mechanism in the ferroxidase center of heavy chain ferritin has been studied extensively. However, the actual production of H2O2 was found to be substantially lower than expected at low flux of Fe(II) to ferritin subunits. Here, we demonstrated that H2O2 could interact with the di-iron nuclear center, leading to the production of hydroxyl radicals and oxygen. Two reaction intermediates were captured in the ferroxidase center by using the time-lapse crystallographic techniques in a shellfish ferritin. The crystal structures revealed the binding of H2O2 as a µ -1,2-peroxo-diferric species and the binding of O2 to the diferric structure. This investigation sheds light on the reaction between the di-iron nuclear center and H2O2 and provides insights for the exploitation of metalloenzymes.

Zinc binding strength of proteins dominants zinc uptake in Caco-2 cells.

Zinc plays a vital role in structural, catalysis, and signal regulation in the human body. Zinc deficiency leads to the dysfunction of many organs and immunity systems. Diet proteins have distinct effects on zinc uptake. However, the mechanisms are uncovered. Here we select three principal components from whey protein: alpha-lactalbumin, beta-lactoglobulin, and bovine serum albumin, which bind with zinc at different affinities, to evaluate the relationship between their potential zinc uptake and protein binding. The experimental data shows that beta-lactoglobulin could promote zinc uptake, alpha-lactalbumin has minor effects, whereas bovine serum albumin reduced zinc uptake in Caco-2 cell lines. Zinc binding effects on protein structure were thoroughly inspected through fluorescent spectroscopy and X-ray crystallography. Isothermal titration calorimetry revealed that three proteins have different binding affinities toward zinc ions. We speculate that protein binding eliminates toxic effects from free zinc, and the binding strength dominates zinc uptake.

Analysis of immune related gene expression profiles and immune cell components in patients with Barrett esophagus.

Barrett's esophagus (BE) is a well-known precancerous condition of esophageal adenocarcinoma. However, the immune cells and immune related genes involved in BE development and progression are not fully understood. Therefore, our study attempted to investigate the roles of immune cells and immune related genes in BE patients. The raw gene expression data were downloaded from the GEO database. The limma package in R was used to screen differentially expressed genes (DEGs). Then we performed the least absolute shrinkage and selection operator (LASSO) and random forest (RF) analyses to screen key genes. The proportion of infiltrated immune cells was evaluated using the CIBERSORT algorithm between BE and normal esophagus (NE) samples. The spearman index was used to show the correlations of immune genes and immune cells. Receiver operating characteristic (ROC) curves were used to assess the diagnostic value of key genes in BE. A total of 103 differentially expressed immune-related genes were identified between BE samples and normal samples. Then, 7 genes (CD1A, LTF, FABP4, PGC, TCF7L2, INSR,SEMA3C) were obtained after Lasso analysis and RF modeling. CIBERSORT analysis revealed that resting CD4 T memory cells and gamma delta T cells were present at significantly lower levels in BE samples. Moreover, plasma cell and regulatory T cells were present at significantly higher levels in BE samples than in NE samples. INSR had the highest AUC values in ROC analysis. We identified 7 immune related genes and 4 different immune cells in our study, that may play vital roles in the occurrence and development of BE. Our findings improve the understanding of the molecular mechanisms of BE.

[Study on the temperature characteristics of fast capacitance in patch clamp experiments].

Patch clamp is a technique that can measure weak current in the level of picoampere (pA). It has been widely used for cellular electrophysiological recording in fundamental medical researches, such as membrane potential and ion channel currents recording, etc. In order to obtain accurate measurement results, both the resistance and capacitance of the pipette are required to be compensated. Capacitance compensations are composed of slow and fast capacitance compensation. The slow compensation is determined by the lipid bilayer of cell membrane, and its magnitude usually ranges from a few picofarads (pF) to a few microfarads (µF), depending on the cell size. The fast capacitance is formed by the distributed capacitance of the glass pipette, wires and solution, mostly ranging in a few picofarads. After the pipette sucks the cells in the solution, the positions of the glass pipette and wire have been determined, and only taking once compensation for slow and fast capacitance will meet the recording requirements. However, when the study needs to deal with the temperature characteristics, it is still necessary to make a recognition on the temperature characteristic of the capacitance. We found that the time constant of fast capacitance discharge changed with increasing temperature of bath solution when we studied the photothermal effect on cell membrane by patch clamp. Based on this phenomenon, we proposed an equivalent circuit to calculate the temperature-dependent parameters. Experimental results showed that the fast capacitance increased in a positive rate of 0.04 pF/℃, while the pipette resistance decreased. The fine data analysis demonstrated that the temperature rises of bath solution determined the kinetics of the fast capacitance mainly by changing the inner solution resistance of the glass pipette. This result will provide a good reference for the fine temperature characteristic study related to cellular electrophysiology based on patch clamp technique.

