Hepatitis B Virus X Protein Stimulates Virus Replication Via DNA Methylation of the C-1619 in Covalently Closed Circular DNA.

Methylation of HBV cccDNA has been detected in vivo and in vitro; however, the mechanism and its effects on HBV replication remain unclear. HBx derived from a 1.2-mer HBV replicon upregulated protein levels and enzyme activities of DNA methyltransferase 1 (DNMT1), 3a, and 3b, resulting in methylation of the negative regulatory region (NRE) in cccDNA, while none of these effects were observed with an HBx-null mutant. The HBx-positive HBV cccDNA expressed higher levels of HBc and produced about 4-fold higher levels of HBV particles than those from the HBx-null counterpart. For these effects, HBx interrupted the action of NRE binding protein via methylation of the C-1619 within NRE, resulting in activation of the core promoter. Treatment with 5-Aza-2'dC or DNMT1 knock-down drastically impaired the ability of HBx to activate the core promoter and stimulate HBV replication in 1.2-mer HBV replicon and in vitro infection systems, indicating the positive role of HBx-mediated cccDNA methylation in HBV replication.

Hepatitis B virus X protein activates proteasomal activator 28 gamma expression via upregulation of p53 levels to stimulate virus replication.

Proteasomal activator gamma (PA28γ), frequently overexpressed in hepatocellular carcinoma, is believed to play important roles in tumourigenesis. However, the underlying mechanism of PA28γ overexpression and its possible roles in hepatitis B virus (HBV) replication are largely unknown. In the present study, we found that hepatitis B virus X protein (HBx) activates PA28γ expression by upregulating p53 levels in human hepatoma cells. The elevated PA28γ levels in turn repressed seven in absentia homologue 1 expression via downregulation of p53 levels, thereby inhibiting ubiquitin-dependent proteasomal degradation of HBx, which ultimately led to upregulation of HBx levels. The correlation among HBx, p53 and PA28γ was exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, knockdown of either p53 or PA28γ in HepG2 cells downregulated HBx levels and thereby inhibited HBV replication, whereas overexpression of p53 or PA28γ in Hep3B cells upregulated HBx levels, which stimulated HBV replication, indicating that p53 and PA28γ act as activators of HBV replication. In conclusion, HBx levels are upregulated via a positive feedback loop involving p53 and PA28γ to stimulate HBV propagation.

Hepatitis B virus X protein activates E3 ubiquitin ligase Siah-1 to control virus propagation via a negative feedback loop.

The seven in absentia homologue 1 (Siah-1) protein is an E3 ubiquitin ligase that induces ubiquitin-dependent proteasomal degradation of HBx, the principal regulatory protein of hepatitis B virus (HBV); however, its role in HBV propagation remains unknown. Here, we found that HBx upregulates Siah-1 levels in HepG2 but not in Hep3B cells, in which p53 is absent. For this effect, HBx sequentially activated ataxia telangiectasia mutated kinase and checkpoint kinase 2 via phosphorylation at the Ser-1981 and Thr-68 residues, respectively, which led to the activation of p53 via phosphorylation at the Ser-15 and Ser-20 residues. As a result, HBx was heavily ubiquitinated by Siah-1 and degraded by the ubiquitin-proteasome system in HepG2 cells, whereas this effect was marginal or undetectable in Hep3B cells. Knock-down of p53 in HepG2 cells downregulated Siah-1 levels and subsequently upregulated HBx levels, whereas ectopic p53 expression in Hep3B cells upregulated Siah-1 levels and subsequently downregulated HBx levels. In addition, Siah-1 knock-down impaired the ubiquitination and proteasomal degradation of HBx in HepG2 cells, whereas ectopic Siah-1 expression induced ubiquitin-dependent proteasomal degradation of HBx in Hep3B cells. The effects of HBx on p53 and Siah-1 were exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, Siah-1 knock-down upregulated the levels of HBx derived from the HBV replicon, resulting in an increase in HBV production. In conclusion, HBx modulates its own protein level via a negative feedback loop involving p53 and Siah-1 to control HBV propagation.

The Measurement of Palpebral Fissure Height Using the Intersection Angle (the Réal Angle) Between the Meeting Points of the Upper Eyelid and the Edge of the Cornea.

BACKGROUND: We evaluated a new palpebral fissure height measurement to evaluate medial, lateral, and overall ptosis. METHODS: We photographed 250 Koreans (44 males, 206 females) and evaluated their Réal 1 angle (angle between the meeting points of the upper eyelid and the corneal edge), Réal 2 angle (angle between the meeting point of the upper eyelid, medial corneal edge and a vertical line through the center of the pupil), Réal 3 angle (angle between the meeting point of the upper eyelid, lateral corneal edge and a vertical line through the center of the pupil), and Réal 4 angle (Réal 2-Réal 3). Angles were compared between sexes and age groups. We then evaluated the Réal angles of 13 Korean actresses. RESULTS: Mean age was 31.85 ± 14.60 years; Réal 1 was 129.01° ± 14.23°, Réal 2 was 68.20° ± 7.49°, Réal 3 was 60.80° ± 9.65°. There was no significant difference between the sexes in Réal 1, Réal 2, and Réal 3 angles. Réal 1 increased with age, and Réal 4 decreased with age. All Réal angles were significantly different between age groups. The actresses' mean age was 30.66 ± 8.01 years; Réal 1 was 102.84° ± 10.16°, Réal 2 was 57.87° ± 6.10°, and Réal 3 was 44.97° ± 8.74°. CONCLUSION: This simple measurement of palpebral fissure height using Réal angles consistently evaluated the amount of medial, lateral, and general ptosis. For average Korean eyes, the lateral portion of the upper eyelid is slightly higher than the medial portion; however, this lateral portion droops with age. Korean actresses have vertically higher eyes than average Korean women. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

DNA methyltransferase inhibitor assay system based on the HBx-induced DNA methylation of E-cadherin.

