Increasing GSH-Px Activity and Activating Wnt Pathway Promote Fine Wool Growth in FGF5-Edited Sheep.

Fibroblast growth factor 5 (FGF5) plays key roles in promoting the transition from the anagen to catagen during the hair follicle cycle. The sheep serves as an excellent model for studying hair growth and is frequently utilized in various research processes related to human skin diseases. We used the CRISPR/Cas9 system to generate four FGF5-edited Dorper sheep and only low levels of FGF5 were detected in the edited sheep. The density of fine wool in GE sheep was markedly increased, and the proportion of fine wool with a diameter of 14.4-20.0 µm was significantly higher. The proliferation signal in the skin of gene-edited (GE) sheep was stronger than in wild-type (WT) sheep. FGF5 editing decreased cortisol concentration in the skin, further activated the activity of antioxidant enzymes such as Glutathione peroxidase (GSH-Px), and regulated the expression of Wnt signaling pathways containing Wnt agonists (Rspondins, Rspos) and antagonists (Notum) in hair regeneration. We suggest that FGF5 not only mediates the activation of antioxidant pathways by cortisol, which constitutes a highly coordinated microenvironment in hair follicle cells, but also influences key signals of the Wnt pathway to regulate secondary hair follicle (SHF) development. Overall, our findings here demonstrate that FGF5 plays a significant role in regulating SHF growth in sheep and potentially serves as a molecular marker of fine wool growth in sheep breeding.

Improving the Efficiency of Precise Genome Editing with CRISPR/Cas9 to Generate Goats Overexpressing Human Butyrylcholinesterase.

The CRISPR/Cas9 system is widely used for genome editing in livestock production, although off-target effects can occur. It is the main method to produce genome-edited goats by somatic cell nuclear transfer (SCNT) of CRISPR/Cas9-mediated genome-edited primary goat fetal fibroblast cells (GFFs). Improving the double-strand break (DSB) efficiency of Cas9 in primary cells would improve the homologous repair (HR) efficiency. The low efficiency of HR remains a major hurdle in CRISPR/Cas9-mediated precise genome editing, increasing the work required to screen the genome-edited primary cell clones. In this study, we modified several essential parameters that affect the efficiency of the CRISPR/Cas9-mediated knock-in GFF cloning system, including establishing a high-efficiency transfection system for primary cells via nucleofection and optimizing homology arm (HA) length during HR. Here, we specifically inserted a recombinant human butyrylcholinesterase gene (rhBChE) into the goat fibroblast growth factor (FGF)-5 locus through the CRISPR/Cas9 system, thereby achieving simultaneous rhBChE insertion and FGF5 knock-out. First, this study introduced the Cas9, FGF5 knock-out small guide RNA, and rhBChE knock-in donors into GFFs by electroporation and obtained positive cell clones without off-target effects. Then, we demonstrated the expression of rhBChE in GFF clones and verified its function. Finally, we obtained a CRISPR/Cas9-mediated rhBChE-overexpression goat.

Shifts in intestinal microbiota and improvement of sheep immune response to resist Salmonella infection using Toll-like receptor 4 (TLR4) overexpression.

Introduction: Toll-like receptor 4 (TLR4) identifies Gram-negative bacteria or their products and plays a crucial role in host defense against invading pathogens. In the intestine, TLR4 recognizes bacterial ligands and interacts with the immune system. Although TLR4 signaling is a vital component of the innate immune system, the influence of TLR4 overexpression on innate immune response and its impact on the composition of the intestinal microbiota is unknown. Methods: Here, we obtained macrophages from sheep peripheral blood to examine phagocytosis and clearance of Salmonella Typhimurium (S. Typhimurium) in macrophages. Meanwhile, we characterized the complex microbiota inhabiting the stools of TLR4 transgenic (TG) sheep and wild-type (WT) sheep using 16S ribosomal RNA (rRNA) deep sequencing. Results: The results showed that TLR4 overexpression promoted the secretion of more early cytokines by activating downstream signaling pathways after stimulation by S. Typhimurium. Furthermore, diversity analysis demonstrated TLR4 overexpression increased microbial community diversity and regulated the composition of intestinal microbiota. More importantly, TLR4 overexpression adjusted the gut microbiota composition and maintained intestinal health by reducing the ratio of Firmicutes/Bacteroidetes and inflammation and oxidative stress-producing bacteria (Ruminococcaceae, Christensenellaceae) and upregulating the abundance of Bacteroidetes population and short-chain fatty acid (SCFA)-producing bacteria, including Prevotellaceae. These dominant bacterial genera changed by TLR4 overexpression revealed a close correlation with the metabolic pathways of TG sheep. Discussion: Taken together, our findings suggested that TLR4 overexpression can counteract S. Typhimurium invasion as well as resist intestinal inflammation in sheep by regulating intestinal microbiota composition and enhancing anti-inflammatory metabolites.

