FokI-RYdCas9 Mediates Nearly PAM-Less and High-Precise Gene Editing in Human Cells.

The demand for high-precision CRISPR/Cas9 systems in biomedicine is experiencing a notable upsurge. The editing system fdCas9 employs a dual-sgRNA strategy to enhance editing accuracy. However, the application of fdCas9 is constrained by the stringent requirement for two protospacer adjacent motifs (PAMs) of Cas9. Here, we devised an optimized editor, fRYdCas9, by merging FokI with the nearly PAM-less RYdCas9 variant, and two fRYdCas9 systems formed a dimer in a proper spacer length to accomplish DNA cleavage. In comparison to fdCas9, fRYdCas9 demonstrates a substantial increase in the number of editable genomic sites, approximately 330-fold, while maintaining a comparable level of editing efficiency. Through meticulous experimental validation, we determined that the optimal spacer length between two FokI guided by RYdCas9 is 16 base pairs. Moreover, fRYdCas9 exhibits a near PAM-less feature, along with no on-target motif preference via the library screening. Meanwhile, fRYdCas9 effectively addresses the potential risks of off-targets, as analyzed through whole genome sequencing (WGS). Mouse embryonic editing shows fRYdCas9 has robust editing capabilities. This study introduces a potentially beneficial alternative for accurate gene editing in therapeutic applications and fundamental research.

CRISPR Screen Identifies the RNA-Binding Protein Eef1a1 as a Key Regulator of Myogenesis.

Skeletal muscle myogenesis hinges on gene regulation, meticulously orchestrated by molecular mechanisms. While the roles of transcription factors and non-coding RNAs in myogenesis are widely known, the contribution of RNA-binding proteins (RBPs) has remained unclear until now. Therefore, to investigate the functions of post-transcriptional regulators in myogenesis and uncover new functional RBPs regulating myogenesis, we employed CRISPR high-throughput RBP-KO (RBP-wide knockout) library screening. Through this approach, we successfully identified Eef1a1 as a novel regulatory factor in myogenesis. Using CRISPR knockout (CRISPRko) and CRISPR interference (CRISPRi) technologies, we successfully established cellular models for both CRISPRko and CRISPRi. Our findings demonstrated that Eef1a1 plays a crucial role in promoting proliferation in C2C12 myoblasts. Through siRNA inhibition and overexpression methods, we further elucidated the involvement of Eef1a1 in promoting proliferation and suppressing differentiation processes. RIP (RNA immunoprecipitation), miRNA pull-down, and Dual-luciferase reporter assays confirmed that miR-133a-3p targets Eef1a1. Co-transfection experiments indicated that miR-133a-3p can rescue the effect of Eef1a1 on C2C12 myoblasts. In summary, our study utilized CRISPR library high-throughput screening to unveil a novel RBP, Eef1a1, involved in regulating myogenesis. Eef1a1 promotes the proliferation of myoblasts while inhibiting the differentiation process. Additionally, it acts as an antagonist to miR-133a-3p, thus modulating the process of myogenesis.

PHF7 is a novel histone H2A E3 ligase prior to histone-to-protamine exchange during spermiogenesis.

Epigenetic regulation, including histone-to-protamine exchanges, controls spermiogenesis. However, the underlying mechanisms of this regulation are largely unknown. Here, we report that PHF7, a testis-specific PHD and RING finger domain-containing protein, is essential for histone-to-protamine exchange in mice. PHF7 is specifically expressed during spermiogenesis. PHF7 deletion results in male infertility due to aberrant histone retention and impaired protamine replacement in elongated spermatids. Mechanistically, PHF7 can simultaneously bind histone H2A and H3; its PHD domain, a histone code reader, can specifically bind H3K4me3/me2, and its RING domain, a histone writer, can ubiquitylate H2A. Thus, our study reveals that PHF7 is a novel E3 ligase that can specifically ubiquitylate H2A through binding H3K4me3/me2 prior to histone-to-protamine exchange.

