DDX17-regulated alternative splicing that produced an oncogenic isoform of PXN-AS1 to promote HCC metastasis.

BACKGROUND AND AIMS: The mechanism underlying HCC metastasis remains unclear, many oncogenes are known to regulate this process. However, the role of alternative splicing (AS) in pro-metastatic HCC is poorly understood. APPROACH AND RESULTS: By performing RNA sequencing on nine pairs of primary HCC tissues with extrahepatic metastasis (EHMH) and nine pairs of metastasis-free HCC (MFH) tissues, we depicted the AS landscape in HCC and found a higher frequency of AS events in EHMH compared with MFH. Moreover, 28 differentially expressed splicing regulators were identified in EHMH compared with MFH. Among these, DEAD-box RNA helicase 17 (DDX17) was significantly up-regulated in EHMH and was strongly associated with patient outcome. Functional studies indicated that DDX17 knockout inhibited the degradation of the extracellular matrix, and diminished the invasive ability of HCC cells. A significant reduction in lung metastasis induced by DDX17 deficiency was also demonstrated in a diethylnitrosamine-induced DDX17HKO mouse model. Mechanistically, high DDX17 induced intron 3 retention of PXN-AS1 and produced a transcript (termed PXN-AS1-IR3). The transcript PXN-AS1-IR3 acted as an important promoter of HCC metastasis by inducing MYC transcription activation via recruiting the complex of testis expressed 10 and p300 to the MYC enhancer region, which led to transcriptional activation of several metastasis-associated downstream genes. Finally, the PXN-AS1-IR3 level was significantly higher in serum and HCC tissues with extrahepatic metastasis. CONCLUSIONS: DDX17 and PXN-AS1-IR3 act as important metastatic promoters by modulating MYC signaling, suggesting that DDX17 and PXN-AS1-IR3 may be potential prognostic markers for metastatic HCC.

An accurate, rapid, and sensitive method for simultaneous determination of four typical heterocyclic amines in roasted pork patties: Application in the study of inhibitory effects of astaxanthin.

Heterocyclic aromatic amines, as a group of mutagenic and carcinogenic compounds, have gained worldwide concern. In this study, an accurate, rapid, and sensitive confirmation and quantification method of four major heterocyclic aromatic amines in roasted pork was developed based on Q-Orbitrap along with Quick, Easy, Cheap, Effective, Rugged, and Safe extraction. The limit of detections and limit of quantitations were found to be 0.2-1.2 µg/kg and 0.6-3.5 µg/kg, respectively, revealing the high sensitivity of this method. Obtained results showed recoveries ranging from 78.1 to 97.4%, depending on the different heterocyclic aromatic amines and spiked levels. Precision was in the range of 2.6-4.5% for four heterocyclic aromatic amines at different levels. In addition, the developed method had been applied to investigate the inhibitory effects of astaxanthin on the above-mentioned heterocyclic aromatic amines in roasted pork. The amount of astaxanthin with the best inhibitory effects was 7.5 mg (0.0375%), which led to significant reduction in heterocyclic aromatic amines levels over 50%.

Fast construction of core-shell structured magnetic covalent organic framework as sorbent for solid-phase extraction of zearalenone and its derivatives prior to their determination by UHPLC-MS/MS.

Magnetic covalent organic framework nanocomposite denoted as Fe3O4@TAPB-Tp with core-shell structure was fabricated via a simple template-mediated precipitation polymerization method at mild conditions. The polyimine network shell was created through the polymerization of 1,3,5-tris(4-aminophenyl)-benzene (TAPB) and 1,3,5-triformyl-phloroglucinol (Tp) in tetrahydrofuran (THF) by the Schiff-base reaction. Featuring with large specific surface area (163.19 m2 g-1), good solution dispersibility, and high stability, the obtained Fe3O4@TAPB-Tp exhibited high adsorption capacities and fast adsorption for zearalenone and its derivatives (ZEAs). The adsorption isotherms showed multilayer adsorption dominated at low concentration and monolayer adsorption at high concentration between the interface of ZEAs and Fe3O4@TAPB-Tp. With the Fe3O4@TAPB-Tp as sorbent, a magnetic solid-phase extraction-ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was established for simultaneous adsorption and detection of five ZEAs in complex samples. The proposed method displayed favorable linearity, low limits of detection (0.003 ~ 0.018 µg kg-1), and good repeatability (2.37~10.4%). The developed method has been applied for real sample analysis, with recoveries of 81.27~90.26%. These results showed that Fe3O4@TAPB-Tp has a good application potential for the adsorption of ZEAs in food samples. Magnetic covalent organic framework nanocomposite (Fe3O4@TAPB-Tp) were quickly fabricated at mild conditions and used as effective adsorbent for magnetic solid-phase extraction of zearalenone and its derivatives (ZEAs) from food samples prior to ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis.

