Mechanistic study of heat shock protein 60-mediated apoptosis in DF-1 cells.

Heat shock proteins (HSP) are a group of highly conserved molecular chaperones found in various organisms and have been associated with tumorigenesis, tumor progression, and metastasis. However, the relationship between HSP60 and apoptosis remains elusive. The aim of this study was to explore the role and regulatory mechanisms of apoptosis in response to altered HSP60 expression. We generated DF-1 cell lines of both HSP60 overexpression and knockdown and assessed their impact on apoptosis levels using ELISA and flow cytometry analyses. Additionally, we examined the transcription and protein expression levels of apoptosis-related signaling factors using fluorescence quantitative PCR (qPCR) and Western blotting analyses. Heat shock proteins 60 overexpression led to a significant decrease in apoptosis levels in DF-1 cells, which could be attributed to the downregulation of BAX and BAK expression, the upregulation of Bcl-2, and the decreased expression of Caspase 3. Conversely, HSP60 knockdown led to a substantial increase in apoptosis levels in DF-1 cells, facilitated by the downregulation of BAX and Bcl-2 expression, and the upregulation of BAK expression, which increased Caspase 3 levels, thereby promoting apoptosis. The findings of our study provide the first evidence of the inhibitory effect of HSP60 on apoptosis in DF-1 cells. These observations have significant implications for disease progression and cancer research, with potential medical applications.

Preparation and Characterization of Multilayer pH-Responsive Hydrogel Loaded Ganoderma lucidum Peptides.

To develop a safe, targeted, and efficient assembly of a stable polypeptide delivery system, in this work, chitosan, sodium alginate, and sodium tripolyphosphate were used as materials for the preparation of hydrogels. M-SCT hydrogels were prepared by ionic gelation and the layer-by-layer (LBL) method. The composite hydrogels exhibited excellent pH sensitivity and Ganoderma lucidum peptides (GLP) loading capacity. The prepared hydrogels were characterized and evaluated. The internal three-dimensional network structure of the hydrogel was observed by scanning electron microscopy (SEM), and Fourier transform infrared (FT-IR) spectroscopy confirmed the electrostatic interactions among the components. X-ray diffraction (XRD) was used to observe the crystal structure of the hydrogel. The maximum peptide encapsulation efficiency was determined to be 81.73%. The digestion stability and thermal stability of M-SCT hydrogels loaded GLP were demonstrated to be improved. The amount of peptides released from the GLP/M-SCT-0.75 hydrogels in simulated gastric fluid was lower than 30%. In addition, the ABTS assays showed that the free radical scavenging ability of the GLP/M-SCT-0.75 hydrogels confirmed the efficacy of the hydrogels in retaining the antioxidant activity of GLP. The study suggested the M-SCT-0.75 hydrogels had a great deal of potential as a peptide carrier for oral delivery.

Non-Structural Protein 3 of Duck Tembusu Virus Induces Autophagy via the ERK and PI3K-AKT-mTOR Signaling Pathways.

Despite autophagy's pivotal role in the replication of viruses such as duck Tembusu virus (DTMUV), which has caused massive economic losses to the poultry industry in the world, the specific relationships between DTMUV and cellular autophagy remain largely unknown. In response, we investigated the interactions between autophagy and DTMUV, the effects of the structural and non-structural proteins of DTMUV on autophagy, and the autophagy-related signaling pathways induced by DTMUV. Among the results, DTMUV increased the autophagy flux in duck embryo fibroblasts (DEF) and BHK-21 cells, while autophagy facilitated viral replication. After we pharmacologically induced autophagy with rapamycin (RAPA), the replication of DTMUV increased by 15.23-fold compared with the control group of DEF cells. To identify which DTMUV protein primarily induced autophagy, all three structural proteins and seven non-structural proteins of DTMUV were transfected into cells, and the results showed that non-structural protein 3 (NS3) induced significant autophagy in DEF cells. By means of Western blot, immunofluorescence, and transmission electron microscopy, we confirmed that NS3 protein could significantly induce autophagy and autophagy flux. Furthermore, we showed that NS3 induced autophagy in DEF cells through extracellular signal-regulated kinase 2 (ERK2) and phosphatidylinositol-3-kinase (PI3K)/AKT and the mammalian target of rapamycin (mTOR) signaling pathways using specific inhibitors and RNA interference assays. Finally, autophagy induced by NS3 promoted DTMUV replication. These results provide novel insight into the relationship between DTMUV and autophagy, broadening the current understanding of the molecular pathogenesis of DTMUV.

Knockout of p53 leads to a significant increase in ALV-J replication.