Transcriptome analysis revealed differences in the microenvironment of spermatogonial stem cells in seminiferous tubules between pre-pubertal and adult buffaloes.

The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs.

Maturation of buffalo oocytes in vitro with acetyl-L-carnitine improves cryotolerance due to changes in mitochondrial function and the membrane lipid profile.

The effects of acetyl-l-carnitine (ALC) supplementation during IVM on subsequently vitrified buffalo oocytes were evaluated, followed by determination of the mitochondrial DNA copy number, measurement of mitochondrial membrane potential (MMP) and identification of the lipid profile of oocyte membranes as markers of oocyte quality after vitrification. Supplementation with ALC during IVM significantly improved the rates of oocyte cleavage and morula and blastocyst formation, and increased MMP after vitrification compared with unsupplemented vitrified oocytes (P<0.05). Using a bidirectional orthogonal projection to latent structures discriminant analysis based on positive ion matrix-assisted laser desorption ionisation time-of-flight mass spectrometry data, five phospholipid ions (m/z 728.7 (phosphatidylcholine (PC) 32:3), 746.9 (PC 32:5), 760.6 (PC 34:1), 768.8 (PC P-36:3) and 782.6 (PC 36:4); P<0.05) were identified as significantly more abundant in fresh oocytes than in unsupplemented vitrified oocytes. Meanwhile, three phospholipid ions (m/z 734.6 (PC 32:0), 760.6 (PC 34:1), and 782.6 (PC 36:4); P<0.05) were more abundant in ALC-supplemented vitrified oocytes than in unsupplemented vitrified oocytes. Therefore, supplementation with ALC during IVM may improve buffalo oocyte quality after vitrification by enhancing mitochondrial function and altering the phospholipid composition of vitrified oocyte membranes.

The effects of growth factors on proliferation of spermatogonial stem cells from Guangxi Bama mini-pig.

The objective of this study was to investigate the effects of different growth factors on the proliferation of Bama mini-pig spermatogonial stem cells (SSCs) in vitro. The growth factors glial cell line-derived neurotrophic factor (GDNF), leukaemia inhibitory factor (LIF), GDNF family receptor alpha-1 (GFRα1) and basic fibroblast growth factor (bFGF) were investigated. The SSCs were seeded on SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder layers. Cultivation of the cells were subjected to a factorial design of the growth factors GDNF + bFGF, GDNF + bFGF + GFRα1, LIF + bFGF and LIF + bFGF + GFRα1. The SSCs could propagate for 25 passages in the medium adding GDNF + bFGF + GFRα1, 22 passages in the medium adding GDNF + bFGF, 6 passages in the medium adding LIF + bFGF, or LIF + bFGF + GFRα1. qRT-PCR analysis showed that the highest mRNA expression levels of NANOG, POU5F, DDX4, GFRα1 and UCHL1 were detected in the group adding GDNF + bFGF + GFRα1. The SSCs from the group adding GDNF + bFGF + GFRα1 also showed UCHL1-, DBA- and CDH1-positive staining. Moreover, Stra8 and Scp3 expression, and haploid peak were detected after induction of the SSCs from the group adding GDNF + bFGF + GFRα1. In conclusion, pig SSCs could be maintained for long term in the presence of GDNF, bFGF, and GFRα1.

ZPAC is required for normal spermatogenesis in mice.

The ubiquitin-proteasome pathway, involved in genetic recombination and sex-chromosome silencing during meiosis, plays critical roles in the specification of germ-line stem cells and the differentiation of gametes from gonocytes. Zygote-specific proteasome assembly chaperone (ZPAC) is expressed in the early mouse embryo, where it is important for progression of the mouse maternal-to-zygotic transition. The role of ZPAC during spermatogenesis in the adult gonads, however, remains unknown. In this study, rapid amplification of cDNA ends was used to determine the Zpac cDNA sequence, a 1584-bp transcript that includes a putative 1122-bp open reading frame coding for a 373 amino acid protein. Western blot and immunohistochemistry revealed that ZPAC was specifically expressed in gonads. To further dissect the function of ZPAC during spermatogenesis, we employed PiggyBac-based RNA interference vectors for transgenesis combined with cell transplantation to deplete Zpac during spermatogenesis. This RNAi-mediate depletion in Zpac expression disrupted normal spermatogenesis from spermatogonial stem cells. Two independent yeast two-hybrid screens further revealed an interaction between ZPAC and SYCE1. Together, these data suggest that ZPAC is required for normal spermatogenesis in mice.

