Low Temperature Plasma Suppresses Lung Cancer Cells Growth via VEGF/VEGFR2/RAS/ERK Axis.

Low temperature plasma (LTP) is a promising cancer therapy in clinical practice. In this study, dielectric barrier discharge plasma with helium gas was used to generate LTP. Significant increases in extracellular and intracellular reactive species were found in lung cancer cells (CALU-1 and SPC-A1) after LTP treatments. Cells viability and apoptosis assays demonstrated that LTP inhibited cells viability and induced cells death, respectively. Moreover, Western blotting revealed that the growth of CALU-1 cells was suppressed by LTP via the VEGF/VEGFR2/RAS/ERK axis for the first time. The results showed that LTP-induced ROS and RNS could inhibit the growth of lung cancer cells via VEGF/VEGFR2/RAS/ERK axis. These findings advance our understanding of the inhibitory mechanism of LTP on lung cancer and will facilitate its clinical application.

Bioaccumulation of uranium by Candida utilis: Investigated by water chemistry and biological effects.

The bioaccumulation of hexavalent uranium (U(VI)) on Candida utilis (C. utilis) and its biological effects were investigated via batch and biologic techniques. The bioaccumulation mechanism of U(VI) and C. utilis were characterized by SEM, TEM, FT-IR and XPS. The batch results showed that C. utilis had a high adsorption capacity (41.15 mg/g wet cells at pH 5.0) and high equilibrium rate (~100% within 3.5 h). The analysis of intracellular hydrogen peroxides and malondialdehyde suggested that the growth of C. utilis was inhibited under different concentrations of U(VI) due to the abundant production of reactive oxide species. The activity of intracellular antioxidants (e.g., super oxide dismutase and glutathione) was significantly enhanced under U(VI) stress, indicating the anti-toxic effect of C. utilis cells under low U(VI) stress. These results indicated that C. utilis is an ideal biosorbent for removing radionuclides in environmental remediation.

Quality evaluation of Houttuynia cordata Thunb. by high performance liquid chromatography with photodiode-array detection (HPLC-DAD).

A new, validated method, developed for the simultaneous determination of 16 phenolics (chlorogenic acid, scopoletin, vitexin, rutin, afzelin, isoquercitrin, narirutin, kaempferitrin, quercitrin, quercetin, kaempferol, chrysosplenol D, vitexicarpin, 5-hydroxy-3,3',4',7-tetramethoxy flavonoids, 5-hydroxy-3,4',6,7-tetramethoxy flavonoids and kaempferol-3,7,4'-trimethyl ether) in Houttuynia cordata Thunb. was successfully applied to 35 batches of samples collected from different regions or at different times and their total antioxidant activities (TAAs) were investigated. The aim was to develop a quality control method to simultaneously determine the major active components in H. cordata. The HPLC-DAD method was performed using a reverse-phase C18 column with a gradient elution system (acetonitrile-methanol-water) and simultaneous detection at 345 nm. Linear behaviors of method for all the analytes were observed with linear regression relationship (r(2)>0.999) at the concentration ranges investigated. The recoveries of the 16 phenolics ranged from 98.93% to 101.26%. The samples analyzed were differentiated and classified based on the contents of the 16 characteristic compounds and the TAA using hierarchical clustering analysis (HCA) and principal component analysis (PCA). The results analyzed showed that similar chemical profiles and TAAs were divided into the same group. There was some evidence that active compounds, although they varied significantly, may possess uniform anti-oxidant activities and have potentially synergistic effects.

Decitabine combined with cold atmospheric plasma induces pyroptosis via the ROS/Caspase-3/GSDME signaling pathway in Ovcar5 cells.

