Novel type I interferon IL-28A suppresses hepatitis C viral RNA replication

Interferon alpha (IFN-α)-based therapy is the currently approved treatment for chronic hepatitis C viral infection. The sustained antiviral response rate is approximately 50% for genotype-1 infection. The major challenge to the HCV community is to improve antiviral efficacy and to reduce the side effects typically seen in IFNα-based therapy. One of the strategies is to identify new interferons, which may have better efficacy and less undesirable side effects. In this report, we examined the role of IL-28A (IFN λ2), a novel type I IFN, in suppression of human hepatitis C viral RNA replication. We have cloned both the human genomic DNA and cDNA of IL-28A, and evaluated their biological activity using HCV RNA replicon cell culture system. The results show that IL-28A effectively inhibits HCV subgenomic RNA replication in a dose-dependent manner. Treatment of human hepatoma cells with IL-28A activates the JAK-STAT signaling pathway and induces the expression of some interferon-stimulated genes (ISGs), such as 6–16 and 1–8U. We also demonstrate that IL-28A induces expression of HLA class I antigens in human hepatoma cells. Moreover, IL-28A appears to specifically suppress HCV IRES-mediated translation. Although IL-28A receptor shares one subunit with the IL-10 receptor, IL-10 treatment has no detectable effect on IL-28A-induced antiviral activity. Interestingly, IL-28A can synergistically enhance IFNα antiviral efficacy. Our results suggest that IL-28A antiviral activity is associated with the activation of the JAK-STAT signaling pathway and expression of ISGs. The effectiveness of IL-28A antiviral activity and its synergistic effect on IFN-α indicate that IL-28A may be potentially used to treat HCV chronic infection

Aqua­bis­(methacrylato-κO)bis(pyridine-κN)copper(II)

In the crystal structure of the title complex, [Cu(C4H5O2)2(C5H5N)2(H2O)], the CuII cation is located on a twofold rotation axis and coordinated by two methyl­acrylate anions, two pyridine ligands and one water mol­ecule in a distorted square-pyramidal geometry. The coordinated water mol­ecule is also located on the twofold axis. In the crystal structure O—H⋯O hydrogen bonds link the mol­ecules, forming chains along the c axis

Innate Host Response in Primary Human Hepatocytes with Hepatitis C Virus Infection

The interaction between hepatitis C virus (HCV) and innate antiviral defense systems in primary human hepatocytes is not well understood. The objective of this study is to examine how primary human hepatocytes response to HCV infection.An infectious HCV isolate JFH1 was used to infect isolated primary human hepatocytes. HCV RNA or NS5A protein in the cells was detected by real-time PCR or immunofluorescence staining respectively. Apoptosis was examined with flow cytometry. Mechanisms of HCV-induced IFN-β expression and apoptosis were determined.Primary human hepatocytes were susceptible to JFH1 virus and released infectious virus. IFN-α inhibited viral RNA replication in the cells. IFN-β and interferon-stimulated genes were induced in the cells during acute infection. HCV infection induced apoptosis of primary human hepatocytes through the TRAIL-mediated pathway. Silencing RIG-I expression in primary human hepatocytes inhibited IFN-β and TRAIL expression and blocked apoptosis of the cells, which facilitated viral RNA replication in the cells. Moreover, HCV NS34A protein inhibited viral induced IFN-β expression in primary human hepatocytes.Innate host response is intact in HCV-infected primary human hepatocytes. RIG-I plays a key role in the induction of IFN and TRAIL by viruses and apoptosis of primary human hepatocytes via activation of the TRAIL-mediated pathway. HCV NS34A protein appears to be capable of disrupting the innate antiviral host responses in primary human hepatocytes. Our study provides a novel mechanism by which primary human hepatocytes respond to natural HCV infection

LmbU, a Cluster-Situated Regulator for Lincomycin, Consists of a DNA-Binding Domain, an Auto-Inhibitory Domain, and Forms Homodimer

Few studies were reported about the regulatory mechanism of lincomycin biosynthesis since it was found in 1962. Although we have proved that a cluster-situated regulator (CSR) LmbU (GenBank Accession No. ABX00623.1) positively modulates lincomycin biosynthesis in Streptomyces lincolnensis NRRL 2936, the molecular mechanism of LmbU regulation is still unclear. In this study, we demonstrated that LmbU binds to the target lmbAp by a central DNA-binding domain (DBD), which interacts with the binding sites through the helix-turn-helix (HTH) motif. N-terminal of LmbU includes an auto-inhibitory domain (AID), inhibiting the DNA-binding activity of LmbU. Without the AID, LmbU variant can bind to its own promoter. Interestingly, compared to other LmbU homologs, the homologs within the biosynthetic gene clusters (BGCs) of known antibiotics generally contain N-terminal AIDs, which offer them the abilities to play complex regulatory functions. In addition, cysteine 12 (C12) has been proved to be mainly responsible for LmbU homodimer formation in vitro. In conclusion, LmbU homologs naturally exist in hundreds of actinomycetes, and belong to a new regulatory family, LmbU family. The present study reveals the DBD, AID and dimerization of LmbU, and sheds new light on the regulatory mechanism of LmbU and its homologs

