Intestinal microbiota via NLRP3 inflammasome dependent neuronal pyroptosis mediates anxiety-like behaviour in mice exposed to 3.5 GHz radiofrequency radiation.

The rapid development of 5G communication technology has increased public concern about the potential adverse effects on human health. Till now, the impacts of radiofrequency radiation (RFR) from 5G communication on the central nervous system and gut-brain axis are still unclear. Therefore, we investigated the effects of 3.5 GHz (a frequency commonly used in 5G communication) RFR on neurobehavior, gut microbiota, and gut-brain axis metabolites in mice. The results showed that exposure to 3.5 GHz RFR at 50 W/m2 for 1 h over 35 d induced anxiety-like behaviour in mice, accompanied by NLRP3-dependent neuronal pyroptosis in CA3 region of the dorsal hippocampus. In addition, the microbial composition was widely divergent between the sham and RFR groups. 3.5 GHz RFR also caused changes in metabolites of feces, serum, and brain. The differential metabolites were mainly enriched in glycerophospholipid metabolism, tryptophan metabolism, and arginine biosynthesis. Further correlation analysis showed that gut microbiota dysbiosis was associated with differential metabolites. Based on the above results, we speculate that dysfunctional intestinal flora and metabolites may be involved in RFR-induced anxiety-like behaviour in mice through neuronal pyroptosis in the brain. The findings provide novel insights into the mechanism of 5G RFR-induced neurotoxicity.

Pan-cancer analysis of LncRNA XIST and its potential mechanisms in human cancers.

Background: X-inactive specific transcript (XIST), it has been found, is abnormal expression in various neoplasms. This work aims to explore its potential molecular mechanisms and prognostic roles in types of malignancies. Methods: This research comprehensively investigated XIST transcription across cancers from Oncomine, TIMER 2.0 and GEPIA2. Correlations of XIST expression with prognosis, miRNAs, interacting protens, immune infiltrates, checkpoint markers, mutations of tumor-associated genes and promoter methylation were also analyzed by public databases. In addition, 98 BRCA samples were collected to investigate XIST expression and evaluate its clinicopathological value. Results: In public databases, compared to normal tissues, XIST was lower in BRCA, CESC, COAD and so on, but increased in KIRC and PRAD. Databases also showed that XIST was a good indicator of prognosis in BRCA, COAD and so on, but a bad one in KIRC, KIRP and so on. From starBase, we found 29 proteins interacting with XIST, and identified 4 miRNAs which might be sponged by XIST in cancers. Furthermore, XIST was linked with immune infiltration, especially T cell CD4+, and was related to over 20 immune checkpoint markers. Moreover, several tumor-associated gene mutations and promoter methylation were negatively related to its expression. In addition, IHC showed that XIST in BRCA was obviously lower in comparison of normal tissues and was negatively related to lymph node invasion and TNM stage. Conclusion: In summary, abnormal expression of XIST influenced prognosis, miRNAs and immune infiltration across cancers, especially BRCA.

Discovery and characterization of amentoflavone as a naturally occurring inhibitor against the bile salt hydrolase produced by Lactobacillus salivarius.

Bile salt hydrolases (BSHs), a group of cysteine-hydrolases produced by gut microbes, play a crucial role in the hydrolysis of glycine- or taurine-conjugated bile acids and have been validated as key targets to modulate bile acid metabolism. This study aims to discover one or more efficacious inhibitors against a BSH produced by Lactobacillus salivarius (lsBSH) from natural products and to characterize the mechanism of the newly identified BSH inhibitor(s). Following screening of the inhibition potentials of more than 100 natural compounds against lsBSH, amentoflavone (AMF), a naturally occurring biflavone isolated from various medicinal plants, was discovered to be an efficacious BSH inhibitor (IC50 = 0.34 µM). Further investigation showed that AMF could strongly inhibit the lsBSH-catalyzed hydrolytic reaction in living gut microbes. Inhibition kinetic analyses demonstrated that AMF reversibly inhibited the lsBSH-catalyzed hydrolytic reaction in a mixed-inhibition manner, with an apparent Ki value of 0.65 µM. Fluorescence quenching assays suggested that AMF could quench the fluorescence of lsBSH via a static quenching procedure. Docking simulations suggested that AMF could be fitted into lsBSH at two distinct ligand-binding sites, mainly via hydrophobic interactions and hydrogen bonding, which explained well the mixed inhibition mode of this agent. Animal tests showed that the hydrolytic activities of BSHs in mice feces could be significantly blocked by AMF. In summary, this study reports that AMF is a strong, naturally occurring inhibitor of lsBSH, which offers a promising lead compound to develop novel agents for modulating bile acid metabolism in the host via targeting BSHs.

