Description: One-half of the genes in the human genome contain alternative promoters, some of which generate products with opposing functions. Aberrant silencing or activation of such alternative promoters is associated with multiple diseases, including cancer, but little is known regarding the molecular mechanisms that control alternative promoter choice. The SHC1 gene encodes p46 Shc /p52 Shc and p66 Shc , proteins oppositely regulating anchorage-independent growth that are produced by transcription initiated from the upstream and downstream tandem promoters of SHC1 , respectively. Here we demonstrate that activation of these promoters is mutually exclusive on separate alleles in single primary endothelial cells in a heritable fashion, ensuring expression of both transcripts by the cell. Peripheral blood lymphocytes that do not transcribe p66 Shc transcribed p52 Shc biallelically. This distinct monoallelic transcription pattern is established by allele-specific chromosomal looping between tandem promoters, which silences the upstream promoter. Our results reveal a new mechanism to control alternative promoter usage through higher-order chromatin structure.

Description: Due to enzootic infections in poultry and persistent human infections in China, influenza A (H7N9) virus has remained a public health threat. The Yangtze River Delta region, which is located in eastern China, is well recognized as the original source for H7N9 outbreaks. Based on the evolutionary analysis of H7N9 viruses from all three outbreak waves since 2013, we identified the Pearl River Delta region as an additional H7N9 outbreak source. H7N9 viruses are repeatedly introduced from these two sources to the other areas, and the persistent circulation of H7N9 viruses occurs in poultry, causing continuous outbreak waves. Poultry movements may contribute to the geographic expansion of the virus. In addition, the AnH1 genotype, which was predominant during wave 1, was replaced by JS537, JS18828, and AnH1887 genotypes during waves 2 and 3. The establishment of a new source and the continuous evolution of the virus hamper the elimination of H7N9 viruses, thus posing a long-term threat of H7N9 infection in humans. Therefore, both surveillance of H7N9 viruses in humans and poultry and supervision of poultry movements should be strengthened. IMPORTANCE Since its occurrence in humans in eastern China in spring 2013, the avian H7N9 viruses have been demonstrating the continuing pandemic threat posed by the current influenza ecosystem in China. As the viruses are silently circulated in poultry, with potentially severe outcomes in humans, H7N9 virus activity in humans in China is very important to understand. In this study, we identified a newly emerged H7N9 outbreak source in the Pearl River Delta region. Both sources in the Yangtze River Delta region and the Pearl River Delta region have been established and found to be responsible for the H7N9 outbreaks in mainland China.

Description: Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that is highly prevalent in almost all human populations and is associated with many human cancers, such as nasopharyngeal carcinoma (NPC), Hodgkin's disease, and gastric carcinoma. However, in these EBV-associated cancers, only NPC exhibits remarkable ethnic and geographic distribution. We hypothesized that EBV genomic variations might contribute to the pathogenesis of different human cancers in different geographic areas. In this study, we collected 18 NPC biopsy specimens from the Hunan Province in southern China and de novo assembled 18 NPC biopsy specimen-derived EBV (NPC-EBV) genomes, designated HN1 to HN18. This was achieved through target enrichment of EBV DNA by hybridization, followed by next-generation sequencing, to reveal sequence diversity. These EBV genomes harbored 20,570 variations totally, including 20,328 substitutions, 88 insertions, and 154 deletions, compared to the EBV reference genome. Phylogenetic analysis revealed that all NPC-EBV genomes were distinct from other EBV genomes. Furthermore, HN1 to HN18 had some nonsynonymous variations in EBV genes including genes encoding latent, early lytic, and tegument proteins, such as substitutions within transmembrane domains 1 and 3 of LMP1, FoP_duplication, and zf-AD domains of ENBA1, in addition to aberrations in noncoding regions, especially in BamHI A rightward transcript microRNAs. These variations might have potential biological significance. In conclusion, we reported a genome-wide view of sequence variation in EBV isolated from primary NPC biopsy specimens obtained from the Hunan Province. This might contribute to further understanding of how genomic variations contribute to carcinogenesis, which would impact the treatment of EBV-associated cancer. IMPORTANCE Nasopharyngeal carcinoma (NPC) is highly associated with Epstein-Barr virus (EBV) infection and exhibits remarkable ethnic and geographic distribution. Hunan Province in southern China has a high incidence rate of NPCs. Here, we report 18 novel EBV genome sequences from viruses isolated from primary NPC biopsy specimens in this region, revealing whole-genome sequence diversity.

