The N-linked glycosylation modifications in the hepatitis B surface protein impact cellular autophagy, HBV replication, and HBV secretion.

N-linked glycosylation is a pivotal post-translational modification that significantly influences various aspects of protein biology. Autophagy, a critical cellular process, is instrumental in cell survival and maintenance. The hepatitis B virus (HBV) has evolved mechanisms to manipulate this process to ensure its survival within host cells. Significantly, post-translational N-linked glycosylation in the large surface protein of HBV (LHBs) influences virion assembly, infectivity, and immune evasion. This study investigated the role of N-linked glycosylation of LHBs in autophagy, and its subsequent effects on HBV replication and secretion. LHBs plasmids were constructed by incorporating single-, double-, and triple-mutated N-linked glycosylation sites through amino acid substitutions at N4, N112, and N309. In comparison to the wild-type LHBs, N-glycan mutants, including N309Q, N4-309Q, N112-309Q, and N4-112-309Q, induced autophagy gene expression and led to autophagosome accumulation in hepatoma cells. Acridine orange staining of cells expressing LHBs mutations revealed impaired lysosomal acidification, suggesting potential blockage of autophagic flux at later stages. Furthermore, N-glycan mutants increased the mRNA expression of HBV surface antigen (HBsAg). Notably, N309Q significantly elevated HBx oncogene level. The LHBs mutants, particularly N309Q and N112-309Q, significantly enhanced HBV replication, whereas N309Q, N4-309Q, and N4-112-309Q markedly increased HBV progeny secretion. Remarkably, our findings demonstrated that autophagy is indispensable for the impact of N-linked glycosylation mutations in LHBs on HBV secretion, as evidenced by experiments with a 3-methyladenine (3-MA) inhibitor. Our study provides pioneering insights into the interplay between N-linked glycosylation mutations in LHBs, host autophagy, and the HBV life cycle. Additionally, we offer a new clue for further investigation into carcinogenesis of hepatocellular carcinoma (HCC). These findings underscore the potential of targeting either N-linked glycosylation modifications or the autophagic pathway for the development of innovative therapies against HBV and/or HCC.

Antibiofilm activity and bioactive phenolic compounds of ethanol extract from the Hericium erinaceus basidiome.

Biofilm formation has become a serious health and environmental problem. Mushrooms are now considered a valuable source of bioactive compounds with antimicrobial properties. The lion's mane mushroom (Hericium erinaceus [HE]) has been used as an antimicrobial for ulcers and gastritis in East Asian countries. However, studies on the antibiofilm activities of HE basidiome against biofilm-forming pathogenic bacteria and their bioactive compound profiles are still limited. The purpose of this study was to determine the antibiofilm activity of HE and to identify its phenolic compound profile. The HE inhibitory activities against bacterial growth and biofilm formation were performed against Pseudomonas aeruginosa, Salmonella Typhimurium, Proteus mirabilis, and Staphylococcus aureus. Remarkably, P. mirabilis was the most susceptible bacteria to HE. The total phenolic content (TPC) of HE was 1652 ± 1.06 µg/ml, with protocatechuic acid and p-coumaric acid being the most abundant phenolic compounds as determined by high-performance liquid chromatography-mass spectrophotometry (HPLC-MS). This research highlights the possibility of HE as an antibiofilm agent that can be developed as a nutraceutical and natural food preservative.

Autophagy related protein 9A increase in hepatitis B virus-associated hepatocellular carcinoma and the role in apoptosis.

The majority of hepatocellular carcinoma (HCC) cases are associated with the hepatitis B virus (HBV) infection. Autophagy related protein 9A (ATG9A) is a transmembrane protein required for autophagosome formation. In order to investigate the role of ATG9A in HBV-associated HCC, ATG9A protein expression was determined in tumor liver tissues and compared with adjacent nontumor tissues from HCC patients with or without HBV infection. In HBV-associated HCC tissues, ATG9A protein level was increased in tumor liver tissues, but not in cases of non-HBV HCC. Our findings suggested that ATG9A might be involved in HBV and cancer cell survival. Therefore, we aimed to analyze the function of ATG9A in HBV replication using RNA interference to evaluate the HBV DNA level using real-time PCR. In the present study, there were no significant differences between shATG9A-transfected HepG2.2.15 cells and the mock control. However, we found that silencing ATG9A affected apoptosis in HepG2.2.15 and HepG2 cell lines. Our results indicated that ATG9A might be partly involved in the survival of HCC. Thus, the inhibition of ATG9A together with other targets might be a potential drug target for HCC treatment.

