Mining key circRNA-associated-ceRNA networks for milk fat metabolism in cows with varying milk fat percentages.

BACKGROUND: Cow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular RNAs (circRNAs) necessary for mammary gland development and lactation in mammals. METHOD: The present study analyzed circRNA expression profiling data in mammary epithelial cells (MECs) from cows with highly variable milk fat percentage (MFP) using differential expression analysis and weighted gene co-expression network analysis (WGCNA). RESULTS: A total of 309 differentially expressed circRNAs (DE-circRNAs) were identified in the high and low MFP groups. WGCNA analysis revealed that the pink module was significantly associated with MFP (r = - 0.85, P = 0.007). Parental genes of circRNAs in this module were enriched mainly in lipid metabolism-related signaling pathways, such as focal adhesion, ECM-receptor interaction, adherens junction and AMPK. Finally, six DE-circRNAs were screened from the pink module: circ_0010571, circ_0007797, circ_0002746, circ_0003052, circ_0004319, and circ_0012840. Among them, circ_0002746, circ_0003052, circ_0004319, and circ_0012840 had circular structures and were highly expressed in mammary tissues. Subcellular localization revealed that these four DE-circRNAs may play a regulatory role in the mammary glands of dairy cows, mainly as competitive endogenous RNAs (ceRNAs). Seven hub target genes (GNB1, GNG2, PLCB1, PLCG1, ATP6V0C, NDUFS4, and PIGH) were obtained by constructing the regulatory network of their ceRNAs and then analyzed by CytoHubba and MCODE plugins in Cytoscape. Functional enrichment analysis revealed that these genes are crucial and most probable ceRNA regulators in milk fat metabolism. CONCLUSIONS: Our study identified several vital circRNAs and ceRNAs affecting milk fat synthesis, providing new research ideas and a theoretical basis for cow lactation, milk quality, and breed improvement.

Non-targeted metabolomics identifies biomarkers in milk with high and low milk fat percentage.

Milk is widely recognized as an important food source with health benefits. Different consumer groups have different requirements for the content and proportion of milk fat; therefore, it is necessary to investigate the differential metabolites and their regulatory mechanisms in milk with high and low milk fat percentages (MFP). In this study, untargeted metabolomics was performed on milk samples from 13 cows with high milk fat percentage (HF) and 13 cows with low milk fat percentage (LF) using ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS). Forty-eight potential differentially labeled compounds were screened using the orthogonal partial least squares-discriminant analysis (OPLS-DA) combined with the weighted gene co-expression network analysis (WGCNA) method. Amino acid metabolism was the key metabolic pathway with significant enrichment of L-histidine, 5-oxoproline, L-aspartic acid, and L-glutamic acid. The negative correlation with MFP differentiated the HF and LF groups. To further determine the potential regulatory role of these amino acids on milk fat metabolism, the expression levels of marker genes in the milk fat synthesis pathway were explored. It was noticed that L-histidine reduced milk fat concentration primarily by inhibiting the triglycerides (TAG) synthesis pathway. L-aspartic acid and L-glutamic acid inhibited milk fat synthesis through the fatty acid de novo and TAG synthesis pathways. This study provides new insights into the mechanism underlying milk fat synthesis and milk quality improvement.

RNA N6-methyladenosine profiling reveals differentially methylated genes associated with intramuscular fat metabolism during breast muscle development in chicken.

Intramuscular fat (IMF) is an important indicator for determining meat quality, and IMF deposition during muscle development is regulated by a complex molecular network involving multiple genes. The N6-methyladenosine (m6A) modification of mRNA plays an important regulatory role in muscle adipogenesis. However, the distribution of m6A and its role in IMF metabolism in poultry has not been reported. In the present study, a transcriptome-wide m6A profile was constructed using methylated RNA immunoprecipitation sequence (MeRIP-seq) and RNA sequence (RNA-seq) to explore the potential mechanism of regulating IMF deposition in the breast muscle based on the comparative analysis of IMF differences in the breast muscles of 42 (group G), 126 (group S), and 180-days old (group M) Jingyuan chickens. The findings revealed that the IMF content in the breast muscle increased significantly with the increase in the growth days of the Jingyuan chickens (P < 0.05). The m6A peak in the breast muscles of the 3 groups was highly enriched in the coding sequence (CDS) and 3' untranslated regions (3' UTR), which corresponded to the consensus motif RRACH. Moreover, we identified 129, 103, and 162 differentially methylated genes (DMGs) in the breast muscle samples of the G, S, and M groups, respectively. Functional enrichment analyses revealed that DMGs are involved in many physiological activities of muscle fat anabolism. The m6A-induced ferroptosis pathway was identified in breast muscle tissue as a new target for regulating IMF metabolism. In addition, association analysis demonstrated that LMOD2 and its multiple m6A negatively regulated DMGs are potential regulators of IMF differential deposition in muscle. The findings of the present study provide a solid foundation for further investigation into the potential role of m6A modification in regulating chicken fat metabolism.