Human carboxylesterase 1A plays a predominant role in the hydrolytic activation of remdesivir in humans.

Remdesivir, an intravenous nucleotide prodrug, has been approved for treating COVID-19 in hospitalized adults and pediatric patients. Upon administration, remdesivir can be readily hydrolyzed to form its active form GS-441524, while the cleavage of the carboxylic ester into GS-704277 is the first step for remdesivir activation. This study aims to assign the key enzymes responsible for remdesivir hydrolysis in humans, as well as to investigate the kinetics of remdesivir hydrolysis in various enzyme sources. The results showed that remdesivir could be hydrolyzed to form GS-704277 in human plasma and the microsomes from human liver (HLMs), lung (HLuMs) and kidney (HKMs), while the hydrolytic rate of remdesivir in HLMs was the fastest. Chemical inhibition and reaction phenotyping assays suggested that human carboxylesterase 1 (hCES1A) played a predominant role in remdesivir hydrolysis, while cathepsin A (CTSA), acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) contributed to a lesser extent. Enzymatic kinetic analyses demonstrated that remdesivir hydrolysis in hCES1A (SHUTCM) and HLMs showed similar kinetic plots and much closed Km values to each other. Meanwhile, GS-704277 formation rates were strongly correlated with the CES1A activities in HLM samples from different individual donors. Further investigation revealed that simvastatin (a therapeutic agent for adjuvant treating COVID-19) strongly inhibited remdesivir hydrolysis in both recombinant hCES1A and HLMs. Collectively, our findings reveal that hCES1A plays a predominant role in remdesivir hydrolysis in humans, which are very helpful for predicting inter-individual variability in response to remdesivir and for guiding the rational use of this anti-COVID-19 agent in clinical settings.

Nucleolar protein 6 promotes cell proliferation and acts as a potential novel prognostic marker for hepatocellular carcinoma.

BACKGROUND: Nucleolar protein 6 (NOL6) is a nucleolar RNA-associated protein that is highly conserved between species. It has been proved to be associated with the prognosis of liver cancer. However, the underlying mechanism has not been fully established. This study aimed to assess the relationship between NOL6 and liver cancer prognosis. METHODS: We constructed an NOL6-short hairpin RNA (shRNA)-expressing lentivirus. Through viral transfection, cell growth assay and fluorescence-activated cell sorting, we evaluated the effect of shRNA-mediated NOL6 knockdown on the proliferation, colony formation, and apoptosis of hepatocellular carcinoma (HCC) cells. The relationship between NOL6 expression and HCC patient survival has been established through bioinformatics analysis. We also explored the downstream molecular regulatory network of NOL6 in HCC by performing an Ingenuity Pathway Analysis in the database. RESULTS: Increased NOL6 expression was detected in HCC cells compared to normal controls; HCC patients with high NOL6 expression had poorer prognoses than those with low expression. NOL6 knockdown inhibited HCC cell proliferation, apoptosis, and colony formation. Also, MAPK8, CEBPA, and FOSL1 were selected as potential downstream genes of NOL6. CONCLUSIONS: NOL6 up-regulates HCC cell proliferation and affects downstream expression of related genes. Moreover, NOL6 is considered to be associated with poor prognosis in HCC patients.

Flavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CLpro: Inhibition potentials, covalent binding sites and inhibitory mechanisms.

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.

Roles of eIF3m in the tumorigenesis of triple negative breast cancer.

