Guanidinoacetic acid ameliorates hepatic steatosis and inflammation and promotes white adipose tissue browning in middle-aged mice with high-fat-diet-induced obesity.

Guanidinoacetic acid (GAA) is a naturally occurring amino acid derivative that plays a critical role in energy metabolism. In recent years, a growing body of evidence has emerged supporting the importance of GAA in metabolic dysfunction. Hence, we aimed to investigate the effects of GAA on hepatic and adipose tissue metabolism, as well as systemic inflammatory responses in obese middle-aged mice models and attempted to explore the underlying mechanism. We found that dietary supplementation of GAA inhibited inguinal white adipose tissue (iWAT) hypertrophy in high-fat diet (HFD)-fed mice. In addition, GAA supplementation observably decreased the levels of some systemic inflammatory factors, including IL-4, TNF-α, IL-1ß, and IL-6. Intriguingly, GAA supplementation ameliorated hepatic steatosis and lipid deposition in HFD-fed mice, which was revealed by decreased levels of TG, TC, LDL-C, PPARγ, SREBP-1c, FASN, ACC, FABP1, and APOB and increased levels of HDL-C in the liver. Moreover, GAA supplementation increased the expression of browning markers and mitochondrial-related genes in the iWAT. Further investigation showed that dietary GAA promoted the browning of the iWAT via activating the AMPK/Sirt1 signaling pathway and might be associated with futile creatine cycling in obese mice. These results indicate that GAA has the potential to be used as an effective ingredient in dietary interventions and thus may play an important role in ameliorating and preventing HFD-induced obesity and related metabolic diseases.

Vitamin a potentiates sheep myoblasts myogenic differentiation through BHLHE40-modulated ID3 expression.

BACKGROUND: Vitamin A and retinoic acid (RA, a metabolite of vitamin A), are inextricably involved to the development of skeletal muscle in animals. However, the mechanisms regulating skeletal muscle development by vitamin A remain poorly reported. The current study designed to investigate the underlying mechanism of vitamin A affecting myogenic differentiation of lamb myoblasts through transcriptome sequencing (RNA-Seq) and gene function validation experiments. It provides a theoretical basis for elucidating the regulation of vitamin A on skeletal muscle development as well as for improving the economic benefits of the mutton sheep industry. RESULTS: Newborn lambs were injected with 7,500 IU vitamin A, and longissimus dorsi (LD) muscle tissue was surgically sampled for RNA-Seq analysis and primary myoblasts isolation at 3 weeks of age. The results showed that a total of 14 down-regulated and 3 up-regulated genes, were identified between control and vitamin A groups. Among them, BHLHE40 expression was upregulated in vitamin A group lambs. Furthermore, BHLHE40 expression is significantly increased after initiation of differentiation in myoblasts, and RA addition during differentiation greatly promoted BHLHE40 mRNA expression. In vitro, RA inhibited myoblasts proliferation and promoted myoblasts myogenic differentiation through BHLHE40. Moreover, BHLHE40 was proved to inhibit the expression of the DNA binding inhibitor 3 (ID3), and meanwhile, ID3 could effectively promote myoblasts proliferation and inhibit myoblasts myogenic differentiation. CONCLUSIONS: Taken together, our results suggested that vitamin A inhibited myoblasts proliferation and promoted myoblasts myogenic differentiation by inhibiting ID3 expression through BHLHE40.

Vitamin A regulates mitochondrial biogenesis and function through p38 MAPK-PGC-1α signaling pathway and alters the muscle fiber composition of sheep.