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.

Description: Pneumonia is one of the most prevalent Staphylococcus aureus -mediated diseases, and the treatment of this infection is becoming challenging due to the emergence of multidrug-resistant S. aureus , especially methicillin-resistant S. aureus (MRSA) strains. It has been reported that LysGH15, the lysin derived from phage GH15, displays high efficiency and a broad lytic spectrum against MRSA and that apigenin can markedly diminish the alpha-hemolysin of S. aureus . In this study, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. No mutual adverse influence was detected between LysGH15 and apigenin in vitro . In animal experiments, the combination therapy showed a more effective treatment effect than LysGH15 or apigenin monotherapy ( P 〈 0.05). The bacterial load in the lungs of mice administered the combination therapy was 1.5 log units within 24 h after challenge, whereas the loads in unprotected mice or mice treated with apigenin or LysGH15 alone were 10.2, 4.7, and 2.6 log units, respectively. The combination therapy group showed the best health status, the lowest ratio of wet tissue to dry tissue of the lungs, the smallest amount of total protein and cells in the lung, the fewest pathological manifestations, and the lowest cytokine level compared with the other groups ( P 〈 0.05). With regard to its better protective efficacy, the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA. This paper reports the combination therapy of lysin and natural products derived from traditional Chinese medicine.

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.

Description: The development of a safe and efficient dengue vaccine represents a global challenge in public health. Chimeric dengue viruses (DENV) based on an attenuated flavivirus have been well developed as vaccine candidates by using reverse genetics. In this study, based on the full-length infectious cDNA clone of the well-known Japanese encephalitis virus live vaccine strain SA14-14-2 as a backbone, a novel chimeric dengue virus (named ChinDENV) was rationally designed and constructed by replacement with the premembrane and envelope genes of dengue 2 virus. The recovered chimeric virus showed growth and plaque properties similar to those of the parental DENV in mammalian and mosquito cells. ChinDENV was highly attenuated in mice, and no viremia was induced in rhesus monkeys upon subcutaneous inoculation. ChinDENV retained its genetic stability and attenuation phenotype after serial 15 passages in cultured cells. A single immunization with various doses of ChinDENV elicited strong neutralizing antibodies in a dose-dependent manner. When vaccinated monkeys were challenged with wild-type DENV, all animals except one that received the lower dose were protected against the development of viremia. Furthermore, immunization with ChinDENV conferred efficient cross protection against lethal JEV challenge in mice in association with robust cellular immunity induced by the replicating nonstructural proteins. Taken together, the results of this preclinical study well demonstrate the great potential of ChinDENV for further development as a dengue vaccine candidate, and this kind of chimeric flavivirus based on JE vaccine virus represents a powerful tool to deliver foreign antigens.

