[FU Li-xin's experience in treatment of vertigo with "regulating the middle jiao, opening gate and relaxing tendon" method of acupuncture].

The paper introduces professor FU Li-xin's theoretic ideas and experience in treatment of vertigo. Professor FU believes that this disease is closely related to the blockage of qi movement in the middle jiao, opening-closing disarrangement in the pivot, "gate" obstruction, malnutrition of brain orifice and decreased blood flow in the nape. Based on the holistic idea of qi movement in traditional Chinese medicine and the circulatory theory of western medicine, the characteristics of the specific acupuncture therapy for "regulating the middle jiao, opening gate and relaxing tendon" are summarized. Using the layered needling technique at Zhongwan (CV 12) and "gate points" in the neck region, the tendon-bone needling technique with modified "dark tortoise seeking hole" at local tendon blockage points, vertigo is cured through regulating qi in the middle jiao, opening gate and nourishing marrow, relaxing tendon and harmonizing the mind.

Exploring DNA Methylation Profiles Altered in Cryptogenic Hepatocellular Carcinomas by High-Throughput Targeted DNA Methylation Sequencing: A Preliminary Study for Cryptogenic Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) includes cryptogenic hepatocellular carcinomas (CR-HCC) that lack a defined cause. Specific DNA methylation patterns and comparisons of the aberrant alterations in DNA methylation between CR-HCC and adjacent peritumor tissues (APTs) have not yet been reported. METHODS: The SureSelectXT Methyl-Seq Target Enrichment System was used to sequence targeted DNA methylation in three paired CR-HCC tissues and APTs. Gene Ontology (GO) enrichment and KEGG pathway analysis were performed to investigate the DNA methylation mechanism of CR-HCC. The mRNA expression levels of HOXB-AS3, HOXB6, HOXB3, USP18, MAP3K6, TIRAP, TNNI2, SHC3, CTTN, and TFAP2A, selected from the identified signaling pathways, were evaluated by quantitative real-time PCR (qPCR). RESULTS: A total of 1728 differentially methylated regions (DMRs) were identified in tumor tissues compared with non-tumor tissues, of which 868 DMRs were hypermethylated and 860 were hypomethylated. The DMRs were mapped within 2091 DMR-associated genes (DMGs). The mRNA expression of HOXB-AS3, HOXB3, and MAP3K6 was downregulated in CR-HCC tissues compared to the APTs. However, the mRNA expression of TIRAP, SHC3, and CTTN was upregulated in the CR-HCC tissues. Differences between the mRNA expression of HOXB6, USP18, TNNI2, and TFAP2A in the CR-HCC and APTS tissues were not statistically significant. GO analysis showed that the molecular functions of "binding", "protein binding", and "cytoskeletal protein binding" were the main categories for the hypermethylated DMGs. The hypomethylated DMGs were mostly enriched in the molecular functions "binding", "protein binding", "calcium ion binding", among others. KEGG pathway analysis showed that the hypermethylated DMGs were enriched in several pathways such as "estrogen signaling pathway", while hypomethylated DMGs were enriched in several pathways such as "proteoglycans in cancer", suggesting that epigenetic modifications play important roles in the cryptogenic hepatocarcinogenesis. CONCLUSION: These results provide useful information for future work to characterize the functions of epigenetic mechanisms on CR-HCC.

Complete chloroplast genome sequence of Chimonanthus praecox link (Calycanthaceae): an endemic plant species in China.

Chimonanthus praecox, a deciduous shrub tree, is endemic to China and widely cultivated in the world as a popular garden and ornamental plant. Here, we have reported its complete chloroplast genome with a length of 153,181 bp, containing a large single copy (LSC) region of 86,916 bp, a small single copy (SSC) region of 19,767 bp and two identical inverted repeat regions (IRs) of 23,249 bp. The overall GC contents of the plastome were 39.27%. A total of 114 unique genes were successfully annotated consisting of 80 protein-coding genes, 30 tRNA genes and four rRNA genes. Sixteen genes each possessed one intron and three genes had two introns. The ML phylogenetic analysis supports Chimonanthus as sister to Calycanthus. This result will be helpful for genetic breeding and population genetics of C. praecox, DNA barcoding of Chimonanthus, and phylogenetic studies of Calycanthaceae.

Research progress on circadian clock genes in common abdominal malignant tumors.

The circadian clock refers to the inherent biological rhythm of an organism, which, is accurately regulated by numerous clock genes. Studies in recent years have reported that the abnormal expression of clock genes is ubiquitous in common abdominal malignant tumors, including liver, colorectal, gastric and pancreatic cancer. In addition, the abnormal expression of certain clock genes is closely associated with clinical tumor parameters or patient prognosis. Studies in clock genes may expand the knowledge about the mechanism of occurrence and development of tumors, and may provide a new approach for tumor therapy. The present study summarizes the research progress in this field.

HBx protein promotes oval cell proliferation by up-regulation of cyclin D1 via activation of the MEK/ERK and PI3K/Akt pathways.

