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Abstract Dynamic metabolic reprogramming occurs at different stages of myogenesis and contributes to the fate determination of skeletal muscle satellite cells (MuSCs). Accumulating evidence suggests that mutations in myostatin (MSTN) have a vital role in regulating muscle energy metabolism. Here, we explored the metabolic reprogramming in MuSCs and myotube cells in MSTN and FGF5 dual-gene edited sheep models prepared previously, and also focused on the metabolic alterations during myogenic differentiation of MuSCs. Our study revealed that the pathways of nucleotide metabolism, pantothenate and CoA biosynthesis were weakened, while the unsaturated fatty acids biosynthesis were strengthened during myogenic differentiation of sheep MuSCs. The MSTN and FGF5 dual-gene editing mainly inhibited nucleotide metabolism and biosynthesis of unsaturated fatty acids in sheep MuSCs, reduced the number of lipid droplets in per satellite cell, and promoted the pentose phosphate pathway, and the interconversion of pentose and glucuronate. The MSTN and FGF5 dual-gene editing also resulted in the inhibition of nucleotide metabolism and TCA cycle pathway in differentiated myotube cells. The differential metabolites we identified can be characterized as biomarkers of different cellular states, and providing a new reference for MSTN and FGF5 dual-gene editing in regulation of muscle development. It may also provide a reference for the development of muscle regeneration drugs targeting biomarkers.

Abstract Ferroptosis, which is driven by iron-dependent lipid peroxidation, plays an essential role in liver ischemia-reperfusion injury (IRI) during liver transplantation (LT). Gp78, an E3 ligase, has been implicated in lipid metabolism and inflammation. However, its role in liver IRI and ferroptosis remains unknown. Here, hepatocyte-specific gp78 knockout (HKO) or overexpressed (OE) mice were generated to examine the effect of gp78 on liver IRI, and a multi-omics approach (transcriptomics, proteomics, and metabolomics) was performed to explore the potential mechanism. Gp78 expression decreased after reperfusion in LT patients and mice with IRI, and gp78 expression was positively correlated with liver damage. Gp78 absence from hepatocytes alleviated liver damage in mice with IRI, ameliorating inflammation. However, mice with hepatic gp78 overexpression showed the opposite phenotype. Mechanistically, gp78 overexpression disturbed lipid homeostasis, remodeling polyunsaturated fatty acid (PUFA) metabolism, causing oxidized lipids accumulation and ferroptosis, partly by promoting ACSL4 expression. Chemical inhibition of ferroptosis or ACSL4 abrogated the effects of gp78 on ferroptosis and liver IRI. Our findings reveal a role of gp78 in liver IRI pathogenesis and uncover a mechanism by which gp78 promotes hepatocyte ferroptosis by ACSL4, suggesting the gp78-ACSL4 axis as a feasible target for the treatment of IRI-associated liver damage.

Abstract Background Hepatic ischemia–reperfusion (IR) injury is the primary reason for complications following hepatectomy and liver transplantation (LT). Insulin-induced gene 2 (Insig2) is one of several proteins that anchor the reticulum in the cytoplasm and is essential for metabolism and inflammatory responses. However, its function in IR injury remains ambiguous. Methods Insig2 global knock-out (KO) mice and mice with adeno-associated-virus8 (AAV8)-delivered Insig2 hepatocyte-specific overexpression were subjected to a 70% hepatic IR model. Liver injury was assessed by monitoring hepatic histology, inflammatory responses, and apoptosis. Hypoxia/reoxygenation stimulation (H/R) of primary hepatocytes and hypoxia model induced by cobalt chloride (CoCl2) were used for in vitro experiments. Multi-omics analysis of transcriptomics, proteomics, and metabolomics was used to investigate the molecular mechanisms underlying Insig2. Results Hepatic Insig2 expression was significantly reduced in clinical samples undergoing LT and the mouse IR model. Our findings showed that Insig2 depletion significantly aggravated IR-induced hepatic inflammation, cell death and injury, whereas Insig2 overexpression caused the opposite phenotypes. The results of in vitro H/R experiments were consistent with those in vivo. Mechanistically, multi-omics analysis revealed that Insig2 is associated with increased antioxidant pentose phosphate pathway (PPP) activity. The inhibition of glucose-6-phosphate-dehydrogenase (G6PD), a rate-limiting enzyme of PPP, rescued the protective effect of Insig2 overexpression, exacerbating liver injury. Finally, our findings indicated that mouse IR injury could be attenuated by developing a nanoparticle delivery system that enables liver-targeted delivery of substrate of PPP (glucose 6-phosphate). Conclusions Insig2 has a protective function in liver IR by upregulating the PPP activity and remodeling glucose metabolism. The supplementary glucose 6-phosphate (G6P) salt may serve as a viable therapeutic target for alleviating hepatic IR.