[Expression analysis of green fluorescent protein in tissues and organs in α-1,3 galactosyltransferase knockout pigs].

The pig is an ideal source to provide organs because its organ size and physiology are similar to humans. However, an acute rejection will ensue after pig-to-human xenotransplantation. The α-1,3 galactosyltransferase gene knockout (GTKO) pigs were generated in recent years, and could solve the problem of hyperacute rejection. But due to lack of reporting genes, the rejection status of cells and organs post pig-to-human xenotransplantation cannot be visualized. In this study, we introduced the enhanced green fluorescent protein (EGFP) gene driven by the CAG promoter into GTKO porcine ear fibroblasts. Then we produced transgenic pigs expressing the EGFP gene by nuclear transfer technology. Expression levels of EGFP in different tissues and organs of the cloned pig were investigated by Nightsea DFP-1 Fluorescent Protein Flashlight, fluorescence microscope and quantitative PCR assays. The results showed that the protein and transcript of EGFP were expressed in all tissues and organs of the GTKO pig, but the expression was weak in the liver and central nervous system. In conclusion, we have successfully produced the transgenic GTKO pigs expressing EGFP in all tested tissues and organs, which builds up a good basis to track transplanted cells or tissues.

A comprehensive atlas of pig RNA editome across 23 tissues reveals RNA editing affecting interaction mRNA-miRNAs.

RNA editing is a co-transcriptional/post-transcriptional modification that is mediated by the ADAR enzyme family. Profiling of RNA editing is very limited in pigs. In this study, we collated 3813 RNA-seq data from the public repositories across 23 tissues and carried out comprehensive profiling of RNA editing in pigs. In total, 127,927 A-to-I RNA editing sites were detected. Our analysis showed that 98.2% of RNA editing sites were located within repeat regions, primarily within the pig-specific SINE retrotransposon PRE-1/Pre0_SS elements. Subsequently, we focused on analyzing specific editing sites (SESs) in skeletal muscle tissues. Functional enrichment analyses suggested that they were enriched in signaling pathways associated with muscle cell differentiation, including DMD, MYOD1 and CAV1 genes. Furthermore, we discovered that RNA editing event in the 3`UTR of CFLAR mRNA influenced miR-708-5p binding in this region. In this study, the panoramic RNA editing landscape of different tissues of pigs was systematically mapped, and RNA editing sites and genes involved in muscle cell differentiation were identified. In summary, we identified modifications to pig RNA editing sites and provided candidate targets for further validation.

Exploring Multi-Tissue Alternative Splicing and Skeletal Muscle Metabolism Regulation in Obese- and Lean-Type Pigs.

Alternative splicing (AS) is a crucial mechanism in post-transcriptional regulation, contributing significantly to the diversity of the transcriptome and proteome. In this study, we performed a comprehensive AS profile in nine tissues obtained from Duroc (lean-type) and Luchuan (obese-type) pigs. Notably, 94,990 AS events from 14,393 genes were identified. Among these AS events, it was observed that 80% belonged to the skipped exon (SE) type. Functional enrichment analysis showed that genes with more than ten AS events were closely associated with tissue-specific functions. Additionally, the analysis of overlap between differentially alternative splicing genes (DSGs) and differentially expressed genes (DEGs) revealed the highest number of overlapped genes in the heart and skeletal muscle. The novelty of our study is that it identified and validated three genes (PYGM, MAPK11 and CAMK2B) in the glucagon signaling pathway, and their alternative splicing differences were highly significant across two pig breeds. In conclusion, our study offers novel insights into the molecular regulation of diverse tissue physiologies and the phenotypic differences between obese- and lean-type pigs, which are helpful for pig breeding.