PTM-Logo: a program for generation of sequence logos based on position-specific background amino-acid probabilities.

SUMMARY: Identification of the amino-acid motifs in proteins that are targeted for post-translational modifications (PTMs) is of great importance in understanding regulatory networks. Information about targeted motifs can be derived from mass spectrometry data that identify peptides containing specific PTMs such as phosphorylation, ubiquitylation and acetylation. Comparison of input data against a standardized 'background' set allows identification of over- and under-represented amino acids surrounding the modified site. Conventionally, calculation of targeted motifs assumes a random background distribution of amino acids surrounding the modified position. However, we show that probabilities of amino acids depend on (i) the type of the modification and (ii) their positions relative to the modified site. Thus, software that identifies such over- and under-represented amino acids should make appropriate adjustments for these effects. Here we present a new program, PTM-Logo, that generates representations of these amino acid preferences ('logos') based on position-specific amino-acid probability backgrounds calculated either from user-input data or curated databases. AVAILABILITY AND IMPLEMENTATION: PTM-Logo is freely available online at http://sysbio.chula.ac.th/PTMLogo/ or https://hpcwebapps.cit.nih.gov/PTMLogo/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

AbDesigner3D: a structure-guided tool for peptide-based antibody production.

Summary: We present AbDesigner3D, a new tool for identification of optimal immunizing peptides for antibody production using a peptide-based strategy. AbDesigner3D integrates 3D structural data from the Protein Data Bank (PDB) with UniProt data, which includes basic sequence data, post-translational modification sites, SNP occurrences and more. Other features, such as uniqueness and conservation scores, are calculated based on sequences from UniProt. The 3D visualization capabilities allow an intuitive interface, while an abundance of quantitative output simplifies the process of comparing immunogen peptides. Important quantitative features added in this tool include calculation and display of accessible surface area (ASA) and protein-protein interacting residues (PPIR). The specialized data visualization features of AbDesigner3D will greatly assist users to optimize their choice of immunizing peptides. Availability and implementation: AbDesigner3D is freely available at http://sysbio.chula.ac.th/AbDesigner3D or https://hpcwebapps.cit.nih.gov/AbDesigner3D/. Supplementary information: Supplementary data are available at Bioinformatics online.

Autophagy Related-Protein 16-1 Up-Regulated in Hepatitis B Virus-Related Hepatocellular Carcinoma and Impaired Apoptosis.

BACKGROUND: Hepatocellular carcinoma (HCC) as primary malignancy of the liver has become the most common type of cancer worldwide. HCC development is mainly caused by viruses, especially the hepatitis B virus (HBV). Autophagy is an important defense mechanism against virus infection; however, HBV promotes autophagy mediated by the HBx protein which stimulates its replication. The autophagy-related protein 16-1 (ATG16L1) binds to the ATG12-ATG5 conjugate and forms a large protein autophagosome complex. Previous studies indicated that the ATG12-ATG5 conjugate was involved in HBV-associated HCC. Therefore, the ATG16L1 protein might consistently relate to this condition. METHODS: Accordingly, the ATG16L1 protein expression was determined in tumor and non-tumor liver cell lines and liver tissue samples using immunoblotting, and also investigated in ATG16L1-knockdown cells to further clarify this function. RESULTS: Our results showed that the ATG16L1 protein was up-regulated in HepG2.2.15 and HepG2 cell lines compared to THLE-2 cells. This protein also increased in tumor liver tissues of HCC patients with HBV infection compared to adjacent non-tumor tissues. Silenced-ATG16L1 also significantly promoted apoptosis in HepG2 cells cultured in starvation conditions. CONCLUSIONS: Findings suggested ATG16L1 as an important molecule involved in apoptosis processes for HCC cells. A more profound understanding is required regarding the mechanisms that link autophagy and apoptosis in HCC development.

Increased ATG5-ATG12 in hepatitis B virus-associated hepatocellular carcinoma and their role in apoptosis.

