Integrated Metabolomic and transcriptomic analyses reveal deoxycholic acid promotes transmissible gastroenteritis virus infection by inhibiting phosphorylation of NF-κB and STAT3.

BACKGROUND: Acute diarrhea, dehydration and death in piglets are all symptoms of transmissible gastroenteritis virus (TGEV), which results in significant financial losses in the pig industry. It is important to understand the pathogenesis and identify new antiviral targets by revealing the metabolic interactions between TGEV and host cells. RESULTS: We performed metabolomic and transcriptomic analyses of swine testicular cells infected with TGEV. A total of 1339 differential metabolites and 206 differentially expressed genes were detected post TEGV infection. The differentially expressed genes were significantly enriched in the HIF-1 signaling pathway and PI3K-Akt signaling. Integrated analysis of differentially expressed genes and differential metabolites indicated that they were significantly enriched in the metabolic processes such as nucleotide metabolism, biosynthesis of cofactors and purine metabolism. In addition, the results showed that most of the detected metabolites involved in the bile secretion was downregulated during TGEV infection. Furthermore, exogenous addition of key metabolite deoxycholic acid (DCA) significantly enhanced TGEV replication by NF-κB and STAT3 signal pathways. CONCLUSIONS: We identified a significant metabolite, DCA, related to TGEV replication. It added TGEV replication in host cells by inhibiting phosphorylation of NF-κB and STAT3. This study provided novel insights into the metabolomic and transcriptomic alterations related to TGEV infection and revealed potential molecular and metabolic targets for the regulation of TGEV infection.

The Metabolic Pathway of Bile Secretion Is Vulnerable to Salmonella enterica Exposure in Porcine Intestinal Epithelial Cells.

Pigs can be colonized with Salmonella enterica and become established carriers. However, the mechanisms of the host's response to Salmonella enterica infection are largely unclear. This study was constructed with the Salmonella enterica infection model in vitro using porcine intestinal epithelial cells (IPEC-J2). Transcriptome profiling of IPEC-J2 cells was carried out to characterize the effect of Salmonella enterica infection and lipopolysaccharide (LPS) treatment, in which LPS-induced inflammation was a positive control. At first, Salmonella enterica infection increased the cell apoptosis rate and induced an inflammation response in IPEC-J2. Then, the up-regulated genes were enriched in metabolic pathways, such as those for bile secretion and mineral absorption, while down-regulated genes were enriched in immune-related pathways, such as the Toll-like receptor signaling and p53 signaling pathways. Moreover, we found 368 up-regulated genes and 101 down-regulated genes in common. Then, an integrative analysis of the transcriptomic profile under Salmonella enterica infection and LPS treatment was conducted, and eight up-regulated genes and one down-regulated gene were detected. Among them, AQP8 is one critical gene of the bile secretion pathway, and its mRNA and protein expression were increased significantly under Salmonella enterica infection and LPS treatment. Thus, the AQP8 gene and bile secretion pathway may be important in IPEC-J2 cells under Salmonella enterica infection or LPS treatment.

Identification of the Effects of 5-Azacytidine on Porcine Circovirus Type 2 Replication in Porcine Kidney Cells.

Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome (PMWS), which mainly targets the body's immune system and poses a serious threat to the global pig industry. 5-Azacytidine is a potent inhibitor of DNA methylation, which can participate in many important physiological and pathological processes, including virus-related processes, by inhibiting gene expression. However, the impact of 5-Aza on PCV2 replication in cells is not yet clear. We explored the impact of 5-Aza on PCV2 infection utilizing PK15 cells as a cellular model. Our objective was to gain insights that could potentially offer novel therapeutic strategies for PCV2. Our results showed that 5-Aza significantly enhanced the infectivity of PCV2 in PK15 cells. Transcriptome analysis revealed that PCV2 infection activated various immune-related signaling pathways. 5-Aza may activate the MAPK signaling pathway to exacerbate PCV2 infection and upregulate the expression of inflammatory and apoptotic factors.

