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  • 1
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    Critical Roles of Glucocorticoid-Induced Leucine Zipper in Infectious Bursal Disease Virus (IBDV)-Induced Suppression of Type I Interferon Expression and Enhancement of IBDV Growth in Host Cells via Interaction with VP4 [Virus-Cell Interactions] (2012)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2012-12-29
    Description: Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by IBD virus (IBDV). Although IBDV-induced immunosuppression has been well established, the underlying exact molecular mechanism for such induction is not very clear. We report here the identification of IBDV VP4 as an interferon suppressor by interaction with the glucocorticoid-induced leucine zipper (GILZ) in host cells. We found that VP4 suppressed the expression of type I interferon in HEK293T cells after tumor necrosis factor alpha (TNF-α) treatment or Sendai virus (SeV) infection and in DF-1 cells after poly(I·C) stimulation. In addition, the VP4-induced suppression of type I interferon could be completely abolished by knockdown of GILZ by small interfering RNA (siRNA). Furthermore, knockdown of GILZ significantly inhibited IBDV growth in host cells, and this inhibition could be markedly mitigated by anti-alpha/beta interferon antibodies in the cell cultures ( P 〈 0.001). Thus, VP4-induced suppression of type I interferon is mediated by interaction with GILZ, a protein that appears to inhibit cell responses to viral infection.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 2
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    Complete Genome Sequences of a Novel Reassortant H4N2 Avian Influenza Virus Isolated from a Live Poultry Market in Eastern China [Genome Announcements] (2012)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2012-10-07
    Description: A/duck/Shanghai/28-1/2009(H4N2) (DK28) was isolated from a live poultry market in Shanghai, China. Using PCR and sequencing analysis, we obtained the complete genome sequences of the DK28 virus. The sequence analysis demonstrated that this H4N2 virus was a novel multiple-gene reassortant avian influenza virus (AIV) whose genes originated from H1N1, H1N3, H3N3, H4N2, and H4N6. Knowledge regarding the complete genome sequences of the DK28 virus will be useful for epidemiological surveillance.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 3
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    An Engineered Strong Promoter for Streptomycetes [Biotechnology] (2013)
    The American Society for Microbiology (ASM)
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    Publication Date: 2013-06-21
    Description: Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor , the native kasO p is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasO p was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasO p 3 . Second, another ScbR binding site internal to the kasO p 3 core promoter region was abolished by random mutation and screening of the mutant library to obtain the strongest promoter, kasO p* (where the asterisk is used to distinguish the engineered promoter from the native promoter). The activities of kasO p* were compared with those of two known strong promoters, ermE p* and SF14p, in three Streptomyces species. kasO p* showed the highest activity at the transcription and protein levels in all three hosts. Furthermore, relative to ermE p* and SF14p, kasO p* was shown to confer the highest actinorhodin production level when used to drive the expression of actII -ORF4 in S. coelicolor . Therefore, kasO p* is a simple and well-defined strong promoter useful for gene overexpression in streptomycetes.
    Print ISSN: 0099-2240
    Electronic ISSN: 1098-5336
    Topics: Biology
    Published by The American Society for Microbiology (ASM)
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  • 4
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    Novel Recombinant Hepatitis B Virus Vectors Efficiently Deliver Protein and RNA Encoding Genes into Primary Hepatocytes [Gene Delivery] (2013)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2013-05-29
    Description: Hepatitis B virus (HBV) has extremely restricted host and hepatocyte tropism. HBV-based vectors could form the basis of novel therapies for chronic hepatitis B and other liver diseases and would also be invaluable for the study of HBV infection. Previous attempts at developing HBV-based vectors encountered low yields of recombinant viruses and/or lack of sufficient infectivity/cargo gene expression in primary hepatocytes, which hampered follow-up applications. In this work, we constructed a novel vector based on a naturally occurring, highly replicative HBV mutant with a 207-bp deletion in the preS1/polymerase spacer region. By applying a novel insertion strategy that preserves the continuity of the polymerase open reading frame (ORF), recombinant HBV (rHBV) carrying protein or small interfering RNA (siRNA) genes were obtained that replicated and were packaged efficiently in cultured hepatocytes. We demonstrated that rHBV expressing a fluorescent reporter (DsRed) is highly infective in primary tree shrew hepatocytes, and rHBV expressing HBV-targeting siRNA successfully inhibited antigen expression from coinfected wild-type HBV. This novel HBV vector will be a powerful tool for hepatocyte-targeting gene delivery, as well as the study of HBV infection.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 5
