The autophagy-related degradation of MDA5 by Tembusu virus nonstructural 2B disrupts IFNß production.

Duck Tembusu virus (TMUV) is a serious avian pathogen causing a decline in egg production, but the mechanism of the virus that breaks through the innate immune system is poorly understood. Here, we show that TMUV inhibits poly(I:C)-induced interferon (IFN) production. Because poly(I:C) transfection can specifically activate the MDA5 pathway in duck primary cells, we found that infection with TMUV can specifically target MDA5 and lead to its degradation. MDA5 downregulation could be blocked by the autophagy inhibitor 3-methyladenine (3-MA) but not a proteasome inhibitor, strongly implicating MDA5 degradation as an autophagy-related degradation pathway. Pretreatment with 3-MA enhanced the expression of MDA5 and inhibited TMUV replication. To screen TMUV proteins that degraded MDA5, the TMUV replicon and MDA5-Flag were cotransfected into cells, and the western blot analysis showed that nonstructural 2B (NS2B) can degrade MDA5 in a dose-dependent manner. Dual-luciferase assays indicate that NS2B alone inhibits MDA5- or poly(I:C)-mediated IFN production. NS2B binds MDA5 in the presence of 3-MA. The deletion of the amino acids of NS2B from residues 51 to 92 (hydrophilic area) restored the expression of MDA5 and relieved the MDA5-mediated IFNß production inhibition by NS2B, indicating that the hydrophilic area of NS2B is important for its interaction with host innate immunity.

Identification of duck GSDME: Tissue distribution, proteolysis and cellular location.

Gasdermin E (GSDME) is a member of the gasdermin family. Cleavage of mammalian GSDME by apoptotic caspases or granzyme proteases liberates the N-terminal effector domain (GSDME-N), which is capable of forming membrane pores and executing inflammation and cell death. Herein, duck GSDME was first cloned with a total length of 1500 bp and encoding 499 amino acids (aa), which is most evolutionally related to the chicken GSDME. The tissue-distribution profiles of GSDME showed that relatively high levels of GSDME mRNA were detected in immune tissues of duckling and adult ducks. Additionally, GSDME mRNA was significantly upregulated in duck primary embryo fibroblasts (DEFs) and duck primary ovary cells after duck Tembusu virus (DTMUV) infection. Intriguingly, when duck caspase-3 was coexpressed, the duck GSDME produced two GSDME-N fragments with molecular weights of 25 kDa and 30 kDa. Furthermore, both GSDME and cleaved GSDME were observed to be located in the cytoplasm by indirect immunofluorescence assay (IFA). Taken together, our research data show that duck GSDME has similar biological characteristics to mammals. These findings highlight the role of duck GSDME in TMUV infection, indicating that cooperation between GSDME and caspase-3 promotes the proteolytic process.

Roles of galectin-7 and S100A9 in cervical squamous carcinoma: Clinicopathological and in vitro evidence.

In our study, we for the first time assessed the association of galectin-7 and S100A9 with clinicopathological variables and survival outcomes in cervical squamous carcinoma patients and explored the underlying molecular mechanisms in cervical squamous carcinoma cell lines. Immunohistochemical analysis of 243 patient samples showed that the positive staining rate for galectin-7 and S100A9 gradually decreased from normal cervical tissue to intraepithelial neoplasia and to cervical squamous carcinoma. Both galectin-7 and S100A9 showed significant negative association with lymph node metastasis and staging of cervical squamous carcinoma. Cervical squamous carcinoma patients with negative staining of galectin-7 or S100A9 showed significantly lower 5-year overall survival rate than those with positive staining. Multivariate analysis with the Cox's proportional hazards model indicated that both galectin-7 and S100A9 had significant protective effect on cervical squamous carcinoma patients. Subsequent in vitro study in SiHa and C-33A human cervical squamous carcinoma cell lines revealed that knocking down galectin-7 or S100A9 enhanced tumor cell invasion and tumor cell viability against paclitaxel-induced apoptotic stress, likely through increasing the matrix metalloproteinase-9 expression and activating the phosphatidylinositol 3-kinase/Akt signaling pathway, respectively. Knocking down both galectin-7 and S100A9 produced a synergistic effect, with galectin-7 displaying more significant and consistent protective effects than S100A9 on cervical squamous carcinoma cells. In summary, our study for the first time provides clinicopathological and in vitro evidence showing that both galectin-7 and S100A9 play important protective roles in cervical squamous carcinoma, which provides fresh insights into the biology of cervical squamous carcinoma.

The G92 NS2B mutant of Tembusu virus is involved in severe defects in progeny virus assembly.

