Transcriptome analysis of Vero cells infected with attenuated vaccine strain CDV-QN-1.

To better understand the interaction between attenuated vaccines and host antiviral responses, we used bioinformatics and public transcriptomics data to analyze the immune response mechanisms of host cells after canine distemper virus (CDV) infection in Vero cells and screened for potential key effector factors. In this study, CDV-QN-1 infect with Vero cells at an MOI of 0.5, and total RNA was extracted from the cells 24 h later and reverse transcribed into cDNA. Transcriptome high-throughput sequencing perform using Illumina. The results showed that 438 differentially expressed genes were screened, of which 409 were significantly up-regulated and 29 were significantly down-regulated. Eight differentially expressed genes were randomly selected for RT-qPCR validation, and the change trend was consistent with the transcriptomics data. GO and KEGG analysis of differentially expressed genes revealed that most of the differentially expressed genes in CDV-QN-1 infection in the early stage were related to immune response and antiviral activity. The enriched signaling pathways mainly included the interaction between cytokines and cytokine receptors, the NF-kappa B signaling pathway, the Toll-like receptor signaling pathway, and the NOD-like receptor signaling pathway. This study provides a foundation for further exploring the pathogenesis of CDV and the innate immune response of host cells in the early stage of infection.

Understanding Macrophage-Tumor Interactions: Insights from Single-Cell Behavior Monitoring in a Sessile Microdroplet System.

Interaction between tumor-associated macrophages and tumor cells is crucial for tumor development, metastasis, and the related immune process. However, the macrophages are highly heterogeneous spanning from anti-tumorigenic to pro-tumorigenic, which needs to be understood at the single-cell level. Herein, a sessile microdroplet system designed for monitoring cellular behavior and analyzing intercellular interaction, demonstrated with macrophage-tumor cell pairs is presented. An automatic procedure based on the inkjet printing method is utilized for the precise pairing and co-encapsulation of heterotypic cells within picoliter droplets. The sessile nature of microdroplets ensures controlled fusion and provides stable environments conducive to adherent cell culture. The nitric oxide generation and morphological changes over incubation are explored to reveal the complicated interactions from a single-cell perspective. The immune response of macrophages under distinct cellular microenvironments is recorded. The results demonstrate that the tumor microenvironment displays a modulating role in polarizing macrophages from anti-tumorigenic into pro-tumorigenic phenotype. The approach provides a versatile and compatible platform to investigate intercellular interaction at the single-cell level, showing promising potential for advancing single-cell behavior studies.

Chemical Plasma Membrane Perforation Generated by a Microfluidic Probe for Single-Cell Intracellular Protein Delivery.

Efficient and convenient delivery of exogenous molecules into cells is important for cell biology research. However, many intracellular delivery methods require carrier-mediated or physical field assistance, complicating the delivery process. Here, a general, simple, and effective method for in situ single-cell intracellular delivery is reported. A solution containing digitonin and cargo is precisely applied to single cells using a microfluidic probe. Digitonin binds to cholesterol in the plasma membrane to induce perforation, and the cargo enters the cell through the pore. By optimizing parameters, propidium iodide (0.67 kDa) and FITC-dextran (10, 40, and 150 kDa) can be successfully introduced into single cells within 3 min while maintaining cell viability. To prove the potential of this method for cell research, we delivered cytochrome C (13 kDa) and cyclophilin A (18 kDa) into cells by this method. The delivered cytochrome C successfully induces cell apoptosis by activating the caspase pathway, and cyclophilin A performs an antioxidant effect in the cells, which may enhance the drug resistance of glioma cells. It is believed that this method will be an attractive tool for single-cell intracellular delivery.

Quantitative proteomics based on TMT revealed the response of PK15 cells infected PEDV wild strain.

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), is an acute and highly contagious enteric disease with a high mortality rate in suckling piglets. Identification of proteins associated with PEDV infection may provide insights into the pathogenesis of this viral disease. In this study, we employed tandem mass tag (TMT) quantitative protein analysis to investigate proteomic changes in PK15 cells following PEDV infection, and differential protein expression profiles were obtained at 0 h, 24 h, and 48 h post-infection. Overall, a total of 6330 proteins were identified. Applying criteria for fold change >1.5 < 0.67 and p-values <0.05 resulted in the identification of 59 up-regulated proteins and 103 down-regulated proteins that exhibited significant alterations in the H24 group compared to the H0 group. The H48 group demonstrated significant upregulation of 110 proteins and downregulation of 144 proteins compared to the H0 group; additionally, there were also 10 upregulated and 30 downregulated proteins in the H48 group when compared to the H24 group. These differentially expressed proteins (DEPs) were involved in immune response regulation, signal transduction, lipid transport and metabolism processes as well as cell apoptosis pathways. Based on these DEPs, we propose that PEDV may disrupt signal transduction pathways along with lipid transport and metabolism processes leading to maximal viral replication, it may also trigger inflammatory cascades accordingly. These findings could provide valuable information for elucidating specific pathogenesis related to PEDV infection while contributing towards developing new antiviral strategies.

Fabrication and Performance of Bubble-Containing Multicompartmental Particles: Novel Self-Orienting Carriers.

In this work, a class of bubble-containing multicompartmental particles with self-orienting capability is developed, where a single bubble is enclosed at the top of the super-segmented architecture. Such bubbles, driven by potential energy minimization, cause the particles to have a bubble-upward preferred orientation in liquid, enabling efficient decoding of their high-density signals in an interference-resistant manner. The particle preparation involves bubble encapsulation via the impact of a multicompartmental droplet on the liquid surface and overall stabilization via rational crosslinking. The conditions for obtaining these particles are systematically investigated. Methodological compatibility with materials is demonstrated by different hydrogel particles. Finally, by encapsulating cargoes of interest, these particles have found broad applications in actuators, multiplexed detection, barcodes, and multicellular systems.