BACKGROUND: High methylation of the DFNA5 gene results in the absence of GSDME, a key protein that mediates pyroptosis, while decitabine demethylates the DFNA5 gene, resulting in high expression of the GSDME protein. Cold atmospheric plasma (CAP) is a novel anti-cancer method that induces tumor cell death. METHODS: The pyroptosis induced by decitabine in combination with CAP in Ovcar5 cells was evaluated. In particular, mitochondrial membrane potential was estimated by JC-1 staining, dehydrogenase (LDH) release was assessed by ELISA, Annexin V/PI staining was detected by flow cytometry, the cell cycle changes were evaluated using PI staining followed by detection by flow cytometry, and Caspase-9 cleavage, Caspase-3 cleavage and GSDME expression were evaluated by western blot. RESULTS: Decitabine resulted in high expression of the GSDME in Ovcar5 in a concentration-dependent manner and increased tumor cell sensitivity to CAP. CAP induced mitochondrial damage and activated the Caspase-9/Caspase-3 pathway. Therefore, decitabine combined with CAP induced Ovcar5 cell pyroptosis through Caspase-3 mediated GSDME cleavage. Reactive oxygen species (ROS) generated by CAP treatment played an important role in the CAP/decitabine combination-induced production of ROS, activation of Caspase-9/Caspase-3, GSDME cleavage and pyroptosis that ROS scavenger NAC inhibited all these processes. CONCLUSIONS: CAP combined with decitabine induced Caspase-3 activation, which cleaved decitabine-upregulated GSDME and ediated pyroptosis.

Transcriptome Analysis of the Immortal Human Keratinocyte HaCaT Cell Line Damaged by Tritiated Water.

Radioactive elements, such as tritium, have been released into the ocean in large quantities as a result of the reactor leakage accident. In this study, an MTT assay demonstrated that the viability of HacaT cells decreased after tritiated water treatment. Bioinformatics analysis was used to analyze gene changes in the HacaT cells. The sequencing results showed 267 significantly differentially expressed genes (DEGs), and GO enrichment analysis showed that the DEGs were mainly divided into three parts. The KEGG pathway analysis showed that the up-regulated DEGs were involved in Wnt and other pathways, while the down-regulated DEGs were involved in Jak-STAT and others. A Western blot assay was used to verify the parts of the sequencing results. This study was the first to explore the mechanism of tritiated water on HacaT cells using Transcriptome analysis. The results will provide a theoretical basis for the study of tritiated water hazard mechanisms.

Polysaccharides from Armillariella tabescens mycelia mitigate DSS-induced ulcerative colitis via modulating intestinal microbiota in mice.

Polysaccharides from Armillariella tabescens mycelia (AT) have a potent anti-inflammatory effect. Nevertheless, the impact of AT on dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) has not yet been illustrated. This study aimed to explore AT's impact on experimental UC mice and investigate its underlying mechanisms. Mice were given DSS, 0.2 g/kg AT or 0.4 g/kg AT for seven days, and the symptoms of UC were observed. The serum and colon samples were harvested to analyze the biochemical indices and inflammasome-related proteins. The feces were collected to analyze short-chain fatty acids (SCFAs) and gut microbiota. The present study found that AT improved the symptoms of UC, colonic oxidative stress, and inflammation. AT treatment elevated SCFAs contents and colonic barrier function. Furthermore, AT improved microbial community structure through the selective enrichment of beneficial bacterial species. In conclusion, these results underlined that AT improved DSS-induced colitis and inhibited colonic inflammation via regulating the intestinal microbial community and enhancing the colonic mucosal barrier.

Epigallocatechin-3-gallate ameliorates renal endoplasmic reticulum stress-mediated inflammation in type 2 diabetic rats.

Epigallocatechin-3-gallate (EGCG), an essential polyphenolic constituent found in tea leaves, possesses various potent biological activities. This research was undertaken to investigate the impact of EGCG against endoplasmic reticulum (ER) stress-mediated inflammation and to clarify the underlying molecular mechanism in type 2 diabetic kidneys. The male rats were randomized into four groups: normal, diabetic, low-dose EGCG, and high-dose EGCG. In type 2 diabetic rats, hyperglycemia and hyperlipidemia noticeably caused renal structural damage and dysfunction and aggravated ER stress. Meanwhile, sustained ER stress activated the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome and then upregulated the contents of inflammatory cytokines in the diabetic kidney. Following supplementation with 40 mg/kg and 80 mg/kg EGCG, hyperglycemia, hyperlipidemia, and renal histopathological alterations and dysfunction were noticeably ameliorated; renal ER stress, NLRP3 inflammasome, and inflammatory response were markedly repressed in the EGCG treatment groups. In summary, the current study highlighted the renoprotective effects of EGCG in type 2 diabetes and its mechanisms are mainly associated with the repression of ER stress-mediated NLRP3 inflammasome overactivation.