Bispecific c-Met/PD-L1 CAR-T Cells Have Enhanced Therapeutic Effects on Hepatocellular Carcinoma.

T cells expressing chimeric antigen receptors, especially CD19 CAR-T cells have exhibited effective antitumor activities in B cell malignancies, but due to several factors such as antigen escape effects and tumor microenvironment, their curative potential in hepatocellular carcinoma has not been encouraging. To reduce the antigen escape risk of hepatocellular carcinoma, this study was to design and construct a bispecific CAR targeting c-Met and PD-L1. c-Met/PD-L1 CAR-T cells were obtained by lentiviral transfection, and the transfection efficiency was monitored by flow cytometry analysis. LDH release assays were used to elucidate the efficacy of c-Met/PD-L1 CAR-T cells on hepatocellular carcinoma cells. In addition, xenograft models bearing human hepatocellular carcinoma were constructed to detect the antitumor effect of c-Met/PD-L1 CAR-T cells in vivo. The results shown that this bispecific CAR was manufactured successfully, T cells modified with this bispecific CAR demonstrated improved antitumor activities against c-Met and PD-L1 positive hepatocellular carcinoma cells when compared with those of monovalent c-Met CAR-T cells or PD-L1 CAR-T cells but shown no distinct cytotoxicity on hepatocytes in vitro. In vivo experiments shown that c-Met/PD-L1 CAR-T cells significantly inhibited tumor growth and improve survival persistence compared with other groups. These results suggested that the design of single-chain, bi-specific c-Met/PD-L1 CAR-T is more effective than that of monovalent c-Met CAR-T for the treatment of hepatocellular carcinoma., and this bi-specific c-Met/PD-L1 CAR is rational and implementable with current T-cell engineering technology.

Primary Tumor Resection Improves Survival in Patients With Metastatic Gastrointestinal Stromal Tumors: A Preliminary Population-Based Analysis.

Background: Surgery has been the primary treatment in patients with localized gastrointestinal stromal tumors (GISTs) for many decades, whereas it remains controversial regarding the efficacy of primary tumor resection for metastatic GISTs treated with chemotherapy, and likewise it is unclear who would benefit from the surgical resection. Methods: GISTs patients with distant metastases were identified from the Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2016. Cox proportional hazards regression models were used to identify prognostic factors of overall survival (OS) and cancer-specific survival (CSS). Kaplan-Meier analyses and log-rank tests were conducted to assess the effectiveness of surgery on survival. Results: In total, of 455 patients with metastatic GISTs, 235 patients (51.6%) underwent primary tumor resection and 220 patients (48.4%) did not. Median survival of patients in resection group was 72 (95% CI: 62.90-81.10) months vs. 40 (95% CI: 29.53-50.47) months for those in non-resection group (p < 0.001). Similarly, surgery in conjunction with chemotherapy led to a favorable impact on survival than chemotherapy alone (OS: 72 vs. 40 months, p < 0.001; CSS: 74 vs. 44 months, p < 0.001). Multivariable analysis showed that both OS (HR: 0.581, 95% CI: 0.386-0.874, p = 0.009) and CSS (HR: 0.663, 95% CI: 0.439-0.912, p = 0.042] were dramatically improved in patients with surgical removal of primary site, as well as primary tumor size between 5 and 10 cm, while increasing age was predictive of poorer survival. Stratified analysis revealed that patients with tumor locations in the stomach demonstrated a prolonged survival after surgery, with no significant differential surgical effect between the stomach and small intestine. Conclusions: Our study preliminarily suggests that carefully selected patients with metastatic GISTs might prolong survival after treatment of surgery, especially those with a primary tumor between 5 and 10 cm and a tumor located in the stomach.