Cortical thickness and white matter microstructure predict freezing of gait development in Parkinson's disease.

The clinical applications of the association of cortical thickness and white matter fiber with freezing of gait (FoG) are limited in patients with Parkinson's disease (PD). In this retrospective study, using white matter fiber from diffusion-weighted imaging and cortical thickness from structural-weighted imaging of magnetic resonance imaging, we investigated whether a machine learning-based model can help assess the risk of FoG at the individual level in patients with PD. Data from the Parkinson's Disease Progression Marker Initiative database were used as the discovery cohort, whereas those from the Fujian Medical University Union Hospital Parkinson's Disease database were used as the external validation cohort. Clinical variables, white matter fiber, and cortical thickness were selected by random forest regression. The selected features were used to train the support vector machine(SVM) learning models. The median area under the receiver operating characteristic curve (AUC) was calculated. Model performance was validated using the external validation cohort. In the discovery cohort, 25 patients with PD were defined as FoG converters (15 men, mean age 62.1 years), whereas 60 were defined as FoG nonconverters (38 men, mean age 58.5 years). In the external validation cohort, 18 patients with PD were defined as FoG converters (8 men, mean age 66.9 years), whereas 37 were defined as FoG nonconverters (21 men, mean age 65.1 years). In the discovery cohort, the model trained with clinical variables, cortical thickness, and white matter fiber exhibited better performance (AUC, 0.67-0.88). More importantly, SVM-radial kernel models trained using random over-sampling examples, incorporating white matter fiber, cortical thickness, and clinical variables exhibited better performance (AUC, 0.88). This model trained using the above mentioned features was successfully validated in an external validation cohort (AUC, 0.91). Furthermore, the following minimal feature sets that were used: fractional anisotropy value and mean diffusivity value for right thalamic radiation, age at baseline, and cortical thickness for left precentral gyrus and right dorsal posterior cingulate gyrus. Therefore, machine learning-based models using white matter fiber and cortical thickness can help predict the risk of FoG conversion at the individual level in patients with PD, with improved performance when combined with clinical variables.

A High-Fat Diet Increases the Characteristics of Gut Microbial Composition and the Intestinal Damage Associated with Non-Alcoholic Fatty Liver Disease.

The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing annually, and emerging evidence suggests that the gut microbiota plays a causative role in the development of NAFLD. However, the role of gut microbiota in the development of NAFLD remains unclear and warrants further investigation. Thus, C57BL/6J mice were fed a high-fat diet (HFD), and we found that the HFD significantly induced obesity and increased the accumulation of intrahepatic lipids, along with alterations in serum biochemical parameters. Moreover, it was observed that the HFD also impaired gut barrier integrity. It was revealed via 16S rRNA gene sequencing that the HFD increased gut microbial diversity, which enriched Colidextribacter, Lachnospiraceae-NK4A136-group, Acetatifactor, and Erysipelatoclostridium. Meanwhile, it reduced the abundance of Faecalibaculum, Muribaculaceae, and Coriobacteriaceae-UCG-002. The predicted metabolic pathways suggest that HFD enhances the chemotaxis and functional activity of gut microbiota pathways associated with flagellar assembly, while also increasing the risk of intestinal pathogen colonization and inflammation. And the phosphotransferase system, streptomycin biosynthesis, and starch/sucrose metabolism exhibited decreases. These findings reveal the composition and predictive functions of the intestinal microbiome in NAFLD, further corroborating the association between gut microbiota and NAFLD while providing novel insights into its potential application in gut microbiome research for NAFLD patients.

Integrated of multi-omics and molecular docking reveal PHGDH, PSAT1 and PSPH in the serine synthetic pathway as potential targets of T-2 toxin exposure in pig intestinal tract.