AIM: To investigate autophagy-related genes, particularly ATG12, in apoptosis and cell cycle in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) and non-HBV-HCC cell lines. METHODS: The expression of autophagy-related genes in HBV-associated hepatocellular carcinoma and non-HBV-HCC cell lines and human liver tissues was examined by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting. The silencing of target genes was used to examine the function of various genes in apoptosis and cell cycle progression. RESULTS: The expression of autophagy related genes ATG5, ATG12, ATG9A and ATG4B expression was analyzed in HepG2.2.15 cells and compared with HepG2 and THLE cells. We found that ATG5 and ATG12 mRNA expression was significantly increased in HepG2.2.15 cells compared to HepG2 cells (P < 0.005). Moreover, ATG5-ATG12 protein levels were increased in tumor liver tissues compared to adjacent non-tumor tissues mainly from HCC patients with HBV infection. We also analyzed the function of ATG12 in cell apoptosis and cell cycle progression. The percentage of apoptotic cells increased by 11.4% in ATG12-silenced HepG2.2.15 cells (P < 0.005) but did not change in ATG12-silenced HepG2 cells under starvation with Earle's balanced salt solution. However, the combination blockade of Notch signaling and ATG12 decreased the apoptotic rate of HepG2.2.15 cells from 55.6% to 50.4% (P < 0.05). CONCLUSION: ATG12 is important for HBV-associated apoptosis and a potential drug target for HBV-HCC. Combination inhibition of ATG12/Notch signaling had no additional effect on HepG2.2.15 apoptosis.

Autophagy machinery impaired interferon signalling pathways to benefit hepatitis B virus replication.

BACKGROUND: Autophagy-related genes ATG4B, ATG7, and ATG12 have been identified to play a critical role in viral replication. However, these genes have yet to be identified in hepatitis B virus (HBV). OBJECTIVE: To characterise the role of ATG4B, ATG7, and ATG12 genes in HBV infection. METHODS: The mRNA expression was examined by quantitative real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) of the target gene was used to examine the function of the gene in HBV replication. Evaluation of HBV DNA level was performed by real-time PCR. RESULTS: Our findings revealed that ATG12 gene expression was significantly up-regulated (p < 0.005), whereas ATG7 gene expression was down-regulated (p < 0.0001) in HepG2.2.15 cells when compared to HepG2 cells. However, no significant difference in mRNA level of ATG4B was observed. These results were consistent with protein level findings. Moreover, we analysed the function of ATG12 in HBV replication by using ATG12 shRNA and evaluated HBV DNA level. We found that the amount of HBV was decreased in ATG12-knockdown HepG2.2.15 cells when compared to control HepG2.2.15 cells (P < 0.05). The mRNA expression of interferon-alpha (IFN-α), interferon-beta (IFN-ß), and interferon-inducible genes (IFI) was also investigated. Our results showed that the expression of IFN-α, IFN-ß, and IFI27 genes were increased in ATG12-knockdown cells but not in Mx1 gene when compared to control cells (p < 0.005, p < 0.0001 and p < 0.005, respectively). CONCLUSION: These autophagy-related genes, ATG12 may play a role in HBV replication via impairing IFN pathway. However, the biological significance of other autophagic genes such as ATG7 warrants further study.

PAAQD: Predicting immunogenicity of MHC class I binding peptides using amino acid pairwise contact potentials and quantum topological molecular similarity descriptors.

Prediction of peptide immunogenicity is a promising approach for novel vaccine discovery. Conventionally, epitope prediction methods have been developed to accelerate the process of vaccine production by searching for candidate peptides from pathogenic proteins. However, recent studies revealed that peptides with high binding affinity to major histocompatibility complex molecules (MHCs) do not always result in high immunogenicity. Therefore, it is promising to predict the peptide immunogenicity rather than epitopes in order to discover new vaccines more effectively. To this end, we developed a novel T-cell reactivity predictor which we call PAAQD. Nonapeptides were encoded numerically, using combining information of amino acid pairwise contact potentials (AAPPs) and quantum topological molecular similarity (QTMS) descriptors. Encoded data were used in the construction of our classification model. Our numerical experiments suggested that the predictive performance of PAAQD is at least comparable with POPISK, one of the pioneering techniques for T-cell reactivity prediction. Also, our experiment suggested that the first and eighth positions of nonapeptides are the most important for immunogenicity and most of the anchor residues in epitope prediction were not important in T-cell reactivity prediction. The R implementation of PAAQD is available at http://pirun.ku.ac.th/~fsciiok/PAAQD.rar.

EpicCapo: epitope prediction using combined information of amino acid pairwise contact potentials and HLA-peptide contact site information.