[Culture in vitro and plant regeneration of Panax japonicus].

OBJECTIVE: To study the condition of culture in vitro and plant regeneration of Panax japonicus. METHODS: Embryos, stems and leaves of P. japonicus were used as explants, effects of different hormones for callus induction and plant regeneration were studied and optimized. RESULTS: The optimal way to obtain sterile explant for seeds was sterilized in 75% ethyl alcohol for 60 s then 0.1% HgCL2 for 12 min; Stems and leaves were sterilized in 75% ethyl alcohol for 15 s then 5% NaClO for 5 min. Used MS as basic medium, the optimal hormones combination for callus induction of embryos, stems and leaves were MS + 1.5 mg/L NAA + 1.5 mg/L 2, 4-D + 0.1 mg/L KT; MS + 1.5 mg/L NAA + 1.0 mg/L 2,4-D + 0.1 mg/L KT; MS + 1.5 mg/L NAA + 1.0 mg/L 2,4-D + 0.2 mg/L KT respectindy under the illumination. But under the darkness,the optimal callus induction hormones combination for embryos leaves were MS + 1.0 mg/L NAA + 1.5 mg/L 2,4-D +0.2 mg/L KT; 1.5 mg/L NAA + 1.5 mg/L 2,4-D + 0.1 mg/L KT; MS + 1.5 mg/L NAA + 1.0 mg/L 2,4-D + 0.1 mg/L KT respectivety. The optimal medium for germination was MS + 3.0 mg/L 6-BA + 1.0 mg/L GA3. The optimal medium for roots generation was MS + 1.0 mg/L 6-BA + 3.0 mg/L IBA. CONCLUSION: We establish the system of culture in vitro and plant regeneration for P. japonicus.

Erratum: Ceramide synthase-4 orchestrates the cell proliferation and tumor growth of liver cancer in vitro and in vivo through the nuclear factor-κB signaling pathway.

[This corrects the article DOI: 10.3892/ol.2017.6365.].

Comprehensive circular RNA profiling reveals the regulatory role of circRNA_0007694 in papillary thyroid carcinoma.

PURPOSE: The present study aimed to identify differentially expressed circRNAs in thyroid cancer and verify their potential functions. METHODS: Next-generation sequencing was used to identify differentially expressed circRNAs between papillary thyroid carcinoma (PTC) tissues and paired pericarcinomatous tissues. Polymerase chain reaction and Sanger sequencing methods successfully identified hsa_circ_0007694. A hsa_circ_0007694 over-expression vector was prepared to determine the effect of this circRNA on proliferation, migration, invasion, apoptosis, and the cell cycle in PTC cells. An in vivo animal assay was conducted by injecting PTC cells into the chests of mice. Further, RNA-seq was performed, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, to verify the regulatory mechanism of hsa_circ_0007694. Western blotting was used to verify the genes thought to be involved in the hsa_circ_0007694 regulatory pathways based on KEGG analysis. RESULTS: We identified a circRNA, hsa_circ_0007694 that was down-regulated in PTC tissues compared to pericarcinomatous tissues. Over-expression of hsa_circ_0007694 promoted apoptosis and inhibited proliferation, migration, and invasion in PTC cells in vitro, and decreased tumor growth in vivo. Transcriptome sequence analysis suggested 129 differentially expressed genes between PTC tissue and paired pericarcinomatous tissue. KEGG analysis and western blotting indicated that the PI3K/AKT/mTOR and Wnt signaling networks are most likely to be related to hsa_circ_0007694 in thyroid cancer. CONCLUSION: The circRNA hsa_circ_0007694 is down-regulated in PTC and is therefore a potential therapeutic target.

[Characteristic comparison of mouse primary macrophages cultured in L929 cell conditioned medium].