T-2 toxin (T-2) with a molecular weight of 466.52 g/mol is an inevitable mycotoxin in food products and feeds, posing a significant threat to human and animal health. However, the underlying molecular mechanisms of the cytotoxic effects of T-2 exposure on porcine intestinal epithelial cells (IPEC-J2) remain unclear. Here, we investigated the cytotoxic effects of T-2 exposure on IPEC-J2 through the detection of cell viability, cell morphology, mitochondrial membrane potential, ROS, apoptosis and autophagy. Further transcriptomic and proteomic analyses of IPEC-J2 upon T-2 exposure were performed by using RNA-seq and TMT techniques. A total of 546 differential expressed genes (DEGs) and 269 differentially expressed proteins (DEPs) were detected. Among these, 24 common DEGs/DEPs were involved in IPEC-J2 upon T-2 exposure. Interestingly, molecular docking analysis revealed potential interactions between T-2 and three key enzymes (PHGDP, PSAT1, and PSPH) in the serine biosynthesis pathway. Besides, further experimental showed that PSAT1 knockdown exacerbated T-2-induced oxidative damage. Together, our findings indicated that the serine biosynthesis pathway including PHGDP, PSAT1, PSPH genes probably acts critical roles in the regulation of T-2-induced cell damage. This study provided new insights into the global molecular effects of T-2 exposure and identified the serine biosynthesis pathway as molecular targets and potential treatment strategies against T-2.

Hypomethylated interferon regulatory factor 8 recruits activating protein-2α to attenuate porcine epidemic diarrhea virus infection in porcine jejunum.

Interferon regulatory factor 8 (IRF8) is a key regulator of innate immune receptor signaling that resists pathogen invasion by regulating cell growth and differentiation. Porcine epidemic diarrhea virus (PEDV) targets the intestine and damages the mucosal barrier. However, whether IRF8 regulates PEDV replication remains unclear. We revealed that PEDV infection activated IRF8 expression. Moreover, IRF8 deletion drastically promoted PEDV replication and invasion, increasing the virus copies and titers. Hypomethylation enrichment of activating protein (AP)-2α was significantly negatively correlated with high IRF8 expression, and AP-2α directly targeted the IRF8 promoter to regulate PEDV replication. Furthermore, IRF8 overexpression decreased the cellular reactive oxygen species levels and mitochondrial membrane potential and increased the antioxidant enzyme activities to alleviate PEDV-induced oxidative damage. IRF8 overexpression suppressed apoptotic gene expression, thereby inhibiting apoptosis in response to PEDV stimulation. Taken together, this study demonstrates that AP-2α is involved in PEDV-induced epigenetic modification of IRF8 to reduce cell apoptosis and oxidative stress and facilitate host resistance to PEDV in the intestinal epithelium.

Genome-wide analysis of circRNA regulation during spleen development of Chinese indigenous breed Meishan pigs.

BACKGROUND: Numerous circular RNAs (circRNAs) have been recently identified in porcine tissues and cell types. Nevertheless, their significance in porcine spleen development is yet unelucidated. Herein, we reported an extensive overlook of circRNA expression profile during spleen development in Meishan pigs. RESULTS: Overall, 39,641 circRNAs were identified from 6,914 host genes. Among them, many circRNAs are up- or down-regulated at different time points of pig spleen development. Using WGCNA analysis, we revealed two essential modules for protein-coding genes and circRNAs. Subsequent correlation analysis revealed 67 circRNAs/co-expressed genes that participated in immnue-associated networks. Furthermore, a competing endogenous RNA (ceRNA) network analysis of circRNAs revealed that 12 circRNAs modulated CD226, MBD2, SAMD3, SIT1, SRP14, SYTL3 gene expressions via acting as miRNA sponges. Moreover, the circRNA_21767/miR-202-3p axis regulated SIT1 expression in a ceRNA manner, which is critical for the immune-based regulation of spleen development in Meishan pigs. CONCLUSIONS: Overall, our results demonstrated that the circRNAs were differentially expressed during different stages of porcine spleen development, meanwhile the circRNAs interacted with immune-related genes in a ceRNA-based fashion. Moreover, we presented biomedical researchers with RNAseqTools, a user-friendly and powerful software for the visualization of transcriptome profile data.