Prognostic and clinicopathological significance of SNHG20 in human cancers: a meta-analysis.

BACKGROUND: It has been widely reported that the expression levels of SNHG20 are elevated in diverse types of cancers, indicating that SNHG20 may participate in cancer initiation and development. Besides, accumulating evidence reveals that SNHG20 overexpression is also connected with poor clinical outcomes among cancer patients. Herein, we carry out a systematic meta-analysis to further determine the prognostic and clinical significance of SNHG20 expression in various human cancers. METHODS: Qualifying publications were selected by searching for keywords in PubMed, Embase, Web of Science and Cochrane Library databases, up to September 1, 2019. Pooled hazard ratio (HR) or odds ratio (OR) with corresponding 95% confidence interval (CI) was computed to estimate the strength of association between SNHG20 and survival of cancer patients or clinicopathology using Stata 14.0 software. RESULTS: In total, 15 studies encompassing 1187 patients met the inclusion criteria were ultimately enrolled for analysis. According to the meta-analysis, patients with high SNHG20 expression were markedly linked to poorer overall survival (OS) (pooled HR = 2.47, 95% CI 2.05-2.98, P = 0.000) and disease-free survival/recurrence-free survival/progression-free survival (DFS/RFS/PFS) (pooled HR = 2.37, 95% CI 1.60-3.51, P = 0.000). Additionally, regarding clinicopathology of patients, enhanced SNHG20 was correlated with advanced tumour-node-metastasis (TNM) stage (OR = 2.80, 95% CI 2.00-3.93, P = 0.000), larger tumor size (OR = 3.08, 95% CI 2.11-4.51, P = 0.000), positive lymph nodes metastasis (OR = 2.99, 95% CI 2.08-4.31, P = 0.000), higher tumor stage (OR = 4.51, 95% CI 2.17-9.37, P = 0.000) and worse histological grade (OR = 1.95, 95% CI 1.44-2.63, P = 0.000), but not with gender, smoking status or distant metastasis. CONCLUSIONS: Up-regulated SNHG20 expression is ubiquitous in different kinds of cancers. Moreover, up-regulated SNHG20 expression is capable of serving as an innovative predictive factor of inferior clinical outcomes in cancer patients. Nevertheless, higher-quality multicenter studies are required to corroborate our results.

Clinicopathological significance and prognosis of long noncoding RNA SNHG16 expression in human cancers: a meta-analysis.

BACKGROUND: Recent studies have highlighted the important role of long non-coding RNA SNHG16 in various human cancers. Here, we conducted a meta-analysis to investigate the effect of SNHG16 expression on clinicopathological features and prognosis in patients with different kinds of human cancers. METHODS: We performed a systematic search in electronic databases including PubMed, EMBASE, Cochrane Library and Web of Science, to investigate the potential association between SNHG16 expression and prognostic significance and clinical features in cancer patients. Odds ratios (ORs) or hazards ratios (HRs) with corresponding 95% confidence intervals (95% CIs) were pooled to estimate the prognosis value of SNHG16 by StataSE 15.0 software. RESULTS: A total of 16 eligible studies with 1299 patients were enrolled in our meta-analysis. The results revealed that increased expression level of SNHG16 was significantly associated with larger tumor size (OR: 3.357; 95% CI: 2.173-5.185; P < 0.001), advanced TNM stage (OR: 2.930; 95% CI: 1.522-5.640; P = 0.001) and poor histological grade (OR: 3.943; 95% CI: 1.955-7.952; P < 0.001), but not correlated with smoking status (P = 0.489), sex (P = 0.932), distant metastasis (P = 0.052), or lymph node metastasis (P = 0.155). Moreover, the pooled HR showed that elevated expression SNHG16 was associated with a significantly poorer overall survival (OS) (HR = 1.866, 95% CI: 1.571-2.216, P < 0.001). For the set of cancer types, high expression of SNHG16 was significantly associated with shorter OS in patients with cancers of the urinary system (HR: 2.523, 95% CI:1.540-4.133; P <0.001), digestive system (HR: 2.406, 95% CI:1.556-3.721; P <0.001), and other cancers (including glioma and non-small cell lung cancer) (HR: 1.786, 95% CI:1.406-2.267; P <0.001). CONCLUSIONS: LncRNA SNHG16 overexpression might serve as an unfavorable prognostic factor, which provides a basis for medical workers to evaluate the prognosis of patients and to help the decision-making process.