Simultaneous determination of 22 phthalate esters in polystyrene food-contact materials by ultra-performance convergence chromatography with tandem mass spectrometry.

A method for the determination of 22 phthalate esters in polystyrene food-contact materials has been established using ultraperformance convergence chromatography with tandem mass spectrometry. In this method, 22 phthalate esters were analyzed in <3.5 min on an ACQUITY Tours 1-AA column by gradient elution. The mobile phase, the compensation solvent, the flow rate of mobile phase, column temperature, and automatic back pressure regulator pressure were optimized, respectively. There was a good linearity of 20 phthalate esters with a range of 0.05-10 mg/L, diisodecyl phthalate and diisononyl phthalate were 0.25-10 mg/L, and the correlation coefficients of all phthalates were higher than 0.99 and those of 16 phthalates were higher than 0.999. The limits of detection and the limits of quantification of 15 phthalates were 0.02 and 0.05 mg/kg, meanwhile diallyl phthalate, diisobutyl phthalate, dimethyl phthalate, di-n-butyl phthalate, and di(2-ethylhexyl) phthalate were 0.05 and 0.10 mg/kg, and diisodecyl phthalate and diisononyl phthalate were 0.10 and 0.25 mg/kg. The spiked recoveries were in the range of 76.26-107.76%, and the relative standard deviations were in the range of 1.78-12.10%. Results support this method as an efficient alternative to apply for the simultaneous determination of 22 phthalate esters in common polystyrene food-contact materials.

[Extremely low frequency electromagnetic field induces apoptosis of osteosarcoma cells via oxidative stress].

OBJECTIVE: To investigate the effects of extremely low frequency electromagnetic field (ELF-EMF) on human osteosarcoma cells and its mechanisms. METHODS: Human osteosarcoma MG-63 cells were exposed to 50 Hz,1 mT ELF-EMF for 1, 2 and 3 h in vitro, with or without pretreatment by reactive oxygen species (ROS) inhibitor N acetylcysteine (NAC) or p38MAPK inhibitor SB203580. The proliferation of MG-63 cells was determined by MTT method; the apoptosis rate and ROS level in MG-63 cells were detected by flow cytometry. The expression of p38MAPK in MG-63 cells was determined by Western blotting. RESULTS: ELF-EMF decreased the viability of MG-63 cells, inhibited cell growth, induced cell apoptosis and increased the level of ROS significantly. The apoptosis rate declined significantly after treatment with ROS inhibitor NAC or p38MAPK inhibitor SB203580. After exposure to ELF-EMF, p38MAPK in MG-63 cells was activated, and the phosphorylation level was also inhibited after treatment with NAC. CONCLUSION: ELF-EMF can induce the apoptosis of MG-63 cells. Increased ROS and p38MAPK activation may be involved in the mechanism.

RNA-binding protein RPS7 promotes hepatocellular carcinoma progression via LOXL2-dependent activation of ITGB1/FAK/SRC signaling.

BACKGROUND: Metastasis is one of the leading cause contributes to treatment failure and poor prognosis of hepatocellular carcinoma (HCC) patients. The underlying mechanism of HCC metastasis remains to be determined. Although several RNA binding proteins (RBPs) have been found to participate in tumorigenesis and progression of liver cancer, the role of RBPs in HCC patients with extrahepatic metastases is poorly understood. METHODS: By performing RNA-seq of primary HCC tissues (including HCC with extrahepatic metastasis and those did not develop metastasis), we identified a set of HCC metastasis-associated RBPs candidates. Among which, ribosomal protein S7 (RPS7) was found to be remarkably increased in HCC tissues and be strongly related to HCC poor survival. Overexpression or CRISPR-Cas9-mediated knockout were applied to investigate the role of RPS7 on the metastasis-associated phenotypes of HCC cells. RNA sequencing, RIP, RNA-pull down, dual luciferase reporter assay, nascent RNA capture assay, and RNA decay and so on, were applied to reveal the underlying mechanism of RPS7 induced HCC metastasis. RESULTS: Gain- and loss- of function analyses revealed that RPS7 promoted HCC cells adhesion, migration and invasion capabilities, as well as lung metastasis. Mechanistically, we uncovered that lysyl oxidase-like 2 (LOXL2) was a critical downstream target of RPS7. RPS7 could stabilize LOXL2 mRNA by binding to AUUUA motifs in the 3155-3375 region of the 3'UTR of LOXL2 mRNA, thus increased LOXL2 expression via elevating LOXL2 mRNA abundance. Further research revealed that LOXL2 could accelerate focal adhesion formation through maintaining the protein stability of ITGB1 and activating ITGB1-mediated FAK/SRC signaling pathway, and thereby contribute to the pro-metastasis effect of RPS7. CONCLUSIONS: Taken together, our data reveal a novel function of RPS7 in HCC metastasis, also reveal the critical roles of the RPS7/LOXL2/ITGB1 axis in HCC metastasis and shed new light on the exploration of molecular drugs against HCC.