The purpose of this study is to provide a culture for mouse bone marrow-derived macrophages (BMDM) and peritoneal macrophages (PM) and to characterize their molecular and cellular biology. The cell number and purity from the primary culture were assessed by cell counter and flow cytometry, respectively. Morphological features were evaluated by inverted microscope. Phagocytosis by macrophages was detected by the neutral red dye uptake assay. Phenotypic markers were analyzed by real-time fluorescent quantitative PCR. Our results show that the cell number was much higher from culture of BMDM than PM, while there was no significant difference regarding the percentage of F4/80+CD11b+ cells (98.30%±0.53% vs. 94.83%±1.42%; P>0.05). The proliferation rate of BMDM was significantly higher than PM in the presence of L929 cell conditioned medium, by using CCK-8 assay. However, PM appeared to adhere to the flask wall and extend earlier than BMDM. The phagocytosis capability of un-stimulated BMDM was significantly higher than PM, as well as lipopolysaccharide (LPS)-stimulated BMDM, except the BMDM stimulated by low dose LPS (0.1 µg/mL). Furthermore, Tnfα expression was significantly higher in un-stimulated BMDM than PM, while Arg1 and Ym1 mRNA expression were significantly lower than PM. The expression difference was persistent if stimulated by LPS+IFN-γ or IL-4. Our data indicate that bone marrow can get larger amounts of macrophages than peritoneal cavity. However, it should be aware that the molecular and cellular characteristics were different between these two culture systems.

Involvement of activated astrocyte and microglia of locus coeruleus in cardiac pain processing after acute cardiac injury.

OBJECTIVE: It is still not known whether the glial cell activation of locus coeruleus (LC) is involved in the neurophysiologic mechanism of the acute phase of heart disease. The aim of this study was to investigate whether the glial cell activation of LC responds to acute cardiac injury (ACI). METHODS: In this study, ACI was established by intramyocardial injection of formalin. Afterward, we analysed c-Fos, OX42, GFAP and P2X(4)R expression levels in the LC of the rats by immunofluorescence staining or Western blot analysis. RESULTS: There was no significant difference in the levels of these markers in the LC between the normal control and the sham-operated groups. Following ACI, up-regulation of GFAP, OX42 and P2X(4)R expression levels were observed in locus coeruleus of the rats. The peak expression time was at hour 24. P2X(4)R was colocalized with OX42 in activated microglias, but not with GFAP in activated astrocytes. Compared with the control group, the ACI group showed a high expression level of c-Fos at hour 1 with a peak expression level at hour 2. CONCLUSION: The results showed that LC glia cells, like neurons, could sensitively respond to cardiovascular nociception induced by ACI at different time points. Results of this study may provide insights into the role of glial activation in response to ACI and may represent a potential strategy for investigation of neurophysiologic mechanism of cardiac pain.

Development of rat antigen-presenting cells from pluripotent ecto-mesenchymal stem cells in vitro and in vivo.

Dendritic cells (DC) are specialized cells that capture and present antigen to T cells. Recent advances have been made in understanding their origin, heterogeneity, and the signals that induce their migration and maturation resident microglia are antigen-presenting cells (APC) involved in stimulation or reactivation of CNS-targeted T cells. Generation of DC from microglia, as demonstrated ex vivo, may support GM-CSF-driven differentiation of brain DC from local, likely, microglial progenitors. Here, we report the establishment of long-term cultures of rat ecto-mesenchymal stem cells (EMSCs) using specific supplemented media for induction. These EMSCs share some morphological characteristics and the allostimulatory capacity of classical DCs, and when transplanted into the brain using a rat glioma model survive within the cortex, and are morphologically and phenotypically similar to microglia over 7 days. Our findings related to the development and differentiation of microglial progenitors support the view that microglia are derived prenatally from mesodermal progenitors that are distinct from monocytes.

Coagulation factors VII, IX and X are effective antibacterial proteins against drug-resistant Gram-negative bacteria.