Growing evidence has shown that hepatic oval cells, also named liver progenitor cells, play an important role in the process of liver regeneration in various liver diseases. Oval cell proliferation has been reported in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and chronic liver disease. Studies have found expression of HBV surface and core antigens in oval cells in the livers of patients with HCC, suggesting that HBV infection of oval cells could be a mechanism of human hepatocarcinogenesis. In addition, there is evidence of multiplication of HBV in oval cell culture. However, little research has been performed to explore the role of HBV-encoded proteins in the proliferation of hepatic oval cells. Previously, we successfully transfected the HBV x (HBx) gene, one of the four genes in the HBV genome, into a rat LE/6 oval cell line. In this study, we tested whether or not the transfected HBx gene could affect oval cell proliferation in vitro. Our results show that overexpression of HBx promotes the proliferation of oval cells and increases cyclin D1 expression, assessed at both the mRNA and protein levels. We also found that HBx activated the PI-3K/Akt and MEK/ERK1/2 pathways in HBx-transfected oval cells. Furthermore, the HBx-induced increases in cyclin D1 expression and oval cell proliferation were completely abolished by treatment with either MEK inhibitor PD184352 or PI-3K inhibitor LY294002. These results demonstrated that HBx has the ability to promote oval cell proliferation in vitro, and its stimulatory effects on cell proliferation and expression of cyclin D1 depend on the activation of the MEK/ERK and PI3K/Akt signaling pathways in cultured oval cells.

Hepatic oval cell lines generate hepatocellular carcinoma following transfection with HBx gene and treatment with aflatoxin B1 in vivo.

Hepatic oval cells (HOC) are considered to be the stem cells of the liver and have been linked to the development of hepatic malignancies. Studies have demonstrated that chronic hepatitis B virus (HBV) infection and dietary aflatoxin B1 (AFB1) exposure are among the most important risk factors for the development of hepatocellular carcinoma (HCC). However, little research has been done to evaluate the role of oval cells in these two environmental factors on hepatocarcinogenesis. In this study, partial transformation of rat HOC (LE/6) were accomplished by transfected HBV x gene (HBx), and then transfected cells were implanted both intra-hepatically and subcutaneously into nude mice treated with AFB1 in vivo. We found the oval cells produced tumors (4/24 of the animals) in liver following transfection with HBx gene and treatment with AFB1. These intrahepatic tumors included HCC cells (immunopositive for HepParl, ALB, CK8 and AFP) and mesenchymal cells (immunopositive for Vimentin and SMA). Whereas mesenchymal tumors were observed at the subcutaneous tissue with a similar rate in all controls treated with cell lines (10/24 in HBx-oval cells/AFB1 group, 8/20 in HBx-oval cells/non-AFB1 group, 10/20 in non-HBx/AFB1 group; 9/20 in non-HBx/non-AFB1 group). Conversely, none of the controls developed intrahepatic tumors. These results provide an evidence that oval cells have the capacity to generate HCC through the combined effects of the HBx and AFB1 in the liver microenvironment.

[The hyposensitization of oval cell to antiproliferative effect of transforming growth factor ß1 by hepatitis B virus X gene].

OBJECTIVE: To determine whether hepatitis B virus X (HBX) protein expression affect the oval cells' response to anti-proliferative effect of transforming growth factor ß1 (TGFß1) in oval cells. METHODS: Real-time PCR, Western blot analysis were performed to detect the expression of TGFßRII in HBX-transfected oval cells named HBX-EGFP-LE/6, and EGFP-LE/6, LE/6 control cells. In addition, exogenous TGFß1 was added into all three oval cell lines, MTT assay was preformed to clarify different responses to the anti-proliferative effect of TGFß1. RESULTS: The TGFßRII mRNA levels in LE/6 and EGFP-LE/6 cells were (10.2 ± 1.8) and (8.8 ± 0.9) folds of those in HBX-EGFP-LE/6 cells, the difference was significant (P < 0.05). HBX protein expression also reduced the protein levels of TGFßRII in HBX-EGFP-LE/6 oval cells, compared to the control cells. The MTT results exhibited that, after TGFß1 addition, proliferative inhibition rate in the HBX-EGFP-LE/6 cells was 18.1% ± 1.5% while those in control cells were 42.2% ± 2.8% and 41.9% ± 5.0%, the difference was significant (P < 0.01). CONCLUSION: HBX protein expression affects TGFßRII transcriptional activity and protein synthesis, and insensitive oval cells to anti-proliferative effect of TGFß1.

[Construction of pEGFP-HBx gene recombinant plasmid and establishment of stable transfected HBx gene liver oval cell strain].

OBJECTIVE: To construct pEGFP-HBx eukaryotic expression plasmid and establish stable and effective transfected rat oval cell (LE/6) strain expressing EGFP-HBx fusion protein to explore the roles of HBx gene and oval cell in carcinogenesis of hepatocellular carcinoma (HCC). METHODS: HBx gene with EcoRI and Hind III endo-enzyme sites was obtained by using PCR from plasmid pcDNA3.1-HBx. The purified HBx gene fragment was inserted into pEGFP-N1 expression vector, and the recombinant plasmid pEGFP-HBx was identified by restriction endonuclease and DNA sequencing analysis. LE/6 cells were transfected with recombinant pEGFP-HBx by lipofectamine reagent. Resistant to G418 clones were selected, and expression of EGFP-HBx fusion protein in clones were examined directly with fluorescence microscope, and these clones were isolated and proliferated. The expression of HBx was detected by RT-PCR analysis and immunocytochemistry. RESULTS: Plasmid pEGFP-HBx has whole HBx gene base and correct reading frame as indicated by restriction endonuclease and DNA sequencing analysis. After transfecting with pEGFP-HBx plasmid, LE/6 cell clones expressing EGFP-HBx fusion protein were obtained. RT-PCR analysis and immunocytochemistry showed that HBx gene was only expression in transfected pEGFP-HBx cells. CONCLUSIONS: The pEGFP-HBx recombinant expression vector was successfully constructed, and the stable transfected LE/6 strain expressing EGFP-HBx fusion protein was successfully established. It will be helpful in the further study on the roles of HBx and liver oval cell in carcinogenesis of HCC.