Genomic characteristics, pathogenicity and viral shedding of a novel DVEV variant derived from goose.

Duck virus enteritis (DVE), caused by the DVE virus (DVEV), is an acute, septicemic, and contagious disease affecting ducks of different breeds, ages, and sexes. In late spring and summer 2019, several outbreaks of DVE were reported in areas with large waterfowl industries in central and southern China. A goose farm located in Jining County, Shandong Province, was impacted by an acute DVE outbreak in July 2019. The causative DVEV field strain (Goose/DVEV/SDJN/China/2019) was subsequently isolated from the liver specimens collected from acute cases of dead geese, which showed severe hemorrhagic lesions on the esophageal mucosal membranes of specimens collected from all the postmortem cases. Comparison of the genome sequence of this newly isolated field strain (Goose/DVEV/SDJN/China/2019) with the common DVEV strains revealed insertions or mutations in the gB and gC genes, which possibly caused the observed high morbidity and mortality in this acute outbreak. We conducted a trial among geese to evaluate the pathogenicity of this strain. Healthy experimental goslings aged 15 d old were inoculated with 10-5.53 ELD50/0.2 mL doses orally or through intramuscular injection. Clinical signs and esophageal erosion appeared in infected geese. Necropsy revealed hemorrhage and necrosis of the cloacal mucosa and liver. Detection of the virus using real-time PCR in the liver, brain, and spleen indicated that they were the hotspots of DVEV infections. One day after the DVEV infection, virus release and seroconvert were observed in infected geese. Thus, our studies demonstrate that DVEV is highly pathogenic and contagious in geese. To the best of our knowledge, this is the first study on the pathogenicity of mutant duck viral enteritis virus in goslings. This study serves as a foundation for further investigations into the pathophysiology of the recently identified variant DVEV strains.

Neutrophil infiltration associated genes on the prognosis and tumor immune microenvironment of lung adenocarcinoma.

The neutrophils exhibit both anti-tumor and pro-tumor effects in cancers. The correlation between neutrophils and tumor development in lung adenocarcinoma (LUAD) is still uncertain, possibly due to a lack of specific neutrophil infiltration evaluation methods. In this study, we identified 30 hub genes that were significantly associated with neutrophil infiltration in LUAD through data mining, survival analysis, and multiple tumor-infiltrating immune cells (TICs) analysis, including TIMER, CIBERSORT, QUANTISEQ, XCELL, and MCPCOUNTER. Consensus clustering analysis showed that these 30 hub genes were correlated with clinical features in LUAD. We further developed a neutrophil scoring system based on these hub genes. The neutrophil score was significantly correlated with prognosis and tumor immune microenvironment (TIME) in LUAD. It was also positively associated with PD-L1 expression and negatively associated with tumor mutational burden (TMB). When combined with the neutrophil score, the predictive capacity of PD-L1 and TMB for prognosis was significantly improved. Thus, the 30 hub genes might play an essential role in the interaction of neutrophils and LUAD, and the neutrophil scoring system might effectually assess the infiltration of neutrophils. Furthermore, we verified the expression of these 30 genes in the LUAD tumor tissues collected from our department. We further found that overexpressed TNFAIP6 and TLR6 and downregulated P2RY13, SCARF1, DPEP2, PRAM1, CYP27A1, CFP, GPX3, and NCF1 in LUAD tissue might be potentially associated with neutrophils pro-tumor effects. The following in vitro experiments demonstrated that TNFAIP6 and TLR6 were significantly overexpressed, and P2RY13 and CYP27A1 were significantly downregulated in LUAD cell lines, compared to BEAS-2B cells. Knocking down TNFAIP6 in A549 and PC9 resulted in the upregulation of FAS, CCL3, and ICAM-1, and the downregulation of CCL2, CXCR4, and VEGF-A in neutrophils when co-culturing with the conditioned medium (CM) from LUAD cells. Knocking down TNFAIP6 in LUAD also led to an elevated early apoptosis rate of neutrophils. Therefore, overexpressed TNFAIP6 in LUAD cancer cells might lead to neutrophils "N2" polarization, which exhibited pro-tumor effects. Further research based on the genes identified in this pilot study might shed light on neutrophils' effects on LUAD in the future.

Effects of the inoculations using bacteria producing ACC deaminase on ethylene metabolism and growth of wheat grown under different soil water contents.