Mogroside V alleviates the heat stress-induced disruption of the porcine oocyte in vitro maturation.

Heat stress (HS) is a stressor that negatively affect female reproduction. Specially, oocytes are very sensitive to HS. It has been demonstrated that some active compounds can protect oocyte from HS. We previously found that Mogroside V (MV), extracted from Siraitia grosvenorii (Luo Han Guo), can protect oocyte from many kinds of stresses. However, how MV alleviates HS-induced disruption of oocyte maturation remains unknown. In this study, we treated the HS-induced porcine oocytes with MV to examine their maturation and quality. Our findings demonstrate that MV can effectively alleviate HS-induced porcine oocyte abnormal cumulus cell expansion, decrease of first polar body extrusion rate, spindle assembly and chromosome separation abnormalities, indicating MV attenuates oocyte mature defects. We further observed that MV can effectively alleviate HS-induced cortical granule distribution abnormality and decrease of blastocyst formation rate after parthenogenesis activation. In addition, MV treatment reversed mitochondrial dysfunction and lipid droplet content decrease, reduced reactive oxygen species levels, early apoptosis and DNA damage in porcine oocytes after HS. Collectively, this study suggests that MV can effectively protect porcine oocytes from HS.

Methylation characteristics and developmental potential of Guangxi Bama minipig (Sus scrofa domestica) cloned embryos from donor cells treated with trichostatin A and 5-aza-2'-deoxycytidine.

Summary Reprogramming of DNA methylation in somatic cell nuclear transfer (SCNT) embryos is incomplete, and aberrant DNA methylation patterns are related to the inefficiency of SCNT. To facilitate nuclear reprogramming, this study investigated the effect of treating Guangxi Bama minipig donor cells with trichostatin A (TSA), 5-aza-2'-deoxycytine (5-aza-dC), or combination of TSA and 5-aza-dC prior to nuclear transfer. Analyses showed that there were no major changes in cell-cycle status among all groups. We monitored the transcription of DNMT1, DNMT3a, HDAC1 and IGF2 genes in donor cells. Transcription levels of HDAC1 were decreased significantly after treatment with a combination of TSA and 5-aza-dC, along with a significantly increased level of IGF2 (P < 0.05). Although treatment of donor cells with either TSA or 5-aza-dC alone resulted in non-significant effects in blastocyst formation rate and DNA methylation levels, a combination of TSA and 5-aza-dC significantly improved the development rates of minipig SCNT embryos to blastocyst (25.6% vs. 16.0%, P < 0.05). This change was accompanied by decreased levels of DNA methylation in somatic cells and blastocyst (P < 0.05). Thus in combination with TSA, lower concentrations of 5-aza-dC may produce a potent demethylating activity, and lead to the significantly enhanced blastocyst development percentage of Bama minipig SCNT embryos.

Mogroside-rich extract from Siraitia grosvenorii fruits protects against heat stress-induced intestinal damage by ameliorating oxidative stress and inflammation in mice.