Development of Magnetic Molecularly Imprinted Polymer Coupled Nanospray Ion Source for Analysis of Cephalosporin Antibiotics in Food Samples.

A magnetic molecularly imprinted polymer (MMIP) coupled nanospray ion source was developed for analysis of cephalosporin antibiotics in food samples. MIP coated Fe3O4 nanospheres were prepared for magnetic solid-phase extraction (MSPE) of the antibiotics in the extract of samples and then integrated into the nanospray capillary for further desorption and mass spectrometry analysis. The developed device combines the advantages of high extraction efficiency of MSPE, unique selectivity of MIPs, and fast analysis speed of ambient ionization mass spectrometry (AIMS). Five cephalosporin antibiotics in milk, egg, and beef samples were analyzed using the developed methods. High sensitivities with limits of detection (LODs) from 0.3 to 0.5 µg kg-1 were achieved for cephalosporin antibiotics in milk, egg, and beef samples, respectively. Good linearity, determination coefficient values (R2 > 0.992), and precision (RSD < 15%) with recoveries ranging from 72.6% to 115.5% were obtained using the spiked milk, egg, and beef sample matrices.

HBx Mediated Increase of DDX17 Contributes to HBV-Related Hepatocellular Carcinoma Tumorigenesis.

HBV is strongly associated with HCC development and DEAD-box RNA helicase 17 (DDX17) is a very important member of the DEAD box family that plays key roles in HCC development by promoting cancer metastasis. However, the important role of DDX17 in the pathogenesis of HBV-related HCC remains unclear. In this study, we investigated the role of DDX17 in the replication of HBV and the development of HBV-associated HCC. Based on data from the GEO database and HBV-infected cells, we found that DDX17 was upregulated by the HBV viral protein X (HBx). Mechanistically, increased DDX17 expression promoted HBV replication and transcription by upregulating ZWINT. Further study showed that DDX17 could promote HBx-mediated HCC metastasis. Finally, the promotive effect of DDX17 on HBV and HBV-related HCC was confirmed in vivo. In summary, the results revealed the novel role of DDX17 in the replication of HBV and the metastasis of HBV-associated HCC.

KAT2A Promotes Hepatitis B Virus Transcription and Replication Through Epigenetic Regulation of cccDNA Minichromosome.

Hepatitis B virus (HBV) infection remains a major health problem worldwide. Sufficient maintenance of the HBV covalently closed circular DNA (cccDNA), which serves as a template for HBV transcription, is responsible for the failure of antiviral therapies. While accumulating evidence suggests that cccDNA transcription is regulated by epigenetic machinery, particularly the acetylation and methylation of cccDNA-bound histone 3 (H3) and histone 4 (H4), the potential contributions of histone succinylation and related host factors remain obscured. Here, by screening a series of succinyltransferases and desuccinylases, we identified KAT2A as an important host factor of HBV transcription and replication. By using HBV-infected cells and mouse models with HBV infection, KAT2A was found to affect the transcriptional activity of cccDNA but did not affect cccDNA production. Mechanism studies showed that KAT2A is mainly located in the nucleus and could bind to cccDNA through interaction with HBV core protein (HBc). Moreover, we confirmed histone H3K79 succinylation (H3K79succ) as a histone modification on cccDNA minichromosome by using the cccDNA ChIP-Seq approach. Importantly, KAT2A silencing specifically reduced the level of cccDNA-bound succinylated H3K79. In conclusion, KAT2A promotes HBV transcription and replication through epigenetic machinery, and our findings may provide new insight into the treatment of HBV infection.