Sodium Butyrate Ameliorates Deoxynivalenol-Induced Oxidative Stress and Inflammation in the Porcine Liver via NR4A2-Mediated Histone Acetylation.

Mycotoxin-induced liver injury is often accompanied by oxidative stress (OS) and inflammation. This research aimed to explore the potential mechanism of sodium butyrate (NaBu) in modulating hepatic anti-oxidation and anti-inflammation pathways in deoxynivalenol (DON)-exposed piglets. The results show that DON induced liver injury, increased mononuclear cell infiltration, and decreased serum total protein and albumin concentrations. Transcriptomic analysis revealed that reactive oxygen species (ROS) and TNF-α pathways were highly activated upon DON exposure. This is associated with disturbed antioxidant enzymes and increased inflammatory cytokines secretion. Importantly, NaBu effectively reversed the alterations caused by DON. Mechanistically, the ChIP-seq result revealed that NaBu strongly depressed DON-increased enrichment of histone mark H3K27ac at the genes involved in ROS and TNF-α-mediated pathways. Notably, we demonstrated that nuclear receptor NR4A2 was activated by DON and remarkably recovered with the treatment of NaBu. In addition, the enhanced NR4A2 transcriptional binding enrichments at the promoter regions of OS and inflammatory genes were hindered by NaBu in DON-exposed livers. Consistently, elevated H3K9ac and H3K27ac occupancies were also observed at the NR4A2 binding regions. Taken together, our results indicated that a natural antimycotic additive, NaBu, could mitigate hepatic OS and inflammatory responses, possibly via NR4A2-mediated histone acetylation.

LncRNA446 Regulates Tight Junctions by Inhibiting the Ubiquitinated Degradation of Alix after Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea (PED) is a highly contagious disease, caused by porcine epidemic diarrhea virus (PEDV), which causes huge economic losses. Tight junction-associated proteins play an important role during virus infection; therefore, maintaining their integrity may be a new strategy for the prevention and treatment of PEDV. Long noncoding RNAs (lncRNAs) participate in numerous cellular functional activities, yet whether and how they regulate the intestinal barrier against viral infection remains to be elucidated. Here, we established a standard system for evaluating intestinal barrier integrity and then determined the differentially expressed lncRNAs between PEDV-infected and healthy piglets by lncRNA-seq. A total of 111 differentially expressed lncRNAs were screened, and lncRNA446 was identified due to significantly higher expression after PEDV infection. Using IPEC-J2 cells and intestinal organoids as in vitro models, we demonstrated that knockdown of lncRNA446 resulted in increased replication of PEDV, with further damage to intestinal permeability and tight junctions. Mechanistically, RNA pulldown and an RNA immunoprecipitation (RIP) assay showed that lncRNA446 directly binds to ALG-2-interacting protein X (Alix), and lncRNA446 inhibits ubiquitinated degradation of Alix mediated by TRIM25. Furthermore, Alix could bind to ZO1 and occludin and restore the expression level of the PEDV M gene and TJ proteins after lncRNA446 knockdown. Additionally, Alix knockdown and overexpression affects PEDV infection in IPEC-J2 cells. Collectively, our findings indicate that lncRNA446, by inhibiting the ubiquitinated degradation of Alix after PEDV infection, is involved in tight junction regulation. This study provides new insights into the mechanisms of intestinal barrier resistance and damage repair triggered by coronavirus. IMPORTANCE Porcine epidemic diarrhea is an acute, highly contagious enteric viral disease severely affecting the pig industry, for which current vaccines are inefficient due to the high variability of PEDV. Because PEDV infection can lead to severe injury of the intestinal epithelial barrier, which is the first line of defense, a better understanding of the related mechanisms may facilitate the development of new strategies for the prevention and treatment of PED. Here, we demonstrate that the lncRNA446 directly binds one core component of the actomyosin-tight junction complex named Alix and inhibits its ubiquitinated degradation. Functionally, the lncRNA446/Alix axis can regulate the integrity of tight junctions and potentially repair intestinal barrier injury after PEDV infection.