Global warming makes humans and animals more vulnerable to heat stress. Heat stress can cause multiorgan dysfunction, especially in the intestine, primarily via oxidative stress and inflammation. Mogroside-rich extract (MGE) is the active ingredient of Siraitia grosvenorii and has significant antioxidant and anti-inflammatory activity. However, whether MGE can alleviate the intestinal damage caused by heat stress has not been explored. In this study, mice were given 600 mg kg-1 MGE followed by exposure to high temperature (40 °C for 2 h per day), and the structures and molecular changes in the ileum were examined. Our findings showed that body weight was decreased by heat stress, while the activity of serum superoxide dismutase (SOD) was increased. We further found that heat stress impaired the intestinal barrier by reducing the number of goblet cells and mRNA levels of the tight junction proteins zona occludens protein 1 (ZO-1), Occludin (OCLD) and recombinant mucin 2 (MUC2 mucin), but it increased the mRNA level of trefoil factor 3 (TFF3). Interestingly, MGE treatment reversed these changes. Furthermore, heat stress increased the activity of SOD in the intestine, downregulated the expression of the oxidative stress-related genes glutathione peroxidase 1 (GPX1), SOD2 and nuclear factor erythroid 2-related factor 2 (NRF2), and upregulated the expression of catalase (CAT). Moreover, heat stress increased tumor necrosis factor-α (TNF-α) levels in the intestine and upregulated the expression of the inflammation-related genes interleukin 10 (IL-10), TNF-α, Interferon-γ (IFN-γ), toll like receptor 4 (TLR4) and nuclear factor-kappa B (NF-kB). However, MGE treatment effectively reduced TNF-α levels and restored the normal activity of SOD and normal mRNA levels for both oxidative stress-related and inflammation-related genes. In summary, our results showed that MGE can protect against heat stress-induced intestinal damage by ameliorating inflammation and oxidative stress.

Resveratrol ameliorates the defects of meiotic maturation in lipopolysaccharide exposed porcine oocytes.

Lipopolysaccharide (LPS), a significant virulence factor of gram-negative bacteria, adversely affects female reproduction, especially the maturation and early embryonic development of oocytes, through inducing of inflammatory and oxidative stress-associated toxic responses. Resveratrol (Res), a potent antioxidant, exhibits many beneficial effects on the maturation and developmental competence of oocytes. However, it is unclear whether Res can restore LPS-induced defects in the maturation of oocytes during meiosis. In this study, we used porcine oocytes to explore the protective effects of Res and its underlying mechanism against the toxic impacts of LPS exposure on meiotic maturation and developmental competence of oocytes during meiosis. The oocytes were randomly assigned to a control, LPS-exposed or Res-supplemented group. Nuclear and cytoplasmic maturation was assessed after 26 h (MI) or 44 h (MII) of in vitro maturation (IVM). Our results showed that 10 µM Res significantly improved the rates of oocyte maturation and blastocyst formation after exposure to 15 µg/mL LPS. In addition, Res preserved the normal spindle/chromosome structure and maintained acetylated tubulin levels, actin polymerization and cortical granules (CGs) distribution. Additionally, Res protected mitochondrial content and function, scavenges reactive oxygen species (ROS), and reduced DNA damage and apoptosis in LPS-exposed oocytes. Furthermore, inhibition of SIRT1 by its specific inhibitor EX527 suppressed the recovery of ROS levels, mitochondrial content, and spindle/chromosome structure by Res supplementation. In summary, this study shows that Res can alleviate the impacts of LPS-induced toxicity on meiosis in porcine oocytes by upregulating SIRT1, which ameliorates oxidative stress and increasing mitochondrial content.

Integrative metabolomic and transcriptomic analysis reveals difference in glucose and lipid metabolism in the longissimus muscle of Luchuan and Duroc pigs.