BACKGROUND: Vitamin A (VA) and its metabolite, retinoic acid (RA), are of great interest for their wide range of physiological functions. However, the regulatory contribution of VA to mitochondrial and muscle fiber composition in sheep has not been reported. METHOD: Lambs were injected with 0 (control) or 7,500 IU VA palmitate into the biceps femoris muscle on d 2 after birth. At the age of 3 and 32 weeks, longissimus dorsi (LD) muscle samples were obtained to explore the effect of VA on myofiber type composition. In vitro, we investigated the effects of RA on myofiber type composition and intrinsic mechanisms. RESULTS: The proportion of type I myofiber was greatly increased in VA-treated sheep in LD muscle at harvest. VA greatly promoted mitochondrial biogenesis and function in LD muscle of sheep. Further exploration revealed that VA elevated PGC-1α mRNA and protein contents, and enhanced the level of p38 MAPK phosphorylation in LD muscle of sheep. In addition, the number of type I myofibers with RA treatment was significantly increased, and type IIx myofibers was significantly decreased in primary myoblasts. Consistent with in vivo experiment, RA significantly improved mitochondrial biogenesis and function in primary myoblasts of sheep. We then used si-PGC-1α to inhibit PGC-1α expression and found that si-PGC-1α significantly abrogated RA-induced the formation of type I myofibers, mitochondrial biogenesis, MitoTracker staining intensity, UQCRC1 and ATP5A1 expression, SDH activity, and enhanced the level of type IIx muscle fibers. These data suggested that RA improved mitochondrial biogenesis and function by promoting PGC-1α expression, and increased type I myofibers. In order to prove that the effect of RA on the level of PGC-1α is caused by p38 MAPK signaling, we inhibited the p38 MAPK signaling using a p38 MAPK inhibitor, which significantly reduced RA-induced PGC-1α and MyHC I levels. CONCLUSION: VA promoted PGC-1α expression through the p38 MAPK signaling pathway, improved mitochondrial biogenesis, and altered the composition of muscle fiber type.

Transcription Factor SATB2 Regulates Skeletal Muscle Cell Proliferation and Migration via HDAC4 in Pigs.

Skeletal muscle development remarkably affects meat production and growth rate, regulated by complex regulatory mechanisms in pigs. Specific AT sequence-binding protein 2 (SATB2) is a classic transcription factor and chromatin organizer, which holds a profound effect in the regulation of chromatin remodeling. However, the regulation role of SATB2 concerning skeletal muscle cell fate through chromatin remodeling in pigs remains largely unknown. Here, we observed that SATB2 was expressed higher in the lean-type compared to the obese-type pigs, which also enriched the pathways of skeletal muscle development, chromatin organization, and histone modification. Functionally, knockdown SATB2 led to decreases in the proliferation and migration markers at the mRNA and protein expression levels, respectively, while overexpression SATB2 had the opposite effects. Further, we found histone deacetylase 4 (HDAC4) was a key downstream target gene of SATB2 related to chromatin remodeling. The binding relationship between SATB2 and HDAC4 was confirmed by a dual-luciferase reporter system and ChIP-qPCR analysis. Besides, we revealed that HDAC4 promoted the skeletal muscle cell proliferation and migration at the mRNA and protein expression levels, respectively. In conclusion, our study indicates that transcription factor SATB2 binding to HDAC4 positively contributes to skeletal muscle cell proliferation and migration, which might mediate the chromatin remodeling to influence myogenesis in pigs. This study develops a novel insight into understanding the molecular regulatory mechanism of myogenesis, and provides a promising gene for genetic breeding in pigs.

Sea buckthorn oil regulates primary myoblasts proliferation and differentiation in vitro.

Skeletal muscle is the main edible part of meat products, and its development directly affects the yield and palatability of meat. Sea buckthorn oil (SBO) contains plenty of bioactive substances and has been recognized as a potential functional food product. The study aimed to explore the effects and possible mechanisms of SBO on sheep primary myoblast proliferation and myogenic differentiation. The results implied that SBO exhibited a pro-proliferative effect on primary myoblasts, along with up-regulated proliferating cell nuclear antigen (PCNA) and Cyclin D1/cyclin-dependent kinase 4 (CDK4) abundances. And, SBO promoted myotube formation by increasing the expression of myogenin. Meanwhile, we found that SBO inhibited the expression of miRNA-292a. Moreover, the regulatory effect of SBO on myogenic differentiation of myoblasts was attenuated by miRNA-292a mimics. Of note, SBO activated protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway and augmented glucose uptake and glucose transporter 4 (GLUT4) content, which might be attributed to AMP-activated protein kinase (AMPK) activation. Additionally, the results were shown that SBO increased the abundance of antioxidative enzymes, including glutathione peroxidase 4 (Gpx4) and catalase. In summary, these data suggested that SBO regulated the proliferation and myogenic differentiation of sheep primary myoblasts in vitro, which might potentiate the application of SBO in muscle growth.