Exploring the role of protein DJ-1 in quality of pale, soft and exudative (PSE) and red, firm and non-exudative (RFN) pork during post-mortem aging.

The purpose of this study was to investigate the changes of oxidative attributes, protein DJ-1 expression, oxidized form (oxDJ-1), and cellular localization in RFN and PSE pork meat during the post-mortem aging. The longissimus thoracis of RFN and PSE groups were collected and classified by determination of pH, color and purge loss and then aged for 1, 3 and 7 d at 4 °C postmortem. Results showed that the content of DJ-1 and oxDJ-1 was continuously increased during 7 d of postmortem aging. A relatively higher protein DJ-1, oxDJ-1, oxygen reactive species and disulfide bond contents were found in PSE meat in comparison to RFN meat. Immunostaining showed that protein DJ-1 was located in cytoplasm, membrane and some nucleus of muscle cells. DJ-1 was shown to correlate with meat quality and oxidative attributes, suggesting a regulatory role in resisting oxidative stress and meat quality formation during post-mortem aging.

Association analysis of DNA methylation and the tissue/developmental expression of the FUT3 gene in Meishan pigs.

Porcine α-1,3-fucosyltransferase (FUT3), as a member of the fucosyltransferase family, plays an important role in the resistance of the piglet intestine to pathogenic microbial infection. To further investigate the tissue/developmental expression of FUT3 and its regulatory mechanism, we analyzed changes in the expression of FUT3 in the duodenal tissues of Meishan pigs at different ages and found that the expression of FUT3 showed a decreasing trend with increasing age. In addition, bisulfite sequencing identified nine methylated CpG sites in the FUT3 core promoter (-500 âˆ¼ -206) region. Therein, the methylation level at the mC-9 site located in FUT3 showed a significantly negative association with mRNA expression (P < 0.05). A further dual-luciferase assay demonstrated that methylation at the mC-9 site of the FUT3 promoter inhibited its transcriptional activity. Then, we confirmed the binding of Sp1 to the FUT3 promoter using RNA knockdown and a ChIP-qPCR assay. Our findings indicate that DNA methylation at the mC-9 site may inhibit the binding of the transcription factor Sp1, thus regulating the developmental expression of the FUT3 gene in the duodenum, providing some theoretical basis for the FUT3 gene as an important candidate marker of disease resistance in Meishan pigs.

Sodium butyrate alleviates deoxynivalenol-induced hepatic cholesterol metabolic dysfunction via RORγ-mediated histone acetylation modification in weaning piglets.

BACKGROUND: Cholesterol is an essential component of lipid rafts in cell plasma membrane, which exerts a hepatoprotective role against mycotoxin exposure in pigs, and cholesterol metabolism is vulnerable to epigenetic histone acetylation. Therefore, our present study aimed to investigate whether a histone deacetylase inhibitor (sodium butyrate [NaBu]) could protect the porcine liver from deoxynivalenol (DON) exposure by modulating cholesterol metabolism. Herein, we randomly divided 28 pigs into four groups, which were fed an uncontaminated basal diet, contaminated diet (4 mg DON/kg), uncontaminated diet supplemented with 0.2% NaBu or 4 mg/kg DON contaminated diet (4 mg DON/kg) supplemented with 0.2% NaBu for 28 d. RESULTS: We found that the serum alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) were all increased in pigs exposed to DON, indicative of significant liver injury. Furthermore, the cholesterol content in the serum of DON-exposed pigs was significantly reduced, compared to the healthy Vehicle group. Transcriptome analysis of porcine liver tissues revealed that the cholesterol homeostasis pathway was highly enriched due to DON exposure. In which we validated by qRT-PCR and western blotting that the cholesterol program was markedly activated. Importantly, NaBu effectively restored parameters associated with liver injury, along with the cholesterol content and the expression of key genes involved in the cholesterol biosynthesis pathway. Mechanistically, we performed a ChIP-seq analysis of H3K27ac and showed that NaBu strongly diminished DON-increased H3K27ac genome-wide enrichment. We further validated that the elevated H3K27ac and H3K9ac occupancies on cholesterol biosynthesis genes were both decreased by NaBu, as determined by ChIP-qPCR analysis. Notably, nuclear receptor RORγ, a novel regulator of cholesterol biosynthesis, was found in the hyperacetylated regions. Again, a remarkable increase of RORγ at both mRNA and protein levels in DON-exposed porcine livers was drastically reduced by NaBu. Consistent with RORγ expression, NaBu also hindered RORγ transcriptional binding enrichments on these activated cholesterol biosynthesis genes like HMGCR, SQLE, and DHCR24. Furthermore, we conducted an in vitro luciferase reporter assay to verify that porcine RORγ directly bonds to the promoters of the above target genes. CONCLUSIONS: Collectively, our results demonstrate the utility of the natural product NaBu as a potential anti-mycotoxin nutritional strategy for regulating cholesterol metabolism via RORγ-mediated histone acetylation modification.

