All-trans retinoic acid downregulates HBx levels via E6-associated protein-mediated proteasomal degradation to suppress hepatitis B virus replication.

All-trans retinoic acid (ATRA), recognized as the principal and most biologically potent metabolite of vitamin A, has been identified for its inhibitory effects on hepatitis B virus (HBV) replication. Nevertheless, the underlying mechanism remains elusive. The present study reveals that ATRA induces E6-associated protein (E6AP)-mediated proteasomal degradation of HBx to suppress HBV replication in human hepatoma cells in a p53-dependent pathway. For this effect, ATRA induced promoter hypomethylation of E6AP in the presence of HBx, which resulted in the upregulation of E6AP levels in HepG2 but not in Hep3B cells, emphasizing the p53-dependent nature of this effect. As a consequence, ATRA augmented the interaction between E6AP and HBx, resulting in substantial ubiquitination of HBx and consequent reduction in HBx protein levels in both the HBx overexpression system and the in vitro HBV replication model. Additionally, the knockdown of E6AP under ATRA treatment reduced the interaction between HBx and E6AP and decreased the ubiquitin-dependent proteasomal degradation of HBx, which prompted a recovery of HBV replication in the presence of ATRA, as confirmed by increased levels of intracellular HBV proteins and secreted HBV levels. This study not only contributes to the understanding of the complex interactions between ATRA, p53, E6AP, and HBx but also provides an academic basis for the clinical employment of ATRA in the treatment of HBV infection.

Reactive Oxygen Species Induction by Hepatitis B Virus: Implications for Viral Replication in p53-Positive Human Hepatoma Cells.

Hepatitis B virus (HBV) infects approximately 300 million people worldwide, causing chronic infections. The HBV X protein (HBx) is crucial for viral replication and induces reactive oxygen species (ROS), leading to cellular damage. This study explores the relationship between HBx-induced ROS, p53 activation, and HBV replication. Using HepG2 and Hep3B cell lines that express the HBV receptor NTCP, we compared ROS generation and HBV replication relative to p53 status. Results indicated that HBV infection significantly increased ROS levels in p53-positive HepG2-NTCP cells compared to p53-deficient Hep3B-NTCP cells. Knockdown of p53 reduced ROS levels and enhanced HBV replication in HepG2-NTCP cells, whereas p53 overexpression increased ROS and inhibited HBV replication in Hep3B-NTCP cells. The ROS scavenger N-acetyl-L-cysteine (NAC) reversed these effects. The study also found that ROS-induced degradation of the HBx is mediated by the E3 ligase Siah-1, which is activated by p53. Mutations in p53 or inhibition of its transcriptional activity prevented ROS-mediated HBx degradation and HBV inhibition. These findings reveal a p53-dependent negative feedback loop where HBx-induced ROS increases p53 levels, leading to Siah-1-mediated HBx degradation and HBV replication inhibition. This study offers insights into the molecular mechanisms of HBV replication and identifies potential therapeutic targets involving ROS and p53 pathways.

Pleiotropic functions of SscA on the asexual spore of the human pathogenic fungus Aspergillus fumigatus.

Asexual spores, called conidia, are key reproductive fungal particles that enable survival in harsh environmental conditions or host systems. The conidia can infect humans, animals, and plants to cause various fungal diseases. Transcription factors, including VosA, WetA, and SscA, have key roles in conidia formation and long-term survival in Aspergillus nidulans. Herein, we report the pleiotropic functions of SscA in the conidia of the human pathogen A. fumigatus. The deletion of sscA increased conidia formation despite decreased fungal growth. Absence of sscA impaired long-term survival and reduced spore resistance to various stresses, including heat, UV, and oxidation. Transcriptomic analyses showed that SscA involved the mRNA expression of cell wall organisation-related genes. Importantly, the sscA deletion mutant conidia contained an increased amount of ß-glucan and chitin compared to wild type conidia. In addition, conidial gliotoxin production was decreased in the sscA deletion strain. Overall, SscA has pleiotropic roles in conidia formation, maturation and dormancy and mycotoxin production in A. fumigatus.

pH-Responsive Cellulose/Silk/Fe3O4 Hydrogel Microbeads Designed for Biomedical Applications.