Intramuscular vitamin A injection in newborn lambs enhances antioxidant capacity and improves meat quality.

Introduction: Vitamin A (VA) and its metabolite, retinoic acid (RA) possess several biological functions. This report investigated whether neonatal intramuscular VA injection affected antioxidative activity and meat quality in longissimus dorsi (LD) muscle of lambs. Methods: Lambs were injected with 0 (control) or 7,500 IU VA palmitate into the biceps femoris muscle on day 2 after birth. At 3, 12, and 32 weeks of age, blood samples were collected in the jugular vein for serum levels of RA and muscle samples were collected in the biceps femoris for analysis of relative mRNA expression of enzyme contributors to retinoid metabolism. All animals were harvested at 32 weeks of age and muscle samples were collected to explore the role of VA on the meat quality and antioxidant capacity of lambs. Results and discussion: Our results indicated that VA increased the redness, crude protein, and crude fat (p < 0.05), without affecting moisture, ash, and amino acid composition in LD muscle (p > 0.05). In addition, VA increased catalase (CAT) activity and decreased malondialdehyde (MDA) levels in LD muscle (p < 0.05). Meanwhile, greater levels of CAT and NRF2 mRNA and protein contents with VA treatment were observed in LD muscle (p < 0.05), partly explained by the increased level of RA (p < 0.05). Collectively, our findings indicated that VA injection at birth could improve lamb meat quality by elevating the redness, crude protein, crude fat, and antioxidative capacity in LD muscle of lambs.

Progenitor cell isolation from mouse epididymal adipose tissue and sequencing library construction.

Here, we present a protocol to isolate progenitor cells from mouse epididymal visceral adipose tissue and construct bulk RNA and assay for transposase-accessible chromatin with sequencing (ATAC-seq) libraries. We describe steps for adipose tissue collection, cell isolation, and cell staining and sorting. We then detail procedures for both ATAC-seq and RNA sequencing library construction. This protocol can also be applied to other tissues and cell types directly or with minor modifications. For complete details on the use and execution of this protocol, please refer to Liu et al. (2023).1.

Guanidinoacetic Acid Attenuates Adipogenesis through Regulation of miR-133a in Sheep.

Guanidinoacetic acid (GAA) is an amino acid derivative, previously described in the skeletal muscle of vertebrates, that serves as an important regulator of cellular bioenergetics and has been widely used as a feed additive. Nevertheless, the effect of GAA on adipose tissue growth remains unclear. Here, we hypothesized that dietary GAA negatively affected adipose tissue development in lambs. Lambs were individually fed diets with (0.09%) or without GAA for 70 d ad libitum, and the subcutaneous adipose tissues were sampled for analysis. The results showed that dietary GAA supplementation decreased the girth rib (GR) value (p < 0.01) of lamb carcasses. Both real-time PCR and Western blot analysis suggested that dietary GAA inhibited the expression of adipogenic markers, including peroxisome proliferator-activated receptor γ (PPARγ, p < 0.05), CCAAT/enhancer-binding protein α (C/EBPα, p < 0.01) and sterol-regulatory-element-binding protein 1c (SREBP1C, p < 0.01) in subcutaneous adipose tissue. In vitro, GAA inhibited sheep stromal vascular fraction (SVF) cell proliferation, which was associated with downregulation of proliferating cell nuclear antigen (PCNA, p < 0.05), cyclin-dependent kinase 4 (CDK 4, p < 0.05) and cyclin D1 (p < 0.01). GAA suppressed adipogenesis of SVF cells. Furthermore, miRNA sequencing revealed that GAA affected the miRNA expression profile, and real-time PCR analysis confirmed that miR-133a expression in both subcutaneous adipose tissue and SVF cell was downregulated by GAA. Meanwhile, miR-133a promoted adipogenic differentiation of SVF cells by targeting Sirt1. miR-133a mimics alleviated the inhibitory effect of GAA on SVF cells' adipogenic differentiation. In summary, GAA attenuated adipogenesis of sheep SVF cells, which might occur through miR-133a-modulated Sirt1 expression.

