Insights into speckled eggs provided by single-cell transcriptomics in high-yield laying hens.

Lipopolysaccharide (LPS) is one of the main virulence factors of Gram-negative bacteria. In the process of waterfowl breeding, an inflammatory reaction due to LPS infection is easily produced, which leads to a decline in waterfowl performance. The liver plays a vital role in the immune response and the removal of toxic components. Therefore, it is necessary to study the mechanism of liver injury induced by LPS in goose. In this study, a total of 100 1-day-old goslings were randomly divided into a control group and LPS group after 3 days of pre-feeding. On days 21, 23, and 25 of the formal experiment, the control group was intraperitoneally injected with 0.5 mL normal saline, and the LPS group was intraperitoneally injected with LPS 2 mg/(kg body weight) once a day. On day 25 of the experiment, liver samples were collected 3 h after the injection of saline and LPS. The results of histopathology and biochemical indexes showed that the livers of the LPS group had liver morphological structure destruction and inflammatory cell infiltration, and the levels of ALT and AST were increased. Next, RNA sequencing analysis was used to determine the abundances and characteristics of the transcripts, as well as the associated somatic mutations and alternative splicing. We screened 727 differentially expressed genes (DEGs) with p < 0.05 and |log2(Fold Change)| ≥ 1, as the thresholds; GO and KEGG enrichment analysis showed that LPS-induced liver injury may be involved in the Toll-like receptor signaling pathway, MAPK signaling pathway, NOD-like receptor signaling pathway, FoxO, and PPAR signaling pathway. Finally, we intersected the genes enriched in the key pathway of LPS-induced liver injury with the top 50 key genes in protein−protein interaction networks to obtain 28 more critical genes. Among them, 17 genes were enriched in Toll-like signaling pathway and MAPK signaling pathway. Therefore, these results suggest that LPS-induced liver injury in geese may be the result of the joint action of Toll-like receptor, MAPK, NOD-like receptor, FoxO, and PPAR signaling pathway. Among them, the TLR7-mediated MAPK signaling pathway plays a major role.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by damage of motor neurons. Recent reports indicate that inflammatory responses occurring within the central nervous system contribute to the pathogenesis of ALS. We aimed to investigate disease-specific gene expression associated with neuroinflammation by conducting transcriptome analysis on fibroblasts from three patients with sporadic ALS and three normal controls. Several pathways were found to be upregulated in patients with ALS, among which the toll-like receptor (TLR) and NOD-like receptor (NLR) signaling pathways are related to the immune response. Genes—toll-interacting protein (TOLLIP), mitogen-activated protein kinase 9 (MAPK9), interleukin-1β (IL-1β), interleukin-8 (IL-8), and chemokine (C-X-C motif) ligand 1 (CXCL1)—related to these two pathways were validated using western blotting. This study validated the genes that are associated with TLR and NLR signaling pathways from different types of patient-derived cells. Not only fibroblasts but also induced pluripotent stem cells (iPSCs) and neural rosettes from the same origins showed similar expression patterns. Furthermore, expression of TOLLIP, a regulator of TLR signaling pathway, decreased with cellular aging as judged by changes in its expression through multiple passages. TOLLIP expression was downregulated in ALS cells under conditions of inflammation induced by lipopolysaccharide. Our data suggest that the TLR and NLR signaling pathways are involved in pathological innate immunity and neuroinflammation associated with ALS and that TOLLIP, MAPK9, IL-1β, IL-8, and CXCL1 play a role in ALS-specific immune responses. Moreover, changes of TOLLIP expression might be associated with progression of ALS.

BackgroundCurrently, clinical treatments for ALI include vasodilators, glucocorticoids, and mechanical ventilation; however, these therapies are associated with various adverse reactions. Therefore, there is a need to develop safer and more effective treatment options. Traditional Chinese Medicine (TCM), particularly Qingwen Hufei Granules (QHG), has demonstrated efficacy in treating respiratory disorders, including upper respiratory tract infections and influenza. However, the active components and mechanism of action of QHG remain unclear.MethodsLipopolysaccharide (LPS) induced ALI mouse model. We analyzed the main pharmacodynamic components of QHG by HPLC, determined the effects of QHG on the cell viability and inflammatory factors of RAW264.7 cells, and recorded the body weight and lung tissue wet/dry (W/D) of ALI mice. Inflammatory factors in bronchoalveolar lavage fluid (BALF), serum, and lung tissue were determined using ELISA, and the protective effect of hematoxylin and eosin (H&E) staining of lung tissue was studied. Transcriptomic, qRT-PCR, Western blotting, immunohistochemistry, and immunofluorescence analyses were used to analyze QHG and explore the mechanism of ALI damage reduction.ResultsThe findings demonstrated that QHG effectively mitigated inflammatory cell invasion and pulmonary edema. Moreover, it diminished the concentrations of IL-6, TNF-α, and IL-1β in lung tissue, serum, and BALF. Furthermore, QHG notably decreased the levels of NO, reactive oxygen species(ROS), IL-6, IL-1β, and TNF-α in the supernatants of RAW264.7 cells. Transcriptomic analysis of the lung tissue revealed that QHG primarily enriched the NOD-like receptor (NLR) signaling pathway. qRT-PCR, Western blotting, immunohistochemistry, and immunofluorescence experiments confirmed that QHG mitigated ALI damage through the NLR signaling pathway. Furthermore, seven key components in QHG were determined by using HPLC.ConclusionOur study demonstrated that QHG effectively mitigated LPS-induced ALI, and provided preliminary insights into its mechanism of action and material basis.