Abstract Background Sorafenib resistance greatly reduces the efficacy of treatments in advanced hepatocellular carcinoma (HCC) patients, but the underlying mechanisms are not thoroughly understood. All-trans retinoic acid (ATRA), an anti-leukaemia agent, has attracted considerable attention due to its role in sensitizing cells to other anticancer treatments. We aimed to investigate the combined effect of ATRA and Sorafenib on HCC and the underlying mechanisms. Methods CCK-8, cell sphere formation, trans-well migration, and wound-healing assays were used to analyse the biological behaviours of HCC cells in vitro. Western blotting and qRT-PCR analysis were conducted to measure the expression of p21 activated kinase 1 (PAK1) and phospho-p21 activated kinase 1 (pPAK1). Xenograft models were established to confirm the synergistic effects of ATRA and Sorafenib in vivo. TUNEL assays and immunohistochemistry were utilized to determine apoptosis, proliferation, PAK1 and pPAK1 levels in tumour tissues. Results We observed that PAK1 was overexpressed in HCC, and its expression was negatively correlated with the survival of patients. PAK1 promoted the proliferation, self-renewal and epithelial-mesenchymal transition of HCC cells. Correlation analysis indicated that the IC50 of Sorafenib was positively correlated with the level of pPAK1 in HCC cell lines. ATRA inhibited the progression of HCC and sensitized HCC response to Sorafenib by downregulation of PAK1, as shown by the calculated coefficient of drug interaction and the data obtained from xenograft models. Conclusions Our findings indicated that instead of treatment with Sorafenib alone, the combination of ATRA and Sorafenib provides a more effective treatment for HCC patients. Video Abstract

Spermatogenesis in mammalian testes is essential for male fertility, ensuring a continuous supply of mature sperm. The testicular microenvironment finely tunes this process, with retinoic acid, an active metabolite of vitamin A, serving a pivotal role. Retinoic acid is critical for various stages, including the differentiation of spermatogonia, meiosis in spermatogenic cells, and the production of mature spermatozoa. Vitamin A deficiency halts spermatogenesis, leading to the degeneration of numerous germ cells, a condition reversible with retinoic acid supplementation. Although retinoic acid can restore fertility in some males with reproductive disorders, it does not work universally. Furthermore, high doses may adversely affect reproduction. The inconsistent outcomes of retinoid treatments in addressing infertility are linked to the incomplete understanding of the molecular mechanisms through which retinoid signaling governs spermatogenesis. In addition to the treatment of male reproductive disorders, the role of retinoic acid in spermatogenesis also provides new ideas for the development of male non-hormone contraceptives. This paper will explore three facets: the synthesis and breakdown of retinoic acid in the testes, its role in spermatogenesis, and its application in male reproduction. Our discussion aims to provide a comprehensive reference for studying the regulatory effects of retinoic acid signaling on spermatogenesis and offer insights into its use in treating male reproductive issues.

CRISPR-Cas9-mediated genome editing in sheep is of great use in both agricultural and biomedical applications. While targeted gene knockout by CRISPR-Cas9 through non-homologous end joining (NHEJ) has worked efficiently, the knockin efficiency via homology-directed repair (HDR) remains lower, which severely hampers the application of precise genome editing in sheep. Here, in sheep fetal fibroblasts (SFFs), we optimized several key parameters that affect HDR, including homology arm (HA) length and the amount of double-stranded DNA (dsDNA) repair template; we also observed synchronization of SFFs in G2/M phase could increase HDR efficiency. Besides, we identified three potent small molecules, RITA, Nutlin3, and CTX1, inhibitors of p53-MDM2 interaction, that caused activation of the p53 pathway, resulting in distinct G2/M cell-cycle arrest in response to DNA damage and improved CRISPR-Cas9-mediated HDR efficiency by 1.43- to 4.28-fold in SFFs. Furthermore, we demonstrated that genetic knockout of p53 could inhibit HDR in SFFs by suppressing the expression of several key factors involved in the HDR pathway, such as BRCA1 and RAD51. Overall, this study offers an optimized strategy for the usage of dsDNA repair template, more importantly, the application of MDM2 antagonists provides a simple and efficient strategy to promote CRISPR/Cas9-mediated precise genome editing in sheep primary cells.

Abstract Background SCNT (somatic cell nuclear transfer) is of great significance to biological research and also to the livestock breeding. However, the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods. This indicates the potential epigenetic variations between them. DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences. In this study, ASMT (acetylserotonin-O-methyltransferase) ovarian overexpression transgenic goat was produced by using SCNT. To investigate whether there are epigenetic differences between cloned and WT (wild type) goats, WGBS (whole-genome bisulfite sequencing) was used to measure the whole-genome methylation of these animals. Results It is observed that the different mCpG sites are mainly present in the intergenic and intronic regions between cloned and WT animals, and their CG-type methylation sites are strongly correlated. DMR (differentially methylated region) lengths are located around 1000 bp, mainly distributed in the exonic, intergenic and intronic functional domains. A total of 56 and 36 DMGs (differentially methylated genes) were identified by GO and KEGG databases, respectively. Functional annotation showed that DMGs were enriched in biological-process, cellular-component, molecular-function and other signaling pathways. A total of 10 identical genes related to growth and development were identified in GO and KEGG databases. Conclusion The differences in methylation genes among the tested animals have been identified. A total of 10 DMGs associated with growth and development were identified between cloned and WT animals. The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT. These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology, particularly in goats.

The declines in laying performance during the late production period have adverse effects on the length of the production cycle. Improving the nutrition of laying hens is a crucial measure to reverse this declination. This study investigated the effect of selenium yeast (SY) on egg production, ileal gene expression and microbiota, as well as elucidating their associations in aged laying hens. A total of 375 Jinghong laying hens at 76 weeks old were randomly assigned into 5 dietary treatments, which included a selenium-deficient basal diet based on corn-soybean meal, and dietary supplementation of SY at 0.15, 0.30 and 0.45 mg/kg, and sodium selenite at 0.45 mg/kg. The results showed that SY ameliorated the depression in aged laying performance in the 0.30 mg/kg group (P