BACKGROUND: Without targets, triple negative breast cancer (TNBC) has the worst prognosis in all subtypes of breast cancer (BC). Recently, eukaryotic translation initiation factor 3 m (eIF3m) has been declared to be involved in the malignant progression of various neoplasms. The aim of this study is to explore biological functions of eIF3m in TNBC. METHODS: Multiple databases, including Oncomine, KM-plotter and so on, were performed to analyze prognosis and function of eIF3m in TNBC. After transfection of eIF3m-shRNA lentivirus, CCK-8, colony formation assay, cell cycle analysis, wound healing assay, transwell assays, mitochondrial membrane potential assay and cell apoptosis analysis were performed to explore the roles of eIF3m in TNBC cell bio-behaviors. In addition, western blotting was conducted to analyze the potential molecular mechanisms of eIF3m. RESULTS: In multiple databases, up-regulated eIF3m had lower overall survival, relapse-free survival and post progression survival in BC. EIF3m expression in TNBC was obviously higher than in non-TNBC or normal breast tissues. Its expression in TNBC was positively related to differentiation, lymph node invasion and distant metastasis. After knockdown of eIF3m, cell proliferation, migration, invasion and levels of mitochondrial membrane potential of MDA-MB-231 and MDA-MB-436 were all significantly suppressed, while apoptosis rates of them were obviously increased. In addition, eIF3m could regulate cell-cycle, epithelial-mesenchymal transition and apoptosis-related proteins. Combined with public databases and RT-qPCR, 14 genes were identified to be modulated by eIF3m in the development of TNBC. CONCLUSIONS: eIF3m is an unfavorable indicator of TNBC, and plays a vital role in the process of TNBC tumorigenesis.

Roles and correlation of FOXA1 and ZIC1 in breast cancer.

The aim of this study was to evaluate the prognostic role of Forkhead box A1 (FOXA1) in breast cancer and determine the relationship between FOXA1 and zinc finger of the cerebellum 1 (ZIC1). BCIP, GEPIA, and Oncomine databases were used to detect expression of FOXA1 and assess prognostic roles of FOXA1 and ZIC1 in invasive breast tumors. A total of 113 female invasive breast cancer cases were collected to investigate FOXA1 and ZIC1 expression via immunohistochemistry. Twenty pairs of frozen-thawed tumors were used to select reliable indicators via western blotting and real-time quantitative polymerase chain reaction. In addition, Kaplan-Meier curves and Cox regression analysis were performed to analyze the overall survival (OS) and relapse-free survival. Multiple databases showed that FOXA1 expression was elevated in invasive breast cancer and negatively related to ZIC1. BCIP database also displayed a poor prognosis of high FOXA1 and low ZIC1. FOXA1 was positively associated with tumor size, grading, lymph node metastasis, and Tumor Node Metastasis (TNM) staging, while ZIC1 expression was negatively related to grading, lymph node metastasis, and TNM staging. In Kaplan-Meier and Cox regression analysis, FOXA1 negative group and ZIC1 positive group had better OS rate and recurrence-free survival rate. In addition, a joint evaluation showed that "FOXA1- ZIC1+" had the highest OS and relapse-free survival, but "FOXA1+ ZIC1-" had the lowest ones. FOXA1 was negatively related to ZIC1 in breast cancer and they had different roles in clinicopathology and prognosis. Combined examination of FOXA1 and ZIC1 could bring more benefit to breast cancer patients.

High Survivin and Low Zinc Finger of the Cerebellum 1 Expression Indicates Poor Prognosis in Triple-negative Breast Carcinoma.

PURPOSE: Triple-negative breast carcinoma (TNBC) is accompanied with high risk of metastasis and recurrence. The present study aimed to explore the clinicopathological and prognostic roles of putative tumor-related genes in patients with TNBC. METHODS: Thirty pairs of frozen-thawed tumors were used to select reliable indicators via real-time quantitative polymerase chain reaction (RT-qPCR). Then, 150 pathology specimens were used to evaluate the expression of proteins in TNBC through immunohistochemistry. In addition, Kaplan-Meier curves and Cox regression analysis were also performed to analyze the overall survival and disease-free survival. RESULTS: RT-qPCR results indicated that among all the proteins analyzed using fresh-frozen TNBC samples, the expression levels of only Survivin and zinc finger of the cerebellum 1 (ZIC1) were obviously different from those in the corresponding normal tissues. Survivin and ZIC1 expression had opposite effects on the clinicopathological diagnosis and prognostic assessment in TNBC patients. Further, there was a negative correlation between Survivin and ZIC1 expression. In addition, the "Survivin-positive ZIC1-negative group" was associated with histologic grade, lymph node metastasis, and TNM staging (p < 0.001) and this was also an independent factor for evaluating the prognosis of TNBC in patients. CONCLUSION: In summary, the expression levels of Survivin and ZIC1 in TNBC are different from those in normal tissues and are negatively correlated mutually. The combined detection of Survivin and ZIC1 expression levels could allow better comprehensive diagnosis and prognostic evaluation for TNBC patients.

Downregulation of microRNA-4295 enhances cisplatin-induced gastric cancer cell apoptosis through the EGFR/PI3K/Akt signaling pathway by targeting LRIG1.