Crop growth and productivity are often impacted by the increased ethylene content induced by adverse environmental conditions such drought. Inoculations with bacteria producing ACC deaminase is considered as a potential biological approach to improve the growth and tolerance of stressed plants by lowering endogenous ethylene level. In this study, germinated wheat seeds were inoculated using three species of the rhizobacteria, which were isolated from the rhizosphere of wheat growing in dryland, and sown in pots. After three weeks, wheat seedlings were exposed to non-limiting water condition, medium drought and severe drought, respectively, for six weeks. The results showed that, irrespective of rhizobacterial inoculations, decreased soil water contents stimulated wheat ethylene metabolism, which was reflected by the significantly increased activity of ACC synthetase and ACC oxidase, besides an increased content of ACC both in the roots and leaves, and an enhanced capacity of leaves to release ethylene, concomitant with a significant decline in shoot and roots biomass. The inoculations of all three rhizobacterial species under each water condition reduced ACC content in wheat leaves, but effects of the inoculations on ACC synthase and ACC oxidase activity in the leaves and roots, ACC content in the roots, the capacity of leaves to release ethylene, and wheat growth varied with water conditions and bacterial species. Hence, both soil water conditions and rhizobacterial inoculations acted on all the processes of ethylene metabolism, with the former being dominant. The inoculations under non-limiting water condition and medium drought promoted shoot and root growth of wheat plants.

Liriopesides B inhibited cell growth and decreased CA125 level in human ovarian cancer A2780 cells.

To study the effect of liriopesides B on cell growth curve, cell doubling time, the activity of tumour marker CA125 and alkaline phosphatase (AKP) in human ovarian cancer A2780 cells. Both cell growth curve and doubling time were studied by MTT assay, CA125 level and AKP activity were determined by respective kits. Results showed that liriopesides B could shift down the A2780 cells growth curve in a dose-time-dependent manner and inhibit the proliferation in A2780 cells with the maximum inhibitory rate 94.462% at 120 h, the doubling time was prolonged too. CA125 level was decreased in a dose-dependent way as well as AKP activity. Liriopesides B exhibited potential anticancer activity against human ovarian cancer A2780 cells.

[Methylation Status of miR-182 Promoter in Lung Cancer Cell Lines].

BACKGROUND AND OBJECTIVE: It has been proven that the abnormal expression of miR-182 was related to the occurrence and development of tumors. The aim of this study is to explore the relationship between the methylation of miR-182 promoter and its expression in lung cancer cell lines. METHODS: Real-time quantitative PCR and methylation-specific PCR were used to detect the expression level of miR-182 and its promoter methylation status in five lung cancer cell lines (A549, L9981, NL9980, 95C and 95D). DNA sequencing was used to confirm the methylation results. RESULTS: The level of miR-182 expression significantly differs among these lung cancer cell lines. The highly metastatic human lung cancer cell lines, namely, A549 and L9981, demonstrate a relatively lower expression level of miR-182 compared with the lowly metastatic human lung cancer cell line 95C. Methylation-specific PCR and DNA sequencing assay results indicate that these lung cancer cell lines present different levels of miR-182 promoter methylation, and the highest methylation level is observed in A549 cells. Furthermore, the expression of miR-182 in these cell lines significantly increases when treated with 10 µM 5'-Aza-dC. CONCLUSIONS: DNA methylation occurs in the miR-182 promoter region in lung cancer cell lines. This methylation can regulate the expression level of miR-182. Further study must be conducted to explore the function of miR-182 promoter methylation in lung cancer occurrence and development.
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Involvement of transient receptor potential melastatin-8 (TRPM8) in menthol-induced calcium entry, reactive oxygen species production and cell death in rheumatoid arthritis rat synovial fibroblasts.

Rheumatoid arthritis is most prominently characterized by synoviocyte hyperplasia which therefore serves as an important target for clinical therapy. In the present study, it was observed that menthol, the specific agonist of transient receptor potential melastatin subtype 8 (TRPM8), could induce sustained increases of cytosolic calcium concentration ([Ca(2+)]c) in synoviocytes isolated from collagen-induced arthritis rats in dose-dependent manner, which was evidently blocked by applying an extracellular Ca(2+)-free buffer. Menthol-induced [Ca(2+)]c increase was also significantly inhibited by potent TRPM8 antagonist capsazepine (CZP), indicating that this [Ca(2+)]c elevation was mostly attributed to TRPM8-mediated Ca(2+) entry. Besides, RT-PCR indeed demonstrated presence of TRPM8 in the synoviocytes. Meanwhile, it was found that menthol evoked production of intracellular reactive oxygen species, which could be abolished by Ca(2+) free solutions or CZP. Further experiments showed that menthol reduced the cell numbers and survival of synoviocytes. This reduction was associated with apoptosis as suggested by mitochondrial membrane depolarization, nuclear condensation and a caspase 3/7 apoptotic assay. Menthol-induced death and apoptosis of synoviocytes both were obviously inhibited by CZP, intracellular calcium chelator BAPTA-AM, and reactive oxygen species inhibitor diphenylene iodonium, respectively. Taken together, our data indicated that menthol resulted in synoviocyte death associated with apoptosis via calcium entry and reactive oxygen species production depending on TRPM8 activation.