We here report a simple assay system for DNA methyltransferase (DNMT) inhibitors based on the HBx-induced DNA methylation of E-cadherin. A stable cell line named G1 was generated by co-transfecting E-cadherin luciferase reporter and HBx-expression plasmid into HepG2 cells. Treatment of G1 cells with DNMT inhibitors, 5-azacytidine, 5-aza-2'-deoxycytidine, and procainamaid, dose-dependently inhibited DNA methylation of E-cadherin promoter in the reporter, resulting in up-regulation of luciferase levels and its enzyme activity. Treatment with all-trans retinoic acid that is known to inhibit DNMT expression, also induced similar effects. Our system can be useful for development of epi-drugs targeting DNA methylation in malignancies.

Effects in the upper face of far east Asians after Oriental blepharoplasty: a scientific perspective on why Oriental blepharoplasty is essential.

BACKGROUND: Oriental blepharoplasty is the most frequently performed aesthetic surgery among far east Asians (i.e., Korean, Chinese, Japanese, and Taiwanese). Numerous studies on oriental blepharoplasty have been published, but only a few, if any, have reported on the measurable effects of such surgeries on the upper face. To scientifically evaluate the changes in the upper face of young Korean adults after oriental blepharoplasty, a periocular morphometry study was conducted. METHODS: A retrospective image analysis was used to identify the changes in the upper face after oriental blepharoplasty. Preoperative and postoperative digital facial images of 612 patients (598 women and 14 men) who underwent oriental blepharoplasty were examined. The height of the palpebral fissure (HPF), the distance between the highest point of the palpebral fissure of the upper eyelid and the eyebrow (DEE), and the distance between the eyebrow and the hairline (DEH) were measured to analyze the effects of oriental blepharoplasty in the upper face. RESULTS: After oriental blepharoplasty, the HPF and DEH increased in length by 11.8 and 4.9 %, respectively, and the DEE decreased in length by 14.0 %. Patients who originally did not possess double eyelids showed greater changes than those who originally did possess such eyelids. Such changes were characterized by a significant inverse correlation between the DEE and DEH. In addition, when postoperative changes were measured before and after 6 months, the palpebral fissure and forehead showed an even greater increase. CONCLUSIONS: Oriental blepharoplasty can induce substantial changes in the proportions of the upper face. It can not only increase the size of the eyes, but also induce changes in eyebrow position resulting in a more refreshed appearance.

Combination effect of paclitaxel and hyaluronic acid on cancer stem-like side population cells.

Cancer recurrence is the main cause of chemotherapeutic treatment failure. The mechanisms driving cancer recurrence may be due to very rare subpopulation cells, cancer stem-like cells (CSCs). Therefore, the early detection and better treatment of cancer stem-like cells are of great interest. In this study, we investigated how to eliminate the side population cells (SP), which have the characteristics of cancer stem-like cells, and also show chemotherapy resistance. Fluorescence-activated cell sorting (FACS) were used to sort SP and non-SP cells from human liver cancers, Huh-7 Hyaluronic acid (HA), which is an abundant component in the extracellular matrix, is known to involve in proliferation of normal and cancer cells. Herein, we investigated the effect of HA component on chemotherapy against SP cells. Cell growth inhibitory effects of the paclitaxel (PTX) chemotherapy combined with the HA component on SP cells of Huh-7 was determined using the trypan blue dye exclusion test. PTX combined with HA was found to show more increased inhibition of cell growth in both SP and non-SP cells, compared to free PTX treatment. In conclusion, SP cells of Huh-7 shows chemotherapeutic drug resistance due to the over-expressed efflux pumps. HA proposed one of possibilities to overcome the limitation of chemotherapy against cancer stem-like cells.

Hepatitis C virus Core protein overcomes all-trans retinoic acid-induced cell growth arrest by inhibiting retinoic acid receptor-ß2 expression via DNA methylation.

Aberrant promoter methylation of tumor suppressor genes including retinoic acid receptor-ß2 (RAR-ß2) is frequently detected in hepatitis C virus (HCV)-associated hepatocellular carcinoma; however, the mechanism and its significance are relatively unknown. Here, we showed that HCV Core induced promoter hypermethylation of RAR-ß2 to inhibit its expression via up-regulation of DNA methyltransferases 1 and 3b. Under the condition, all-trans retinoic acid (ATRA) failed to activate p16 expression and thus could not inactivate the Rb-E2F pathway. Accordingly, Core-expressing cells exhibited resistance to ATRA-induced growth inhibition. Taken together, HCV Core antagonizes ATRA, a natural anti-cancer compound, to stimulate cell growth via epigenetic down-regulation of RAR-ß2.