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.

MicroRNA-2285f regulates milk fat metabolism by targeting MAP2K2 in bovine mammary epithelial cells.

In this study, Holstein dairy cows raised in Ningxia were selected as the research object. Mammary epithelial cells (BMECs) were extracted from the milk of eight Holstein cows with significantly different milk fat expression rates and transcribed for sequencing. Bioinformatics analysis was used to analyse the correlation of fat milk percentage, and the critical miR-2285f regulating milk fat was screened out. The target gene binding sites were predicted, and 293T cells and mammary epithelial cells were used as miRNA and target gene models for functional verification in vitro. The tissue difference of miR-2285f Holstein cows was quantitatively analysed by transfecting miR-2285f mimic and inhibitor. Assay (dual luciferase reporter gene assay) and quantitative real-time PCR (quantitative real-time PCR, qRT-PCR), triglyceride (TAG) detection, oil red O detection of lipid droplets, Western Blot assay, Edu and Flow cytometry, The molecular regulatory effects of miR-2285f and target gene MAP2K2 on milk fat metabolism of Holstein dairy cows were studied. The wild-type vector and mutant vector of map2k2-3'utr were constructed, and double luciferase reporting experiments were conducted to verify that MAP2K2 was one of the target genes of miR-2285f. According to qRT-PCR and Western Blot analysis, miR-2285f mainly regulates the expression of MAP2K2 protein in BMECs at the translation level. Bta-miR-2285f can promote cell proliferation and slow cell apoptosis by regulating MAP2K2. Bta-miR-2285f can promote triglyceride (TAG) and lipid droplet accumulation in mammary epithelial cells by targeting MAP2K2. Bta-miR-2285f can regulate protein levels of fat milk marker gene PPARG by targeting MAP2K2. In conclusion, miR-2285f can target the expression of the MAP2K2 gene, promote the proliferation of dairy mammary epithelial cells, inhibit cell apoptosis and regulate the milk fat metabolism in dairy mammary epithelial cells. The results of this study revealed the function of miR-2285f in regulating the differential expression of fat milk in Holstein dairy cows at the cellular level. They provided a theoretical and experimental basis for analysing the regulation network of milk fat synthesis of Holstein dairy cows and the molecular breeding of dairy cows.

Identification of critical lncRNAs for milk fat metabolism in dairy cows using WGCNA and the construction of a ceRNAs network.

As key regulators, long non-coding RNAs (lncRNAs) play a crucial role in the ruminant mammary gland. However, the function of lncRNAs in milk fat synthesis from dairy cows is largely unknown. In this study, we used the weighted gene co-expression network analysis (WGCNA) to comprehensive analyze the expression profile data of lncRNAs from the group's previous Illumina PE150 sequencing results based on bovine mammary epithelial cells from high- and low-milk-fat-percentage (MFP) cows, and identify core_lncRNAs significantly associated with MFP by module membership (MM) and gene significance (GS). Functional enrichment analysis (Gene Ontology, Kyoto Encyclopedia of Genes and Genomes) of core_lncRNA target genes (co-localization and co-expression) was performed to screen potential lncRNAs regulating milk fat metabolism and further construct an interactive regulatory network of lipid metabolism-related competing endogenous RNAs (ceRNAs). A total of 4876 lncRNAs were used to construct the WGCNA. The MEdarkturquoise module among the 19 modules obtained was significantly associated with MFP (r = 0.78, p-value <0.05) and contained 64 core_lncRNAs (MM > 0.8, GS > 0.4). Twenty-four lipid metabolism-related lncRNAs were identified by core_lncRNA target gene enrichment analysis. TCONS_00054233, TCONS_00152292, TCONS_00048619, TCONS_00033839, TCONS_00153791 and TCONS_00074642 were key candidate lncRNAs for regulating milk fat synthesis. The 22 ceRNAs most likely to be involved in milk fat metabolism were constructed by interaction network analysis, and TCONS_00133813 and bta-miR-2454-5p were located at the network's core. TCONS_00133813_bta-miR-2454-5p_TNFAIP3, TCONS_00133813_bta-miR-2454-5p_ARRB1 and TCONS_00133813_bta-miR-2454-5p_PIK3R1 are key candidate ceRNAs associated with milk fat metabolism. This study provides a framework for the co-expression module of MFP-related lncRNAs in ruminants, identifies several major lncRNAs and ceRNAs that influence milk fat synthesis, and provides a new understanding of the complex biology of milk fat synthesis in dairy cows.

Modulation of broiler plasma metabolic spectrum by the addition of lysine residue to the diet.