Anti-inflammatory effects of potato extract on a rat model of cigarette smoke-induced chronic obstructive pulmonary disease.

OBJECTIVE: This study aimed to evaluate the therapeutic effects of potato extract (PE) on cigarette smoke (CS)-induced chronic obstructive pulmonary disease (COPD). METHODS: PE was first prepared by frozen centrifugation, and its amino acid composition was detected. Toxicity of PE was analyzed by changes in morphology, behavior, routine blood indexes, and biochemical criteria of mice. Then, the COPD rat model was established by CS exposure, and PE, doxofylline, and prednisolone acetate were used to treat these rats. After 45 days of treatment, the morphology and behavior of rats were recorded. In addition, the histopathology of lung tissue was evaluated by chest x-ray and hematoxylin and eosin staining. The expression of interleukine-10 (IL-10), tumor necrosis factor-α (TNF-α), and granulocyte colony-stimulating factor (G-CSF) was detected in serum and lung tissue by enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. RESULTS: Various amino acids were identified in PE, and no toxicity was exhibited in mice. The CS-induced COPD rat model was successfully established, which exhibited significant thickened and disordered lung markings on 90% of the rats. After administering doxofylline and prednisolone acetate, inflammation symptoms were improved. However, side effects such as emaciation, weakness, and loosening of teeth appeared. In the PE group, obviously improved histopathology was observed in lung tissues. Meanwhile, it was revealed that PE could increase the expression of IL-10 and reduce the expression of TNF-α and G-CSF in COPD rats, and doxofylline and prednisolone acetate also elicited similar results. CONCLUSION: Our study suggests PE might be effective in the treatment of CS-induced COPD by inhibiting inflammation.

Molecularly imprinted microspheres and nanospheres for di(2-ethylhexyl)phthalate prepared by precipitation polymerization.

Molecularly imprinted microspheres (MIMs, >3 microm) and nanospheres (MINs, approximately 450 nm) for the environmental endocrine disruptor di(2-ethylhexyl)phthalate (DEHP) were prepared by a precipitation polymerization (PP) procedure. The effect of the dispersive solvents acetonitrile (ACN) and cyclohexane (CH), the cross-linkers ethylene glycol dimethacrylate (EDMA) and trimethylpropane trimethacrylate (TRIM), and the template on particle size and morphology of polymers was investigated in detail by scanning electron microscopy (SEM) and BET adsorption isotherm determination. When used as HPLC stationary phase, the microspheres exhibited strong affinity for the template DEHP with an imprint factor (IF) higher than 8.0 in ACN/water (60:40, v/v) as mobile phase. Furthermore, baseline separation of DEHP from benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) could be achieved. In contrast, no or only poor separation could be observed with non-imprinted polymeric polymers (NIPs) or imprinted bulk polymers (MIB), respectively. Similarly, the obtained MINs exhibited an imprinting effect in pure ACN, i.e. the bond amount of DEHP was significantly higher compared to NIPs, as was shown in rebinding experiments. Besides their use as an HPLC stationary phase, MIMs might further be applicable for SPE sample cleanup, while MINs could be used as a recognition layer on sensor surfaces.

Molecularly imprinted polymer with salicylaldehyde-Cu(OAc)2 as template.

In the report molecularly imprinted polymer (MIP) with salicylaldehyde-Cu(OAc)(2) as the template was synthesized and characterized by SEM, porosity and elemental analysis. Copper acetate was introduced since salicylaldehyde alone cannot display imprinting effect for its intramolecular hydrogen bond. The strong coordination interaction between salicylaldehyde and copper acetate made the complex have high retention on the HPLC column based on the SAD-Cu(OAc)(2) imprinted polymer. Several structural analogues such as salicylaldoxime, sulfosalicylic acid, p-hydroxybenzaldehyde and their complexes with copper acetate were chosen to study the selectivity of the MIPs. The influence of acetic acid and H(2)O in methanol mobile phase was studied. The experimental results showed that small amount of either acetic acid or H(2)O in mobile phase would weaken the interaction between the complex and the polymer, therefore, the retention of the complex was lowered to a large extent, but that of salicylaldehyde remained almost unchanged. The polymer imprinted with the complex showed high selectivity to both the acetate and copper (II). In addition, the MIP showed an enhanced selectivity to its template compared with the polymer prepared without copper acetate.