RSL3 Inhibits Porcine Epidemic Diarrhea Virus Replication by Activating Ferroptosis.

Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that induces diarrhea and death in neonatal piglets, resulting in substantial economic losses to the global swine industry. The mechanisms of PEDV infection and the roles of host factors are still under exploration. In this study, we used the ferroptosis pathway downstream target activator (1S,3R)-RSL3 compound as a starting point, combined with the interactions of N-acetylcysteine and deferoxamine, to elucidate the effects of a series of compounds on PEDV proliferation. We also established glutathione peroxidase 4 (GPX4) gene overexpression to further elucidate the relationship between the ferroptosis pathway and PEDV. (1S,3R)-RSL3 inhibited PEDV replication in Vero cells, while N-acetylcysteine and deferoxamine promoted its proliferation. In addition, (1S,3R)-RSL3 mainly affected the replication stage of PEDV. Overexpression of GPX4 promoted PEDV proliferation, indicating that the ferroptosis pathway could influence PEDV replication in Vero cells. This study focused on the mechanism of (1S,3R)-RSL3 inhibition on PEDV, laying the foundation for exploring the pathogenic mechanisms of PEDV and drug development.

Comparative transcriptomic analysis of PK15 cells infected with a PRV variant and the Bartha-K/61 vaccine strain.

Introduction: Pseudorabies virus (PRV) is a herpesvirus that can infect domestic animals, such as pigs, cattle and sheep, and cause fever, itching (except pigs), and encephalomyelitis. In particular, the emergence of PRV variants in 2011 have resulted in serious economic losses to the Chinese pig industry. However, the signaling pathways mediated by PRV variants and their related mechanisms are not fully understood. Methods: Here, we performed RNA-seq to compare the gene expression profiling between PRV virulent SD2017-infected PK15 cells and Bartha-K/61-infected PK15 cells. Results: The results showed that 5,030 genes had significantly different expression levels, with 2,239 upregulated and 2,791 downregulated. GO enrichment analysis showed that SD2017 significantly up-regulated differentially expressed genes (DEGs) were mainly enriched in the binding of cell cycle, protein and chromatin, while down-regulated DEGs were mainly enriched in ribosomes. KEGG enrichment analysis revealed that the pathways most enriched for upregulated DEGs were pathways in cancer, cell cycle, microRNAs in cancer, mTOR signaling pathway and autophagy-animal. The most down-regulated pathways of DEGs enrichment were ribosome, oxidative phosphorylation, and thermogenesis. These KEGG pathways were involved in cell cycle, signal transduction, autophagy, and virus-host cell interactions. Discussion: Our study provides a general overview of host cell responses to PRV virulent infection and lays a foundation for further study of the infection mechanism of PRV variant strain.

Erastin inhibits porcine epidemic diarrhea virus replication in Vero cells.

Background: Porcine epidemic diarrhea virus (PEDV), an intestinal pathogenic coronavirus, has caused significant economic losses to the swine industry worldwide. At present, there are several treatment methods, but there is still a lack of clinically effective targeted drugs, new antiviral mechanisms and drugs need to be explored. Methods: In this study, we established a model of erastin versus ferrostatin-1 treatment of Vero cells, and then detected virus proliferation and gene expression by RT-qPCR through PEDV infection experiments. Results: We demonstrated for the first time that erastin significantly inhibited the replication of PEDV upon entry into cells; Vero treated with erastin significantly regulated the expression of three genes, NRF2, ACSL4 and GPX4, notably erastin regulated the expression of these three genes negatively correlated with the expression induced by PEDV virus infection. Conclusions: Since NRF2, ACSL4 and GPX4 are classical Ferroptosis genes, this study speculates that erastin may inhibit the replication of PEDV in Vero cells in part through the regulation of ferroptosis pathway, and erastin may be a potential drug for the treatment of PEDV infection.

[Effects of different orthodontic treatments on sputum chemokines CX3CL1, RANKL/OPG levels in patients with malocclusion].

PURPOSE: To investigate the effects of different orthodontic treatments on gingival crevicular fluid chemokine CX3CL1, nuclear factor κB receptor activating factor ligand/osteoprotegerin(RANKL/OPG) levels in patients with malocclusion. METHODS: Ninety-six patients with malocclusion who were scheduled to undergo orthodontic treatment were randomly divided into four groups. All patients were treated with square wire appliance, and 0, 50, 150, 250 g of far-distal orthodontic force were given respectively. The levels of CX3CL1 and RANKL/OPG in gingival crevicular fluid were detected in four groups after 1, 2, 3, and 4 weeks of treatment. SPSS 25.0 software package was used for statistical analysis of the date. RESULTS: The levels of CX3CL1, RANKL and RANKL/OPG in the gingival crevicular fluid of the four groups were continuously increased after treatment for 1-3 weeks, and decreased after 4 weeks of treatment (P<0.05). The OPG in the gingival crevicular fluid was at a low level after 1-3 weeks of treatment. There was an increase after 4 weeks of treatment (P<0.05). The levels of CX3CL1, RANKL, OPG and RANKL/OPG in gingival crevicular fluid increased gradually in group A, B, C and D (P<0.05), and the differences between the groups were statistically significant (P<0.05). CONCLUSIONS: The levels of CX3CL1 and RANKL/OPG in gingival crevicular fluid are closely related to orthodontic force and treatment time, and can be used as an index to evaluate orthodontic treatment of alveolar bone remodeling.