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    Novel cis-Acting Element within the Capsid-Coding Region Enhances Flavivirus Viral-RNA Replication by Regulating Genome Cyclization [Genome Replication and Regulation of Viral Gene Expression] (2013)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2013-05-29
    Description: cis -Acting elements in the viral genome RNA (vRNA) are essential for the translation, replication, and/or encapsidation of RNA viruses. In this study, a novel conserved cis -acting element was identified in the capsid-coding region of mosquito-borne flavivirus. The d ownstream of 5' c yclization s equence (5'CS) p seudo k not (DCS-PK) element has a three-stem pseudoknot structure, as demonstrated by structure prediction and biochemical analysis. Using dengue virus as a model, we show that DCS-PK enhances vRNA replication and that its function depends on its secondary structure and specific primary sequence. Mutagenesis revealed that the highly conserved stem 1 and loop 2, which are involved in potential loop-helix interactions, are crucial for DCS-PK function. A predicted loop 1-stem 3 base triple interaction is important for the structural stability and function of DCS-PK. Moreover, the function of DCS-PK depends on its position relative to the 5'CS, and the presence of DCS-PK facilitates the formation of 5'-3' RNA complexes. Taken together, our results reveal that the cis -acting element DCS-PK enhances vRNA replication by regulating genome cyclization, and DCS-PK might interplay with other cis -acting elements to form a functional vRNA cyclization domain, thus playing critical roles during the flavivirus life cycle and evolution.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 6
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    Rational Design of a Flavivirus Vaccine by Abolishing Viral RNA 2'-O Methylation [Vaccines and Antiviral Agents] (2013)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2013-04-25
    Description: Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'- O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'- O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'- O methylation is replicative, but its viral RNA lacks 2'- O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'- O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'- O methylation was stable in cell culture after being passaged for 〉30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo . A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'- O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'- O methyltransferases.
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    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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  • 7
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    Evaluation of Meropenem Regimens Suppressing Emergence of Resistance in Acinetobacter baumannii with Human Simulated Exposure in an In Vitro Intravenous-Infusion Hollow-Fiber Infection Model [Experimental Therapeutics] (2014)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2014-10-17
    Description: The emergence of resistance to carbapenems in Pseudomonas aeruginosa can be suppressed by optimizing the administration of meropenem. However, whether the same is true for Acinetobacter baumannii is not fully understood. We assessed the bactericidal activity of meropenem and its potency to suppress the emergence of resistance in A. baumannii with human simulated exposure in an in vitro intravenous-infusion hollow-fiber infection model (HFIM). Two clinical strains of carbapenem-susceptible multidrug-resistant A. baumannii (CS-MDRAB), CSRA24 and CSRA91, were used, and their MICs and mutant prevention concentrations (MPCs) were determined. Six meropenem dosage regimens (0.5, 1.0, or 2.0 g given every 8 h [q8h] with a 0.5-h or 3-h infusion for seven consecutive days) were simulated and then evaluated in the HFIM. Both the total population and resistant subpopulations of the two strains were quantified. Drug concentrations were measured by high-performance liquid chromatography. All dosage regimens, except for the lowest dosage (0.5 g for both the 0.5-h and 3-h infusions), showed 3-log CFU/ml bacterial killing. Dosage regimens of 2.0 g with 0.5-h and 3-h infusions exhibited an obvious bactericidal effect and suppressed resistance. Selective amplification of subpopulations with reduced susceptibility to meropenem was suppressed with a percentage of the dosage interval in which meropenem concentrations exceeded the MPC ( T 〉MPC) of ≥20% or with a ratio of T 〉MPC to the percentage of the dosage interval in which drug concentrations are within the mutant selection window of ≥0.25. Our in vitro data support the use of a high dosage of meropenem (2.0 g q8h) for the treatment of severe infection caused by CS-MDRAB.