Global impact of aberrant splicing on human gene expression levels.

Alternative splicing (AS) is pervasive in human genes, yet the specific function of most AS events remains unknown. It is widely assumed that the primary function of AS is to diversify the proteome, however AS can also influence gene expression levels by producing transcripts rapidly degraded by nonsense-mediated decay (NMD). Currently, there are no precise estimates for how often the coupling of AS and NMD (AS-NMD) impacts gene expression levels because rapidly degraded NMD transcripts are challenging to capture. To better understand the impact of AS on gene expression levels, we analyzed population-scale genomic data in lymphoblastoid cell lines across eight molecular assays that capture gene regulation before, during, and after transcription and cytoplasmic decay. Sequencing nascent mRNA transcripts revealed frequent aberrant splicing of human introns, which results in remarkably high levels of mRNA transcripts subject to NMD. We estimate that ~15% of all protein-coding transcripts are degraded by NMD, and this estimate increases to nearly half of all transcripts for lowly-expressed genes with many introns. Leveraging genetic variation across cell lines, we find that GWAS trait-associated loci explained by AS are similarly likely to associate with NMD-induced expression level differences as with differences in protein isoform usage. Additionally, we used the splice-switching drug risdiplam to perturb AS at hundreds of genes, finding that ~3/4 of the splicing perturbations induce NMD. Thus, we conclude that AS-NMD substantially impacts the expression levels of most human genes. Our work further suggests that much of the molecular impact of AS is mediated by changes in protein expression levels rather than diversification of the proteome.

Structural and Biological Evaluations of a Non-Nucleoside STING Agonist Specific for Human STINGA230 Variants.

Previously we identified a non-nucleotide tricyclic agonist BDW568 that activates human STING (stimulator of interferon genes) gene variant containing A230 in a human monocyte cell line (THP-1). STINGA230 alleles, including HAQ and AQ, are less common STING variants in human population. To further characterize the mechanism of BDW568, we obtained the crystal structure of the C-terminal domain of STINGA230 complexed with BDW-OH (active metabolite of BDW568) at 1.95 Å resolution and found the planar tricyclic structure in BDW-OH dimerizes in the STING binding pocket and mimics the two nucleobases of the endogenous STING ligand 2',3'-cGAMP. This binding mode also resembles a known synthetic ligand of human STING, MSA-2, but not another tricyclic mouse STING agonist DMXAA. Structure-activity-relationship (SAR) studies revealed that all three heterocycles in BDW568 and the S-acetate side chain are critical for retaining the compound's activity. BDW568 could robustly activate the STING pathway in human primary peripheral blood mononuclear cells (PBMCs) with STINGA230 genotype from healthy individuals. We also observed BDW568 could robustly activate type I interferon signaling in purified human primary macrophages that were transduced with lentivirus expressing STINGA230, suggesting its potential use to selectively activate genetically engineered macrophages in macrophage-based approaches, such as chimeric antigen receptor (CAR)-macrophage immunotherapies.

Vitamin A injection at birth improves muscle growth in lambs.