In this study, cellulose/Fe3O4 hydrogel microbeads were prepared through the sol-gel transition of a solvent-in-oil emulsion using various cellulose-dissolving solvents and soybean oil without surfactants. Particularly, 40% tetrabutylammonium hydroxide (TBAH) and 40% tetrabutylphosphonium hydroxide (TBPH) dissolved cellulose at room temperature and effectively dispersed Fe3O4, forming cellulose/Fe3O4 microbeads with an average diameter of ~15 µm. Additionally, these solvents co-dissolved cellulose and silk, allowing for the manufacture of cellulose/silk/Fe3O4 hydrogel microbeads with altered surface characteristics. Owing to the negatively charged surface characteristics, the adsorption capacity of the cellulose/silk/Fe3O4 microbeads for the cationic dye crystal violet was >10 times higher than that of the cellulose/Fe3O4 microbeads. When prepared with TBAH, the initial adsorption rate of bovine serum albumin (BSA) on the cellulose/silk/Fe3O4 microbeads was 18.1 times higher than that on the cellulose/Fe3O4 microbeads. When preparing TBPH, the equilibrium adsorption capacity of the cellulose/silk/Fe3O4 microbeads for BSA (1.6 g/g) was 8.5 times higher than that of the cellulose/Fe3O4 microbeads. The pH-dependent BSA release from the cellulose/silk/Fe3O4 microbeads prepared with TBPH revealed 6.1-fold slower initial desorption rates and 5.2-fold lower desorption amounts at pH 2.2 than those at pH 7.4. Cytotoxicity tests on the cellulose and cellulose/silk composites regenerated with TBAH and TBPH yielded nontoxic results. Therefore, cellulose/silk/Fe3O4 microbeads are considered suitable pH-responsive supports for orally administered protein pharmaceuticals.

Development of Blended Biopolymer-Based Photocatalytic Hydrogel Beads for Adsorption and Photodegradation of Dyes.

Blended biopolymer-based photocatalytic hydrogel beads were synthesized by dissolving the biopolymers in 1-ethyl-3-methylimidazolium acetate ([Emim][Ac]), adding TiO2, and reconstituting the beads with ethanol. The incorporation of modifying biopolymer significantly enhanced the adsorption capacity of the cellulose/TiO2 beads. Cellulose/carrageenan/TiO2 beads exhibited a 7.0-fold increase in adsorption capacity for methylene blue (MB). In contrast, cellulose/chitosan/TiO2 beads showed a 4.8-fold increase in adsorption capacity for methyl orange (MO) compared with cellulose/TiO2 beads. In addition, cellulose/TiO2 microbeads were prepared through the sol-gel transition of the [Emim][Ac]-in-oil emulsion to enhance photodegradation activity. These microbeads displayed a 4.6-fold higher adsorption capacity and 2.8-fold higher photodegradation activity for MB than the millimeter-sized beads. Furthermore, they exhibited superior dye removal efficiencies for various dyes such as Congo red, MO, MB, crystal violet, and rhodamine B, surpassing the performance of larger beads. To expand the industrial applicability of the microbeads, biopolymer/TiO2 magnetic microbeads were developed by incorporating Fe2O3. These magnetic microbeads outperformed millimeter-sized beads regarding the efficiency and time required for MB removal from aqueous solutions. Furthermore, the physicochemical properties of magnetic microbeads can be easily controlled by adjusting the type of biopolymer modifier, the TiO2 and magnetic particle content, and the ratio of each component based on the target molecule. Therefore, biopolymer-based photocatalytic magnetic microbeads have great potential not only in environmental fields but also in biomedical fields.

All-trans Retinoic Acid Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation.

All-trans retinoic acid (ATRA), the most biologically active metabolite of vitamin A, is known to abolish the potential of HBx to downregulate the levels of p14, p16, and p21 and to stimulate cell growth during hepatitis B virus (HBV) infection, contributing to its chemopreventive and therapeutic effects against HBV-associated hepatocellular carcinoma. Here, we demonstrated that ATRA antagonizes HBx to inhibit HBV replication. For this effect, ATRA individually or in combination with HBx upregulated p53 levels, resulting in upregulation of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induces ubiquitination and proteasomal degradation of HBx in the presence of ATRA. The ability of ATRA to induce Siah-1-mediated HBx degradation and the subsequent inhibition of HBV replication was proven in an in vitro HBV replication model. The effects of ATRA became invalid when either p53 or Siah-1 was knocked down by a specific shRNA, providing direct evidence for the role of p53 and Siah-1 in the negative regulation of HBV replication by ATRA.