    Print ISSN: 0066-4804
    Electronic ISSN: 1098-6596
    Topics: Biology , Medicine
    Published by The American Society for Microbiology (ASM)
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  • 8
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    Development of a Gene Knockout System Using Mobile Group II Introns (Targetron) and Genetic Disruption of Acid Production Pathways in Clostridium beijerinckii [Physiology] (2013)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2013-09-07
    Description: Clostridium beijerinckii is a well-known solvent-producing microorganism with great potential for biofuel and biochemical production. To better understand and improve the biochemical pathway to solvents, the development of genetic tools for engineering C. beijerinckii is highly desired. Based on mobile group II intron technology, a targetron gene knockout system was developed for C. beijerinckii in this study. This system was successfully employed to disrupt acid production pathways in C. beijerinckii , leading to pta (encoding phosphotransacetylase)- and buk (encoding butyrate kinase)-negative mutants. In addition to experimental characterization, the mutant phenotypes were analyzed in the context of our C. beijerinckii genome-scale model. Compared to those of the parental strain ( C. beijerinckii 8052), acetate production in the pta mutant was substantially reduced and butyrate production was remarkably increased, while solvent production was dependent on the growth medium. The pta mutant also produced much higher levels of lactate, suggesting that disrupting pta influenced the energy generation and electron flow pathways. In contrast, acetate and butyrate production in the buk mutant was generally similar to that of the wild type, but solvent production was consistently 20 to 30% higher and glucose consumption was more rapid and complete. Our results suggest that the acid and solvent production of C. beijerinckii can be effectively altered by disrupting the acid production pathways. As the gene disruption method developed in this study does not leave any antibiotic marker in a disrupted allele, multiple and high-throughput gene disruption is feasible for elucidating genotype and phenotype relationships in C. beijerinckii .
    Print ISSN: 0099-2240
    Electronic ISSN: 1098-5336
    Topics: Biology
    Published by The American Society for Microbiology (ASM)
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  • 9
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    Transactivation of Atg4b by C/EBP{beta} Promotes Autophagy To Facilitate Adipogenesis [Articles] (2013)
    The American Society for Microbiology (ASM)
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    Publication Date: 2013-07-26
    Description: Autophagy is a highly conserved self-digestion pathway involved in various physiological and pathophysiological processes. Recent studies have implicated a pivotal role of autophagy in adipocyte differentiation, but the molecular mechanism for its role and how it is regulated during this process are not clear. Here, we show that CCAAT /enhancer-binding protein β (C/EBPβ), an important adipogenic factor, is required for the activation of autophagy during 3T3-L1 adipocyte differentiation. An autophagy-related gene, Atg4b , is identified as a de novo target gene of C/EBPβ and is shown to play an important role in 3T3-L1 adipocyte differentiation. Furthermore, autophagy is required for the degradation of Klf2 and Klf3, two negative regulators of adipocyte differentiation, which is mediated by the adaptor protein p62/SQSTM1. Importantly, the regulation of autophagy by C/EBPβ and the role of autophagy in Klf2/3 degradation and in adipogenesis are further confirmed in mouse models. Our data describe a novel function of C/EBPβ in regulating autophagy and reveal the mechanism of autophagy during adipocyte differentiation. These new insights into the molecular mechanism of adipose tissue development provide a functional pathway with therapeutic potential against obesity and its related metabolic disorders.
    Print ISSN: 0270-7306
    Electronic ISSN: 1098-5549
    Topics: Biology , Medicine
    Published by The American Society for Microbiology (ASM)
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  • 10
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    Later Passages of Neural Progenitor Cells from Neonatal Brain Are More Permissive for Human Cytomegalovirus Infection [Pathogenesis and Immunity] (2013)
    The American Society for Microbiology (ASM)
    Details
    Publication Date: 2013-09-21
    Description: Congenital human cytomegalovirus (HCMV) infection is the most frequent infectious cause of birth defects, primarily neurological disorders. Neural progenitor/stem cells (NPCs) are the major cell type in the subventricular zone and are susceptible to HCMV infection. In culture, the differentiation status of NPCs may change with passage, which in turn may alter susceptibility to virus infection. Previously, only early-passage (i.e., prior to passage 9) NPCs were studied and shown to be permissive to HCMV infection. In this study, NPC cultures derived at different gestational ages were evaluated after short (passages 3 to 6) and extended (passages 11 to 20) in vitro passages for biological and virological parameters (i.e., cell morphology, expression of NPC markers and HCMV receptors, viral entry efficiency, viral gene expression, virus-induced cytopathic effect, and release of infectious progeny). These parameters were not significantly influenced by the gestational age of the source tissues. However, extended-passage cultures showed evidence of initiation of differentiation, increased viral entry, and more efficient production of infectious progeny. These results confirm that NPCs are fully permissive for HCMV infection and that extended-passage NPCs initiate differentiation and are more permissive for HCMV infection. Later-passage NPCs being differentiated and more permissive for HCMV infection suggest that HCMV infection in fetal brain may cause more neural cell loss and give rise to severe neurological disabilities with advancing brain development.
    Print ISSN: 0022-538X
    Electronic ISSN: 1098-5514
    Topics: Medicine
    Published by The American Society for Microbiology (ASM)
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