Description: 3,5,6-Trichloro-2-pyridinol (TCP) is a widespread pollutant. Some bacteria and fungi have been reported to degrade TCP, but the gene clusters responsible for TCP biodegradation have not been characterized. In this study, a fragment of the reduced flavin adenine dinucleotide (FADH 2 )-dependent monooxygenase gene tcpA was amplified from the genomic DNA of Ralstonia sp. strain T6 with degenerate primers. The tcpA disruption mutant strain T6- tcpA could not degrade TCP but could degrade the green intermediate metabolite 3,6- d i h ydroxy p yridine-2,5- d ione (DHPD), which was generated during TCP biodegradation by strain T6. The flanking sequences of tcpA were obtained by self-formed adaptor PCR. tcpRXA genes constitute a gene cluster. TcpR and TcpX are closely related to the LysR family transcriptional regulator and flavin reductase, respectively. T6- tcpA -com, the complementation strain for the mutant strain T6- tcpA , recovered the ability to degrade TCP, and the strain Escherichia coli DH10B- tcpRXA , which expressed the tcpRXA gene cluster, had the ability to transform TCP to DHPD, indicating that tcpA is a key gene in the initial step of TCP degradation and that TcpA dechlorinates TCP to DHPD. A library of DHPD degradation-deficient mutants of strain T6 was obtained by random transposon mutagenesis. The fragments flanking the Mariner transposon were amplified and sequenced, and the dhpRIJK gene cluster was cloned. DhpJ could transform DHPD to yield an intermediate product, 5- a mino-2,4,5- t ri o xo p entanoic a cid (ATOPA), which was further degraded by DhpI. DhpR and DhpK are closely related to the AraC family transcriptional regulator and the MFS family transporter, respectively.

Description: KLF5 is an essential basic transcriptional factor that regulates a number of physiopathological processes. In this study, we tested whether and how KLF5 modulates the epithelial-mesenchymal transition (EMT). Using transforming growth factor β (TGF-β)- and epidermal growth factor (EGF)-treated epithelial cells as an established model of EMT, we found that KLF5 was downregulated during EMT and that knockdown of KLF5 induced EMT even in the absence of TGF-β and EGF treatment, as indicated by phenotypic and molecular EMT properties. Array-based screening suggested and biochemical analyses confirmed that the microRNA 200 (miR-200) microRNAs, a group of well-established EMT repressors, were transcriptionally activated by KLF5 via its direct binding to the GC boxes in miR-200 gene promoters. Functionally, overexpression of miR-200 prevented the EMT induced by KLF5 knockdown or by TGF-β and EGF treatment, and ectopic expression of KLF5 attenuated TGF-β- and EGF-induced EMT by rescuing the expression of miR-200. In mouse prostates, knockout of Klf5 downregulated the miR-200 family and induced molecular changes indicative of EMT. These findings indicate that KLF5 maintains epithelial characteristics and prevents EMT by transcriptionally activating the miR-200 family in epithelial cells.

Description: Hepatitis C virus (HCV) infection is a major cause of severe liver disease. Interferon (IFN)/ribavirin treatment remains the standard therapeutic regimen for HCV infection in most countries. IFN-stimulated genes are believed to contribute to antiviral effects. However, emerging evidence suggests that microRNAs (miRNAs), a class of noncoding small RNAs, are involved in the control of viral infection. Here, we systematically profiled the hepatocyte expression of a set of 750 miRNAs in response to alpha interferon (IFN-α) and interleukin-28B (IL-28B) treatments. The anti-HCV activity of differentially expressed miRNAs was evaluated using cell culture-derived HCV in vitro . The results demonstrate that let-7b had a significant anti-HCV effect by inhibiting HCV replication and viral protein translation in human hepatoma cells. In particular, we show that the inhibition of let-7b attenuated the anti-HCV effects of IFN-α and IL-28B. Furthermore, we show that the host factor insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is a target of let-7b. IGF2BP1 was required for HCV replication, and its expression was downregulated by IFN-α and IL-28B. Deletion of the wild-type seed region of let-7b abolished its antiviral activity. Finally, we demonstrate that other let-7 family miRNAs were able to inhibit HCV and to suppress IGF2BP1 expression. In conclusion, we provide an example of a host miRNA regulated by type I and type III IFNs that inhibits HCV replication and infectivity by targeting host targets. These results highlight the important role of miRNAs in the host antiviral immune response and provide a novel candidate for anti-HCV therapy.