The expression, function, and network regulation of circDNAJB6 in chicken macrophages under lipopolysaccharide (LPS) stimulation.

Circular RNAs (circRNA) originate from back-splicing events that link a downstream 5'splice site to an upstream 3' splice site. Circular RNA has been shown to be involved in gene expression, interacting with microRNA and RNA binding proteins to affect transcription, splicing, translation, and other processes. However, little is known about the potential function of chicken circRNAs that trigger the pathogenesis. In a previous study, a circular RNA DNAJB6 (circDNAJB6) was identified as a typical covalently closed circular RNA that is abundant in chicken macrophages upon bacterial infection. It was identified that circDNAJB6 was formed by reverse splicing of exons 2 to 5 of the DNAJB6 gene by PCR amplification, Sanger sequencing, and RNase R exonuclease treatment. Moreover, circDNAJB6 had ability to exacerbate the lipopolysaccharides (LPS) induced cellular injury via reducing cell viability, increasing NO product and pro-inflammatory cytokines. In addition, bioinformatic analysis showed that five miRNAs were identified to interact with circDNAJB6, potentially targeting 75 genes, which were significantly enriched in the pathways of autophagy-animal and MAPK signaling. This study has provided and broadened a better understanding the function of circDNAJB6, which may exert potential biomarkers and act as potential targets for the treatment of bacterial infection.

Functional analysis of circSTX8 in chicken macrophages under lipopolysaccharide stimulation.

Circular RNAs (circRNAs) have a regulatory role in gene expression, development, differentiation, and immune response. In a previous study, circular RNA STX8 (circSTX8) exhibited low expression in chicken lungs during lipopolysaccharide (LPS) stimulation. PCR amplification and Sanger sequencing showed that circSTX8 was created by back-splicing of exons 5 to 6 of STX8. RNase R exonuclease treatment indicated that circSTX8 was a stable circular RNA. RT-qPCR showed that circSTX8 was highly expressed in cecum, spleen, harderian gland, stomach, thymus, liver, small intestine, and lung instead of that in muscle, cerebrum, and cerebellum (n = 8). Chicken macrophages were then divided into four groups: control, overexpression of circSTX8 group, LPS group, and overexpression of circSTX8 + LPS group. CCK8 and RT-qPCR showed that circSTX8 can exacerbate the cellular injury induced by LPS, resulting in a reduction of cell viability and an increase of the pro-inflammatory cytokines expression. In addition, four miRNAs were identified to interact with circSTX8, potentially targeting 914 genes, which were significantly enriched in the pathways of Tight junction, mTOR signaling pathway, MAPK signaling pathway, TGF-beta signaling pathway, Notch signaling pathway, ErbB signaling pathway, and Cell adhesion molecules. These findings showed that circSTX8 was able to regulate the LPS induced cellular immune and inflammatory response.

Identification and characterization of microRNAs, especially gga-miR-181b-5p, in chicken macrophages associated with avian pathogenic E. coli infection.

Avian pathogenic E. coli (APEC) is a common pathogen in the poultry industry, which can cause substantial economic losses. Recently, emerging evidence showed that miRNAs were involved in various viral and bacterial infections. To elucidate the role of miRNAs in chicken macrophages in response to APEC infection, we attempted to investigate the miRNAs expression pattern upon APEC infection via miRNA-seq, and to identify the molecular mechanism of the important miRNAs by using RT-qPCR, western blotting, dual-luciferase reporter assay, and CCK-8. The results showed that a total of 80 differentially expressed (DE) miRNAs were identified in comparison of APEC vs. wild-type group, which corresponded to 724 target genes. Moreover, the target genes of the identified DE miRNAs were mainly significantly enriched in the MAPK signalling pathway, autophagy-bird, mTOR signalling pathway, ErbB signalling pathway, Wnt signalling pathway, and TGF-beta signalling pathway. Remarkably, gga-miR-181b-5p is able to participate in host immune and inflammatory responses against APEC infection via targeting of TGFBR1 to modulate the activation of TGF-beta signalling pathway. Collectively, this study provides a perspective of miRNA expression patterns in chicken macrophages upon APEC infection. These findings provide insight into miRNAs against APEC infection, and gga-miR-181b-5p might be a potential target for treating APEC infection.

Analysis of circRNA expression in chicken HD11 cells in response to avian pathogenic E.coli.