Flavour is an important factor in evaluating meat quality, and amino acids and fats are important components affecting meat flavour. In this study, we evaluated the relationship between the variation of lysine residue addition and the slaughter performance and meat quality of broilers, which decreased with the addition of lysine residues but improved the meat quality of the broilers. 10% lysine residue addition was the most beneficial for reducing feed cost and improving meat quality. Meanwhile, the plasma metabolites of broilers fed increasing concentrations of lysine residue supplemented feeds were analysed using liquid chromatography-mass spectrometry (LC-MS). Principal component analysis (PCA) and partial least square discriminant analysis (OPLS-DA) were used screen, the differential metabolites induced by lysine residue. In the broilers 29, 37, 63, 87, 80 and 111 differential metabolites were detected (p < 0.05). Amongst them, 3-iodotyrosine, N-methyl-L-glutamic acid, coumaraldehyde, 2-dimethylphenol, N-methylnicotinamide and L-erythrone were the common differential metabolites between group A and groups B, C, D, E, F and G. The addition of lysine residue was positively correlated with alanine aminotransferase (p < 0.05, r = 0.942) and high-density lipoprotein cholesterol (p < 0.05, r = 0.798) and negatively correlated with aspartate aminotransferase (p < 0.05, r = 0.822). According to the classification of differential metabolites and their enriched pathway analysis, differential metabolites mainly caused changes in amino acid and lipid metabolism. Our study shows that a certain proportion of lysine residue in diet affects the specific metabolic pathway of broilers, which may affect amino acid and fat metabolism by regulating alanine aminotransferase, aspartate aminotransferase and high-density lipoprotein cholesterol, ultimately affecting the flavour.

Association of D-dimer and acute kidney injury associated with rhabdomyolysis in patients with exertional heatstroke: an over 10-year intensive care survey.

Patients with rhabdomyolysis (RM) following exertional heatstroke (EHS) are often accompanied by dysfunction of coagulation and acute kidney injury (AKI). The purpose of this study was to investigate the relationship between D-dimer and AKI in patients with RM following EHS. A retrospective study was performed on patients with EHS admitted to the intensive care unit over 10-year. Data including baseline clinical information at admission, vital organ dysfunction, and 90-day mortality were collected. A total of 84 patients were finally included, of whom 41 (48.8%) had AKI. AKI patients had more severe organ injury and higher 90-day mortality (34.1 vs.0.0%, p < 0.001) than non-AKI patients. Multivariate logistic analysis showed that D-dimer (OR 1.3, 95% CI 1.1-1.7, p = 0.018) was an independent risk factor for AKI with RM following EHS. Curve fitting showed a curve relationship between D-dimer and AKI. Two-piecewise linear regression showed that D-dimer was associated with AKI in all populations (OR 1.3, 95% CI 1.2-1.5, p < 0.001) when D-dimer <10.0 mg/L, in RM group (OR 1.3, 95% CI 1.1-1.5, p < 0.001) when D-dimer >0.4 mg/L, in the non-RM group (OR 6.4, 95% CI 1.7-23.9, p = 0.005) when D-dimer <1.3 mg/L and D-dimer did not increase the incidence of AKI in the non-RM group when D-dimer >1.3 mg/L. AKI is a life-threatening complication of RM following EHS. D-dimer is associated with AKI in critically ill patients with EHS. The relationship between D-dimer and AKI depends on whether RM is present or not.

Life threatening biphasic adverse reactions to desmopressin: case report and review of the literature.

Treatment with desmopressin diacetate arginine vasopressin (DDAVP) and its withdrawal are associated with side effects. We present a rare case of severe biphasic adverse reactions induced by DDAVP and its withdrawal in a 63-year-old female patient. A lump in the left axillary region was biopsied, and she received DDAVP after surgery. The following day, she lost consciousness, with foaming at the mouth and seizures. Hypotonic encephalopathy was considered. DDAVP was ceased, and she received electrolytes. On day 1, she displayed low blood pressure and increased urine output. She received DDAVP and dopamine as well as electrolytes. The patient was ambulatory on day 7 and was discharged without brain abnormalities on MRI. In conclusion, severe hyponatremia induced by DDAVP and massive polyuria and hypovolemic shock induced by DDAVP withdrawal are life-threatening conditions. This case underlines the need to be vigilant when administering DDAVP and to monitor for any side effects.

Intermittent Stretching and Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells via the p38MAPK-Osterix Signaling Pathway.

AIMS: The relationship between the p38MAPK signaling pathway and osterix in osteogenic differentiation of BMMSCs subjected to intermittent stretching was investigated. METHODS: BMMSCs derived from C57BL/6J mice were divided into the following groups: 1) control, 2) stretch, and 3) SB203580+stretch (SB203580 is a p38MAPK signal pathway inhibitor). BMMSCs were exposed to an intermittent mechanical strain of 0.8% (8000µ strain) at 0.5 Hz, twice a day for 30 min each application. BMMSCs were harvested on days 1, 3, and 5 post-treatment. The expression of ALP, COL I, OCN, and osterix mRNA was assessed utilizing RT-PCR while the expression of P-p38MAPK and osterix protein was assessed by Western blot analysis. The osterix gene in mouse BMMSCs was knocked down using RNAi technology and its protein expression was also assessed by Western blot. RT-PCR was used to detect ALP, COL I, and OCN mRNA expression. RESULTS: Intermittent stretching was found to promote expression of ALP, COL I, OCN, and osterix mRNA. Silencing the osterix gene was found to reduce levels of ALP, COL I, and OCN mRNA. Western blot analysis demonstrated that the levels of osterix and P-p38MAPK proteins in the stretch group were significantly higher than in the control group (P<0.05). There was less expression of ALP, COL I, OCN, and osterix mRNA in the SB203580+stretch group than in the control and stretch groups. CONCLUSIONS: Data demonstrate that intermittent stretching promotes osteogenic differentiation of BMMSCs, and the p38MAPK-osterix pathway has an important role in the control of osteogenesis-related gene expression.

