Impaired HBsAg release and antiproliferative/antioxidant cell regulation by HBeAg-negative patient isolates reflects an evolutionary process.

BACKGROUND: The hepatitis B e antigen (HBeAg)-negative infection Phase 3 is characterized by no or minimal signs of hepatic inflammation and the absence of hepatic fibrosis. However, underlying molecular mechanisms leading to this benign phenotype are poorly understood. METHODS: Genotype A, B and D HBeAg-negative patient isolates with precore mutation G1896A from Phase 3 were analysed in comparison with respective HBeAg-positive rescue mutant and HBeAg-positive wild-type reference genomes regarding differences in viral replication, morphogenesis, infectivity and impact on NF-E2-related factor 2 (Nrf2)/antioxidant response element (ARE)-dependent gene expression and cellular kinome. RESULTS: In comparison with reference genomes, the patient isolates are characterized by a lower intra- and extracellular hepatitis B surface antigen (HBsAg)-amount, and HBsAg-retention in the endoplasmic reticulum. Rescue of HBeAg expression increased HBsAg-amount but not its release. Expression of the isolated genomes is associated with a higher Nrf2/ARE-dependent gene expression as compared to reference genomes independent of HBeAg expression. Kinome analyses revealed a decreased activity of receptors involved in regulation of proliferative pathways for all patient isolates compared to the reference genomes. No specific conserved mutations could be found between all genomes from Phase 3. CONCLUSIONS: HBeAg-negative genomes from Phase 3 exhibit distinct molecular characteristics leading to lower HBsAg synthesis and release, enhanced oxidative stress protection and decreased activity of key kinases, triggering an antiproliferative stage, which might contribute to the lower probability of hepatocellular carcinoma. The observed differences cannot be associated with loss of HBeAg or specific mutations common to all analysed isolates, indicating the phenotype of Phase 3 derived genomes to be the result of a multifactorial process likely reflecting a conserved natural selection process.

Diverse Transformation of Vinyl Azides with 2,2,6,6-Tetramethyl- N-oxopiperidinium.

A 2,2,6,6-tetramethyl- N-oxopiperidinium (TEMPO+)-mediated three-component diverse transformation of vinyl azides under metal-free conditions is described. The reaction protocols are operationally simple and conducted at ambient temperature, allowing to access various TEMPO-trapped ketones, amides, and α-alkoxyalkyl azides. Preliminary mechanistic studies indicate that an alkene radical cation-mediated radical-radical cross-coupling C-O bond formation could be involved.

Radical alkylation of para-quinone methides with 4-substituted Hantzsch esters/nitriles via organic photoredox catalysis.

A novel photocatalytic protocol is herein described for the preparation of functionalized phenols via radical alkylation of para-quinone methides under transition-metal-free conditions. The reaction is external oxidant free and performed at ambient temperature upon visible light irradiation, allowing the access to various desired products in satisfactory yields. The readily available 4-alkyl-1,4-dihydropyridines serve as the effective alkyl radical precursors.

Identification of Four SNPs in LHB Gene and Their Associations with Sperm Qualities of Chinese Buffaloes.

Luteinizing hormone beta polypeptide (LHB) gene has been considered important for sexual behavior and has associations with sperm quality. In this study, four SNPs (g.276 T>C, g.377A>C, g.401T>C, and g.412A>G) were detected in the LHB gene of 165 water buffaloes by direct sequencing and identification of overlap peaks, each of which was associated with at least one sperm quality trait of ejaculate volume, sperm concentration, post-thaw sperm motilities, and sperm abnormalities by chi-square analysis. Among them, g.276 T>C was associated with ejaculate volume (F = 2.857, p < 0.05), sperm concentration (F = 2.052, p < 0.05), and post-thaw sperm motilities (F = 3.480, p < 0.05); g.377A>C was related to ejaculate volume (F = 4.178, p < 0.05), g.401T>C had a marker effect on sperm abnormalities (F = 3.332, p < 0.05), g.412A>G was associated with sperm concentration (F = 3.579, p < 0.05), and sperm abnormalities (F = 3.408, p < 0.05). Furthermore, four haplotypes (H1: ACG, H2: CCG, H3: CTA, H4: CCA) were generated by linkage disequilibrium analysis, which composed seven genotypes. Among them, the buffaloes with combined genotype H2H2 had the higher ejaculate volume and the individuals with the combined haplotypes H1H4 had higher sperm concentration. In summary, our study showed that there was a significant association between SNPs of LHB gene and Chinese water buffalo sperm quality traits. To the best of our knowledge, this is the first report addressing the associations between the SNPs in the LHB gene and the sperm qualities of Chinese buffaloes.