Avian pathogenic E. coli (APEC), one of the widespread zoonotic-pathogen, can cause a series of diseases collectively known as colibacillosis. This disease can cause thousands of million dollars economic loss each year in poultry industry and threaten to human health via meat or egg contamination. However, the detailed molecular mechanism underlying APEC infection is still not fully understood. Circular RNAs, a new type of endogenous noncoding RNA, have been demonstrated to involve in various biological processes. However, it is still not clear whether the circRNAs participate in host response against APEC infection. Herein, we utilized the high-throughput sequence technology to identify the circRNA expression profiles in APEC infected HD11 cells. A total of 49 differentially expressed (DE) circRNAs were detected in the comparison of APEC infected HD11 cells vs. wild type HD11 cells, which were involved in MAPK signaling pathway, Endocytosis, Focal adhesion, mTOR signaling pathway, and VEGF signaling pathway. Specifically, the source genes (BRAF, PPP3CB, BCL2L13, RAB11A, and TSC2) and their corresponding DE circRNAs may play a significant role in APEC infection. Moreover, based on ceRNA regulation, we constructed the circRNA-miRNA network and identified a couple of important regulatory relationship pairs related to APEC infection, including circRAB11A-gga-miR-125b-3p, circRAB11A-gga-miR-1696, and circTSC2-gga-miR-1649-5p. Results indicate that the aforementioned specific circRNAs and circRNA-miRNA network might have important role in regulating host immune response against APEC infection. This study is the first time to investigate the circRNAs expression profile and the biological function of the source genes of the identified DE circRNAs after APEC infection of chicken HD11 cells. These results would contribute to a better understanding of the molecular mechanisms in host response against APEC infection.

Transcriptional Regulation of RIP2 Gene by NFIB Is Associated with Cellular Immune and Inflammatory Response to APEC Infection.

Avian pathogenic E. coli (APEC) can cause localized or systemic infection, resulting in large economic losses per year, and impact health of humans. Previous studies showed that RIP2 (receptor interacting serine/threonine kinase 2) and its signaling pathway played an important role in immune response against APEC infection. In this study, chicken HD11 cells were used as an in vitro model to investigate the function of chicken RIP2 and the transcription factor binding to the RIP2 core promoter region via gene overexpression, RNA interference, RT-qPCR, Western blotting, dual luciferase reporter assay, CHIP-PCR, CCK-8, and flow cytometry assay following APEC stimulation. Results showed that APEC stimulation promoted RIP2 expression and cells apoptosis, and inhibited cells viability. Knockdown of RIP2 significantly improved cell viability and suppressed the apoptosis of APEC-stimulated cells. Transcription factor NFIB (Nuclear factor I B) and GATA1 (globin transcription factor 1) binding site was identified in the core promoter region of RIP2 from -2300 bp to -1839 bp. However, only NFIB was confirmed to be bound to the core promoter of RIP2. Overexpression of NFIB exacerbated cell injuries with significant reduction in cell viability and increased cell apoptosis and inflammatory cytokines levels, whereas opposite results were observed in NFIB inhibition treatment group. Moreover, RIP2 was up-regulated by NFIB overexpression, and RIP2 silence mitigated the effect of NFIB overexpression in cell apoptosis, inflammation, and activation of NFκB signaling pathways. This study demonstrated that NFIB overexpression accelerated APEC-induced apoptosis and inflammation via up-regulation of RIP2 mediated downstream pathways in chicken HD11 cells.

Research on Thermal Decomposition Kinetics and Thermal Safety for a New Epoxiconazole Crystal.

To clarify the thermal safety inherent in a new epoxiconazole crystal, differential scanning calorimetry (DSC) and adiabatic accelerating rate calorimetry (ARC) were used for testing and research. The Friedman method and model method were used to analyze thermal decomposition kinetics based on the DSC data, and the N-order and autocatalytic decomposition reaction kinetic models were established. The double scan method was utilized to verify the autocatalytic effect during the decomposition process. The Friedman method, N-order, and autocatalytic model methods were used to study the substance's thermal decomposition characteristics. ARC data are utilized to verify the aforementioned prediction results and the kinetic parameters that were obtained based on ARC data from N-order and autocatalytic model methods that concur with the simulation results. This paper applies the N-order and autocatalytic model to the kinetic model to further predict thermal safety parameter time to maximum rate under adiabatic conditions.