The Characteristic Changes in Hepatitis B Virus X Region for Hepatocellular Carcinoma: A Comprehensive Analysis Based on Global Data

Objectives

Mutations in hepatitis B virus (HBV) X region (HBx) play important roles in hepatocarcinogenesis while the results remain controversial. We sought to clarify potential hepatocellular carcinoma (HCC)-characteristic mutations in HBx from HBV genotype C-infected patients and the distribution of those mutations in different disease phases and genotypes.

Methods

HBx sequences downloaded from an online global HBV database were screened and then classified into Non-HCC or HCC group by diagnosis information. Patients'' data of patient age, gender, country or area, and viral genotype were also extracted. Logistic regression was performed to evaluate the effects of mutations on HCC risk.

Results

1) Full length HBx sequences (HCC: 161; Non-HCC: 954) originated from 1115 human sera across 29 countries/areas were extracted from the downloaded 5956 HBx sequences. Genotype C occupied 40.6% of Non-HCC (387/954) and 89.4% of HCC (144/161). 2) Sixteen nucleotide positions showed significantly different distributions between genotype C HCC and Non-HCC groups. 3) Logistic regression showed that mutations A1383C (OR: 2.32, 95% CI: 1.34-4.01), R1479C/T (OR: 1.96, 95% CI: 1.05-3.64; OR: 5.15, 95% CI: 2.53-10.48), C1485T (OR: 2.40, 95% CI: 1.41-4.08), C1631T (OR: 4.09, 95% CI: 1.41-11.85), C1653T (OR: 2.58, 95% CI: 1.59-4.19), G1719T (OR: 2.11, 95% CI: 1.19-3.73), and T1800C (OR: 23.59, 95% CI: 2.25-247.65) were independent risk factors for genotype C HBV-related HCC, presenting different trends among individual disease phases. 4) Several genotype C HCC risk mutations pre-existed, even as major types, in early disease phases with other genotypes.

Conclusions

Mutations associated with HCC risk were mainly located in HBx transactivation domain, viral promoter, protein/miRNA binding sites, and the area for immune epitopes. Furthermore, the signatures of these mutations were unique to disease phases leading to HCC, suggesting molecular counteractions between the virus and host during hepatocarcinogenesis.     

Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4⁺CD8⁺ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5⁺K8⁺) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5⁺K8⁺ TEC persisted. Interestingly, a surgical lesion of the inner ear’s vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living.     

Demonstration of pollinator‐mediated competition between two native Impatiens species,Impatiens noli‐tangere and I. textori (Balsaminaceae)

Plant–plant interspecific competition via pollinators occurs when the flowering seasons of two or more plant species overlap and the pollinator fauna is shared. Negative sexual interactions between species (reproductive interference) through improper heterospecific pollen transfer have recently been reported between native and invasive species demonstrating pollination‐driven competition. We focused on two native Impatiens species (I. noli‐tangere and I. textori) found in Japan and examined whether pollinator‐mediated plant competition occurs between them. We demonstrate that I. noli‐tangere and I. textori share the same pollination niche (i.e., flowering season, pollinator fauna, and position of pollen on the pollinator's body). In addition, heterospecific pollen grains were deposited on most stigmas of both I. noli‐tangere and I. textori flowers that were situated within 2 m of flowers of the other species resulting in depressed fruit set. Further, by hand‐pollination experiments, we show that when as few as 10% of the pollen grains are heterospecific, fruit set is decreased to less than half in both species. These results show that intensive pollinator‐mediated competition occurs between I. noli‐tangere and I. textori. This study suggests that intensive pollinator‐mediated competition occurs in the wild even when interacting species are both native and not invasive.     

Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction

Homoserine dehydrogenase (HSD; 305 amino acid residues) catalyzes an NAD(P)-dependent reversible reaction between l-homoserine and aspartate 4-semialdehyde and is involved in the aspartate pathway. HSD from the hyperthermophilic archaeon Sulfolobus tokodaii was markedly activated (2.5-fold) by the addition of 0.8 mM dithiothreitol. The crystal structure of the homodimer indicated that the activation was caused by cleavage of the disulfide bond formed between two cysteine residues (C303) in the C-terminal regions of the two subunits.     

Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.     

Effects of nitric oxide scavengers on thermoinhibition of seed germination in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Plant seeds sometimes do not germinate at elevated temperature. The thermoinhibition mechanisms of seed germination have yet not revealed. Here we describe a chemical approach to improve seed germination at high temperature. We compared the temperature response of germination between wild-type Arabidopsis thaliana and its T-DNA insertion mutant ΔAtGLB3 that lacks a functional gene encoding GLB3, a homologue of bacterial truncated Hb (trHb). Under optimal temperature conditions (e.g. 22°C), the seeds of ΔAtGLB3 and the wild type germinated at a frequency near 100%. In contrast, at 32°C the seeds of ΔAtGLB3 did not germinate while wild-type seeds retained the same high germination frequency. The germination of ΔAtGLB3 at 32°C was partially restored by supplementation with the nitric oxide-specific scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO; cPTIO), 3-(3,4-dihydroxycinnamoyl)quinic acid, bovine serum Hb, or isoprene. The results presented in this study suggest that chemical scavengers for reactive nitrogen species potentially improve seed germination at high temperature.     

Molecular basis for the differences in lipid and lipoprotein binding properties of human apolipoproteins E3 and E4

Human apolipoprotein (apo) E4 binds preferentially to very low-density lipoproteins (VLDLs), whereas apoE3 binds preferentially to high-density lipoproteins (HDLs), resulting in different plasma cholesterol levels for the two isoforms. To understand the molecular basis for this effect, we engineered the isolated apoE N-terminal domain (residues 1-191) and C-terminal domain (residues 192-299) together with a series of variants containing deletions in the C-terminal domain and assessed their lipid and lipoprotein binding properties. Both isoforms can bind to a phospholipid (PL)-stabilized triolein emulsion, and residues 261-299 are primarily responsible for this activity. ApoE4 exhibits better lipid binding ability than apoE3 as a consequence of a rearrangement involving the segment spanning residues 261-272 in the C-terminal domain. The strong lipid binding ability of apoE4 coupled with the VLDL particle surface being ～60% PL-covered is the basis for its preference for binding VLDL rather than HDL. ApoE4 binds much more strongly than apoE3 to VLDL but less strongly than apoE3 to HDL(3), consistent with apoE-lipid interactions being relatively unimportant for binding to HDL. The preference of apoE3 for binding to HDL(3) arises because binding is mediated primarily by interaction of the N-terminal helix bundle domain with the resident apolipoproteins that cover ～80% of the HDL(3) particle surface. Thus, the selectivity in the binding of apoE3 and apoE4 to HDL(3) and VLDL is dependent upon two factors: (1) the stronger lipid binding ability of apoE4 relative to that of apoE3 and (2) the differences in the nature of the surfaces of VLDL and HDL(3) particles, with the former being largely covered with PL and the latter with protein.     

Ethanol production from cellulosic materials using cellulase-expressing yeast

We demonstrate direct ethanol fermentation from amorphous cellulose using cellulase-co-expressing yeast. Endoglucanases (EG) and cellobiohydrolases (CBH) from Trichoderma reesei, and β-glucosidases (BGL) from Aspergillus aculeatus were integrated into genomes of the yeast strain Saccharomyces cerevisiae MT8-1. BGL was displayed on the yeast cell surface and both EG and CBH were secreted or displayed on the cell surface. All enzymes were successfully expressed on the cell surface or in culture supernatants in their active forms, and cellulose degradation was increased 3- to 5-fold by co-expressing EG and CBH. Direct ethanol fermentation from 10 g/L phosphoric acid swollen cellulose (PASC) was also carried out using EG-, CBH-, and BGL-co-expressing yeast. The ethanol yield was 2.1 g/L for EG-, CBH-, and BGL-displaying yeast, which was higher than that of EG- and CBH-secreting yeast (1.6 g/L ethanol). Our results show that cell surface display is more suitable for direct ethanol fermentation from cellulose.