Missense mutations in the signal peptide of the porcine GH gene affect cellular synthesis and secretion.

CONTEXT: In previous investigations, we have demonstrated the mutations in the signal peptide of porcine GH gene were associated with the body size. METHODS: In this study, the fusion gene expression vectors which consisted of eight signal peptide mutants of GH gene and EGFP gene were constructed according to three missense mutations (p.Val9Ala, p.Gln22Arg and p.Asp25Gly), and they were transfected into the GH3 cell line. RESULTS: The inhibition levels of EGFP gene transcriptions with different signal peptide mutants were significantly different. Typically, the allelic variants carrying Val in codon nine showed higher protein synthesis (P < 0.05), and the allelic variants carrying neutral Gln in codon 22 and Gly in codon 25 showed higher secretion proportion (P < 0.05) compared with the other groups as assessed by western blotting. In silico RNA folding prediction indicated that the mutations gave rise to different RNA secondary structures, suggesting that they might affect translation and protein synthesis. CONCLUSION: We conclude that the missense mutations within the signal sequence influence the expression and the secretion of the protein. To the best of our knowledge, this is the first report addressing the functional consequences of the mutations in the signal peptide of porcine GH gene.

Interplay between curvature and lateral organization of lipids and peptides/proteins in model membranes.

Membrane curvature plays a crucial role in the realization of many cellular membrane functions such as signaling and trafficking. Here, using coarse-grained molecular dynamics (MD) simulation, we present an effective method of producing curved model membranes and systematically investigated the interplay between the curvature and lateral sorting of lipids and transmembrane (TM) peptides/proteins in the model membranes. We first confirmed the experimental results of the lateral organization of lipid domains in curved ternary membranes. Then, we focused on exploring the lateral sorting of TM peptides/proteins with symmetric shape in the curved membranes. The results showed that the lateral inhomogeneous packing of lipids induced by the curvature and/or the component heterogeneity drives the peptides/proteins to accumulate in the curved regions in both the unary and ternary membranes. However, whether the peptides/proteins can stably and compactly reside in the curved regions is determined by their final packing configuration, which may be influenced by the membrane curvature in the curved regions. Additionally, the insertion of peptides/proteins may enhance the membrane curvature. This work provided some theoretical insights into understanding the mechanism of the interplay of membrane curvature and lateral organization (especially the lateral sorting of the peptides/proteins with symmetric shape) in the biomembrane in some biological processes.

Effect of hydrophobic mismatch on domain formation and peptide sorting in the multicomponent lipid bilayers in the presence of immobilized peptides.

In the plasma membranes, many transmembrane (TM) proteins/peptides are anchored to the underlying cytoskeleton and/or the extracellular matrix. The lateral diffusion and the tilt of these proteins/peptides may be greatly restricted by the anchoring. Here, using the coarse-grained molecular dynamics simulation, we investigated the domain formation and peptide sorting in the ternary lipid bilayers in the presence of the immobilized peptide-grid and peptide-cluster. We mainly focused on examining the combining effect of the peptide immobilization and hydrophobic mismatch on the domain formation and peptide sorting in the lipid bilayers. Compared to the lipid bilayers inserted with free TM peptides, our results showed that, because of the tilt restriction imposed on the peptides, the hydrophobic mismatch effect more significantly influences the domain size, the dynamics of domain formation, and the peptide sorting in our systems. Our results provide some theoretical insights into understanding the formation of nanosized lipid rafts, the protein sorting in the lipid rafts and the interaction between the cytoskeleton, the extracellular matrix, and the plasma membranes.

Indirect treatment of plasma-processed air to decrease decay and microbiota of strawberry fruit caused by mechanical damage.