Antithrombin III is an important anticoagulant factor with anti-inflammatory properties. However, few studies have explored its anti-inflammatory actions in ATIII overexpressed transgenic animals. In this study, the dairy goats with mammary overexpression of ATIII were used to investigate their general health, milk quality and particularly their response to inflammatory challenge. The results showed that transgenic goats have a normal phenotype regarding their physiological and biochemical parameters, including whole blood cells, serum protein levels, total cholesterol, urea nitrogen, uric acid, and total bilirubin, compared to the WT. In addition, the quality of milk also improved in transgenic animals compared to the WT, as indicated by the increased milk fat and dry matter content and the reduced somatic cell numbers. Under the stimulation of an LPS injection, the transgenic goats had elevated contents of IGA, IGM and superoxide dismutase SOD, and had reduced proinflammatory cytokine release, including IL-6, TNF-α and IFN-β. A 16S rDNA sequencing analysis also showed that the transgenic animals had a similar compositions of gut microbiota to the WT goats under the stimulation of LPS injections. Mammary gland ATIII overexpression in dairy goats is a safe process, and it did not jeopardize the general health of the transgenic animals; moreover, the compositions of their gut microbiota also improved with the milk quality. The LPS stimulation study suggests that the increased ATIII expression may directly or indirectly suppress the inflammatory response to increase the resistance of transgenic animals to pathogen invasion. This will be explored in future studies.

IntroductionMyostatin (MSTN) negatively regulates skeletal muscle development. However, its function in reproductive performance and visceral organs has not been thoroughly investigated. Previously, we prepared a MSTN and fibroblast growth factor 5 (FGF5) double-knockout sheep, which was a MSTN and FGF5 dual-gene biallelic homozygous (MF−/−) mutant.MethodsTo understand the role of MSTN and FGF5 in reproductive performance and visceral organs, this study evaluated the ejaculation amount, semen pH, sperm motility, sperm density, acrosome integrity, rate of teratosperm, and seminal plasma biochemical indicators in adult MF−/− rams. We also compared the overall morphology, head, head-neck junction, middle segment and the transection of middle segment of spermatozoa between wildtype (WT) and MF−/− rams.ResultsOur results showed that the seminal plasma biochemical indicators, sperm structure and all sperm indicators were normal, and the fertilization rate also has no significant difference between WT and MF−/− rams, indicating that the MF−/− mutation did not affect the reproductive performance of sheep. Additional analysis evaluated the histomorphology of the visceral organs, digestive system and reproductive system of MF+/− sheep, the F1 generation of MF−/−, at the age of 12 months. There was an increased spleen index, but no significant differences in the organ indexes of heart, liver, lung, kidney and stomach, and no obvious differences in the histomorphology of visceral organs, digestive system and reproductive system in MF+/− compared with WT sheep. No MF+/− sheep were observed to have any pathological features.DiscussionIn summary, the MSTN and FGF5 double-knockout did not affect reproductive performance, visceral organs and digestive system in sheep except for differences previously observed in muscle and fat. The current data provide a reference for further elucidating the application of MSTN and FGF5 double-knockout sheep.

Base editors are genome engineering tools that can generate nucleotide substitutions without introducing double-stranded breaks. Here the authors show that a phage-derived peptidyl CRISPR inhibitor can be employed to modulate the activity and targeting scope of CRISPR base editor for precision base editing applications.

Gram-negative bacterial infections pose a significant threat to public health. Toll-like receptor 4 (TLR4) recognizes bacterial lipopolysaccharide (LPS) and induces innate immune responses, autophagy, and cell death, which have major impacts on the body’s physiological homeostasis. However, the role of TLR4 in bacterial LPS-induced autophagy and apoptosis in large mammals, which are closer to humans than rodents in many physiological characteristics, remains unknown. So far, few reports focus on the relationship between TLR, autophagy, and apoptosis in large mammal levels, and we urgently need more tools to further explore their crosstalk. Here, we generated a TLR4-enriched mammal model (sheep) and found that a high-dose LPS treatment blocked autophagic degradation and caused strong innate immune responses and severe apoptosis in monocytes/macrophages of transgenic offspring. Excessive accumulation of autophagosomes/autolysosomes might contribute to LPS-induced apoptosis in monocytes/macrophages of transgenic animals. Further study demonstrated that inhibiting TLR4 downstream NF-κB or p38 MAPK signaling pathways reversed the LPS-induced autophagy activity and apoptosis. These results indicate that the elevated TLR4 aggravates LPS-induced monocytes/macrophages apoptosis by leading to lysosomal dysfunction and impaired autophagic flux, which is associated with TLR4 downstream NF-κB and MAPK signaling pathways. This study provides a novel TLR4-enriched mammal model to study its potential effects on autophagy activity, inflammation, oxidative stress, and cell death. These findings also enrich the biological functions of TLR4 and provide powerful evidence for bacterial infection.

Abstract Cytokeratin 19-positive (CK19+) hepatocellular carcinoma (HCC) is an aggressive subtype characterized by early recurrence and chemotherapy tolerance. However, there is no specific therapeutic option for CK19+ HCC. The correlation between tumor recurrence and expression status of CK19 were studied in 206 patients undergoing liver transplantation for HCC. CK19−/+ HCC cells were isolated to screen effective antitumor drugs. The therapeutic effects of regorafenib were evaluated in patient-derived xenograft (PDX) models from 10 HCC patients. The mechanism of regorafenib on CK19+ HCC was investigated. CK19 positiveness indicated aggressiveness of tumor and higher recurrence risk of HCC after liver transplantation. The isolated CK19+ HCC cells had more aggressive behaviors than CK19− cells. Regorafenib preferentially increased the growth inhibition and apoptosis of CK19+ cells in vitro, whereas sorafenib, apatinib, and 5-fluorouracil did not. In PDX models from CK19−/+ HCC patients, the tumor control rate of regorafenib achieved 80% for CK19+ HCCs, whereas 0% for CK19− HCCs. RNA-sequencing revealed that CK19+ cells had elevated expression of mitochondrial ribosomal proteins, which are essential for mitochondrial function. Further experiments confirmed that regorafenib attenuated the mitochondrial respiratory capacity in CK19+ cells. However, the mitochondrial respiration in CK19− cells were faint and hardly repressed by regorafenib. The mitochondrial respiration was regulated by the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which was inhibited by regorafenib in CK19+ cells. Hence, CK19 could be a potential marker of the therapeutic benefit of regorafenib, which facilitates the individualized therapy for HCC. STAT3/mitochondria axis determines the distinct response of CK19+ cells to regorafenib treatment.