Excavation and characterization of key circRNAs for milk fat percentage in Holstein cattle.

Milk fat percentage is one of the significant indicators governing the price and quality of milk and is regulated by a variety of non-coding RNAs. We used RNA sequencing (RNA-seq) techniques and bioinformatics approaches to explore potential candidate circular RNAs (circRNAs) regulating milk fat metabolism. After analysis, compared with low milk fat percentage (LMF) cows, 309 circRNAs were significantly differentially expressed in high milk fat percentage (HMF) cows. Functional enrichment and pathway analysis revealed that the main functions of the parental genes of differentially expressed circRNAs (DE-circRNAs) were related to lipid metabolism. We selected four circRNAs (Novel_circ_0000856, Novel_circ_0011157, novel_circ_0011944, and Novel_circ_0018279) derived from parental genes related to lipid metabolism as key candidate DE-circRNAs. Their head-to-tail splicing was demonstrated by linear RNase R digestion experiments and Sanger sequencing. However, the tissue expression profiles showed that only Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 were expressed with high abundance in breast tissue. Based on the subcellular localization found that Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 mainly function as competitive endogenous RNAs (ceRNAs) in the cytoplasm. Therefore, we constructed their ceRNA regulatory networks, and the five hub target genes (CSF1, TET2, VDR, CD34, and MECP2) in ceRNAs were obtained by CytoHubba and MCODE plugins in Cytoscape, as well as tissue expression profiles analysis of target genes. These genes play a key role as important target genes in lipid metabolism, energy metabolism, and cellular autophagy. The Novel_circ_0000856, Novel_circ_0011157, and Novel_circ_0011944 regulate the expression of hub target genes through interaction with miRNAs and constitute key regulatory networks that may be involved in milk fat metabolism. The circRNAs obtained in this study may act as miRNA sponges and thus influence mammary gland development and lipid metabolism in cows, which improves our understanding of the role of circRNAs in cow lactation.

Milk is an important food source, consisting of a complex mixture of lipids, proteins, carbohydrates, and other factors, of which milk fat not only affects the flavor and nutritional value of milk but also plays an important role in the metabolism of nutrients during human growth and development. To dig for potential circular RNAs (circRNAs) and their key regulatory networks that regulate milk fat, we used RNA sequencing (RNA-seq) to identify 309 circRNAs that are differentially expressed between the mammary epithelial cells (MECs) of cows with high and low milk fat percentage. We screened key circRNAs and their circRNA-miRNA-mRNA regulatory networks affecting milk fat by bioinformatic methods. It provides a new way to study lactation, milk quality, and breed improvement in dairy cows.

CircRNA screening and ceRNA network construction for milk fat metabolism in dairy cows.

Background: Milk fat is one of the main reference elements for evaluating milk quality and is a primary objective trait in dairy cattle breeding. In recent years, circular RNAs (circRNAs) have been found to play crucial roles in many biological processes. However, the function and expression profiles of circRNAs in milk fat synthesis in cows are not completely understood. We performed RNA sequencing to analyze the genome-wide expression of circRNA transcripts in bovine mammary epithelial cells (BMECs) from cows with extreme differences in milk fat percentage. We identified candidate differential circRNAs associated with milk fat metabolism using functional enrichment analysis and constructed a lipid metabolism-related competing endogenous RNA (ceRNA) interactive regulatory network. Results: A total of 290 circRNAs were significantly differentially expressed (DE-circRNAs) in high milk fat percentage (HMF) cows compared to that in low milk fat percentage (LMF) cows. Of the 290 circRNAs, 142 were significantly upregulated and 148 were significantly downregulated. Enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) identified four DE-circRNAs (circ_0001122, circ_0007367, circ_0018269, and circ_0015179) that potentially regulate milk fat metabolism. Among them, circ_0001122, circ_0007367, and circ_0015179 had relatively high expression levels in cow mammary gland tissue compared to other tissues (heart, liver, kidney, uterus, ovaries, and small intestine) of cows. The regulatory networks circ_0001122:miR-12043:LIPG, circ_0007367:miR-331-3p:CIDEA/PML, and circ_0018269:miR-11989:RORC/HPX are potential networks to explore the mechanism of milk fat regulation. Conclusions: These results reveal the possible role of circRNAs in milk fat metabolism in dairy cows. Several important circRNAs and ceRNAs affecting milk fat synthesis were identified, providing insights into the complex biology of milk fat synthesis as well as a novel theoretical perspective for future research on lactation, milk quality, and breed improvement in dairy cows.

Regulatory role of RNA N6-methyladenosine modifications during skeletal muscle development.