Due to the soft texture of strawberry fruit, it is highly susceptible to mechanical damage during postharvest supply chains, resulting in decay and quality deterioration. Urgent investigation is needed on the treatment techniques to mitigate the impact of postharvest mechanical damage of strawberry fruit. In the present study, the effect of indirect plasma-processed air (PPA) pretreatment to decrease decay and microbiota of strawberry fruit caused by mechanical damage was investigated. The results show PPA pretreatment reduced the total counts of indigenous microbiota on the surface of intact and mechanical damaged strawberry fruit by 4.29 and 3.76 log10CFU/g at day 0, respectively, and reduced the counts of S. aureus and E. coli inoculated on strawberry fruit by 3.05-3.16 and 3.55-3.56 log10CFU/g, respectively. The disease incidence of fruit inoculated with Botrytis cinerea was also decreased by 6.67 %-18.89 % during storage. Besides, PPA pretreatment reduced the decay rate of strawberry fruit by 5.56 %-21.11 % during storage and did not significantly affect the firmness, color index of red grapes (CIRG) and total soluble solids (TSS) content of strawberry fruit. DHHP results indicate that the antioxidant activity of the strawberry fruit was increased. After PPA pretreatment, 39 metabolites were differentially accumulated in strawberry fruits, 37 of which were up-regulated, including flavonoids, phenolic acids and organic acid.

Therapeutic efficacy and underlying mechanisms of Gastrodia elata polysaccharides on dextran sulfate sodium-induced inflammatory bowel disease in mice: Modulation of the gut microbiota and improvement of metabolic disorders.

Inflammatory bowel disease (IBD) is a chronic inflammatory gastrointestinal disease, and an imbalance in the gut microbiota is a critical factor in its development. Gastrodia elata (G. elata), an Orchidaceae plant, is recognized for its nutritional and medicinal value. Studies have shown that G. elata polysaccharides (GBP) have anti-inflammatory properties that may ameliorate IBD. However, the therapeutic effects of GBP on gut microbiota metabolism remain unknown. Therefore, we aimed to examine the therapeutic potential of G. elata extract and GBP in dextran sulfate sodium (DSS)-induced IBD mice. GBP demonstrated the best therapeutic effect by reducing IBD symptoms in mice to the greatest extent. Administering GBP resulted in significant increases in the relative abundances of bacteria with potential anti-inflammatory effects, such as Ligilactobacillus and Alloprevotella, and decreases in the levels of bacteria associated with proinflammatory responses, such as Bacteroides and Escherichia-Shigella. Furthermore, 36 significant differential metabolites between the model and GBP groups were identified in feces, which were mainly enriched in amino acid metabolism, including tryptophan and cysteine, vitamin B6 metabolism and steroid hormone biosynthesis. Consequently, investigating the metabolic regulation of the gut microbiota is a promising approach to evaluate the therapeutic effect of GBP on IBD.

Presence of Intact Hepatitis B Virions in Exosomes.

BACKGROUND & AIMS: Hepatitis B virus (HBV) was identified as an enveloped DNA virus with a diameter of 42 nm. Multivesicular bodies play a central role in HBV egress and exosome biogenesis. In light of this, it was studied whether intact virions wrapped in exosomes are released by HBV-producing cells. METHODS: Robust methods for efficient separation of exosomes from virions were established. Exosomes were subjected to limited detergent treatment for release of viral particles. Electron microscopy of immunogold labeled ultrathin sections of purified exosomes was performed for characterization of exosomal HBV. Exosome formation/release was affected by inhibitors or Crispr/Cas-mediated gene silencing. Infectivity/uptake of exosomal HBV was investigated in susceptible and non-susceptible cells. RESULTS: Exosomes could be isolated from supernatants of HBV-producing cells, which are characterized by the presence of exosomal and HBV markers. These exosomal fractions could be separated from the fractions containing free virions. Limited detergent treatment of exosomes causes stepwise release of intact HBV virions and naked capsids. Inhibition of exosome morphogenesis impairs the release of exosome-wrapped HBV. Electron microscopy confirmed the presence of intact virions in exosomes. Moreover, the presence of large hepatitis B virus surface antigen on the surface of exosomes derived from HBV expressing cells was observed, which conferred exosome-encapsulated HBV initiating infection in susceptible cells in a , large hepatitis B virus surface antigen/Na+-taurocholate co-transporting polypeptide-dependent manner. The uptake of exosomal HBV with low efficiency was also observed in non-permissive cells. CONCLUSION: These data indicate that a fraction of intact HBV virions can be released as exosomes. This reveals a so far not described release pathway for HBV.