Reswellable alginate/activated carbon/carboxymethyl cellulose hydrogel beads for ibuprofen adsorption from aqueous solutions.

In this study, alginate (Alg) composite beads were prepared by blending with activated carbon (AC) to enhance adsorption capacity for ibuprofen and carboxymethyl cellulose (CMC) to create a reswellable hydrogel. The dried Alg/AC/CMC composite beads could be recovered to sizes and morphologies similar to the initial hydrogel states via a simple reswelling process; however, the dried Alg/AC composite beads without CMC could not be recovered to the initial hydrogel state. Following the reswelling process, the dried Alg/AC/CMC beads demonstrated an 86 % recovery (qe = 34.0 mg/g) in the adsorption capacity for ibuprofen compared to the initial hydrogel beads (qe = 39.6). In contrast, the reswelled Alg/AC beads exhibited only 18 % (qe = 8.6) of the initial adsorption capacity (qe = 48.1). We elucidated the effects of the substitution degree of CMC, AC content, and solution pH on the reswelling property and ibuprofen adsorption capacity of the Alg/AC/CMC composite beads. The adsorption kinetics and isotherms of the prepared composite beads in the hydrogel and reswelled states fit the pseudo-second-order and Langmuir models, respectively. Furthermore, the reswelled Alg composite beads exhibited high adsorption capacity (>93 %) after 10 cycles. Taken together, our findings indicate that the Alg/AC/CMC composite beads can be used as adsorbents without a considerable decrease in adsorption performance by reswelling the beads with distilled water after long-term storage in a dry state.

Hepatitis B Virus X Protein Stimulates Hepatitis C Virus (HCV) Replication by Protecting HCV Core Protein from E6AP-Mediated Proteasomal Degradation.

Most clinical and experimental studies have suggested that hepatitis C virus (HCV) is dominant over hepatitis B virus (HBV) during coinfection, although the underlying mechanism remains unclear. In this study, we found that the HBV X protein (HBx) upregulates the levels of the HCV core protein to stimulate HCV replication during coinfection in human hepatoma cells. For this purpose, HBx upregulated both the protein levels and enzyme activities of cellular DNA methyltransferase 1 (DNMT1) and DNMT3b, and this subsequently reduced the expression levels of the E6-associated protein (E6AP), an E3 ligase of the HCV core protein, via DNA methylation. The ubiquitin-dependent proteasomal degradation of the HCV core protein was severely impaired in the presence of HBx, whereas this effect was not observed when E6AP was either ectopically expressed or restored by treatment with 5-aza-2'dC or DNMT1 knockdown. The effect of HBx on the HCV core protein was accurately reproduced in HBV/HCV coinfection systems, which were established by either monoinfection by HCV in Huh7D cells transfected with a 1.2-mer HBV replicon or coinfection by HBV and HCV in Huh7D-Na+-taurocholate cotransporting polypeptide cells, providing evidence for the stimulation of HCV replication by HBx. The present study may provide insights into understanding HCV dominance during HBV/HCV coinfection in patients. IMPORTANCE Hepatitis B virus (HBV) and hepatitis C virus (HCV) are major human pathogens that cause a substantial proportion of liver diseases worldwide. As the two hepatotropic viruses have the same modes of transmission, coinfection is often observed, especially in areas and populations where HBV is endemic. High-risk populations include people who inject drugs. Both clinical and experimental studies have shown that HCV is more dominant than HBV during coinfection, but the underlying mechanism remains unclear. In this study, we show that HBV X protein (HBx) stimulates HCV replication by inhibiting the expression of E6-associated protein (E6AP) via DNA methylation, thereby protecting the HCV core protein from proteasomal degradation, which can contribute to HCV dominance during HBV/HCV coinfection.

Tumor Suppressor p53 Inhibits Hepatitis B Virus Replication by Downregulating HBx via E6AP-Mediated Proteasomal Degradation in Human Hepatocellular Carcinoma Cell Lines.