With aging, the progressive loss of skeletal muscle will have negative effect on multiple physiological parameters, such as exercise, respiration, thermoregulation, and metabolic homeostasis. Accumulating evidence reveals that oxidative stress and inflammation are the main pathological characteristics of skeletal muscle during aging. Here, we focus on aging-related sarcopenia, summarize the relationship between aging and sarcopenia, and elaborate on aging-mediated oxidative stress and oxidative damage in skeletal muscle and its critical role in the occurrence and development of sarcopenia. In addition, we discuss the production of excessive reactive oxygen species in aging skeletal muscle, which reduces the ability of skeletal muscle satellite cells to participate in muscle regeneration, and analyze the potential molecular mechanism of ROS-mediated mitochondrial dysfunction in aging skeletal muscle. Furthermore, we have also paid extensive attention to the possibility and potential regulatory pathways of skeletal muscle aging and oxidative stress mediate inflammation. Finally, in response to the abnormal activity of oxidative stress and inflammation during aging, we summarize several potential antioxidant and anti-inflammatory strategies for the treatment of sarcopenia, which may provide beneficial help for improving sarcopenia during aging.

Abstract Background Gram-negative bacterial infections have a major economic impact on both the livestock industry and public health. Toll-like receptor 4 (TLR4) plays a crucial role in host defence against Gram-negative bacteria. Exploring the defence mechanism regulated by TLR4 may provide new targets for treatment of inflammation and control of bacterial infections. In a previous study, we generated transgenic sheep overexpressing TLR4 by microinjection to improve disease resistance. The defence mechanism through which TLR4 overexpression protected these sheep against pathogens is still not fully understood. Results In the present study, we used Escherichia coli to infect monocytes isolated from peripheral blood of the animal model. The overexpression of TLR4 strongly enhanced the percentage of endocytosis and capacity of elimination in monocytes during the early stages of infection. This phenomenon was mainly due to overexpression of TLR4 promoting caveolae-mediated endocytosis. Pretreatment of the transgenic sheep monocytes with inhibitors of TLR4, Src signalling, or the caveolae-mediated endocytosis pathway reduced the internalization of bacteria, weakened the ability of the monocytes to eliminate the bacteria, and increased the pH of the endosomes. Conclusion Together, our results reveal the effects of TLR4 on the control of E. coli infection in the innate immunity of sheep and provide crucial evidence of the caveolae-mediated endocytosis pathway required for host resistance to invading bacteria in a large animal model, providing theoretical support for breeding disease resistance in the future. Furthermore, Src and caveolin 1 (CAV1) could be potentially valuable targets for the control of infectious diseases.

Staphylococcus aureus infections pose a potential threat to livestock production and public health. A novel strategy is needed to control S. aureus infections due to its adaptive evolution to antibiotics. Autophagy plays a key role in degrading bacteria for innate immune cells. In order to promote S. aureus clearance via Toll-like receptor (TLR)-induced autophagy pathway, the domain fusion TLR2-4 with the extracellular domain of TLR2, specific recognizing S. aureus, and transmembrane and intracellular domains of TLR4 is assembled, then the goat expressing TLR2-4 is generated. TLR2-4 substantially augments the removal of S. aureus within macrophages by elevating autophagy level. Phosphorylated JNK and ERK1/2 promote LC3-puncta in TLR2-4 macrophages during S. aureus-induced autophagy via MyD88 mediated the TAK1 signaling cascade. Meantime, the TRIF-dependent TBK1-TFEB-OPTN signaling is involved in TLR2-4-triggered autophagy after S. aureus challenge. Moreover, the transcript of ATG5 and ATG12 is significantly increased via cAMP-PKA-NF-κB signaling, which facilitates S. aureus-induced autophagy in TLR2-4 macrophages. Overall, the novel receptor TLR2-4 enhances the autophagy-dependent clearance of S. aureus in macrophages via TAK1/TBK1-JNK/ERK, TBK1-TFEB-OPTN, and cAMP-PKA-NF-κB-ATGs signaling pathways, which provide an alternative approach for resistant against S. aureus infection.

B cells are a class of professional antigen-presenting cells that produce antibodies to mediate humoral immune response and participate in immune regulation. m6A modification is the most common RNA modification in mRNA; it involves almost all aspects of RNA metabolism and can affect RNA splicing, translation, stability, etc. This review focuses on the B-cell maturation process as well as the role of three m6A modification-related regulators—writer, eraser, and reader—in B-cell development and B-cell-related diseases. The identification of genes and modifiers that contribute to immune deficiency may shed light on regulatory requirements for normal B-cell development and the underlying mechanism of some common diseases.