Improving Iturin A Production of Bacillus amyloliquefaciens by Genome Shuffling and Its Inhibition Against Saccharomyces cerevisiae in Orange Juice

Genome shuffling is an effective method for the rapid improvement of the production of secondary metabolites. This study used the principle of gene shuffling to enhance the yield of iturin A produced by Bacillus amyloliquefaciens LZ-5. Improvements in lipopeptide yield were evident among four strains subjected to recursive protoplast fusion. The four strains were obtained through mutagenesis processes: nitrosoguanidine, ultraviolet irradiation, and atmospheric and room temperature plasma. A high yield strain with 179.22 mg/l of iturin A was obtained after two rounds of genome shuffling, which was a 2.03-fold increase compared with the wild strain. To evaluate the efficacy of iturin A for control of spoilage yeast in food, the anti-yeast efficacy of iturin A was evaluated in orange juice incubated with Saccharomyces cerevisiae. The juice treated with 0.76 mg/ml iturin A showed a significant (p < 0.05) control on yeast population during the storage, similar to that of the 0.30 mg/ml natamycin. In addition, iturin A showed a tiny effect on chemical-physical characteristics of orange juice. Our results provide a basis for the application of antimicrobial lipopeptide in juice products

Acetate and auto-inducing peptide are independent triggers of quorum sensing in Lactobacillus plantarum

The synthesis of plantaricin in Lactobacillus plantarum is regulated by quorum sensing. However, the nature of the extra-cytoplasmic (EC) sensing domain of the histidine kinase (PlnB1) and the ability to recognize the auto-inducing peptide PlnA1 is not known. We demonstrate the key motif Ile-Ser-Met-Leu of auto-inducing peptide PlnA1 binds to the hydrophobic region Phe-Ala-Ser-Gln-Phe of EC loop 2 of PlnB1 via hydrophobic interactions and hydrogen bonding. Moreover, we identify a new inducer, acetate, that regulates the synthesis of plantaricin by binding to a positively charged region (Arg-Arg-Tyr-Ser-His-Lys) in loop 4 of PlnB1 via electrostatic interaction. The side chain of Phe143 on loop 4 determined the specificity and affinity of PlnB1 to recognize acetate. PlnA1 activates quorum sensing in log phase growth and acetate in stationary phase to maintain the synthesis of plantaricin under conditions of reduced growth. Acetate activation of PlnB was also evident in four types of PlnB present in different Lb. plantarum strains. Finally, we proposed a model to explain the developmental regulation of plantaricin synthesis by PlnA and acetate. These results have potential applications in improving food fermentation and bacteriocin production

Targeted Delivery of Chemotherapy Agents Using a Liver Cancer-Specific Aptamer

Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general

Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

The aim of this study was to examine the mechanisms of IFN induction and viral escape. In order to accomplish the goal we compared our new hepatoma cell line LH86, which has intact TLR3 and RIG-I expression and responds to HCV by inducing IFN, with Huh7.5 cells which lack those features.The initial interaction of LH86 cells, Huh7.5 cells or their transfected counter parts (LH86 siRIG-I, siTLR3 or siTLR7 and Huh7.5 RIG-I, TLR3 or TLR7) after infection with HCV (strain JFH-1) was studied by measuring the expression levels of IFNβ, TRAIL, DR4, DR5 and their correlation to viral replication.HCV replicating RNA induces IFN in LH86 cells. The IFN induction system is functional in LH86, and the expression of the RIG-I and TLR3 in LH86 is comparable to the primary hepatocytes. Both proteins appear to play important roles in suppression of viral replication. We found that innate immunity against HCV is associated with the induction of apoptosis by RIG-I through the TRAIL pathway and the establishment of an antiviral state by TLR3. HCV envelope proteins interfere with the expression of TLR3 and RIG-I.These findings correlate with the lower expression level of PRRs in HCV chronic patients and highlight the importance of the PRRs in the initial interaction of the virus and its host cells. This work represents a novel mechanism of viral pathogenesis for HCV and demonstrates the role of PRRs in viral infection