Infections caused by drug-resistant "superbugs" pose an urgent public health threat due to the lack of effective drugs; however, certain mammalian proteins with intrinsic antibacterial activity might be underappreciated. Here, we reveal an antibacterial property against Gram-negative bacteria for factors VII, IX and X, three proteins with well-established roles in initiation of the coagulation cascade. These factors exert antibacterial function via their light chains (LCs). Unlike many antibacterial agents that target cell metabolism or the cytoplasmic membrane, the LCs act by hydrolyzing the major components of bacterial outer membrane, lipopolysaccharides, which are crucial for the survival of Gram-negative bacteria. The LC of factor VII exhibits in vitro efficacy towards all Gram-negative bacteria tested, including extensively drug-resistant (XDR) pathogens, at nanomolar concentrations. It is also highly effective in combating XDR Pseudomonas aeruginosa and Acinetobacter baumannii infections in vivo. Through decoding a unique mechanism whereby factors VII, IX and X behave as antimicrobial proteins, this study advances our understanding of the coagulation system in host defense, and suggests that these factors may participate in the pathogenesis of coagulation disorder-related diseases such as sepsis via their dual functions in blood coagulation and resistance to infection. Furthermore, this study may offer new strategies for combating Gram-negative "superbugs".

Maxillary second molar with four roots and five canals.

In this case report, we present a maxillary second molar variant, which had two palatal roots with two canals and two buccal roots with three canals, including a second mesiobuccal canal. A 44-year-old female patient complained about a tooth crown fracture and severe pain in her right maxillary second molar. A clinical intraoral inspection and radiography were carried out on the tooth, and a diagnosis of chronic apical periodontitis was made. Four roots (two buccal and two palatal) and five canals (three buccal and two palatal) were found. The anatomical variation of the tooth was further confirmed by cone-beam computed tomography, a cone-fit procedure, and a radiograph with a shifted projection angle. Root-canal treatment was performed under an endodontic microscope.

Knockdown of angiopoietin-like 4 inhibits the development of human gastric cancer.

Human gastric cancer (GC) is the second most common cause of cancer-related deaths worldwide and is one of the most common metastatic cancers. Tumor proliferation, apoptosis, metastasis and invasion are important predictors of the invasiveness of GC and are key factors in cancer-induced death. Angiopoietin-like 4 (ANGPTL4) is a secreted protein that belongs to the angiopoietin (ANGPTL) family and is involved in the regulation of cancer metastasis. However, whether ANGPTL4 plays a role in the progression of GC remain unclear. In the present study, immunoreactivity of ANGPTL4 demonstrated that ANGPTL4 expression was upregulated in GC tissues with the development of GC. The siRNA targeting ANGPTL4 effectively knocked down ANGPTL4 in the SNU­1 and BGC823 cell lines at the mRNA and protein levels. Following ANGPTL4 downregulation, the proliferation and invasion abilities of GC cell lines were suppressed as determined by MTT and Transwell assays, and cell apoptosis level and sensitivity to cisplatin were increased as determined by flow cytometry and MTT assay. In conclusion, these findings suggest that ANGPTL4 may be a new potential therapeutic target for GC.

(-)-Epigallocatechin-3-Gallate Ameliorates Atherosclerosis and Modulates Hepatic Lipid Metabolic Gene Expression in Apolipoprotein E Knockout Mice: Involvement of TTC39B.