Although the role of the flavivirus non-structural 2B (NS2B) as a viral protease cofactor has been investigated, its role in viral proliferation has rarely been studied. Moreover, important details on the involvement of the flavivirus NS2B in the virus replication cycle, including attachment, entry, RNA replication, assembly and release, remain unknown. Here, an alignment of flavivirus NS2B was performed to select conserved amino acids for the site-directed mutagenesis study. G92, which is not totally conserved to mutation, was also used. Certain mutations (P32A, G36A and G37A) completely blocked viral RNA synthesis, four mutations (D25A, A43Y, V44A, G92A and P112A) showed a slight defect in virus replication, and only the G92A mutant affected virus proliferation by blocking virus release but not RNA replication. By taking advantage of the tembusu virus (TMUV) replicon that represents viral genome RNA replication, we showed that the G92A mutation did not obviously impair viral replication. Naturally, the NS2B3 G92A mutation protein did not affect the self-cleavage ability or NS2B3 protein-mediated cleavage of the NS2AB G92A mutant. These results indicated that the G92A mutation did not affect viral protease activity. By using the TMUV infection clone and the replicon package system, the effect of G92 on the virus lifecycle was narrowed to viral resemblance or release. Subcellular fractionation further confirmed that G92A attenuated TMUV by reducing virus assembly. Collectively, these studies extend the list of NS2B functions beyond protease activity as a cofactor, show that transmembrane region amino acids play an important role in viral replication and indicate that G92 is involved in the assembly of infectious TMUV particles.

Caveolin-1 enhances resveratrol-mediated cytotoxicity and transport in a hepatocellular carcinoma model.

BACKGROUND: Resveratrol (RES), an estrogen analog, is considered as a potential cancer chemo-preventive agent. However, it remains unclear how RES is transported into cells. In this study, we observed that Caveolin-1(CAV1) expression can increase the cytotoxic and pro-apoptotic activity of RES in a dose- and time-dependent manner both in vitro and in vivo in a Hepatocellular Carcinoma animal model. METHODS: High performance liquid chromatography (HPLC) demonstrated that RES intra-cellular concentration is increased about 2-fold in cells stably expressing CAV1 or CAVM1 (a scaffolding domain (81-101AA)-defective CAV1 mutant) compared to the untransduced human Hepatoblastoma cell line (HepG2) or after transduction with the green fluorescent protein (GFP) control vector. The increased intra-cellular transport of RES was abolished in cells stably expressing CAVM2 (a cholesterol shuttle domain (143-156AA)-defective CAV1 mutant) or CAVRNAi. In order to further characterize CAV1-dependent RES transport, we synthesized RES-dansyl chloride derivatives as fluorescent probes to visualize the transport process, which demonstrated a distribution consistent with that of CAV1 in HepG2 cells. RESULTS: In addition, RES endocytosis was not mediated by estrogen receptor (ER) alpha and beta, as suggested by lack of competitive inhibition by estrogen or Tamoxifen. Pathway analysis showed that RES can up-regulate the expression of endogenous CAV1; this activates further the MAPK pathway and caspase-3 expression. DISCUSSION: This study provides novel insights about the role played by CAV1 in modulating cellular sensitivity to RES through enhancement of its internalization and trafficking.

Screening for differentially expressed genes between left- and right-sided colon carcinoma by microarray analysis.

Left-sided colon carcinoma (LSCC) and right-sided colon carcinoma (RSCC) differ in their genetic susceptibilities to neoplastic transformation. The present study identified 11 genes that were differentially expressed in LSCC and RSCC by expression profiling with microarray analysis. Compared with RSCC, the human genes for L-lactate dehydrogenase B chain (LDHB), cyclin-dependent kinase 4 inhibitor D (CDKN2D), phosphatidylinositol-4-phosphate-3-kinase C2 domain-containing subunit α (PI3KC2α), protocadherin fat 1 (FAT; a human protein that closely resembles the Drosophila tumor suppressor, fat) and dual specificity protein phosphatase 2 (DUSP2) were upregulated in LSCC. By contrast, genes for ubiquitin D (UBD), casein kinase-1 binding protein (CK1BP), synaptotagmin-13 (SYT1), zinc finger protein 560 (ZNF560), pleckstrin homology domain-containing family B member 2 (PLEKHB2) and IgGFc-binding protein (FCGBP) were downregulated in LSCC compared with RSCC. A quantitative polymerase chain reaction (qPCR) analysis revealed that the mRNA levels of UBD and CK1BP in LSCC were significantly lower compared with those in RSCC (P=0.033 and P= 0.005, respectively), whereas the mRNA levels of LDHB and CDKN2D in LSCC were significantly higher compared with those in RSCC (P=0.008 and P=0.017, respectively). Western blot and immunohistochemical analyses demonstrated that the expression of CDKN2D in LSCC was significantly higher compared with that in RSCC, while the expression of UBD in LSCC was significantly lower compared with that in RSCC. The present study provides important insights into the understanding of the molecular genetic basis for the different biological behaviors observed between LSCC and RSCC. These insights may therefore serve as a basis for the identification of novel colon cancer markers and therapeutic targets.