Pterostilbene Ameliorates Fumonisin B1-Induced Cytotoxic Effect by Interfering in the Activation of JAK/STAT Pathway.

Fumonisin B1 (FB1) is a mycotoxin that poses a great threat to agricultural production and the health of humans and animals. Pterostilbene (PTE) is a natural plant polyphenolic compound with good anti-inflammatory, antioxidant and cell regeneration effects, yet its effectiveness in treating FB1-induced cytotoxicity remains to be explored. In this study, we used porcine alveolar macrophages (3D4/21) as a model to characterize the cytotoxicity induced by FB1, and to investigate the potential alleviating effect of PTE on FB1-induced cytotoxicity. We demonstrate that FB1 induces cytotoxicity, apoptosis, pro-inflammatory cytokine production and mitochondrial damage, which can be largely recovered by PTE treatment, suggesting the promising application of PTE to treat FB1-induced damage. Mechanistically, FB1 activates the JAK/STAT signaling pathway, while PTE attenuates FB1-induced cytotoxicity through the inhibition of key JAK/STAT genes such as JAK2 and STAT3. Overall, our study characterized the molecular mechanism for FB1-induced cytotoxicity and found PTE to be a promising component which can alleviate FB1-induced cytotoxicity by interfering in the activation of JAK/STAT pathway.

Effect and Mechanism Analysis of Pig FUT8 Gene on Resistance to Escherichia coli F18 Infection.

Post-weaning diarrhea caused by enterotoxigenic Escherichia coli F18 (E. coli F18) causes significant economic losses for pig producers. Fucosyltransferase 8 (FUT8) is a glycosyltransferase that catalyzes core fucosylation; however, its role in mediating the resistance to E. coli F18 infection in pigs remains unknown. In this study, we systematically verified the relationship between FUT8 expression and E. coli resistance. The results showed that FUT8 was expressed in all detected tissues of Meishan piglets and that its expression was significantly increased in the duodenum and jejunum of E. coli F18-sensitive individuals when compared to E. coli F18-resistant individuals. FUT8 expression increased after exposure to E. coli F18 (p < 0.05) and decreased significantly after LPS induction for 6 h (p < 0.01). Then, the IPEC-J2 stable cell line with FUT8 interference was constructed, and FUT8 knockdown decreased the adhesion of E. coli F18ac to IPEC-J2 cells (p < 0.05). Moreover, we performed a comparative transcriptome study of IPEC-J2 cells after FUT8 knockdown via RNA-seq. In addition, further expression verification demonstrated the significant effect of FUT8 on the glycosphingolipid biosynthesis and Toll-like signaling pathways. Moreover, the core promoter of FUT8, which was located at −1213 bp to −673 bp, was identified via luciferase assay. Interestingly, we found a 1 bp C base insertion mutation at the −774 bp region, which could clearly inhibit the transcriptional binding activity of C/EBPα to an FUT8 promoter. Therefore, it is speculated that FUT8 acts in a critical role in the process of E. coli infection; furthermore, the low expression of FUT8 is conducive to the enhancement of E. coli resistance in piglets. Our findings revealed the mechanism of pig FUT8 in regulating E. coli resistance, which provided a theoretical basis for the screening of E. coli resistance in Chinese local pig breeds.