Abstract Background Toll-like receptor 4 (TLR4) plays an important role in the elimination of Gram-negative bacteria infections and the initiation of antiinflammatory response. Using the technology of pronuclear microinjection, genetically modified (GM) sheep with TLR4 overexpression were generated. Previous studies have shown that these GM sheep exhibited a higher inflammatory response to Gram-negative bacteria infection than wild type (WT) sheep. In order to evaluate the gene expression of GM sheep and study the co-expressed and downstream genes for TLR4, peripheral blood mononuclear cells (PBMC) from TLR4-overexpressing (Tg) and wild type (WT) sheep were selected to discover the transcriptomic differences using RNA-Seq. Result An average of 18,754 and 19,530 known genes were identified in the Tg and WT libraries, respectively. A total of 338 known genes and 85 novel transcripts were found to be differentially expressed in the two libraries (p

There are three main types of cancer in the female reproductive system, specifically ovarian cancer (OVCA), endometrial cancer (EC), and cervical cancer (CC). They are common malignant tumors in women worldwide, with high morbidity and mortality. In recent years, androgen receptors (ARs) have been found to be closely related to the occurrence, progression, prognosis, and drug resistance of these three types of tumors. This paper summarizes current views on the role of AR in female reproductive system cancer, the associations between female reproductive system cancers and AR expression and polymorphisms. AR regulates the downstream target genes transcriptional activity and the expression via interacting with coactivators/corepressors and upstream/downstream regulators and through the gene transcription mechanism of “classical A/AR signaling” or “non-classical AR signaling”, involving a large number of regulatory factors and signaling pathways. ARs take part in the processes of cancer cell proliferation, migration/invasion, cancer cell stemness, and chemotherapeutic drug resistance. These findings suggest that the AR and related regulators could target the treatment of female reproductive system cancer.

RNA plays an important role in biology, and more than 170 RNA modifications have been identified so far. Post-transcriptional modification of RNA in cells plays a crucial role in the regulation of its stability, transport, processing, and gene expression. So far, the research on RNA modification and the exact role of its enzymes is becoming more and more comprehensive. Human immunodeficiency virus 1 (HIV-1) is an RNA virus and the causative agent of acquired immunodeficiency syndrome (AIDS), which is one of the most devastating viral pandemics in history. More and more studies have shown that HIV has RNA modifications and regulation of its gene expression during infection and replication. This review focuses on several RNA modifications and their regulatory roles as well as the roles that different RNA modifications play during HIV-1 infection, in order to find new approaches for the development of anti-HIV-1 therapeutics.

With a broad range of hosts, Salmonella enterica serovar Typhimurium (S. Typhimurium) is the major cause of gastroenteritis in human beings and systemic disease in susceptible mice strains. However, different S. Typhimurium strains differ in regard to virulence and host adaptation. Here, C57BL/6 mice were infected, respectively, with different S. Typhimurium strains SL1344 (calf), CVCC541 (chicken) and CMCC50115 (mutton) to determine their virulence and host immune responses. It was found that mice were less susceptible to infection by S. Typhimurium CVCC541 and CMCC50115 strains, with lower lethality and decreased bacterial burden in liver and spleen. Besides, S. Typhimurium strains CVCC541 and CMCC50115 enhanced host innate immune responses by increased frequencies of macrophages and neutrophils 3 days after infection. But SL1344 strain evaded immune response by inducing apoptosis of macrophages. Moreover, CVCC541 could elicit adaptive immune responses of host 11 days after infection upon examination of the proliferation and activation of CD4+ T cells. In addition, 125 and 138 unique mutant coding genes, respectively, in S. Typhimurium strains CVCC541 and CMCC50115 and 78 shared mutant coding genes were annotated by genomic alignment to SL1344 genome and the signal pathways involving these genes were further analyzed. The acquired results indicate that different original S. Typhimurium strains show differential virulence and may induce diverse immune responses in the same host infected.

Angiogenesis is involved in physiological and pathological processes in the body. Tumor angiogenesis is a key factor associated with tumor growth, progression, and metastasis. Therefore, there is great interest in developing antiangiogenic strategies. Hypoxia is the basic initiating factor of tumor angiogenesis, which leads to the increase of vascular endothelial growth factor (VEGF), angiopoietin (Ang), hypoxia-inducible factor (HIF-1), etc. in hypoxic cells. The pathways of VEGF and Ang are considered to be critical steps in tumor angiogenesis. A number of antiangiogenic drugs targeting VEGF/VEGFR (VEGF receptor) or ANG/Tie2, or both, are currently being used for cancer treatment, or are still in various stages of clinical development or preclinical evaluation. This article aims to review the mechanisms of angiogenesis and tumor angiogenesis and to focus on new drugs and strategies for the treatment of antiangiogenesis. However, antitumor angiogenic drugs alone may not be sufficient to eradicate tumors. The molecular chaperone heat shock protein 90 (HSP90) is considered a promising molecular target. The VEGFR system and its downstream signaling molecules depend on the function of HSP90. This article also briefly introduces the role of HSP90 in angiogenesis and some HSP90 inhibitors.

Animal cloning is of great importance to the production of transgenic and genome-edited livestock. Especially for multiple gene-editing operations, recloning is one of the most feasible methods for livestock. In addition, a multiple-round cloning method is practically necessary for animal molecular breeding. However, cloning efficiency remains extremely low, especially for serial cloning, which seriously impedes the development of livestock breeding based on genome editing technology. The incomplete reprogramming and failure in oocyte activation of some pluripotent factors were deemed to be the main reason for the low efficiency of animal recloning. Here, to overcome this issue, which occurred frequently in the process of animal recloning, we established a reporter system in which fluorescent proteins were driven by pig OCT4 or SOX2 promoter to monitor the reprogramming process in cloned and recloned pig embryos. We studied the effect of different histone deacetylase (HDAC) inhibitors on incomplete reprogramming. Our results showed that Trichostatin A (TSA) could activate pluripotent factors and significantly enhance the development competence of recloned pig embryos, while the other two inhibitors, valproic acid (VPA) and Scriptaid, had little effect on that. Furthermore, we found no difference in OCT4 mRNA abundance between TSA-treated and untreated embryos. These findings suggest that TSA remarkably improves the reprogramming state of pig recloned embryos by restoring the expression of incompletely activated pluripotent genes OCT4 and SOX2.

