Overexpression of Toll-Like Receptor 4 Contributes to Phagocytosis of Salmonella Enterica Serovar Typhimurium via Phosphoinositide 3-Kinase Signaling in Sheep

Background/Aims: Phagocytosis of bacteria by monocytes/macrophages can trigger the immune response and the clearance of bacteria. This innate immune response involves Toll-like receptor 4 (TLR4). However, much remains unknown about the mechanism of TLR4-regulated phagocytosis of Salmonella enterica serovar Typhimurium (S. typhimurium) within sheep monocytes/macrophages. Here, we aimed to address these knowledge gaps by infecting transgenic sheep overexpressing TLR4 with S. typhimurium and examining the phagocytic mechanisms involved. Methods: Transgenic sheep were generated by microinjection of the constructed plasmids into fertilized eggs. Monocytes/macrophages isolated from sheep blood were stimulated with LPS and S. typhimurium. Phagocytosis-related factor expression, phagocytic ability, and adhesion were then determined. TLR4/phosphatidylinositide 3-kinase (PI3K) signaling was inhibited to investigate if this pathway is involved in changes in bacterial internalization in sheep. Results: We found that TLR4 overexpression effectively activated the PI3K signaling pathway and upregulated the expression of scavenger receptors. Additionally, actin polymerization and adhesive capacity were both enhanced in TLR4-overexpressing sheep monocytes/macrophages. TLR4 inhibition decreased S. typhimurium phagocytosis by reducing the actin polymerization and adhesive capacity of cells. Furthermore, inhibition of PI3K markedly impaired TLR4-dependent phagocytosis by restraining actin polymerization and scavenger receptor expression and reduced the adhesive capacity of the monocytes/macrophages. Conclusion: Our findings indicate that overexpression of TLR4 enhances phagocytosis through PI3K signaling and the subsequent activation of actin polymerization and scavenger receptors in sheep monocytes/macrophages infected with S. typhimurium

Effects of Dietary Supplementation with Mulberry Leaf Powder on the Growth Performance, Lipid Metabolism Parameters, Immunity Indicators, and Gut Microbiota of Dogs

Overfeeding and a lack of exercise are increasingly causing obesity in dogs, which has become a big problem threatening the health of dogs. Therefore, it is necessary to investigate how dietary regulations can help to improve dogs’ body conditions and minimize obesity. This study was carried out to investigate the effects of dietary mulberry leaf powder (MLP) supplementation on the growth performance, lipid metabolism parameters, and gut microbiota of Chinese indigenous dogs. Fifteen Chinese indigenous dogs (6.34 ± 0.56 kg) were randomly assigned to three treatment groups and received either the control diet (CON), high-fat diet (HF), or high-fat diet containing 6% Mulberry leaf powder (MLP) for four weeks. The CON group received a basal diet, the HF group received a basal diet supplemented with 10% lard, and the MLP group received a basal diet supplemented with 10% lard and 6% MLP. The trial lasted for four weeks. The growth performance, lipid metabolism parameters, immune globulins, cytokines, and fecal microbiota were measured. Results showed that there was no significant difference in growth performance. The MLP group appeared to have decreased (p p Alloprevotella, Sarcina, and species belonging to the Bacteroides and Lactobacillus genus. Overall, the dietary supplementation of 6% MLP can improve lipid metabolism conditions and immunity in high-fat-diet-fed dogs, and can alter the gut microbial composition of dogs

Effects of Long-Chain Fatty Acyl-CoA Synthetase 1 on Diglyceride Synthesis and Arachidonic Acid Metabolism in Sheep Adipocytes

Long-chain fatty acyl-CoA synthetase (ACSLs) is an essential enzyme for the synthesis of fatty acyl-CoA. ACSL1 plays a key role in the synthesis of triglycerides, phospholipids, and cholesterol esters. Background: In the current study, triglyceride content did not increase after overexpression of the ACSL1 gene. Methods: RNA-seq and lipid metabolome profiling were performed to determine why triglyceride levels did not change with ACSL1 overexpression. Results: Fatty acyl-CoA produced by ACSL1 was determined to be involved in the diglyceride synthesis pathway, and diglyceride content significantly increased when ACSL1 was overexpressed. Moreover, the arachidonic acid (AA) content in sheep adipocytes significantly increased, and the level of cyclooxygenase 2 (COX2) expression, the downstream metabolic gene, was significantly downregulated. Knocking down the ACSL1 gene was associated with an increase in COX2 mRNA expression, as well as an increase in prostaglandin content, which is the downstream metabolite of AA. Conclusions: The overexpression of the ACSL1 gene promotes the production of AA via downregulation of COX2 gene expression