Endoscopic Submucosal Dissection of Gastrointestinal Stromal Tumours: A Retrospective Cohort Study.

BACKGROUND: Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal neoplasms. Endoscopic submucosal dissection (ESD) has been used to remove submucosal tumours for many years. However, whether ESD can be recommended for the treatment of GISTs is still controversial. Therefore, we evaluated the efficacy and safety of ESD for treating GISTs. PATIENTS AND METHODS: We retrospectively analysed 75 GIST patients who underwent ESD in our hospital from January 2016 to December 2018, and the demographic data, clinical presentation of tumours, operative parameters, postoperative complications and length of hospital stay were analysed. RESULTS: Seventy-five patients successfully underwent en bloc resection, and 74 (98.7%) patients underwent complete resection of the lesions, with an average tumour size of 1.7 cm (range 0.3-6.0 cm). The median operation time was 84.8 min (range 20-180 min). Forty-two (56.0%) patients underwent endoscopic purse-string suture with no conversions to an open operation. The median postoperative length of hospitalization was 6.6 days (range 3-14 days). Out of a total of 75 GIST patients, 48 (64.0%) were considered very low risk, 19 (25.3%) were low risk, 5 (6.7%) were mild risk, and 3 (4.0%) were high risk. The median follow-up was 24.0 months (range 6-45 months). During hospitalization and follow-up, no complications, recurrence or metastasis occurred. CONCLUSION: Based on our study from a medical centre, ESD is a safe and effective method for treating GISTs. However, further studies are needed.

Comparison of endoscopic versus laparoscopic resection for gastric gastrointestinal stromal tumors: A preliminary meta-analysis.

BACKGROUND AND AIM: For localized disease, complete surgical resection is regarded as the "gold standard" therapeutic modality. With the rapid development of endoscopic techniques, endoscopic resection (ESR) has been confirmed as an efficient and safe alternative for the treatment of gastrointestinal stromal tumors (GISTs) in the stomach. Nevertheless, the management of gastric GISTs remains poorly defined. The purpose of this study is to evaluate the security and effectiveness of ESR with laparoscopic resection (LAR) for gastric GISTs. METHODS: A literature search of online databases was conducted to identify relevant comparative studies of ESR and LAR procedures for gastric GISTs published before April 10, 2020. The cumulative data analysis was also performed utilizing the software STATA. RESULTS: In total, 10 studies involving 1165 patients met the inclusion criteria for analysis (651 for ESR and 514 for LAR). From the results of meta-analysis, patients who underwent ESR experienced decreased operative time (P = 0.000), less intraoperative blood loss (P = 0.002), earlier time to diet (P = 0.000), shorter hospital stay (P = 0.000), and lower total charges (P = 0.000) compared with LAR. Moreover, there were no significant differences between these two approaches concerning tumor rupture, conversion rate to other procedure, complete resection rate, postoperative complication rate, recurrence rate, and disease-free survival. CONCLUSIONS: Endoscopic resection, as an effective alternative treatment strategy with satisfactory outcomes, is acceptable for selective patients with gastric GISTs compared with LAR. Further well-designed randomized controlled trials with large samples are warranted to corroborate our observations.

Prognostic and clinicopathological significance of SNHG6 in human cancers: a meta-analysis.