Sunflower seed oil combined with ginseng stem-leaf saponins as an adjuvant to enhance the immune response elicited by Newcastle disease vaccine in chickens

Elizabethkingia miricola is an emerging opportunistic pathogen that is highly pathogenic in both immunocompromised humans and animals. Once the disease occurs, treatment can be very difficult. Therefore, a deep understanding of the pathological mechanism of Elizabethkingia miricola is the key to the prevention and control of the disease. In this study, we isolated the pathogenic bacteria from bullfrogs with dark skin color, weak limbs, wryneck, and cataracts. Via subsequent morphological observations and a 16S rRNA gene sequence analysis, the pathogen was identified as Elizabethkingia miricola. The histopathological and transmission electron microscopy analysis revealed that the brain was the main target organ. Therefore, brain samples from diseased and healthy bullfrogs were used for the RNA-Seq analysis. The comparative transcriptome analysis revealed that the diseased bullfrog brain was characterized by the immune activation and inflammatory response, which were mediated by the "NOD-like receptor signaling pathway" and the "Toll-like receptor signaling pathway". We also performed qRT-PCR to examine the expression profile of inflammation-related genes, which further verified the reliability of our transcriptome data. Based on the above results, it was concluded that the NOD/Toll-like receptor-related networks that dominate the immune activation and inflammatory response were activated in the brain of Elizabethkingia miricola-infected bullfrogs. This study contributes to the search for therapeutic targets for bullfrog meningitis and provides basic information for establishing effective measures to prevent and control bullfrog meningitis.

The differences in molecular mechanisms between osteochondroma and bizarre parosteal osteochondromatous proliferation (BPOP) remain to be fully elucidated. In the present study, the differentially expressed genes between BPOP and osteochondroma were obtained from the Gene Expression Omnibus online database, and the associations among these genes were analyzed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) online bioinformatics software. The results revealed several differentially expressed genes between human BPOP and osteochondroma. These differentially expressed genes were also enriched in different subgroups based on the analysis using DAVID online software, including ‘transforming growth factor β receptor signaling pathway’, ‘BMP signaling pathway’, ‘Wnt receptor signaling pathway’, ‘response to chemical stimulus’, ‘regulation of inflammatory response’, ‘response to stress’, ‘glycosaminoglycan binding’, ‘polysaccharide binding’, ‘extracellular matrix structural constituent’ and ‘growth factors binding’. Taken together, these findings led to the conclusion that different gene regulatory mechanisms exist between BPOP and osteochondroma. Environmental stimulation and inflammation may contribute to BPOP or osteochondroma, and differences in extracellular matrix may contribute to differences in biological characteristics between BPOP and osteochondroma.