HBx, a multifunctional regulatory protein, plays an essential role in the replication and pathogenesis of the hepatitis B virus (HBV). In this study, we found that in human hepatoma cells, the tumor suppressor p53 downregulates HBx via ubiquitin-dependent proteasomal degradation. p53 transcriptional activity that results from HBV infection was not essential for this effect. This was shown by treatment with a potent p53 inhibitor, pifithrin-α. Instead, we found that p53 facilitated the binding of E6-associated protein (E6AP), which is an E3 ligase, to HBx and induced E6AP-mediated HBx ubiquitination in a ternary complex of p53, E6AP, and HBx. The ability of p53 to induce E6AP-mediated downregulation of HBx and inhibit HBV replication was demonstrated in an in vitro HBV infection system. This study may provide insights into the regulation of HBx and HBV replication, especially with respect to p53 status, which may also help in understanding HBV-associated tumorigenesis in patients.

Hepatitis C virus core protein inhibits hepatitis B virus replication by downregulating HBx levels via Siah-1-mediated proteasomal degradation during coinfection.

Most clinical and experimental studies have suggested that hepatitis C virus (HCV) is dominant over hepatitis B virus (HBV) during coinfection, although the mechanism remains unclear. Here, we found that HCV core protein inhibits HBV replication by downregulating HBx levels during coinfection in human hepatoma cells. For this effect, HCV core protein increased reactive oxygen species levels in the mitochondria and activated the ataxia telangiectasia mutated-checkpoint kinase two pathway in the nucleus, resulting in an upregulation of p53 levels. Accordingly, HCV core protein induced p53-dependent activation of seven in absentia homolog one expression, an E3 ligase of HBx, resulting in the ubiquitination and proteasomal degradation of HBx. The effect of the HCV core protein on HBx levels was accurately reproduced in both a 1.2-mer HBV replicon and in vitro HBV infection systems, providing evidence for the inhibition of HBV replication by HCV core protein. The present study may provide insights into the mechanism of HCV dominance in HBV- and HCV-coinfected patients.

Proteasomal activator 28 gamma stabilizes hepatitis B virus X protein by competitively inhibiting the Siah-1-mediated proteasomal degradation.

Proteasomal activator 28 gamma (PA28γ) upregulates the levels of HBx, a regulatory protein of hepatitis B virus (HBV) to stimulate HBV replication; however, the detailed mechanism remains unknown. Here, we found that PA28γ impaired the ability of seven in absentia homolog 1 (Siah-1) as an E3 ubiquitin ligase of HBx. PA28γ competitively inhibited the binding of Siah-1 to HBx in human hepatoma cells. Accordingly, PA28γ increased the stability of HBx and decreased HBx ubiquitination, abolishing the potential of Siah-1 to downregulate HBx levels. PA28γ also executed its role as an antagonist of Siah-1 during HBV replication, as demonstrated by an in vitro HBV replication system. The present study may provide insights into the mechanisms underlying the regulation of HBV replication.

The Link between ENSO-like Forcing and Hydroclimate Variability of Coastal East Asia during the Last Millennium.

Inconsistent reconstructions of East Asian hydroclimate for the last millennium significantly limit our understanding of the mechanisms behind climate variability during the medieval climate anomaly (MCA) and little ice age (LIA) in the region. In this study, we present new high-resolution multiproxy records (diatom, δ13C, C/N, TS) from the Mulyoungari swamp, Jeju Island, South Korea. Our results indicate that El Niño southern oscillation-like variations caused the dry MCA/wet LIA pattern in the study area. Recent paleo-ENSO studies generally support the hypothesis that the MCA was characterized by more persistent El Niño-like conditions. During El Niño events, the genesis of typhoons affecting coastal East Asia tends to diminish because of warm anomalies of eastern tropical Pacific (ETP) SSTs and downward motions over the western tropical Pacific. Therefore, coastal East Asia likely experienced a decline in typhoon-related precipitation during the MCA, in contrast to monsoon-dominated northern China. Our results additionally imply that SST anomalies in the ETP need to be carefully checked to better understand current hydroclimate variability in coastal East Asia, one of the most populated areas on earth.