BACKGROUND: Recently, accumulating evidence has suggested that the aberrant expression of SNHG6 exists in a variety of tumors and has a correlation with poor clinical outcomes across cancer patients. Considering the inconsistent data among published studies, we aim to assess the prognostic effect of SNHG6 on malignancies. METHODS: We retrieved relevant publications in Web of Science, Embase, MEDLINE, PubMed and Cochrane Library based on predefined selection criteria, up to October 1, 2019. Pooled hazard ratios (HRs) and odds ratios (ORs) with 95% confidence intervals (CIs) were utilized to evaluate the correlation between SNHG6 and overall survival (OS), recurrence-free survival (RFS) and progression-free survival (PFS) as well as clinicopathology. RESULTS: In total, 999 patients from 14 articles were enrolled in our meta-analysis. The results revealed that augmented SNHG6 expression was significantly correlated with poor OS (HR = 2.20, 95% CI = 1.76-2.75, P < 0.001) and RFS (HR = 3.10, 95% CI = 1.90-5.07, P < 0.001), but not with PFS (HR = 2.11, 95% CI = 0.82-5.39, P = 0.120). In addition to lung cancer and ovarian cancer, subgroup analysis showed that the prognostic value of SNHG6 across multiple tumors was constant as the tumor type, sample size, and methods of data extraction changed. Moreover, cancer patients with enhanced SNHG6 expression were prone to advanced TNM stage (OR = 3.31, 95% CI = 2.46-4.45, P < 0.001), distant metastasis (OR = 4.67, 95% CI = 2.98-7.31, P < 0.001), lymph node metastasis (OR = 2.59, 95% CI = 1.41-4.77, P = 0.002) and deep tumor invasion (OR = 3.75, 95% CI = 2.10-6.69, P < 0.001), but not associated with gender, histological grade and tumor size. CONCLUSIONS: SNHG6 may serve as a promising indicator in the prediction of prognosis and clinicopathological features in patients with different kinds of tumors.

miR-140-3p Suppresses Cell Growth And Induces Apoptosis In Colorectal Cancer By Targeting PD-L1.

BACKGROUND: A variety of miRNAs have been recently reported to be abnormally expressed in colorectal cancer (CRC). A growing number of studies have demonstrated that aberrantly expressed miRNAs are closely related to the development and progression of CRC. It has been found that miR-140-3p plays a vital role in several cancers. However, its expression, roles and mechanisms in CRC are remain unknown. MATERIALS AND METHODS: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to determine miR-140-3p expression in CRC tissues and cell lines. CCK8, migration, invasion and flow cytometric assays were used to determine the influence of miR-140-3p upregulation on cell proliferation, migration, invasion and apoptosis of CRC cells. Luciferase reporter assays and Western blots were utilized to identify the target genes of miR-140-3p. In addition, the potential mechanism of miR-140-3p action in CRC cells was elucidated. RESULTS: In our study, miR-140-3p expression was significantly decreased in CRC tissues and cell lines. Overexpression of miR-140-3p attenuated proliferation, migration, and invasion and induced the apoptosis of CRC cells. Bioinformatics analyse and luciferase reporter analysis identified PD-L1 as a putative target gene of miR-140-3p. PD-L1 was overexpressed in CRC tissues and inversely correlated with miR-140-3p expression. Suppression of PD-L1 expression in CRC cells generated biological behaviours in CRC cells that were similar to those observed after treated with miR-140-3p mimics. Restoration of PD-L1 expression partially attenuated the inhibitory effect of miR-140-3p on CRC cells. Western blot were used to verify the effect of PD-L1 expression on PI3K/AKT pathway. In addition, overexpression of miR-140-3p could inhibit CRC tumor growth in vivo. CONCLUSION: In general, these data demonstrate that miR-140-3p acts as a tumour suppressor in CRC by directly targeting PD-L1 and inactivating PI3K/AKT pathway, suggesting that miR-140-3p might be a novel target for CRC diagnosis and treatment.

Reversal of sorafenib resistance in hepatocellular carcinoma: epigenetically regulated disruption of 14-3-3η/hypoxia-inducible factor-1α.