Background: Transgenic animal production is an important means of livestock breeding and can be used to model pharmaceutical applications. Methods: In this study, to explore the biological activity of endogenously produced melatonin, Acetylserotonin-O-methyltransferase (ASMT)-overexpressed melatonin-enriched dairy goats were successfully generated through the use of pBC1-ASMT expression vector construction and prokaryotic embryo microinjection. Results: These transgenic goats have the same normal phenotype as the wild-type goats (WT). However, the melatonin levels in their blood and milk were significantly increased (p < 0.05). In addition, the quality of their milk was also improved, showing elevated protein content and a reduced somatic cell number compared to the WT goats. No significant changes were detected in the intestinal microbiota patterns between groups. When the animals were challenged by the intravenous injection of E. coli, the ASMT-overexpressed goats had a lower level of pro-inflammatory cytokines and higher anti-inflammatory cytokines compared to the WT goats. Metabolic analysis uncovered a unique arachidonic acid metabolism pattern in transgenic goats. Conclusions: The increased melatonin production due to ASMT overexpression in the transgenic goats may have contributed to their improved milk quality and enhanced the anti-inflammatory ability compared to the WT goats.

MSTN and FGF5 gene knockout sheep generated by the CRISPR/Cas9 system exhibit the ‘double-muscle’ phenotype, and increased density and length of hairs, providing valuable new breeding material. In a previous study, we obtained MSTN and FGF5 double-knockout sheep of significant breeding value. In this study, we carried out a 90-day feeding study in Wistar rats to assess the safety of genome-edited mutton. Seven rat groups with 10 females and 10 males per group were fed different concentrations (3.75%, 7.5%, and 15%) of double-knockout mutton or wild-type mutton in a conventional commercial diet for 90 days. At the end of the feeding, routine urine and blood tests and measurements of blood biochemical indicators were performed. Furthermore, the major organs of each group of rats were weighed and examined histopathologically. Although there were significant differences among the groups in some parameters, all values were within the normal ranges. Therefore, the 90-day rat feeding study showed that the meat from MSTN and FGF5 double-knockout sheep did not have any long-term adverse effects on rat health. This study also provides valuable reference information for assessing the safety of meat from animals with knockout of multiple genes.

Orf is a widespread contagious epithelial viral disease found particularly in most sheep breeding countries in the world. Recently, an orf virus (ORFV) strain OV-HLJ05 was isolated from an outbreak in northeast China. Three genes of interest including ORFV011 (B2L), ORFV059 (F1L), and ORFV132 (VEGF) of ORFV, were recruited to identify and genetically characterize this newly isolated virus. Amino acid (aa) sequence compared with the ORFV references listed in GenBank, both B2L and F1L of OV-HLJ05 showed less microheterogeneity from their references. In contrast, the VEGF gene was included in the NZ7-VEGF like group as previously considered by Mercer in 2002. Unexpectedly, further multiple VEGF matches were made, using 34 published sequences from China and India, resulting in 27 strains of the NZ7 members. Based on Karki's report in 2020, NZ7-VEGF like viruses are emerging more and more frequently in these two countries, damaging the Asian sheep industry. Obvious heterogeneity with the NZ2, insertion of two oligopeptides TATI(L)QVVVAI(L) and SSSS(S) motif were found in the NZ7-like VEGF protein. These VEGFs are divided mainly into two types and a significant increase in the number of hydrogen bonds within the NZ7-like VEGF dimers was observed. The NZ7-like ORFV apparently favors the goat as a host and an emphasis on this in future epidemiological and pathological studies should be considered, focusing on the NZ7-like virus.

Lipid metabolic disorders have become a major global public health concern. Fatty liver and dyslipidemia are major manifestations of these disorders. Recently, MicroRNA-33 (miR-33), a post-transcriptional regulator of genes involved in cholesterol efflux and fatty acid oxidation, has been considered as a good therapeutic target for these disorders. However, the traditional methods of gene therapy impede their further clinical transformation into a mature treatment system. To counter this problem, in this study we used mesoporous silica nanoparticles (MSNs) as nanocarriers to deliver miR-33 antagomirs developing nanocomposites miR-MSNs. We observed that the hepatocellular uptake of miR-33 antagomirs increased by ∼5 times when they were delivered using miR-MSNs. The regulation effects of miR-MSNs on miR-33 and several genes involved in lipid metabolism were confirmed in L02 cells. In a high-fat diet fed mice, miR-33 intervention via miR-MSNs lowered the serum triglyceride levels remarkably by 18.9% and reduced hepatic steatosis. Thus, our results provide a proof-of-concept for a potential strategy to ameliorate lipid metabolic disorders.