Objective: Chronic obstructive pulmonary disease (COPD) is recognized as a common preventable respiratory disease whose character is incompletely reversible airflow limitation. However, its specific pathogenesis is still not fully understood. Here we sought to identify novel biomarkers or targets for COPD. Methods: We performed expression profiling through gene arrays contained lung tissue data of 14 COPD patients and 5 non-smokers from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) are identified by using the GEO2R tool with standard of the |log(FC)|>2 and P Results: A total of 133 DEGs were discovered from GEO2R, including 78 up-regulated genes and 55 down-regulated genes. GO enrichment analysis showed that up-regulated genes were involved in cell chemotaxis, cytokine activity and external stimuli response, the down-regulated genes had influenced on membrane organization and neurogenesis, and the all DEGs were mainly involved in cell chemotaxis, cytokine activity, external stimuli response and response to bacterium. KEGG pathway analysis showed that up-regulated genes were mainly involved in Toll-like receptors(TLR), phosphoinositide-3-kinase/protein kinase (PI3K-Akt), fork head box class O (FoxO) and tumor necrosis factor (TNF), and down-regulated genes mainly had influenced on estrogen and cell adhesion molecules (CAMs) signaling pathways, and all DEGs were mainly involved in interaction with cytokine and cytokine receptor, Chemokine and IL-17 signaling pathway. A total of 4 hub genes (degree≥9) were obtained from the PPI network, including interleukin6 (IL6), CC chemokine ligand 20 (CCL20), secreted phosphoprotein 1 (SPP1) and epithelial neutrophil activating peptide-78 (CXCL5). When we used the MCODE plug-in in Cytoscape 3.6.0 software, we found an important gene group. Through KEGG analysis of those gene groups, there are signaling pathways stated out, including immune response, inflammatory response, reninangiotensin system, tyrosine kinase-signal transducers and transcriptional activators (Jak-STAT), phagosome and others. Conclusion: This study indicates that IL6, CCL20, SPP1, CXCL5 and the pathways involved were crucial to the COPD pathophysiology. These key genes and pathways are valuable for further investigations. This study provides a better understanding of the gene expression profile in COPD pathophysiology.

Single-cell RNA transcriptome landscape of hepatocytes and non-parenchymal cells in healthy and NAFLD mouse liver

Structural characteristics of speckled chicken eggshells and their effect on reproductive performance

Dynamic immune response in the spleens of rainbow trout (Oncorhynchus mykiss) to infectious hematopoietic necrosis virus revealed by transcriptome and immune-related genes expression analysis

Simple SummaryThe outbreak of Hirame novirhabdovirus is significantly temperature dependent. The aim of this study was to identify differential responses of flounder to HIRRV infection at different temperatures by proteome and phosphoproteome. Post HIRRV infection under 10 °C and 20 °C, the enriched immune-related DEPs were both involved in RLR and NLR signaling pathways, apoptosis, phagosome and lysosome, and the DEPPs were also both enriched in spliceosome, mTOR signaling pathway and RNA transport. Compared with the group under 10 °C, the proteins and phosphoproteins involved in interferon production and signaling showed stronger response to infection under 20 °C. qRT-PCR assay showed that eight antiviral-related mRNA including IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44, MX1 and ISG15 displayed significantly stronger and quicker response at early infection under 20 °C. This study provided a comprehensive understanding of signaling alterations and differential antiviral responses of flounder to HIRRV infection under different temperatures.Hirame novirhabdovirus (HIRRV) is one of most serious viral pathogens causing significant economic losses to the flounder (Paralichthys olivaceus)-farming industry. Previous studies have shown that the outbreak of HIRRV is highly temperature-dependent, and revealed the viral replication was significantly affected by the antiviral response of flounders under different temperatures. In the present study, the proteome and phosphoproteome was used to analyze the different antiviral responses in the HIRRV-infected flounder under 10 °C and 20 °C. Post viral infection, 472 differentially expressed proteins (DEPs) were identified in the spleen of flounder under 10 °C, which related to NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, RNA transport and so on. Under 20 °C, 652 DEPs were identified and involved in focal adhesion, regulation of actin cytoskeleton, phagosome, NOD-like receptor signaling pathway and RIG-I-like receptor signaling pathway. Phosphoproteome analysis showed that 675 differentially expressed phosphoproteins (DEPPs) were identified in the viral infected spleen under 10 °C and significantly enriched in Spliceosome, signaling pathway, necroptosis and RNA transport. Under 20 °C, 1304 DEPPs were identified and significantly enriched to Proteasome, VEGF signaling pathway, apoptosis, Spliceosome, mTOR signaling pathway, mRNA surveillance pathway, and RNA transport. To be noted, the proteins and phosphoproteins involved in interferon production and signaling showed significant upregulations in the viral infected flounder under 20 °C compared with that under 10 °C. Furthermore, the temporal expression profiles of eight selected antiviral-related mRNA including IRF3, IRF7, IKKβ, TBK1, IFIT1, IFI44, MX1 and ISG15 were detected by qRT-PCR, which showed a significantly stronger response at early infection under 20 °C. These results provided fundamental resources for subsequent in-depth research on the HIRRV infection mechanism and the antiviral immunity of flounder, and also gives evidences for the high mortality of HIRRV-infected flounder under low temperature.

iTRAQ-based quantitative proteomics analysis of the spleen reveals innate immunity and cell death pathways associated with heat stress in broilers (Gallus gallus)