Functional cells in embryonic myogenesis and postnatal muscle development undergo multiple stages of proliferation and differentiation, which are strict procedural regulation processes. N6-methyladenosine (m6A) is the most abundant RNA modification that regulates gene expression in specific cell types in eukaryotes and regulates various biological activities, such as RNA processing and metabolism. Recent studies have shown that m6A modification-mediated transcriptional and post-transcriptional regulation plays an essential role in myogenesis. This review outlines embryonic and postnatal myogenic differentiation and summarizes the important roles played by functional cells in each developmental period. Furthermore, the key roles of m6A modifications and their regulators in myogenesis were highlighted, and the synergistic regulation of m6A modifications with myogenic transcription factors was emphasized to characterize the cascade of transcriptional and post-transcriptional regulation during myogenesis. This review also discusses the crosstalk between m6A modifications and non-coding RNAs, proposing a novel mechanism for post-transcriptional regulation during skeletal muscle development. In summary, the transcriptional and post-transcriptional regulatory mechanisms mediated by m6A and their regulators may help develop new strategies to maintain muscle homeostasis, which are expected to become targets for animal muscle-specific trait breeding and treatment of muscle metabolic diseases.

Overexpression of uracil permease and nucleoside transporter from Bacillus amyloliquefaciens improves cytidine production in Escherichia coli.

Cytidine is an important raw material for nucleic acid health food and genetic engineering research. In recent years, it has shown irreplaceable effects in anti-virus, anti-tumor, and AIDS drugs. Its biosynthetic pathway is complex and highly regulated. In this study, overexpression of uracil permease and a nucleoside transporter from Bacillus amyloliquefaciens related to cell membrane transport in Escherichia coli strain BG-08 was found to increase cytidine production in shake flask cultivation by 1.3-fold (0.91 ± 0.03 g/L) and 1.8-fold (1.26 ± 0.03 g/L) relative to that of the original strain (0.70 ± 0.03 g/L), respectively. Co-overexpression of uracil permease and a nucleoside transporter further increased cytidine yield by 2.7-fold (1.59 ± 0.05 g/L) compared with that of the original strain. These results indicate that the overexpressed uracil permease and nucleoside transporter can promote the accumulation of cytidine, and the two proteins play a synergistic role in the secretion of cytidine in Escherichia coli.

Genome-wide assessment of population structure and genetic diversity of Chinese Lou onion using specific length amplified fragment (SLAF) sequencing.

Lou onion (Allium fistulosum L. var. viviparum) is an abundant source of flavonols which provides additional health benefits to diseases. Genome-wide specific length amplified fragment (SLAF) sequencing method is a rapidly developed deep sequencing technologies used for selection and identification of genetic loci or markers. This study aimed to elucidate the genetic diversity of 122 onion accessions in China using the SLAF-seq method. A set of 122 onion accessions including 107 A.fistulosum L. var. viviparum Makino, 3 A.fistulosum L. var. gigantum Makino, 3 A.mongolicum Regel and 9 A.cepa L. accessions (3 whites, 3 reds and 3 yellows) from different regions in China were enrolled. Genomic DNA was isolated from young leaves and prepared for the SLAF-seq, which generated a total of 1,387.55 M reads and 162,321 high quality SNPs (integrity >0.5 and MAF >0.05). These SNPs were used for the construction of neighbor-joining phylogenetic tree, in which 10 A.fistulosum L. var. viviparum Makino accessions from Yinchuan (Ningxia province) and Datong (Qinghai province) had close genetic relationship. The 3 A.cepa L. clusters (red, white and yellow) had close genetic relationship especially with the 97 A.fistulosum L. var. viviparum Makino accessions. Population structure analysis suggested entire population could be clustered into 3 groups, while principal component analysis (PCA) showed there were 4 genetic groups. We confirmed the SLAF-seq approach was effective in genetic diversity analysis in red onion accessions. The key findings would provide a reference to the Lou onion germplasm in China.

Fermentation by Multiple Bacterial Strains Improves the Production of Bioactive Compounds and Antioxidant Activity of Goji Juice.

Microorganisms can be used for enhancing flavors or metabolizing functional compounds. The fermented-food-derived bacterial strains comprising Bacillus velezensis, Bacillus licheniformis, and Lactobacillus reuteri mixed with Lactobacillus rhamnosus and Lactobacillus plantarum were used to ferment goji berry (Lycium barbarum L.) juice in this study. The fermentation abilities and antioxidant capacities of different mixtures of multiple strains in goji juice were compared. The results showed that the lactic acid contents increased 9.24-16.69 times from 25.30 ± 0.71 mg/100 mL in goji juice fermented using the SLV (Lactobacillus rhamnosus, Lactobacillus reuteri, and Bacillus velezensis), SZP (Lactobacillus rhamnosus, Lactobacillus plantarum, and Bacillus licheniformis), and SZVP (Lactobacillus rhamnosus, Lactobacillus plantarum, Bacillus velezensis, and Bacillus licheniformis) mixtures, and the protein contents increased 1.31-2.11 times from 39.23 ± 0.67 mg/100 mL. In addition, their contents of volatile compounds increased with positive effects on aroma in the fermented juices. Conversion of the free and bound forms of phenolic acids and flavonoids in juice was influenced by fermentation, and the antioxidant capacity improved significantly. Fermentation enhanced the contents of lactic acid, proteins, volatile compounds, and phenols. The antioxidant capacity was strongly correlated with the phenolic composition.