Analysis of RIOK2 Functions in Mediating the Toxic Effects of Deoxynivalenol in Porcine Intestinal Epithelial Cells.

Deoxynivalenol (DON) is a type of mycotoxin that threatens human and livestock health. Right open reading frame kinase 2 (RIOK2) is a kinase that has a pivotal function in ribosome maturation and cell cycle progression. This study aims to clarify the role of the RIOK2 gene in DON-induced cytotoxicity regulation in porcine intestinal epithelial cells (IPEC-J2). Cell viability assay and flow cytometry showed that the knockdown of RIOK2 inhibited proliferation and induced apoptosis, cell cycle arrest, and oxidative stress in DON-induced IPEC-J2. Then, transcriptome profiling identified candidate genes and pathways that closely interacted with both DON cytotoxicity regulation and RIOK2 expression. Furthermore, RIOK2 interference promoted the activation of the MAPK signaling pathway by increasing the phosphorylation of ERK and JNK. Additionally, we performed the dual-luciferase reporter and ChIP assays to elucidate that the expression of RIOK2 was influenced by the binding of transcription factor Sp1 with the promoter region. Briefly, the reduced expression of the RIOK2 gene exacerbates the cytotoxic effects induced by DON in IPEC-J2. Our findings provide insights into the control strategies for DON contamination by identifying functional genes and effective molecular markers.

Integrated analysis of lncRNA-mediated ceRNA network involved in immune regulation in the spleen of Meishan piglets.

Meishan pigs are a famous local pig breed in China, with high fertility and early sexual maturity, and stronger immunity compared to other breeds. The spleen is the largest lymphoid organ in pigs and performs essential functions, such as those relating to immunity and haematopoiesis. The invasion of many pathogenic microorganisms in pigs is associated with spleen damage. Long non-coding RNAs participate in a broad range of biological processes and have been demonstrated to be associated with splenic immune regulation. However, the expression network of mRNAs and lncRNAs in the spleen of Meishan pigs remains unclear. This study collected spleen tissues from Meishan piglets at three different ages as a model, and mRNA and lncRNA transcripts were profiled for each sample. Additionally, 1,806 differential mRNAs and 319 differential lncRNAs were identified. A complicated interaction between mRNAs and lncRNAs was identified via WGCNA, demonstrating that lncRNAs are a crucial regulatory component in mRNA. The results show that the modules black and red have similar mRNA and lncRNA transcription patterns and are mainly involved in the process of the immune defense response. The core genes (DHX58 and IFIT1) and key lncRNAs (TCONS-00002102 and TCONS-00012474) of piglet spleen tissue were screened using the ceRNA network. The expression of these genes is related to the immune response of pigs. Our research may contribute to a further understanding of mRNA and lncRNA expression in the spleen of piglets, and provide new ideas to improve the disease resistance of piglets.

Comprehensive Analysis Revealed the Potential Roles of N6-Methyladenosine (m6A) Mediating E. coli F18 Susceptibility in IPEC-J2 Cells.

Post-weaning diarrhea caused by enterotoxigenic Escherichia coli F18 (E. coli F18) causes significant economic losses for pig producers. N6-methyladenosine (m6A) is a highly abundant epitranscriptomic marker that has been found to be involved in regulating the resistance of host cells to pathogenic infection, but its potential role in E. coli F18-exposed intestinal porcine epithelial cells (IPEC-J2) remains undetermined. Here, we demonstrated that m6A and its regulators modulate E. coli F18 susceptibility. Briefly, we revealed that the Wilms' tumor 1-associating protein (WTAP) expressions were markedly elevated in IPEC-J2 cells upon E. coli F18 exposure. WTAP are required for the regulation of E. coli F18 adhesion in IPEC-J2 cells. Additionally, WTAP knockdown significantly suppressed m6A level at N-acetyllactosaminide beta-1,6-N-acetylglucosaminyl-transferase (GCNT2) 3'UTR, resulting in the enhancement of TH N6-methyladenosine RNA binding protein 2 (YTHDF2)-mediated GCNT2 mRNA stability. Subsequently, the altered GCNT2 expressions could inhibit the glycosphingolipid biosynthesis, thus improving resistance to E. coli F18 infection in IPEC-J2. Collectively, our analyses highlighted the mechanism behind the m6A-mediated management of E. coli F18 susceptibility, which will aid in the development of novel approaches that protect against bacterial diarrhea in piglets.