BACKGROUND: Epitope identification is an essential step toward synthetic vaccine development since epitopes play an important role in activating immune response. Classical experimental approaches are laborious and time-consuming, and therefore computational methods for generating epitope candidates have been actively studied. Most of these methods, however, are based on sophisticated nonlinear techniques for achieving higher predictive performance. The use of these techniques tend to diminish their interpretability with respect to binding potential: that is, they do not provide much insight into binding mechanisms. RESULTS: We have developed a novel epitope prediction method named EpicCapo and its variants, EpicCapo(+) and EpicCapo(+REF). Nonapeptides were encoded numerically using a novel peptide-encoding scheme for machine learning algorithms by utilizing 40 amino acid pairwise contact potentials (referred to as AAPPs throughout this paper). The predictive performances of EpicCapo(+) and EpicCapo(+REF) outperformed other state-of-the-art methods without losing interpretability. Interestingly, the most informative AAPPs estimated by our study were those developed by Micheletti and Simons while previous studies utilized two AAPPs developed by Miyazawa & Jernigan and Betancourt & Thirumalai. In addition, we found that all amino acid positions in nonapeptides could effect on performances of the predictive models including non-anchor positions. Finally, EpicCapo(+REF) was applied to identify candidates of promiscuous epitopes. As a result, 67.1% of the predicted nonapeptides epitopes were consistent with preceding studies based on immunological experiments. CONCLUSIONS: Our method achieved high performance in testing with benchmark datasets. In addition, our study identified a number of candidates of promiscuous CTL epitopes consistent with previously reported immunological experiments. We speculate that our techniques may be useful in the development of new vaccines. The R implementation of EpicCapo(+REF) is available at http://pirun.ku.ac.th/~fsciiok/EpicCapoREF.zip. Datasets are available at http://pirun.ku.ac.th/~fsciiok/Datasets.zip.

Association of interferon-gamma gene polymorphisms with susceptibility to oral lichen planus in the Thai population.

OBJECTIVE: Interferon-gamma (IFN-γ) is highly expressed in oral lichen planus (OLP). The IFN-γ (+874 in intron 1, rs2430561) TT genotype, which has been reported to be associated with high IFN-γ production, was hypothesized to be associated with susceptibility to OLP in the Thai population. DESIGN: Genomic DNA samples from 74 OLP and 268 healthy controls were evaluated for IFN-γ polymorphisms by polymerase chain reaction with sequence-specific primers (PCR-SSP) and direct sequencing method. RESULTS: The T allele was significantly associated with an increased risk of OLP development as compared to the A allele (OR=1.76, P=0.004; P(c)=0.02). The effect of the T allele was similar to an autosomal recessive disorder; the presence of TT genotype (when compared to AA and AT) conferred an OR of 2.61, P=0.008; P(c)=0.04. CONCLUSIONS: We found an association between IFN-γ +874A/T polymorphism and susceptibility to OLP. However, an association study utilising a larger sample size and patients from other races apart from the Asian population should be performed to further verify these findings.

Association between CTLA-4 polymorphisms and the susceptibility to systemic lupus erythematosus and Graves' disease in Thai population.

BACKGROUND: Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is a cell surface molecule involved in the regulation of T cells. Single nucleotide polymorphisms (SNPs) of CTLA-4 gene are known to be associated with susceptibility to several autoimmune diseases, including systemic lupus erythematosus (SLE) and Graves' disease (GD). OBJECTIVE: The aim of this study was to determine whether the common SNPs +49A/G on exon1 and CT60A/G in 3'UTR of the CTLA-4 gene are associated with susceptibility to SLE and GD in Thai population. METHODS: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to analyze these two SNPs in 151 patients with SLE, 132 patients with GD and 153 healthy controls. RESULTS: Our study showed that there were no statistically significant differences in the allele and genotype frequencies of +49A/G and CT60A/G SNPs between patients with SLE and healthy controls as well as patients with GD vs. healthy controls (P > 0.05). However, the GG genotypes of +49A/G and CT60A/G were likely to be risk factors (OR >1) for GD but not in SLE. The effect of the +49G allele was similar to that of an autosomal recessive gene in the presence of the GG genotype, when compared to AA and AG, with an OR of 1.58 (95% CI = 0.95-2.61, p = 0.061) in GD. We also observed a dose response effect of the CT60G allele on GD susceptibility with an OR of 1.43 for GG homozygous and 1.17 for AG heterozygous subjects, when compared to the AA genotype, although these were not statistically significant (P > 0.05). CONCLUSION: We found no association between two functional polymorphisms (+49A/G and CT60A/G) of the CTLA-4 gene and susceptibility to SLE and GD. However, the association study utilizing a larger sample size should be performed to confirm this.