Chan-Lam Reaction and Lewis Acid Promoted 1,3-Rearrangement of N-O Bonds to Prepare N-(2-Hydroxyaryl)pyridin-2-ones.

We describe the difunctionalization of arylboronic acids to prepare various N-(2-hydroxyaryl)pyridin-2-ones in good yields using N-hydroxypyridin-2-ones as the oxygen and nitrogen sources through a copper(II)-catalyzed Chan-Lam reaction and subsequent BF3-promoted selective 1,3-rearrangement of N-O bond in a one-pot procedure. Mechanistic studies reveal that the 1,3-rearrangement selectivity is controlled by the formation of the key aryloxypyridinium salt. The obtained products are easily converted to various useful pyridin-2-one scaffolds.

Incivility and Knowledge Hiding in Academia: Mediating Role of Interpersonal Distrust and Rumination.

Workplace incivility is under investigation for the last three decades, and it holds a central position in organizational behavior literature. However, despite the extensive investigations in the past, there exists a missing link between workplace incivility and knowledge hiding in academia. This study aims to tap this missing link for which data were collected from the universities staff. Data were collected in two waves to reduce the common method biases. In the first wave, questions were asked from the respondents regarding their demographic characteristics and exposure to workplace incivility. At this stage, 400 questionnaires were floated and 355 completely filled responses were received back, while in the second wave, those respondents were approached for data collection who have completely filled questionnaires in the first wave. The time interval between the two waves was 1 month. In the second wave, questions related to distrust and knowledge hiding behavior were asked from the respondents. At this stage, 323 questionnaires were received back out of which 290 were filled and these were considered for final data analysis. Collected data were analyzed by applying structural equation modeling (SEM) through SmartPLS. Results indicated that employees tend to hide knowledge when they experience incivility at workplace. Moreover, they develop a sense of distrust in response to workplace incivility which further triggers them to hide knowledge. Limitations and future directions are also discussed.

The N-Terminus Makes the Difference: Impact of Genotype-Specific Disparities in the N-Terminal Part of The Hepatitis B Virus Large Surface Protein on Morphogenesis of Viral and Subviral Particles.

The N-terminus of the hepatitis B virus (HBV) large surface protein (LHB) differs with respect to genotypes. Compared to the amino terminus of genotype (Gt)D, in GtA, GtB and GtC, an additional identical 11 amino acids (aa) are found, while GtE and GtG share another similar 10 aa. Variants of GtB and GtC affecting this N-terminal part are associated with hepatoma formation. Deletion of these amino-terminal 11 aa in GtA reduces the amount of LHBs and changes subcellular accumulation (GtA-like pattern) to a dispersed distribution (GtD-like pattern). Vice versa, the fusion of the GtA-derived N-terminal 11 aa to GtD causes a GtA-like phenotype. However, insertion of the corresponding GtE-derived 10 aa to GtD has no effect. Deletion of these 11aa decreases filament size while neither the number of released viral genomes nor virion size and infectivity are affected. A negative regulatory element (aa 2-8) and a dominant positive regulatory element (aa 9-11) affecting the amount of LHBs were identified. The fusion of this motif to eGFP revealed that the effect on protein amount and subcellular distribution is not restricted to LHBs. These data identify a novel region in the N-terminus of LHBs affecting the amount and subcellular distribution of LHBs and identify release-promoting and -inhibiting aa residues within this motive.

Porcine IGF-1R synonymous mutations in the intracellular domain affect cell proliferation and alter kinase activity.