Identification of a 5-Methylcytosine Site (mC-7) That May Inhibit CXCL11 Expression and Regulate E. coli F18 Susceptibility in IPEC-J2 Cells.

The primary pathogen causing post-weaning diarrhea in piglets is Escherichia coli F18 (E. coli F18), hence it is essential to investigate the mechanism governing E. coli F18 resistance in native pig breeds. Based on the previous RNA-seq results of the duodenum from E. coli F18-resistant and -susceptible Meishan piglets, CXCL11, an important functional gene, was preliminarily screened. In this investigation, in order to further examine the expression regulation mechanism of E. coli F18 in intestinal porcine epithelial cells (IPEC-J2) against E. coli F18 infection, CXCL11 gene expression on IPEC-J2 cells infected by E. coli F18 was detected, which was significantly downregulated (p < 0.01). Secondly, the overexpression on the IPEC-J2 cell line was successfully structured, and a relative quantification method of the PILIN, bacteria enumeration, and immunofluorescence assay indicated that the CXCL11 overexpression significantly reduced the ability of E. coli F18 to interact with IPEC-J2 in vitro. The promoter region of the CXCL11 gene was predicted to contain a CpG island (−619 ~ −380 bp) of which 13 CpG sites in the sequencing region were methylated to varying degrees, and the methylation level of one CPG site (mC-7) positively linked negatively with the expression of the CXCL11 gene (p < 0.05). Meanwhile, a dual luciferase assay detected the mutation of the mC-7 site that significantly inhibited the luciferase activity of the CXCL11 gene promoter (p < 0.01). Transcription factor prediction and expression verification indicated that mC-7 is located in the OSR1-binding domain, and that its expression level is related to E. coli F18 susceptibility. We speculated that methylation modification of the mC-7 site of the CpG island in the promoter region of the CXCL11 gene might inhibit the binding of transcription factor OSR1 with the mC-7 site, and then affect its expression level to regulate the susceptibility to E. coli F18.

Histone Methyltransferase MLL1 Mediates Oxidative Stress and Apoptosis upon Deoxynivalenol Exposure in the Intestinal Porcine Epithelial Cells.

Deoxynivalenol (DON), as a secondary metabolite of fungi, is continually detected in livestock feed and has a high risk to animals and humans. Moreover, pigs are very sensitive to DON. Recently, the role of histone modification has drawn people's attention; however, few studies have elucidated how histone modification participates in the cytotoxicity or genotoxicity induced by mycotoxins. In this study, we used intestinal porcine epithelial cells (IPEC-J2 cells) as a model to DON exposure in vitro. Mixed lineage leukemia 1 (MLL1) regulates gene expression by exerting the role of methyltransferase. Our studies demonstrated that H3K4me3 enrichment was enhanced and MLL1 was highly upregulated upon 1 µg/mL DON exposure in IPEC-J2 cells. We found that the silencing of MLL1 resulted in increasing the apoptosis rate, arresting the cell cycle, and activating the mitogen-activated protein kinases (MAPKs) pathway. An RNA-sequencing analysis proved that differentially expressed genes (DEGs) were enriched in the cell cycle, apoptosis, and tumor necrosis factor (TNF) signaling pathway between the knockdown of MLL1 and negative control groups, which were associated with cytotoxicity induced by DON. In summary, these current results might provide new insight into how MLL1 regulates cytotoxic effects induced by DON via an epigenetic mechanism.