The innate immune system provides a first line of defense against invading pathogens, in which the pattern recognition receptors (PRR) recognize pathogen-associated molecular patterns (PAMP) and initiate the downstream signaling pathways to eliminate the encountered pathogens. There are two main classes of such signaling pathways: NOD-like receptor (NLR) signaling pathway and Toll-like receptor (TLR) signaling pathway. The microbial pathogens under selective pressure have evolved numerous mechanisms to avoid and/or manipulate the NLR and TLR signal transduction for survival and replication. To evade the NLR signaling pathway, pathogens interfere and/or inhibit inflammasome activation in innate immune cells by producing virulence factors or reducing PAMPs expression. The mechanisms for pathogens to evade TLR signaling pathway include: inhibition of mitogen activated protein kinases (MAPKs) cascade reaction, inhibition of NF-КB activation, and interference of down-stream signal transduction by producing Toll/interleukin-1 receptor (TIR)-containing proteins which bind directly with TLRs or adaptor proteins in the signaling pathway.

Dysbiosis of respiratory microbiota is closely related to the pathophysiological processes of asthma, including airway inflammation. Previous studies have shown that Qingfei oral liquid (QF) can alleviate airway inflammation and airway hyper-responsiveness in respiratory syncytial virus-infected asthmatic mice, but its effect on the respiratory microbiota is unknown. We therefore aimed to observe the effects of QF on airway inflammation and respiratory microbiota in ovalbumin (OVA)-induced asthmatic mice. We also explored the potential mechanism of QF in reducing airway inflammation by regulating respiratory microbiota. Hematoxylin and eosin as well as periodic acid-Schiff staining were performed to observe the effects of QF on lung pathology in asthmatic mice. Cytokine levels in bronchoalveolar lavage fluid (BALF) specimens were also measured. Changes in respiratory microbiota were analyzed using 16S rRNA gene sequencing, followed by taxonomical analysis. In order to verify the metagenomic function prediction results, the expression of key proteins related to the MAPK and NOD-like receptor signaling pathways in the lung tissues were detected by immunohistochemistry. The current study found that QF had a significant anti-inflammatory effect in the airways of asthmatic mice. This is mainly attributed to a reduction in lung pathology changes and regulating cytokine levels in BALF. Analysis of the respiratory microbiota in asthmatic mice showed that the abundance of Proteobacteria at the phylum level and Pseudomonas at the genus level increased significantly and QF could significantly regulate the dysbiosis of respiratory microbiota in asthmatic mice. Metagenomic functional prediction showed that QF can downregulate the MAPK and Nod-like receptor signaling pathways. Immunohistochemical results showed that QF could downregulate the expression of p-JNK, p-P38, NLRP3, Caspase-1, and IL-1β, which are all key proteins in the signaling pathway of lung tissue. Our study therefore concluded that QF may reduce airway inflammation in asthmatic mice by regulating respiratory microbiota, and to the possibly downregulate MAPK and Nod-like receptor signaling pathways as its underlying mechanism.

BackgroundVulnerable plaque was associated with recurrent cardiovascular events. This study was designed to explore predictive biomarkers of vulnerable plaque in patients with coronary artery disease.MethodsTo reveal the phenotype-associated cell type in the development of vulnerable plaque and to identify hub gene for pathological process, we combined single-cell RNA and bulk RNA sequencing datasets of human atherosclerotic plaques using Single-Cell Identification of Subpopulations with Bulk Sample Phenotype Correlation (Scissor) and Weighted gene co-expression network analysis (WGCNA). We also validated our results in an independent cohort of patients by using intravascular ultrasound during coronary angiography.ResultsMacrophages were found to be strongly correlated with plaque vulnerability while vascular smooth muscle cell (VSMC), fibrochondrocyte (FC) and intermediate cell state (ICS) clusters were negatively associated with unstable plaque. Weighted gene co-expression network analysis showed that Secreted Phosphoprotein 1 (SPP1) in the turquoise module was highly correlated with both the gene module and the clinical traits. In a total of 593 patients, serum levels of SPP1 were significantly higher in patients with vulnerable plaques than those with stable plaque (113.21 [73.65 - 147.70] ng/ml versus 71.08 [20.64 - 135.68] ng/ml; P < 0.001). Adjusted multivariate regression analysis revealed that serum SPP1 was an independent determinant of the presence of vulnerable plaque. Receiver operating characteristic curve analysis indicated that the area under the curve was 0.737 (95% CI 0.697 - 0.773; P < 0.001) for adding serum SPP1 in predicting of vulnerable plaques.ConclusionElevated serum SPP1 levels confer an increased risk for plaque vulnerability in patients with coronary artery disease.