Luchuan pig, an obese indigenous Chinese porcine breed, has a desirable meat quality and reproductive capacity. Duroc, a traditional western breed, shows a faster growth rate, high feed efficiency and high lean meat rate. Given the unique features these two porcine breeds have, it is of interest to investigate the underlying molecular mechanisms behind their distinctive nature. In this study, the metabolic and transcriptomic profiles of longissimus dorsi muscle from Duroc and Luchuan pigs were compared. A total of 609 metabolites were identified, 77 of which were significantly decreased in Luchuan compared to Duroc, and 71 of which were significantly elevated. Most differentially accumulated metabolites (DAMs) upregulated in Luchuan were glycerophospholipids, fatty acids, oxidized lipids, alcohols, and amines, while metabolites downregulated in Luchuan were mostly amino acids, organic acids and nucleic acids, bile acids and hormones. From our RNA-sequencing (RNA-seq) data we identified a total of 3638 differentially expressed genes (DEGs), 1802 upregulated and 1836 downregulated in Luchuan skeletal muscle compared to Duroc. Combined multivariate and pathway enrichment analyses of metabolome and transcriptome results revealed that many of the DEGs and DAMs are associated with critical energy metabolic pathways, especially those related to glucose and lipid metabolism. We examined the expression of important DEGs in two pathways, AMP-activated protein kinase (AMPK) signaling pathway and fructose and mannose metabolism, using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). Genes related to glucose uptake, glycolysis, glycogen synthesis, fatty acid synthesis (PFKFB1, PFKFB4, MPI, TPI1, GYS1, SLC2A4, FASN, IRS1, ULK1) are more activated in Luchuan, while genes related to fatty acid oxidation, cholesterol synthesis (CPT1A, HMGCR, FOXO3) are more suppressed. Energy utilization can be a decisive factor to the distinctive metabolic, physiological and nutritional characteristics in skeletal muscle of the two breeds we studied. Our research may facilitate future porcine breeding projects and can be used to reveal the potential molecular basis of differences in complex traits between various breeds.

Dietary supplementation with jasmine flower residue improves meat quality and flavor of goat.

Jasmine flower residue (JFR) is a by-product retained in the production process of jasmine tea and can be used as an unconventional feed due to its rich nutrient value. This study aimed to evaluate the effects of the addition of JFR to the diet of goats on their meat quality and flavor. Twenty-four castrated Nubian male goats were randomly divided into two groups and fed a mixed diet containing 10% JFR (JFR, n = 12) or a conventional diet (CON, n = 12) for 45 days. Meat quality and flavor were measured at the end of the treatment. The addition of JFR to the diet could reduce the shear force of the longissimus dorsi muscle, as well as, the cross-sectional area and diameter of muscle fibers, indicating that the addition of JFR improved meat quality. JFR also increased the content of glutamic acid and ω-3 polyunsaturated fatty acid (C18:3n3 and C20:5N3) and reduced the content of C24:1 and saturated fatty acid (C20:0 and C22:0). In addition, the use of JFR increased the content of acetaldehyde and hexanal in the meat. Furthermore, JFR introduced new volatile components in the meat. The umami, saltiness, and richness of the meat also improved. In conclusion, the addition of jasmine flower residue to the diet can improve the meat quality and flavor of goat.

Transcriptomic Analysis of mRNA Expression Profiles in the Microglia of Mouse Brains Infected with Rabies Viruses of Varying Virulence.

Rabies is a lethal encephalitis caused by the rabies virus (RABV) with a fatality rate near 100% after the onset of clinical symptoms in humans and animals. Microglia are resident immune cells in the central nervous system. Few studies have been conducted on the functional role of microglia in RABV infection. Here, we performed a transcriptomic analysis of mRNA expression profiles in the microglia of mouse brains intracerebrally infected with RABV. We successfully isolated single microglial cells from the mouse brains. The survival rate of dissociated microglial cells was 81.91%-96.7%, and the purity was 88.3%. Transcriptomic analysis revealed 22,079 differentially expressed mRNAs identified in the microglia of mouse brains infected with RABV strains (rRC-HL, GX074, and CVS-24) of varying virulence at 4 and 7 days post-infection (dpi) compared to the control group. The numbers of DEGs versus the control at 4 and 7 dpi in mice infected with rRC-HL, GX074, and CVS-24 were 3622 and 4590, 265 and 4901, and 4079 and 6337. The GO enrichment analysis showed that response to stress, response to external stimulus, regulation of response to stimulus, and immune system process were abundant during RABV infection. The KEGG analysis indicated that the Tlr, Tnf, RIG-I, NOD, NF-κB, MAPK, and Jak-STAT signaling pathways were involved in RABV infection at both 4 and 7 dpi. However, some phagocytosis and cell signal transduction processes, such as endocytosis, p53, phospholipase D, and oxidative phosphorylation signaling pathways, were only expressed at 7 dpi. The involvement of the Tnf and Tlr signaling pathways prompted us to construct a protein-protein interaction (PPI) network of these pathways. The PPI revealed 8 DEGs, including Mmp9, Jun, Pik3r1, and Mapk12. Notably, Il-1b interacted with Tnf and Il-6 with combined scores of 0.973 and 0.981, respectively. RABV causes significant changes in mRNA expression profiles in the microglia in mice. 22,079 differentially expressed mRNAs were identified in the microglia of mice infected with RABV strains of varying virulence at 4 and 7 dpi. The DEGs were evaluated using GO, KEGG, and PPI network analysis. Many immune pathways were up-regulated in RABV-infected groups. The findings will help elucidate the microglial molecular mechanisms of cellular metabolism dysregulated by RABV and may provide important information for investigating RABV pathogenesis and therapeutic methods.