Vitamin A and its metabolite, retinoic acid (RA) play important roles in regulating skeletal muscle development. This study was conducted to investigate the effects of early intramuscular vitamin A injection on the muscle growth of lambs. A total of 16 newborn lambs were given weekly intramuscular injections of corn oil (control group, n = 8) or 7,500 IU vitamin A palmitate (vitamin A group, n = 8) from birth to 3 wk of age (4 shots in total). At 3 wk of age and weaning, biceps femoris muscle samples were taken to analyze the effects of vitamin A on the myogenic capacity of skeletal muscle cells. All lambs were slaughtered at 8 months of age. The results suggest that vitamin A treatment accelerated the growth rate of lambs and increased the loin eye area (P < 0.05). Consistently, vitamin A increased the diameter of myofibers in longissimus thoracis muscle (P < 0.01) and increased the final body weight of lambs (P < 0.05). Vitamin A injection did not change the protein kinase B/mammalian target of rapamycin and myostatin signaling (P > 0.05). Moreover, vitamin A upregulated the expression of PAX7 (P < 0.05) and the myogenic marker genes including MYOD and MYOG (P < 0.01). The skeletal muscle-derived mononuclear cells from vitamin A-treated lambs showed higher expression of myogenic genes (P < 0.05) and formed more myotubes (P < 0.01) when myogenic differentiation was induced in vitro. In addition, in vitro analysis showed that RA promoted myogenic differentiation of the skeletal muscle-derived mononuclear cells in the first 3 d (P < 0.05) but not at the later stage (P > 0.05) as evidenced by myogenic gene expression and fusion index. Taken together, neonatal intramuscular vitamin A injection promotes lamb muscle growth by promoting the myogenic potential of satellite cells.

Astragalus polysaccharide promotes sheep satellite cell differentiation by regulating miR-133a through the MAPK/ERK signaling pathway.

Astragalus polysaccharide (APS) possesses extensive biological activities, pharmacological effects, and anti-fatigue function. MiR-133a is a specifically expressed miRNA in skeletal muscle that participates in the regulation of myoblast proliferation and differentiation. However, little is known about the role of APS in the development of sheep skeletal muscle. In this study, we aimed to investigate the underlying mechanism of APS and miR-133a on the differentiation of sheep skeletal muscle satellite cells (SMSCs) and the regulatory relationship between APS and miR-133a. The results suggested that APS plays a positive regulatory role in the proliferation and differentiation of sheep SMSCs. Moreover, miR-133a significantly promotes SMSC differentiation and the activity of the MAPK/ERK signaling pathway. Importantly, we found that APS function requires the mediation of miR-133a in the differentiation of sheep SMSCs. Taken together, our results indicate that APS accelerates SMSC differentiation by regulating miR-133a via the MAPK/ERK signaling pathway in sheep.

Tcf21 marks visceral adipose mesenchymal progenitors and functions as a rate-limiting factor during visceral adipose tissue development.

Distinct locations of different white adipose depots suggest anatomy-specific developmental regulation, a relatively understudied concept. Here, we report a population of Tcf21 lineage cells (Tcf21 LCs) present exclusively in visceral adipose tissue (VAT) that dynamically contributes to VAT development and expansion. During development, the Tcf21 lineage gives rise to adipocytes. In adult mice, Tcf21 LCs transform into a fibrotic or quiescent state. Multiomics analyses show consistent gene expression and chromatin accessibility changes in Tcf21 LC, based on which we constructed a gene-regulatory network governing Tcf21 LC activities. Furthermore, single-cell RNA sequencing (scRNA-seq) identifies the heterogeneity of Tcf21 LCs. Loss of Tcf21 promotes the adipogenesis and developmental progress of Tcf21 LCs, leading to improved metabolic health in the context of diet-induced obesity. Mechanistic studies show that the inhibitory effect of Tcf21 on adipogenesis is at least partially mediated via Dlk1 expression accentuation.

Integrative Analysis of Liver Metabolomics and Transcriptomics Reveals Oxidative Stress in Piglets with Intrauterine Growth Restriction.

The correlation between oxidative stress and liver metabolic dysfunction in piglets with intrauterine growth restriction (IUGR) remains limited. Therefore, the objective of the present study was to investigate potential mechanisms of metabolic characteristics induced by oxidative stress in the livers of IUGR piglets using metabolomic and transcriptomic analysis. Analysis of the phenotypic characteristics showed that the liver weight of the intrauterine growth restriction piglets was significantly lower than that of normal birth weight piglets. Intrauterine growth restriction piglets exhibited disordered hepatic cord arrangement and vacuolization as well as excessive lipid accumulation in hepatocytes. In addition, the activities of antioxidant enzymes were significantly decreased in the liver of the intrauterine growth restriction piglets, whereas the level of the lipid peroxidation marker MDA was significantly increased. Finally, our findings revealed that intrauterine growth restriction piglets were involved in a variety of metabolic abnormalities, including mitochondrial dysfunction, imbalance of fatty acid composition, disruption to sources of one-carbon unit supply, and abnormal galactose conversion, which may be responsible for oxidative stress in the liver. In summary, these data provided a detailed theoretical reference for revealing the hepatic metabolic characteristics of intrauterine growth restriction piglets.