Interleukin-1beta gene polymorphism associated with hepatocellular carcinoma in hepatitis B virus infection.

AIM: To examine the effect of interleukin-1-beta (IL-1beta) promoter region C-511T and IL-1 receptor antagonist (IL-1RN) polymorphism among the patients with chronic hepatitis B virus (HBV) infection (HCC and non-HCC). METHODS: Genomic DNA from 136 Thai patients with chronic HBV infection (HCC=46 and non-HCC=90) and 152 healthy individuals was genotyped for IL-1beta gene polymorphism (-511) using polymerase chain reaction with sequence specific primers (PCR-SSP). The variable number of tandem repeats (VNTR) of IL-1RN gene was assessed by a PCR-based assay. The association between these genes and status of the disease was evaluated by chi2 test. RESULTS: IL-1B-511 genotype C/C was found to be significantly different in patients with HCC when compared with healthy individuals (P=0.036, OR=2.29, 95%CI=1.05-4.97) and patients without HCC (P=0.036, OR=2.52, 95%CI=1.05-6.04). Analysis of allele frequencies of IL-1B-511 showed that IL-1B-511 C allele was also significantly increased in patients with HCC, compared to that in healthy control (P=0.033, OR=1.72, 95%CI=1.04-2.84). However, no significant association in IL-1RN gene was found between the two groups. CONCLUSION: IL-1B-511C allele, which may be associated with high IL-1B production in the liver, is a genetic marker for the development of HCC in chronic hepatitis B patients in Thai population.

Distribution of human leukocyte antigens-E alleles in Thailand.

The aim of this study was to investigate the distribution of human leukocyte antigens (HLA) -E alleles in Thailand. HLA-E alleles were assigned by using a polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) method and direct sequencing in 200 healthy individuals. They comprised 100 Thai, 50 Chinese and 50 Thai-Chinese. From the results, three alleles of HLA-E could be detected in these populations. The E*0101 was the most common allele in Thai and Thai-Chinese with allelic frequencies of 42.5 per cent and 38 per cent, respectively. The other HLA-E allele frequencies of Thai origin were 33 per cent for E*01031 and 24.5 per cent for E*01032, respectively. Among Thai-Chinese, the allele frequencies of HLA-E were 31 per cent for E*01031 and E*01032, respectively. Whereas, the E*01031 was the predominant allele in Chinese origin with a frequency of 39 per cent, followed by E*0101 and E*01032 with 32 per cent and 29 per cent, respectively. No E*01033, E*0102 and E*0104 could be detected in all individuals. When comparing the distribution of HLA-E alleles between each of the populations (Thai vs Chinese, Thai vs Thai-Chinese and Chinese vs Thai-Chinese), no significant difference could be found among these populations. In addition, there was no significant difference of the distribution of HLA-E alleles between the study populations and other populations from Asian countries, reported previously. However, there were significant differences between the populations (Thai, Chinese and Thai-Chinese) and Danish (chi2 = 15.64, p = 0.0004; chi2 = 24.58, p = 0.0000046; chi2 = 14.69, p = 0.00065, respectively).

DRB1*04 subtype in Thai patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) in a Thai population is significantly associated with HLA-DR4. The frequency of DR4 was 43 per cent in RA patients and 20 per cent in the healthy controls (p = 0.00008, OR = 3.06, 95% CI = 1.71, 5.52). To analyze which DR4 alleles were associated with the disease, the authors subtyped 52 DR4-positive RA patients compared to 28 DR4-positive healthy controls by amplification with DR4-specific primers followed by direct sequencing. Six DR4 alleles (DRB1*0401, *0403, *0404, *0405, *0406, and *0410) were found in the RA patient group while 5 alleles (DRB1*0401, *0403, *0405, *0406, and *0407) were found in the control group. Both groups were predominated by DRB11*0405, but there was a significant increase in the frequency of DRB1*0405 in DR4+ RA patients compared to DR4+ healthy controls (84.6% vs 46.4%, p = 0.0008, OR = 6.35, 95% CI = 1.96, 21.08). DR4 which shared epitope alleles (DRB1*0401, *0404, *0405) were observed in 47 (90.3%) DR4+ patients and 15 (53.5%) DR4+ controls (p = 0.0005, OR = 8.15, 95% CI = 2.29, 33.2). In addition, the authors found that DRB1*0403 was significantly decreased in DR4+ RA patients compared to controls (p = 0.0065, OR = 0.07, 95% CI = 0, 0.67).