Differential Analysis of Gene Expression of Toll-like Receptors and Antimicrobial Peptides in Immune Organs and Tissues of Tibetan and Duroc-Landrace-Yorkshire pigs

【Objective】In mammals, Toll-like receptors and antimicrobial peptide genes are important components of the innate immune system, which play a crucial role in fighting against pathogen attacks. The study was conducted to explore the expression differences of Toll-like receptor and antimicrobial peptide genes between different immune organs or tissues in Tibetan pigs and Duroc-Landrace-Yorkshire pigs, with an aim to reveal the potential contribution of these genes to disease resistance and immune response and provide theoretical support for the screening of molecular markers for disease resistance.【Method】The mRNA abundance of Toll-like receptors genes (TLR1-TLR9) and two types of antimicrobial peptide genes (PBD-1 and PR-39) in lungs, mesenteric lymph nodes, inguinal lymph nodes, submandibular lymph nodes and spleens of 6-month-old Tibetan and Duroc-Landrace-Yorkshire pigs were detected by qPCR.【Result】The mRNA expression of Tolllike receptors and antimicrobial peptide genes in most of the immune organs or tissues of Tibetan pigs was significantly higher than that of Duroc-Landrace-Yorkshire pigs. Among them, the mRNA expression of TLR1 and TLR2 in lungs was increased by about 50%, and PR-39 was increased by 2.6 times; the expression of TLR4 in mesenteric lymph nodes was increased by 40%, and the expression of TLR1 and PR-39 was increased by 88% and 3 times, respectively. In the inguinal lymph nodes, the expression of TLR1 and TLR2 was increased by about 2 times, and the expression of TLR9 and PR-39 was increased by 70%, especially, the expression of PR-39 increasing by 7 times; The expression of TLR1, TLR2, TLR4 and TLR7 in submandibular lymph nodes was increased by more than 2 times, and the expression of PR-39 was increased by nearly 7 times, which was similar to that in inguinal lymph nodes; the expression of TLR1 in the spleen rose by 3.5 times, which was similar to that in submandibular lymph nodes. The expression of TLR4 and TLR9 increased by about 50%, and the expression of PR-39 increased by 2.5 times.【Conclusion】Tibetan pigs show higher expression levels of Toll-like receptors and antimicrobial peptide genes in multiple immune organs or tissues compared to Duroc-Landrace-Yorkshire pigs. It is implied that Tibetan pigs may possess stronger innate immunity and be able to generate more effective local or systemic immune responses against pathogenic microbial infections. The results of this study provide important theoretical support for the identification of disease resistance molecular markers and are expected to provide a scientific basis for further improvement of disease resistance in Tibetan pigs and other pig breeds

Application of Gene Editing Technology in Resistance Breeding of Livestock

As a new genetic engineering technology, gene editing can precisely modify the specific gene sequence of the organism’s genome. In the last 10 years, with the rapid development of gene editing technology, zinc-finger nucleases (ZFNs), transcription activator-like endonucleases (TALENs), and CRISPR/Cas9 systems have been applied to modify endogenous genes in organisms accurately. Now, gene editing technology has been used in mice, zebrafish, pigs, cattle, goats, sheep, rabbits, monkeys, and other species. Breeding for disease-resistance in agricultural animals tends to be a difficult task for traditional breeding, but gene editing technology has made this easier. In this work, we overview the development and application of gene editing technology in the resistance breeding of livestock. Also, we further discuss the prospects and outlooks of gene editing technology in disease-resistance breeding

The Effects of Dietary Inclusion of Mulberry Leaf Powder on Growth Performance, Carcass Traits and Meat Quality of Tibetan Pigs