Toll-like receptor 4 (TLR4) is a critical pattern recognition receptor that plays a critical role in the host innate immune system’s recognition of Gram-negative bacteria. Since it is the lipopolysaccharide (LPS) receptor, it links the activated inflammatory response with autophagy and oxidative stress. Autophagy, or type II programmed cell death, was reported to have defensive functions in response to the production of inflammatory cytokines and oxidative stress. To explore the relationship between autophagy, inflammation, and oxidative stress, a TLR4-enriched transgenic (Tg) animal model (sheep) was generated. Autophagy activity in the Tg blood monocytes was significantly higher than in the wild-type animal under LPS stress, and it returned to normal after transfection of TLR4 siRNA. Pretreatment with 3-methyladenine (3-MA) inhibited autophagy and enhanced oxidative stress and the production of TNF-α. The LPS-induced reactive oxygen species (ROS) level was markedly increased in the Tg group at an early stage before quickly returning to normal values. In addition, suppressing ROS production by N-acetyl-L-cysteine down-regulated the number of intracellular autophagosomes and the expression of Beclin-1, ATG5, and cytokines IL-1β, IL-6, and TNF-α. Further mechanistic investigation suggested that the TLR4-associated p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in autophagy and oxidative stress. P38 MAPK promotes intracellular autophagy, ROS production, and inflammatory response. Moreover, TLR4 over-expression suppressed oxidative stress and the production of inflammatory cytokines and increased autophagy activity in vivo. Taken together, our results showed that LPS induced autophagy, which was related to TLR4-mediated ROS production through the p38 MAPK signaling pathway. In addition, our study also provided a novel transgenic animal model to analyze the effects of TLR4 on autophagy, oxidative stress, and inflammatory responses.

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.

Abstract Background Interferon-γ (IFN-γ) is critical for innate and adaptive immunity against viral and bacterial infections. IFN-γ reportedly affects the phagocytic ability of monocytes and macrophages as well as regulates pituitary function in humans and mice. The present study analyzed the impact of IFN-γ on monocyte and macrophage phagocytosis, production performance, and pituitary function in vivo and in vitro (in dwarf chickens). IFN-γ was injected into dwarf chickens through a vein, and then, the laying rate, average egg weight, and levels of follicle-stimulating hormone (FSH) and IFN-γ were measured in treatment and control groups. For the in vitro experiment, the pituitary tissues were supplemented with IFN-γ, and the mRNA expression levels of follicle-stimulating hormone beta subunit (FSH-β), interferon gamma receptor 1 (IFNGR1), and interferon gamma receptor 2 (IFNGR2) in the pituitary were assessed. Results Monocyte and macrophage phagocytosis product (PP) was decreased by IFN-γ treatment in a dose-dependent manner in vitro. In the in vivo experiment, the level of IFN-γ in the treatment group was higher than that in the control group at 7 d (P

It has been well proved that melatonin participates in the regulation of the seasonal reproduction of ewes. However, the effects of short term treatment of melatonin on ewe’s ovulation are still to be clarified. In this study, the effects of melatonin on the number of embryos harvested from superovulation, and the pregnant rate in recipients after embryo transferred have been investigated. Hu sheep with synchronous estrus treatment were given melatonin subcutaneously injection (0, 5, and 10 mg/ewe, respectively). It was found that the estrogen level in the group of 5 mg melatonin was significantly higher than that of other two groups at the time of sperm insemination (p

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.

Background The open pulled straw (OPS) vitrification method has been successfully applied in mouse, pig, and goat embryos as well as in buffalo oocytes, but it has not yet been applied to the microinjected embryos. This study examined the effects of OPS vitrification on embryo development and the reproductive capacity of the transgenic offspring in order to establish a method for preservation of microinjected embryos. Methods Ovine pronuclear embryos were microinjected with the exogenous aralkylamine N-acetyltransferase gene (AANAT), frozen by the OPS method, and subsequently thawed for embryo transplantation. Pregnancy rate, lambing rate, survival rate, average birth weight and transgenic positive rate as well as reproduction efficiency and hormone level of the transgenic offspring were investigated to analyze the effect of OPS vitrification on microinjectd pronuclear embryos. Results No significant differences were observed in the birth rate, lamb survival rate and transgenic positive rate between the frozen and non-frozen AANAT-microinjected pronuclear embryos. The average birth weight of the frozen embryos offspring was greater than that of the non-frozen embryos. Importantly, the transgenic offspring that overexpressed the AANAT gene showed improved ovulation efficiency and lambing rate by regulating their hormone levels. Conclusions The OPS vitrification approach may be a valuable method in microinjected- embryo transfer technology, which could reserve embryos and result in fewer unnecessary animal sacrifices. In addition, the AANAT+ transgenic offspring exhibited improved reproductive capacity on account of regulation effect of melatonin on reproductive hormone. These data may provide available references for human-assisted reproduction.

Salmonella enterica serovar Gallinarum biovar Pullorum ( S. Pullorum ) is responsible for the systemic salmonellosis in different breeds of chickens. Macrophages, as host cells, play a key role in the innate immune response following infection with S. Pullorum . In this study, we first generated macrophages from two breeds of chicken (White Leghorn (WL) and Tibetan Chickens (TC)) peripheral blood monocytes in vitro. Then, we showed that the production of interleukin-1β (IL-1β), macrophage inflammatory protein-1β (MIP-1β) and interleukin-10 (IL-10) in lipopolysaccharide (LPS)-treated macrophages was significantly higher compared with the unstimulated cells in TC. LPS triggered only more expression of IL-10 in WL macrophages. Furthermore, macrophages from TC eliminated intracellular bacteria more efficiently than those from WL after S. Pullorum infection at a multiplicity of infection (MOI) 1. In addition, the variation between individuals and sex had the crucial effect on the immune response to LPS and S. Pullorum invasion.