Miniature pigs are regarded as ideal organ donors for xenotransplantation into humans. Elucidating the formation mechanism of miniature pigs is important. The insulin-like growth factor 1 receptor (IGF-1R) is crucial in the regulation of cell proliferation and organismal growth. According to our previous research, the IGF-1R expression levels between large and miniature pigs showed different profiles in liver and muscle tissues. Here, five synonymous mutations of IGF-1R in the coding sequence (CDS) of intracellular domain (ICD) between large and miniature pigs were analysed by constructing expression vectors of two haplotypes and named pcDNA3.1-LP (with the CDS of IGF-1R ICD of Large White pigs, LP group) and pcDNA3.1-BM (with the CDS of IGF-1R ICD of Bama Xiang pigs, BM group). The IGF-1R of the BM group was expressed lower than that of the LP group in transcription, translation and autophosphorylation levels. The IGF-1R of the BM group also down-regulated the protein levels of p-AKT/p-ERK than that of the LP group. PK-15 and C2C12 cell proliferation were detected to further understand the function of the haplotype. Results showed that the proliferation viability of PK-15 and C2C12 cells weakened in the BM group. Moreover, the mRNA and protein stabilities of the BM group were higher than those of the LP group. Our data indicated that two haplotypes of IGF-1R CDS in ICD between large and miniature pigs altered IGF-1R expression and down-regulated AKT and ERK signalling pathways at translation levels, resulting in an inhibitory effect on PK-15 and C2C12 cell proliferation.

Formation of semi-enveloped particles as a unique feature of a hepatitis B virus PreS1 deletion mutant.

BACKGROUND: Naturally occurring variants with deletions or mutations in the C-terminal PreS1 domain from hepatitis B virus (HBV) chronically infected patients have been shown to promote HBsAg retention, inhibit HBsAg secretion and change the extracellular appearance of PreS1-containing HBV particles (filaments and virions). AIMS: To study the impact of N-terminal deletion in preS1 domain on viral secretion and morphogenesis. METHODS: An HBV mutant with 15 amino acids (aa 25-39) deletion in N-terminal preS1 was isolated. Intracellular and extracellular HBsAg were quantified by Western blot. Subcellular HBsAg distribution was analysed by confocal laser scanning microscopy. The viral morphology was characterised by sucrose density gradient ultracentrifugation, Western blot, electron microscopy, HBV mixed ELISA and HBV particle gel essay. RESULTS: Expression of this mutant genome released higher amounts of HBsAg in the form of shorter filaments. A significant fraction of semi-enveloped virions was observed in the supernatant that has been unprecedented so far. Stepwise insertion of aa 25-31, aa 32-39 and aa 25-39 increased the length of filaments. The rescue of aa 25-31 and aa 25-39 drastically reduced the amounts of extracellular HBsAg and semi-enveloped virions, while such effects could not be observed after insertion of aa 32-39, arguing against a simple spacer function of this region. The deletion and rescued mutants do not differ in subcellular HBsAg distribution and colocalisation with ER, Golgi and multivesicular bodies markers arguing against differences in release pathways. CONCLUSION: N-terminal PreS1-domain (aa 25-31) determines HBsAg secretion and triggers proper assembly of PreS1-containing particles.

Linkage disequilibrium and functional analysis of PRE1 insertion together with SNPs in the promoter region of IGFBP7 gene in different pig breeds.

Polymorphisms in regions upstream of transcription initiation site may modify the transcriptional activity of target genes by changing promoter activity. This study aims to determine whether or not polymorphisms at porcine IGFBP7 promoter region affect gene expression. In this study, eight SNPs and one PRE1 insertion in this region were first confirmed. The PRE1 insertion was widespread in 20 Chinese indigenous breeds, but was not observed in three commercial breeds. A perfect linkage disequilibrium, consisting of six of those SNPs and a PRE1, was observed with two haplotypes (h1 and h2) in five pig breeds. The h1 haplotype had an overwhelming superiority distribution in Large White, Landrace, and Bama mini-pig; in turn, the h2 only existed in the PRE1 presence breeds. As the haplotypes and PRE1 were located at gene promoter regions, we further investigated the transfection of plasmids with three different fragments of IGFBP-7 promoter region (H1, H2, RF). The CMV promoter of the pEGFP-N1 was substituted by these three different fragments, respectively. Different transcriptional and translational activities of EGFP in PK-15 cells were observed in these three recombinant plasmids by quantitative real-time PCR and flow cytometric analysis. The results indicated that H1 had the higher transcriptional and translational activities of EGFP as compared to the H2 (P < 0.05, P < 0.05). As compared to the RF group, EGFP mRNA expression level was significantly higher in H1 groups (P < 0.05). The IGFBP-7 promoter polymorphisms detected in this study may be important functional variants and potential genetic markers for pig population genetic study.