This research was conducted to study the effects of dietary inclusion of mulberry leaf powder (MLP) on growth performance, meat quality, antioxidant activity, and carcass traits of Tibetan pigs. Eighteen Tibetan pigs (33.8 ± 1.1 kg) were assigned to two treatment groups randomly and received either the control diet (CON) or a basal diet containing 8% MLP (MLP) for two months. After the two-month feeding trial, the MLP group showed lower backfat thickness while a higher lean percentage. Compared with CON pigs, MLP pigs had higher serum CAT activity. In addition, dietary MLP supplementation significantly decreased the muscle shear force. Muscle fiber morphology analysis showed that MLP pigs had larger muscle fiber density while smaller muscle fiber cross-sectional area. Up-regulated gene expression of myosin heavy chain (MyHC)IIa was also observed in MLP pigs. These results indicate that the enhanced antioxidant activity and altered muscle fiber type and morphology appeared to contribute to the improvement of meat quality in Tibetan pigs fed diets containing MLP

CircBIRC6 facilitates the malignant progression via miR-488/GRIN2D-mediated CAV1-autophagy signal axis in gastric cancer

Circular RNAs (circRNAs) represent a novel class of non-coding RNAs that play significant roles in tumorigenesis and tumor progression. High-throughput sequencing of gastric cancer (GC) tissues has identified circRNA BIRC6 (circBIRC6) as a potential circRNA derived from the BIRC6 gene, exhibiting significant upregulation in GC tissues. The expression of circBIRC6 is notably elevated in GC patients. Functionally, it acts as a molecular sponge for miR-488, consequently upregulating GRIN2D expression and promoting GC proliferation, migration, and invasion. Moreover, overexpression of circBIRC6 leads to increased GRIN2D expression, which in turn enhances caveolin-1 (CAV1) expression, resulting in autophagy deficiency due to miR-488 sequestration. This cascade of events significantly influences tumorigenesis in vivo. Our findings collectively illustrate that the CircBIRC6-miR-488-GRIN2D axis fosters CAV1 expression in GC cells, thereby reducing autophagy levels. Both circBIRC6 and GRIN2D emerge as potential targets for treatment and independent prognostic factors for GC patients

Effects of <i>AANAT</i> overexpression on the inflammatory responses and autophagy activity in the cellular and transgenic animal levels

<p>To explore the anti-inflammatory activity of endogenous produced melatonin, a melatonin-enriched animal model (goat) with <i>AANAT</i> transfer was successfully generated with somatic cell nuclear transfer (SCNT) technology. Basically, a pIRES2-EGFP-<i>AANAT</i> expression vector was constructed and was transferred into the female fetal fibroblast cells (FFCs) via electrotransfection and then the nuclear of the transgenic FFC was transferred to the eggs of the donor goats. The peripheral blood mononuclear cells (PBMCs) of the transgenic offspring expressed significantly higher levels of <i>AANAT</i> and melatonin synthetic function than those PBMCs from the wild-type (WT) animals. After challenge with lipopolysaccharide (LPS), the transgenic PBMCs had increased autophagosomes and <i>LC3B</i> expression while they exhibited suppressed production of the proinflammatory cytokines, IL1B and IL12 (IL12A-IL12B/p70), compared to their WT. The mechanistic analysis indicated that the anti-inflammatory activity of endogenous melatonin was mediated by MTNR1B (melatonin receptor 1B). MTNR1B stimulation activated the MAPK14 signaling pathway to promote cellular macroautophagy/autophagy, thus, suppressing the excessive inflammatory response of cellular. However, when the intact animals challenged with LPS, the serum proinflammatory cytokines were significantly higher in the transgenic goats than that in the WT. The results indicated that endogenous melatonin inhibited the MAPK1/3 signaling pathway and ROS production, subsequently downregulated gene expression of <i>BECN1, ATG5</i> in PMBCs and then suppressed the autophagy activity of PBMCs and finally elevated levels of serum proinflammatory cytokines in transgenic animals, Herein we provided a novel melatonin-enriched animal model to study the potential effects of endogenously produced melatonin on inflammatory responses and autophagy activity.</p