Chicken embryonic stem cells (cESCs) obtained from stage X embryos provide a novel model for the study of avian embryonic development. A new way to maintain cESCs for a long period in vitro still remains unexplored. We found that the cESCs showed stem cell-like properties in vitro for a long term with the support of DF-1 feeder and basic culture medium supplemented with human basic fibroblast growth factor (hbFGF), mouse stem cell factor (mSCF), and human leukemia inhibitory factor (hLIF). During the long culture period, the cESCs showed typical ES cell morphology and expressed primitive stem cell markers with a relatively stable proliferation rate and high telomerase activity. These cells also exhibited the capability to differentiate into cardiac myocytes, smooth muscle cells, neural cells, osteoblast, and adipocyte in vitro. Chimera chickens were produced by cESCs cultured for 25 passages with this new culture system. The experiments showed that DF-1 was the optimal feeder and hbFGF was an important factor for maintaining the pluripotency of cESCs in vitro.

Background: In normal hearts, capillaries are densely distributed throughout the myocardial tissue, and the cross-talk between myocytes and capillary endothelial cells plays a pivotal role in regulating cardiac development, maturation and function. Although previous studies have suggested a role for the endothelium in the organisation of nearby cardiomyocytes, the underlying mechanism has yet to be illustrated. Methods and Results: Using a transwell coculture system, we studied the paracrine effect of endothelial cells on cardiomyocytes and found that the regulation of cardiomyocyte spatial reorganisation and cytoskeletal dynamics by endothelial cells was coupled with β1-integrin induction. To determine the role of β1-integrin in this process, we preincubated myocytes with a β1-integrin function-blocking antibody before coculture. β1-integrin blockage abolished myocyte chemotactic activity and inhibited microtubule extension and stress fibre assembly. We further evaluated the therapeutic potential of combined endothelial cell-cardiac myocyte transplantation against ischemic cardiomyopathy in an acute myocardial infarction (AMI) mouse model. The results showed that myocytes and endothelial cells synergistically promoted ischemic myocardial repair, as evidenced by the robust engraftment and migration of implanted cells within the infarcted area, as well as the stimulation of angiogenesis, the attenuation of scar tissue and the improvement of cardiac function. Conclusion: Our study demonstrated the necessity of β1-integrin in the interactions between cardiomyocytes and endothelial cells and presented a novel combined transplantation approach that might hold promise for treating ischemic cardiomyopathy.

Skeletal muscle myogenesis and injury-induced muscle regeneration contribute to muscle formation and maintenance. As myogenic stem cells, skeletal muscle satellite cells have the ability to proliferate, differentiate and self-renew, and are involved in muscle formation and muscle injury repair. Accumulating evidence suggests that non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), are widely involved in the regulation of gene expression during skeletal muscle myogenesis, and their abnormal expression is associated with a variety of muscle diseases. From the perspective of the molecular mechanism and mode of action of ncRNAs in myogenesis, this review aims to summarize the role of ncRNAs in skeletal muscle satellite cells’ myogenic differentiation and in muscle disease, and systematically analyze the mechanism of ncRNAs in skeletal muscle development. This work will systematically summarize the role of ncRNAs in myogenesis and provide reference targets for the treatment of various muscle diseases, such as muscle dystrophy, atrophy and aberrant hypertrophy.

Genetic modification offers alternative strategies to traditional animal breeding. However, the food safety of genetically modified (GM) animals has attracted increasing levels of concern. In this study, we produced GM sheep overexpressing TLR4, and the transgene-positive offsprings (F1) were confirmed using the polymerase chain reaction (PCR) and Southern blot. The expression of TLR4 was 2.5-fold compared with that of the wild-type (WT) sheep samples. During the 90-day safety study, Sprague-Dawley rats were fed with three different dietary concentrations (3.75%, 7.5%, and 15% wt/wt) of GM sheep meat, WT sheep meat or a commercial diet (CD). Blood samples from the rats were collected and analyzed for hematological and biochemical parameters, and then compared with hematological and biochemical reference ranges. Despite a few significant differences among the three groups in some parameters, all other values remained within the normal reference intervals and thus were not considered to be affected by the treatment. No adverse diet-related differences in body weights or relative organ weights were observed. Furthermore, no differences were observed in the gross necropsy findings or microscopic pathology of the rats whose diets contained the GM sheep meat compared with rats whose diets contained the WT sheep meat. Therefore, the present 90-day rat feeding study suggested that the meat of GM sheep overexpressing TLR4 had no adverse effect on Sprague-Dawley rats in comparison with WT sheep meat. These results provide valuable information regarding the safety assessment of meat derived from GM animals.

BACKGROUND: Many groups of Gram-negative bacteria cause diseases harmful to sheep. TLR4 is an important Toll-like receptor (TLR) which responds to common Gram-negative bacterial infections. Activation of TLR4 leads to the induction of inflammatory responses, which is a linkage between the innate and adaptive immune systems. A vector pTLR4-3S was constructed to overexpress TLR4 gene in sheep. In this study, effects of TLR4 overexpression on inflammation response under LPS stimulated were addressed in vivo and in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Sheep fetal fibroblasts were transfected with expression vector pTLR4-3S. Transgenic sheep were produced by microinjection of the constructed plasmids into fertilized eggs. Fetal fibroblasts, monocyte-macrophage and fibroblasts isolated from the transgenic sheep were stimulated by LPS. After that immunoactive factors (TNF-α, IL-10, IL-6, IL-8, IFN-γ), nitric oxide, phagocytize ability and adhesion were detected. Furthermore, transgenic sheep were intradermal injected of LPS in ear and observed pathological changes by HE strain. Overexpression of TLR4 gene was observed on transgenic cells and individuals. In vitro, TLR4 overexpression transgenic cells secreted Th1 and Th2 inducing cytokines with a strong LPS mediated inflammation response and promoting the secretion of nitric oxide, and then recovered to initial level. The phagocytosis index of monocyte/macrophage in transgenic sheep was higher than that of non-transgenic sheep (P