2-Mercaptoethanol promotes porcine oocyte maturation in vitro by maintaining autophagy homeostasis.

2-Mercaptoethanol (2-ME) is often used as an antioxidant to optimize culture systems for in vitro oocyte maturation in livestock. However, the relationship between 2-ME and autophagy has not yet been elucidated. In this study, we hypothesized that 2-ME can promote porcine oocyte maturation in vitro by maintaining autophagy homeostasis. To test this hypothesis, we explored the effects of 2-ME on the maturation of porcine oocytes exposed to an autophagy activator (rapamycin) or an autophagy inhibitor (3-methyladenine, i.e., 3-MA) in vitro. Rapamycin-induced autophagy over-activation significantly increased autophagy- and apoptosis-related gene expression, oxidative stress, apoptosis rates, abnormal mitochondrial redistribution, and significantly decreased oocyte first polar body extrusion (PBE) rates, spindle/chromosome integrity and developmental competence. 3-MA-mediated autophagy inhibition exerted similar effects on all these parameters except the expression of genes that promote autophagy and inhibit apoptosis. Importantly, 2-ME supplementation significantly attenuated the detrimental effects of rapamycin and 3-MA. Interestingly, we observed that 44 h of coincubation with rapamycin/3-MA and 2-ME restored autophagy homeostasis in vitro. In conclusion, our study confirmed that 2-ME promotes porcine oocyte maturation and embryo development in vitro by maintaining autophagy homeostasis and lays a foundation for further research on the underlying mechanism.

Mogroside-Rich Extract From Siraitia grosvenorii Fruits Ameliorates High-Fat Diet-Induced Obesity Associated With the Modulation of Gut Microbiota in Mice.

Siraitia grosvenorii is a kind of medicinal food plant. The mogroside-rich extract (MGE) of its fruits can effectively ameliorate obesity, but the underlying mechanisms remain underexplored. In this study, we aimed to determine whether MGE can ameliorate obesity by protecting against the divergences of gut microbiota. Mice were challenged with a high-fat diet (HFD) and treated with MGE by oral gavage. Then, the characteristics of the gut microbiota were determined by 16S rDNA analysis. Our findings showed that MGE could significantly reduce body weight gain and fat tissue weight of the mice fed with HFD. Moreover, MGE markedly attenuated fatty liver, and improved glucose tolerance and insulin sensitivity. We further found that the gut microbiota structures were disturbed by HFD feeding. In particular, the abundance of Firmicutes was increased and the abundance of Bacteroidetes was decreased, resulting in an increased proportion of Firmicutes to Bacteroidetes (F/B), which contributes to obesity. Interestingly, the abnormal proportion of F/B of HFD feeding mice was restored to the level of control mice by MGE treatment. Additionally, the abundances of obesogenic microbiota, such as Ruminiclostridium and Oscillibacter were also decreased after MGE treatment. In summary, our findings demonstrate that MGE can modulate gut microbiota in obese mice and shed new light on how it alleviates obesity.