Processing, mechanical properties and bio-applications of silk fibroin-based high-strength hydrogels.

Hydrogels are an attractive class of materials that possess similar structural and functional characteristics to wet biological tissues and demonstrate a diversity of applications in biomedical engineering. Silk fibroin (SF) is a unique natural polymer due to its fibrous protein nature, versatile formats, biocompatibility, tunable biodegradation and is thus a good hydrogel candidate for bio-applications. Compared to synthetic polymer hydrogels, poor mechanical performance is still a fatal drawback that hinders the application of SF hydrogels as structural materials. Researchers have attempted to develop strategies to construct silk fibroin-based high-strength hydrogels (SF-HSHs). Herein, we firstly provide an overview of the approaches of processing SF-HSHs with a focus on the physical/non-covalent crosslinking mechanisms. The examples of SF-HSHs are discussed in detail under four categories, including physical-crosslinked, dual-crosslinked, double network and composite hydrogels respectively. A brief section follows to elucidate on the gelation mechanisms of SF-HSHs before a description of the utility of SF-HSHs in biomedicine and devices is presented. Finally, the potential challenges and future development of SF-HSHs are briefly discussed. This review aims to enhance our understanding of the structure-mechanical property-function relationships of soft materials made from natural polymers and guide future research of silk fibroin-based hydrogels for biomedical applications. STATEMENT OF SIGNIFICANCE: Silk fibroin (SF) extracted from silk fibres is increasingly applied in the biomedical field, and SF hydrogel has been an emerging area for frontier bio-research. Since SF biopolymer has an intrinsic tendency to form regular ß-sheet stacks, it can be processed into purely physically crosslinked hydrogels, thus avoiding the use of chemical crosslinkers. Nevertheless, akin to other natural polymers, lab-produced SF is variable (i.e. the molecular weight and distribution), and the gelation of SF hydrogel is challenging to control. In addition, hydrogels made from SF are usually weak and brittle, which hinders the wide use of this biofriendly and biodegradable hydrogel. Recently, there is a pressing need for high strength hydrogels from natural polymers for biomedical applications, and SF is proposed as a strong candidate. Therefore, we have studied the literature in the past 10 years and would like to focus on the gelation mechanism and mechanical strength of SF hydrogels for the review.

Highly Stretchable and Tough Physical Silk Fibroin-Based Double Network Hydrogels.

Regenerated silk fibroin (RSF) is a promising biomedical material, but the poor mechanical properties of RSF hydrogels may hinder the use as structural components. Herein, an equilibrium RSF hydrogel is prepared and optimized based on the double network (DN) concept. After sufficient soaking in water and removal of small molecules, the equilibrium RSF DN hydrogels prove stable in water, strong, highly extensible, and tough with 0.26-0.44 MPa tensile strength, 500-900% elongation, and 2 MJ m-3 work of extension. The combination of high strength and extensibility is attributed to the homogeneous morphology and the hydrophobic interactions and hydrogen bonding between the two networks. The strategy in this work overcomes the previous issue of swelling and eventual fracture of as-prepared RSF/SDS DN hydrogels in water. In addition, such mechanically superior RSF DN hydrogels also display low cytotoxicity. It concludes that the elastic and tough RSF DN hydrogels could be engineered by introducing widely used polymer networks, and the hydrogels from inexpensive, environmentally friendly, and biocompatible silk fibroin may hold great potential in biomedical applications.

Replacement of H1 linker histone during bovine somatic cell nuclear transfer.

Linker histone variants are involved in regulation of chromosome organization and gene transcription; several subtypes are expressed in the maturing oocyte and developing embryo. In Xenopus and mice, the transition between linker histone variants occurred following nuclear transfer, and apparently contributed to donor nuclear reprogramming. To determine whether such linker histone replacement occurred after bovine nuclear transfer, red fluorescent protein (RFP) tagged H1e (somatic linker histone H1e) donor cells and Venus tagged H1foo eggs were created, enucleated eggs were injected with donor cells, and embryos were created by fusion. Using fluorescence microscopy, release of H1e in the donor nucleus, acquisition of H1foo by donor chromosomes, and the H1foo-to-H1e transition were observed in live cells. Linker histone replacement occurred more slowly in bovine than murine embryos. Low levels of diffuse red fluorescence (H1e) in the donor nucleus were detected 5 h after fusion, at which time green fluorescence (H1foo) had incorporated into donor chromosomes. However, complete replacement did not occur until 8 h after fusion. We concluded that the linker histone transition was sufficiently conserved among species, which provided further evidence regarding its important role in nuclear reprogramming.