Gastric cancer (GC) is one of the leading causes of cancer-associated mortality worldwide. The aim of the present study was to investigate the mechanism of microRNA-4295 (miR-4295), which regulates cisplatin (DDP)-induced apoptosis in GC cells through the leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1)-mediated epidermal growth factor receptor (EGFR)/phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. Two cell lines were selected, one with the highest expression of miR-4295 and one with the lowest expression of LRIG1, for the experiments. The half maximal inhibitory concentration of DDP in the human GC MKN-28 and MKN-45 cell lines was calculated, and mitochondrial membrane potentials of the GC cells were detected by tetramethylrhodamine, ethyl ester, perchlorate staining. The proliferation and apoptosis of GC cells with or without DDP treatment were assessed by MTT assay and plate colony formation, as well as flow cytometry and TUNEL staining. Western blot analysis and reverse transcription-quantitative polymerase chain reaction were employed to determine the expression of EGFR/PI3K/Akt signaling pathway-related genes and apoptosis-related genes. LRIG1 was identified as a target gene of miR-4295. The expression of miR-4295 was upregulated, and the expression of LRIG1 was downregulated in GC cells. Furthermore, DDP enhanced the decrease in miR-4295 expression and the increase in LRIG1 expression in GC cells. miR-4295 promoted the proliferation and inhibited the DDP-induced apoptosis of GC cells without DDP treatment. In addition, miR-4295 increased the expression levels of EGFR, PI3K, Akt, p-PI3K and p-Akt, suggesting that miR-4295 promotes the activation of the EGFR/PI3K/Akt signaling pathway by targeting LRIG1. miR-4295 targeted and negatively regulated LRIG1 expression to activate the EGFR/PI3K/Akt signaling pathway, thereby promoting the proliferation of the GC cells and inhibiting the apoptosis of the GC cells induced by DDP. Therefore, miR-4295 may be a novel therapeutic target in patients with GC.

High Oct4 predicted worse prognosis of right-sided colon cancer patients.

AIM: This present study was aimed to compare the role of Oct4 in left-sided colon cancer (LCC) with right-sided colon cancer (RCC). PATIENTS & METHODS: One hundred and fifty one pathology specimens, 68 frozen-thawed tumors and cell lines were used to evaluate the role of Oct4 in LCC and RCC through immunohistochemistry, western blot and real-time quantitative PCR. RESULTS: In LCC, positive expression of Oct4 was positively related to differentiation and Dukes stage (p < 0.01). Only in RCC, Oct4 expression was also positively related to lymphatic invasion and survival rates of 'negative group' were significantly higher. CONCLUSION: In summary, Oct4 was related to tumor differentiation and later Dukes stage in colon cancer, and was correlated with invasion of lymphatic only in RCC. In addition, Oct4 was a potential prognostic indicator in RCC.

Combination of TNM staging and pathway based risk score models in patients with gastric cancer.

Due to the complexity and heterogeneity of gastric cancer (GC) in individual patient, current staging system is inadequate for predicting outcome of GC. Comprehensive computational and bioinformatics approach may triumph for the prediction. In this study, GC patients were devided according to stage and treatment: curative surgery plus chemoradiotherapy in stage II, curative surgery plus chemoradiotherapy in stages III, and IV, unresectable metastatic gastric cancer. The training sets were downloaded from GEO datasets (GSE26253 and GSE14208). Gene set enrichment analysis (GSEA) was performed to explore enriched difference between recurrence and nonrecurrence. The core enrichment genes of enriched pathways significantly associated with recurrence or progression were identified using Cox proportional hazards analysis. Thereafter, the risk score models were externally validated in independent datasets-GSE15081 and The Cancer Genome Atlas (TCGA). We generated respective risk score models of patients in different stages and treatment. A five-gene signature comprising FARP1, SGCE, SGCA, LAMA4, and COL9A2 was strongly associated with recurrence of patients with curative surgery plus chemoradiotherapy in stage II. A six-gene signature consisting of SHH, NF1, AP4B1, COMP, MATN3, and CCL8 was correlated with recurrence of patients with curative surgery plus chemoradiotherapy in stages III and IV. And a four-gene signature composing of ABCC2, AHNAK2, RNF43, and GSPT2 was highly related to progression of patients with unresectable metastatic GC. Taking into consideration TNM stage and gene signature reflecting recurrence or progression, the risk score models significantly improved the accuracy in predicting outcome of GC.