Avian leukemia is a common malignant disease, and and its regulatory mechanism is complex. As the most extensive tumor suppressor gene in cancer research, p53 can control multiple functions such as that of DNA repair, induction of apoptosis, cell cycle arrest and so on. In view of the diversity associated with varied function of p53, this study analyzed the possible effect of gene on ALV-J replication and its regulatory mechanism. We successfully constructed a p53 knockout DF-1 cell line (p53-KO-DF-1 cells) by using CRISPR-Cas9 system. When ALV-J was co-infected with DF-1 and p53-KO-DF-1 cells, it was found that compared with wild-type DF-1 cells, the viral copy number of p53-KO-DF-1 cells infected with ALV-J increased significantly 48 h after infection, whereas the expression of innate immune factors such as Il-2,TNF- α, IFN- γ and MX1 decreased significantly. Detection of p53-related tumor genes indicated that after p53 deletion, the expression of c-myc, bcl-2, and bak increased significantly, while the expression of p21 and p27 was noted to be decreased. The cell cycle distribution and apoptosis of the 2 cell lines was detected by flow cytometry analysis. The results showed that p53 knockout prevented G0/G1 and G2 M phase arrest induced by ALV-J, and substantially decreased the rate of apoptosis. Overall, the results indicated that p53 gene can effectively inhibits ALV-J replication by regulating important cellular processes, and p53 gene related proteins involved in cell cycle activity may function as the key targets for the prevention and treatment of ALV-J.

Detection of p53 mutation and serum monitoring alert caused by Marek's disease virus in poultry.

BACKGROUND: Marek's disease (MD) is a chicken neoplastic disease, which brings huge economic losses to the global poultry industry. The wild type p53, a tumor suppressor gene, plays a key role in blocking cell cycle, promoting apoptosis, and maintaining the stability of the genome. However, the mutant p53 losses its tumor inhibitory role and become an oncogene when a mutation has happened. RESULTS: The mutation rate of p53 was 60% in the experimentally and naturally infected chickens. The mutations included point-mutations and deletions, and mostly located in the DNA-binding domain. The mutated p53 was expressed in various tumor tissues in an infected chicken. The mutant P53 proteins were notably accumulated in the cytoplasm due to the loss in the function of nuclear localization. Unlike the study on human cancer, the concentrations of P53 in the serums of MD infected chicken were significantly lower than the control group. CONCLUSIONS: The p53 mutations were apparent in the development of MD. P53 and P53 antibody level in serum could be a useful marker in the diagnosis and surveillance of MD.

Porcine Reproductive and Respiratory Syndrome Virus Structural Protein GP3 Regulates Claudin 4 To Facilitate the Early Stages of Infection.

Claudins (CLDN) are a family of proteins that represent the most important components of tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Several types of viruses make full use of CLDN to facilitate entry into cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogens in the swine industry. In this study, we found that CLDN4 functions as an anti-PRRSV factor by blocking its absorption during the early stages of infection. The small extracellular loop (ECL2) of CLDN4 restricted the viral particles outside cells by binding to GP3. A novel function of GP3-mediated regulation of CLDN4 transcription was suggested. CLDN4 can be decreased through downregulating the level of CLDN4 transcription by ubiquitinating the transcription factor, SP1. The mechanism by which highly pathogenic PRRSV infects the epithelium was proposed. Importantly, ECL2 was found to block PRRSV absorption and infection and neutralize the virus. A more in-depth understanding of PRRSV infection is described, and novel therapeutic antiviral strategies are discussed.IMPORTANCE In the present study, the role of CLDN4 in PRRSV infection was studied. The results showed that CLDN4 blocked absorption into cells and restricted extracellular viral particles via the interaction between the CLDN4 small extracellular loop, ECL2, and the viral surface protein GP3. GP3 was found to downregulate CLDN4 through ubiquitination of the transcription factor SP1 to facilitate viral entry. The mechanism by which highly pathogenic PRRSV infects the epithelium is suggested. A novel function of GP3 in regulating gene transcription was discovered. Moreover, ECL2 could block PRRSV absorption and infection, as well as neutralizing the virus in the supernatant, which may lead to the development of novel therapeutic antiviral strategies.

Florfenicol-induced Mitochondrial Dysfunction Suppresses Cell Proliferation and Autophagy in Fibroblasts.

Florfenicol (FLO) is one of the most popular antibiotics used in veterinary clinic and aquaculture. FLO can inhibit both bacterial and mitochondrial protein synthesis. However, the effects of FLO on mitochondrial function and cellular homeostasis remain unclear. Here we show that FLO inhibits expression of mitochondrial DNA-encoded proteins, decreases mitochondrial membrane potential, and promotes generation of reactive oxygen species (ROS) in vitro. As a result, activities of mitochondrial respiratory chain complex I and IV and the cellular ATP level are decreased and mitochondrial morphology is damaged. FLO represses cell growth and proliferation by suppression of phosphorylation of p70S6K through AMPK/mTOR/p70S6K pathway. Furthermore, FLO also induces G0/G1 cell cycle arrest via increase of p21 levels through activating ROS/p53/p21 pathway. Moreover, the clearance of damaged mitochondria by autophagy is impaired, leading to cell proliferation inhibition and promotes cell senescence. In addition, FLO-induced upregulation of cytosolic p53 may contribute to mitophagy deficiency via regulation of Parkin recruitment. In summary, our data suggest that florfenicol is an inhibitor of mitochondrial protein synthesis that can induce noticeable cytotoxicity. Thus, these findings can be useful for guiding the proper use of FLO and the development of safe drugs.