Background: Aberrant chronic inflammation and excess accumulation of lipids play a pivotal role in the occurrence and progression of atherosclerosis. (-)-Epigallocatechin-3-gallate (EGCG), the major catechins in green tea, displayed anti-atherosclerotic properties in vivo and in vitro. However, the effects and underlying mechanism of EGCG on atherosclerosis remain unclear. Methods: Male apolipoprotein E-knockout (ApoE-/-) mice (7 weeks old) fed with high-fat diet (HFD) were treated with normal saline or EGCG (40 mg/kg/d, i.g.) for 18 weeks. Atherosclerotic plaque and liver lipid accumulation were measured by Oil Red staining. Plasma lipids and cytokines were detected using commercial kits. The expression of protein and mRNA was analyzed by western blot and quantitative real-time reverse transcription-polymerase chain reaction, respectively. Results: EGCG administration markedly attenuated atherosclerotic plaque formation in HFD-fed ApoE-/- mice, which were accompanied by increased plasma interleukin-10 (IL-10) level and decreased plasma IL-6 and tumor necrosis factor-α (TNF-α) levels. In addition, EGCG modulated high-fat-induced dyslipidemia, evidencing by decreased total cholesterol (TC) and low-density lipoprotein levels and increased high-density lipoprotein level. Meanwhile, EGCG treatment alleviated high-fat-mediated liver lipid accumulation and decreased liver TC and triglyceride. Mechanistically, EGCG significantly modulated high-fat-induced hepatic tetratricopeptide repeat domain protein 39B (TTC39B) expression and its related genes (Lxrß, Abcg5, Abcg8, Abca1, Srebf1, Scd1, Scd2, Fas, Elovl5, Mylip) expression in liver from ApoE-/- mice. Notably, EGCG remarkably induced hepatic liver X receptor α (LXRα) and LXRß expression and inhibited both precursor and mature sterol regulatory element binding transcription factor-1 (SREBP-1) expression. Conclusion: Taken together, our data for the first time suggested that TTC39B was involved in EGCG-mediated anti-atherosclerotic effects through modulation of LXR/SREBP-1 pathway.

Corrigendum: (-)-Epigallocatechin-3-Gallate Ameliorates Atherosclerosis and Modulates Hepatic Lipid Metabolic Gene Expression in Apolipoprotein E Knockout Mice: Involvement of TTC39B.

[This corrects the article on p. 195 in vol. 9, PMID: 29593532.].

Ceramide synthase-4 orchestrates the cell proliferation and tumor growth of liver cancer in vitro and in vivo through the nuclear factor-κB signaling pathway.

Liver cancer is one of the leading causes of cancer-associated mortalities worldwide, partly due to the absence of effective therapeutic targets and diagnostic biomarkers. Therefore, novel molecular targets are critical to develop new therapeutic approaches for liver cancer. In the present study, ceramide synthase-4 (CERS4) was investigated as a novel molecular target for liver cancer. High expression of CERS4 in liver cancer tissues was detected by reverse transcription polymerase chain reaction and western blot analysis. Subsequently, CERS4 was silenced by lentivirus-mediated RNA interfere, and the proliferation rates of liver cancer cells were significantly suppressed (P<0.001). In addition, the weight and volume of the tumors were reduced subsequent to silencing of CERS4 in liver cancer cells, revealed by an in vivo study using Balb/c nude mice. In addition, the nuclear factor (NF)-κB signaling pathway was affected following knockdown of CERS4 in liver cancer cells. The present results proposed that CERS4 is an important regulator of liver cancer cell proliferation and indicated that CERS4 may be a potential anticancer therapeutic target and a promising diagnostic biomarker for human liver cancer.

Role of reactive oxygen species-mediated MAPK and NF-κB activation in polygonatum cyrtonema lectin-induced apoptosis and autophagy in human lung adenocarcinoma A549 cells.

Polygonatum cyrtonema lectin (PCL), a mannose/sialic acid-binding lectin isolated from the rhizomes of Polygonatum cyrtonema Hua, has been reported to possess remarkable anti-tumour effects via inducing apoptosis and autophagy. The aim of this study was to investigate the molecular mechanisms mediating PCL-induced apoptosis and autophagy in A549 cells. Herein, we found that the treatment of A549 cells with PCL caused a remarkable generation of reactive oxygen species (ROS) and ROS scavenger N-acetyl-cysteine (NAC) inhibited PCL-induced apoptosis and autophagy. In addition, PCL treatment activated mitogen-activated protein kinase (MAPK) members extracellular signal-regulated kinase (ERK), JNK and p38, JNK inhibitor and p38 inhibitor partially reduced PCL-induced apoptosis and autophagy. Moreover, PCL administration activated NF-κB survival pathway in A549 cells, NF-κB inhibitor Bay11-7082 promoted PCL-induced apoptosis. Importantly, we found PCL may bind to the cell surface in a mannose-specific manner, and was then internalized and accumulated primarily onto the mitochondria. These findings may provide a new perspective of PCL as a potential anti-tumour drug targeting apoptosis and autophagy pathways for future cancer therapeutics.