Modeling the Self-Assembly of Bolaamphiphiles under Nanoconfinement by Coarse-Grained Molecular Dynamics.

Novel and complex nanostructures of amphiphiles assemblies facilitate realizing of many practical applications in nanotechnologies. Under different physical conditions, amphiphiles can form various morphologies. In this article, the self-assembly of bolaamphiphiles was investigated under the cylindrical confinement of carbon nanotubes by coarse-grained molecular dynamics simulation. We found that bolaamphiphiles inside a carbon nanotube with a relatively large diameter tended to self-assemble into curved cylindrical micelles, and further some of these micelles can form double helix structures which were not observed in bulk solutions. We develop an effective method to characterize different morphologies. Our analysis results showed that the formation of double helix and other amorphous morphologies of micelles in carbon nanotubes is not induced by the conformational variation of bolaamphiphiles. Moreover, through examining the potential energies of different structures, we infer that it is entropy which leads micelles to bend even to form double stranded helix structures. In addition, we analyzed the sizes of micelles and find the scission and recombination of micelles during the double helix formation process. This work indicates that the confinement of carbon nanotube is an available method to produce complex nanostructures and provides some new insights of the self-assembly of bolaamphiphiles.

Distribution and linkage disequilibrium analysis of polymorphisms of GH1 gene in different populations of pigs associated with body size.

Growth hormone (GH) has been considered as a candidate gene for growth and body size in pigs. In this study, polymorphisms of the GH1 gene were evaluated for associations with body size traits in 190 pig individuals. Seventeen single-nucleotide polymorphisms (SNPs) were identified in GH1 gene of the large pig breeds and miniature pig breeds using direct sequencing and genotyped by allele-specific PCR approach. Notably, six (g.237A>G, g.283T>C, g.309A>G, g.318A>G, g.540A>G and g.544A>G) of them were significantly associated with body size, of which three loci (g.283T>C, g.309A>G, g.318A>G) located in the signal-peptide coding region of GH1 gene compose a CGG haplotype for large pigs and TAA haplotype for miniature pigs (P <0.001), two loci (g.540A>G and g.544A>G) located in the second intron of GH1 gene compose a GG haplotype for large pigs and AA haplotype for miniature pigs (P < 0.001). Our results demonstrate that these SNPs in GH1 gene are associated with the body size of pigs providing genetic basis for pig breeding with the improved economic benefits.

Expression Profiles of IGF-1R Gene and Polymorphisms of its Regulatory Regions in Different Pig Breeds.

Insulin like growth factor 1 receptor (IGF-1R) is a candidate gene for growth and carcass traits in regulating animal growth, metabolism and endocrine. It is widely expressed in liver, muscle, bone tissues where the IGF-1R functions as a factor that promotes cell growth. In this study, the protein expression level of IGF-1R gene in liver and muscle tissues of three periods (birth, weaning and adult) of three pig breeds (BamaXiang pigs (BM), Tibetan pigs (TM) and Junmu No.1 pigs (JM)) were tested by western blot. SNPs within the regulatory region of pig IGF-1R gene were detected using direct sequencing and then the genotypes were identified through AS-PCR approach. Results showed expression profiles of IGF-1R gene between liver and muscle tissues were different and significant differences were also found among pig breeds. In the same time, four SNPs were detected in the regulatory region of IGF-1R gene, among which the genotype frequency of three (g.-1468G > C, g.-1192 C > T and g.330,424 C > T) were significantly different among the pig breeds. BM tended to heterozygous (GC/CT) of the anterior two loci, while TM and JM preferred the other two homozygotes respectively. For the g.330,424 C > T, all pig breeds were tended to be the heterozygous. In conclusion, the SNPs with different genotype distribution among the three pig breeds may explain the gene expression difference between the different pig breeds.