Cellular Target Deconvolution of Small Molecules Using a Selection-Based Genetic Screening Platform.

Small-molecule drug target identification is an essential and often rate-limiting step in phenotypic drug discovery and remains a major challenge. Here, we report a novel platform for target identification of activators of signaling pathways by leveraging the power of a clustered regularly interspaced short palindromic repeats (CRISPR) knockout library. This platform links the expression of a suicide gene to the small-molecule-activated signaling pathway to create a selection system. With this system, loss-of-function screening using a CRISPR single-guide (sg) RNA library positively enriches cells in which the target has been knocked out. The identities of the drug targets and other essential genes required for the activity of small molecules of interest are then uncovered by sequencing. We tested this platform on BDW568, a newly discovered type-I interferon signaling activator, and identified stimulator of interferon genes (STING) as its target and carboxylesterase 1 (CES1) to be a key metabolizing enzyme required to activate BDW568 for target engagement. The platform we present here can be a general method applicable for target identification for a wide range of small molecules that activate different signaling pathways.

Quantitative proteomic analysis of the microbial degradation of 3-aminobenzoic acid by Comamonas sp. QT12.

A mab cluster associated with 3-aminobenzoic acid (3AB) degradation was identified in Comamonas sp. QT12. However, the cellular response of Comamonas sp. QT12 to 3-aminobenzoic acid remains unclear. In this study, label-free quantitative proteome analysis based on LC-MS/MS was used to study the protein expression difference of strain QT12 under the condition of using 3AB (3AB) and citric acid/ammonium chloride as substrates (3ABCon). A total of 2068 proteins were identified, of which 239 were significantly up-regulated in 3AB group, 124 were significantly down-regulated in 3AB group, 624 were expressed only in 3AB group, and 216 were expressed only in 3ABCon group in 3AB group. KEGG pathway analysis found that 83 pathways were up-regulated and 49 pathways were down-regulated, In GO analysis, 315 paths were up-regulated and 156 paths were down-regulated. There were 6 genes in the mab cluster that were only detected in the 3AB group.The mab cluster was found to be related to degradation of 3AB. By knockout, it was found that the growth rate of the mutant △orf7 and △orf9 were slowed down. HPLC results showed that the mutant △orf7 and △orf9 could still degrade 3AB, it was found that orf7, orf9 were not key genes about 3AB degradation and they could be replaced by other genes in strain QT12. These findings improve our understanding of the molecular mechanisms underlying the cellular response of 3AB degradation in Comamonas bacterium.

Skeletal muscle mass, meat quality and antioxidant status in growing lambs supplemented with guanidinoacetic acid.

Guanidinoacetic acid (GAA) exists naturally as a precursor of creatine, which possesses several biological functions. In the present study, the effects of dietary GAA supplementation on skeletal muscle mass and meat quality of lambs were investigated. The GAA supplementation increased final body weight, promoted muscle mass and changed the distribution of myofiber size. Meanwhile, elevated ultimate pH and water holding capacity (WHC) of resulting meat were observed in GAA fed lambs. Moreover, the total antioxidative capacity was elevated. Dietary GAA accelerated myofibril protein synthesis through regulation with IGF-1/Akt/mTOR signaling pathway and minimized protein breakdown via regulating abundances of myostatin and phosphorylated FoxO1. In vitro, GAA treatment inhibited sheep primary myoblasts proliferation, and enhanced its myogenic potential. Collectively, these results suggested that GAA might be a feed additive for use by the lamb meat industry as it has potential to improve growth performance, antioxidant status and WHC of resulting meat.

Seabuckthorn Reverses High-Fat-Diet-Induced Obesity and Enhances Fat Browning via Activation of AMPK/SIRT1 Pathway.

