Porcine promyelocytic leukemia protein isoforms suppress Japanese encephalitis virus replication in PK15 cells.

BACKGROUND: Promyelocytic leukemia protein (PML) is a primary component of PML nuclear bodies (PML-NBs). PML and PML-NBs play critical roles in processes like the cell cycle, DNA damage repair, apoptosis, and the antiviral immune response. Previously, we identified five porcine PML alternative splicing variants and observed an increase in the expression of these PML isoforms following Japanese encephalitis virus (JEV) infection. In this study, we examined the functional roles of these PML isoforms in JEV infection. METHODS: PML isoforms were either knocked down or overexpressed in PK15 cells, after which they were infected with JEV. Subsequently, we analyzed the gene expression of PML isoforms, JEV, and the interferon (IFN)-ß signaling pathway using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Viral titers were determined through 50% tissue culture infectious dose (TCID50) assays. RESULTS: Our results demonstrated that the knockdown of endogenous PML promoted JEV replication, while the overexpression of PML isoforms 1, 3, 4, and 5 (PML1, PML3, PML4, and PML5) inhibited JEV replication. Further investigation revealed that PML1, PML3, PML4, and PML5 negatively regulated the expression of genes involved in the interferon (IFN)-ß signaling pathway by inhibiting IFN regulatory factor 3 (IRF3) post-JEV infection. CONCLUSIONS: These findings demonstrate that porcine PML isoforms PML1, PML3, PML4, and PML5 negatively regulate IFN-ß and suppress viral replication during JEV infection. The results of this study provide insight into the functional roles of porcine PML isoforms in JEV infection and the regulation of the innate immune response.

Development and students' evaluation of a blended online and offline pedagogy for physical education theory curriculum in China during the COVID-19 pandemic.

The outbreak and continuation of the COVID-19 pandemic has challenged the implementation of physical education theory (PET) curriculums among global colleges and universities. This study aimed to describe the design and students' evaluation of a blended "Sports Multimedia Courseware Design" course among Chinese university students during the COVID-19 pandemic. Using information communication technologies, a 4-month blended course was developed, which consisted of 36 credits (18-credit online self-learning + 18-credit offline group-learning). A total of 1300 Chinese university students who majored in physical education, completed the blended course from Mar to Jun 2020, among which 238 (69.75% males; 21 ± 1.2 years) were randomly recruited to evaluate the course in terms of three aspects: (1) online self-learning, (2) offline group-learning, and (3) overall learning outcomes. A descriptive analysis was conducted using the IBM SPSS 27.0. Students' overall positive evaluation supported a successful development and implementation of the blended course. Over 90% of students fulfilled the learning tasks and satisfied with the online learning resources. About 83% of students indicated high levels of autonomous motivation and engagement in online self-learning. Approximately 88% of students showed positive attitudes to the offline group-learning content, while the participation rate (60%) was relatively lower than of the online self-learning. Over 50% of the students indicated self-improvements in diverse aspects after attending the blended course. Blended online and offline pedagogy shows apparent promise in delivering the PET course among Chinese university students during the COVID-19 pandemic. Further application and comprehensive evaluation are warranted in the future.

Japanese encephalitis virus perturbs PML-nuclear bodies by engaging in interactions with distinct porcine PML isoforms.

Promyelocytic leukemia (PML) protein constitutes an indispensable element within PML-nuclear bodies (PML-NBs), playing a pivotal role in the regulation of multiple cellular functions while coordinating the innate immune response against viral invasions. Simultaneously, numerous viruses elude immune detection by targeting PML-NBs. Japanese encephalitis virus (JEV) is a flavivirus that causes Japanese encephalitis, a severe neurological disease that affects humans and animals. However, the mechanism through which JEV evades immunity via PML-NBs has been scarcely investigated. In the present study, PK15 cells were infected with JEV, and the quantity of intracellular PML-NBs was enumerated. The immunofluorescence results indicated that the number of PML-NBs was significantly reduced in JEV antigen-positive cells compared to viral antigen-negative cells. Subsequently, ten JEV proteins were cloned and transfected into PK15 cells. The results revealed that JEV non-structural proteins, NS2B, NS3, NS4A, NS4B, and NS5, significantly diminished the quantity of PML-NBs. Co-transfection was performed with the five JEV proteins and various porcine PML isoforms. The results demonstrated that NS2B colocalized with PML4 and PML5, NS4A colocalized with PML1 and PML4, NS4B colocalized with PML1, PML3, PML4, and PML5, while NS3 and NS5 interacted with all five PML isoforms. Furthermore, ectopic expression of PML isoforms confirmed that PML1, PML3, PML4, and PML5 inhibited JEV replication. These findings suggest that JEV disrupts the structure of PML-NBs through interaction with PML isoforms, potentially leading to the attenuation of the host's antiviral immune response.

Integrated Transcriptomic and Metabolomic Analysis of the Mechanism of Foliar Application of Hormone-Type Growth Regulator in the Improvement of Grape (Vitis vinifera L.) Coloration in Saline-Alkaline Soil.

(1) Background: To solve the problems of incomplete coloration and quality decline caused by unreasonable use of regulators in grapes, this study clarified the differences in the effects of a hormone-type growth regulator (AUT) and two commercial regulators on grape coloration and quality through field experiments. (2) Methods: The color indexes (brightness (L*), red/green color difference (a*), yellow/blue color difference (b*), and color index for red grapes (CIRG)) of grape fruit were measured using a CR-400 handheld color difference meter. The titratable acid content, total phenol content, and total sugar content were measured using anthrone colorimetry, folinol colorimetry, and NaOH titration, respectively, and the chalcone isomerase activity, phenylalanine ammoniase activity, dihydroflavol reductase activity, and anthocyanin content were measured using a UV spectrophotometer. (3) Results: The a*, total sugar and total phenol contents, and chalcone isomerase (CHI) and phenylalanine ammoniase (PAL) activities of grape fruit in the AUT treatment significantly increased, while the titratable acid content significantly decreased, compared to those in the CK treatment. The expressions of the differentially expressed genes (DEGs) trpB and argJ in AUT treatment were significantly up-regulated. The expressions of the differentially expressed metabolites (DEMs) phenylalanine and 4-oxoproline were significantly up-regulated, while those of 3,4-dihydroxybenzaldehyde and N-acetyl glutamate were significantly down-regulated. The CIRG significantly increased by 36.4% compared to that in the CK, indicating improved fruit coloration. (4) Conclusion: The AUT could shorten the color conversion period of grape fruit and improve the coloration, taste, and tolerance to saline and alkaline stresses.