Sorafenib resistance is one of the main obstacles to the treatment of advanced/recurrent hepatocellular carcinoma (HCC). Here, sorafenib-resistant HCC cells and xenografts in nude mice were used as experimental models. A cohort of patients with advanced recurrent HCC who were receiving sorafenib therapy was used to assess the clinical significance of this therapy. Our data showed that 14-3-3η maintained sorafenib resistance in HCC. An analysis of the underlying molecular mechanisms revealed that 14-3-3η stabilizes hypoxia-inducible factor 1α (HIF-1α) through the inhibition of ubiquitin-dependent proteasome protein degradation, which leads to the maintenance of cancer stem cell (CSC) properties. We further found that microRNA-16 (miR-16) is a competent miRNA that reverses sorafenib resistance by targeting the 3'-UTR of 14-3-3η and thereby inhibits 14-3-3η/HIF-1α/CSC properties. In HCC patients, significant negative correlations were found between the expression of miR-16 and 14-3-3η, HIF-1α, or CSC properties. Further analysis showed that low miR-16 expression but high 14-3-3η expression can prognosticate sorafenib resistance and poor survival. Collectively, our present study indicated that miR-16/14-3-3η is involved in sorafenib resistance in HCC and that these two factors could be potential therapeutic targets and biomarkers for predicting the response to sorafenib treatment.

Arsenic trioxide reverses the chemoresistance in hepatocellular carcinoma: a targeted intervention of 14-3-3η/NF-κB feedback loop.

BACKGROUND: Multi-drug resistance (MDR) is one of the main obstacles for treatment of advanced/recurrent hepatocellular carcinoma (HCC). We have previously identified arsenic trioxide (ATO) as an effective metastasis/angiogenesis inhibitor in HCC. Here, we further found that MDR-HCC cells were more sensitive to ATO. METHODS: The MDR-HCC cells were used as experimental models. Biological functions were investigated using cell transfection, polymerase chain reaction, western blot, southwestern blot, immunostaining, immunoprecipitation plus atomic fluorescence spectrometry, and so on. RESULTS: The MDR-HCC cells underwent high oxidative stress condition, and employed adaptive mechanisms for them to survive; while ATO abolished such mechanisms via targeting the 14-3-3η/nuclear factor kappa B (NF-κB) feedback Loop. Briefly, in MDR cells, the increase of ROS activated NF-κB signaling, which transcriptionally activated 14-3-3η. Meanwhile, the activation of NF-κB can be constitutively maintained by 14-3-3η. As a NF-κB inhibitor, ATO transcriptionally inhibited the 14-3-3η mRNA level. Meanwhile, ATO was also validated to directly bind to 14-3-3η, enhancing the degradation of 14-3-3η protein in an ubiquitination-dependent manner. Knockdown of 14-3-3η reduced the ATO-induced reversal extents of drug resistance in MDR cells. CONCLUSION: 14-3-3η/NF-κB feedback loop plays an important role in maintaining the MDR phenotype in HCC. Moreover, via targeting such feedback loop, ATO could be considered as a potential molecular targeted agent for the treatment of HCC.

[Meta-analysis of the role of Argatroban in renal replacement therapy].

OBJECTIVE: To assess the role of direct thrombin inhibitor argatroban in the renal replacement therapy. METHODS: Electronic databases including Cochrane library, PubMed, EMBASE, Highwire, MEDLINE, CBM, CNKI, and CSJD were searched using keywords including "Argatroban", "hemodialysis", "renal function", "renal failure", and "renal replacement therapy". A meta-analysis of all randomized controlled trials(RCTs)comparing argatroban with controls in renal replacement therapy was performed. Both the study selection and the meta-analysis were conducted according to the Cochrane Handbook for systematic reviews. Data were extracted from these trials and analyzed by RevMan 5.0 software. RESULTS: Compared with the control group, argatroban in renal replacement therapy showed no significant difference in mortality(RR=0.97, 95%CI: 0.48-1.97, P=0.93)and bleeding rate(RR=0.71, 95%CI: 0.37-1.34, P=0.29). Argatroban significantly decreased the incidence of new thrombosis in renal replacement therapy for patients with heparin-induced Thrombocytopenia(RR=0.40, 95%CI: 0.21-0.75, P=0.004). Also, argatroban significantly decreased the clotting events in extracorporeal circuit during the renal replacement therapy(RR=0.06, 95%CI: 0.01-0.23, P<0.0001). CONCLUSION Argatroban applied in renal replacement therapy can decrease the incidences of new thrombosis and clotting events in extracorporeal circuit and meanwhile will not increase the mortality and bleeding.