Seabuckthorn possesses various bioactive compounds and exhibits several positive pharmacological activities. The present trial aims to determine the effect of seabuckthorn powder intake on high-fat diet (HFD)-induced obesity prevention in mice. The results suggest that seabuckthorn powder intake decreased body weight, fat mass, and circulating lipid levels, and improved insulin sensitivity in HFD-fed mice. Moreover, dietary seabuckthorn powder alleviated hepatic steatosis and hepatic lipid accumulation induced by the HFD. Furthermore, seabuckthorn exhibited obvious anti-inflammatory capacity in white adipose tissue (WAT) by regulating the abundance of inflammation-related cytokines, such as interleukins 4, 6, and 10; tumor necrosis factor α; and interferon-γ. More importantly, dietary seabuckthorn powder promoted a thermogenic program in BAT and induced beige adipocyte formation in iWAT in HFD-fed mice. Interestingly, we found that seabuckthorn powder effectively restored AMPK and SIRT1 activities in both BAT and iWAT in HFD-fed mice. Collectively, these results potentiate the application of seabuckthorn powder as a nutritional intervention strategy to prevent obesity and related metabolic diseases by promoting thermogenesis in BAT and improving beige adipocyte formation in WAT.

Cross-talk between NOTCH2 and BMP4/SMAD signaling pathways in bovine follicular granulosa cells.

NOTCH and bone morphogenetic protein (BMP)/SMAD signaling play key regulatory roles in mammalian ovarian development. The study aimed to investigate interregulatory mechanisms between NOTCH2 and BMP4/SMAD signaling pathways in bovine follicular granulosa cells (GCs). The results showed that NOTCH2 silence reduced the mRNA expression of SMAD1, SMAD5, SMAD8 (also known as SMAD9) and Mg2+/Mn2+- dependent Protein Phosphatase 1A (PPM1A), which are effectors of BMP/SMAD signaling pathway (P < 0.01). Overexpressing NOTCH2 intracellular sequence increased the mRNA expression of BMPR1A, SMAD1, SMAD4, SMAD5, SMAD8 and PPM1A (P < 0.01). Meanwhile, treating GCs with BMP4 inhibited the mRNA expression of its downstream gene SMAD1 and steroidogenesis genes STAR and CYP11A1 in the presence of follicular stimulating hormone (FSH) (P < 0.01). Moreover, BMP4 inhibited the mRNA expression of NOTCH signaling pathway target gene HES1 (P < 0.05), while the increase in NOTCH2 may be due to negative feedback of HES1. By and large, these results indicated that NOTCH2 up-regulated key genes of BMP/SMAD signaling in bovine follicle GCs, while BMP4 inhibited its downstream signaling factors and NOTCH signaling pathway target gene HES1. This study suggests there are complex synergistic and antagonistic effects between the two signaling pathways, which jointly participate in regulating bovine follicular development through regulating follicular GCs.

CRISPR-Mediated Enzyme Fragment Complementation Assay for Quantification of the Stability of Splice Isoforms.

Small-molecule splicing modulators exemplified by an FDA-approved drug, risdiplam, are a new pharmacological modality for regulating the expression and stability of splice isoforms. We report a CRISPR-mediated enzyme fragment complementation (EFC) assay to quantify the splice isoform stability. The EFC assay harnessed a 42 amino acid split of a ß-galactosidase (designate α-tag), which could be fused at the termini of the target genes using CRISPR/cas9. The α-tagged splice isoform would be quantified by measuring the enzymatic activity upon complementation with the rest of ß-galactosidase. This EFC assay retained all the sequences of introns and exons of the target gene in the native genomic environment that recapitulates the cell biology of the diseases of interest. For a proof-of-concept, we developed a CRISPR-mediated EFC assay targeting the exon 7 of the survival of motor neuron 2 (SMN2) gene. The EFC assay is compatible with 384-well plates and robustly quantified the splicing modulation activity of small molecules. In this study, we also discovered that a coumarin derivative, compound 4, potently modulated SMN2 exon 7 splicing at as low as 1.1 nM.