Biomarkers for assessing mucosal barrier dysfunction induced by chemotherapy: Identifying a rapid and simple biomarker.

BACKGROUND: Chemotherapy-induced mucosal barrier dysfunction is of clinical interest. However, the assessment of mucosal barrier dysfunction still poses challenges. In this study, we compared several biomarkers with the dual sugar gut permeability test for assessing mucosal barrier dysfunction during chemotherapy. METHODS: Forty-two patients with gastric or colorectal cancer underwent chemotherapy, including FAM or FOLFOX4 regimens. Patients were asked to grade and record their symptoms of gastrointestinal toxicity daily. The urinary lactulose-mannitol ratio was measured to assess the intestinal permeability. Plasma levels of citrulline, diamine oxidase (DAO), D-lactic acid, and endotoxin were also measured. Intestinal permeability was observed in the subgroup of patients with diarrhea or constipation. RESULTS: The urinary lactulose-mannitol ratio and plasma citrulline levels increased on the third and sixth post-chemotherapy days, respectively. There were no significant differences in the plasma levels of D-lactic acid, endotoxin or DAO activity compared to their levels before chemotherapy. The urinary lactulose-mannitol ratio in diarrhea patients was significantly higher than in constipation patients. CONCLUSIONS: These results indicate that the urinary lactulose-mannitol ratio and plasma citrulline level are appropriate biomarkers for assessing mucosal barrier dysfunction in patients receiving chemotherapy. Mucosal barrier dysfunction in diarrhea patients was greater than in constipation patients.

3D micro/nano hydrogel structures fabricated by two-photon polymerization for biomedical applications.

Hydrogels are three-dimensional natural or synthetic cross-linked networks composed of polymer chains formed by hydrophilic monomers. Due to the ability to simulate many properties of natural extracellular matrix, hydrogels have been widely used in the biomedical field. Hydrogels can be obtained through a variety of polymerization strategies such as heating and redox. However, photochemistry is one of the most interesting methods for researchers in this field. Gelatin-methacryloyl (GelMA) inherits the biological activity of gelatin and has become one of the gold standards in the field of biomaterials. GelMA, as a photopolymerizable hydrogel precursor, can be used to fabricate 3D porous structures for biomedical applications through two-photon polymerization. We report a new formulation of GelMA-based photoresist and used it to manufacture a series of two-photon polymerization structures, with a maximum resolution less than 120 nm. The influence of process parameters on 3D structures manufacturing is studied by adjusting the scanning speed, laser power, and layer spacing values in two-photon polymerization processing. In vitro biological tests show that the 3D hydrogel produced by two-photon polymerization in this paper is biocompatible and suitable for MC3T3-E1 cell.

Transcriptome analysis reveals the role of Long noncoding RNAs (lncRNA) in specific deposition of inosine monphosphate in Jingyuan chickens.

Inosine monphosphate (IMP) is one of the important indicators for evaluating meat flavor, and long noncoding RNAs (lncRNAs) play an important role in its transcription and post-transcriptional regulation. Currently, there is little information about how lncRNA regulates the specific deposition of IMP in chicken muscle. In this study, we used transcriptome sequencing to analyze the lncRNAs of the breast and leg muscles of the Jingyuan chicken and identified a total of 357 differentially expressed lncRNAs (DELs), of which 158 were up-regulated and 199 were down-regulated. There were 2,203 and 7,377 cis- and trans-regulated target genes of lncRNAs, respectively, and we identified the lncRNA target genes that are involved in NEGF signaling pathway, glycolysis/ glucoseogenesis and biosynthesis of amino acids pathways. Meanwhile, 621 pairs of lncRNA-miRNA-mRNA interaction networks were constructed with target genes involved in purine metabolism, fatty acid metabolism, and biosynthesis of amino acids. Next, three interacting meso-networks gga-miR-1603-LNC_000324-PGM1, gga-miR-1768-LNC_000324-PGM1 and gga-miR-21-LNC_011339- AMPD1 were identified as closely associated with IMP-specific deposition. Both differentially expressed genes (DEGs) PGM1 and AMPD1 were significantly enriched in IMP synthesis and metabolism-related pathways, and participated in the anabolic process of IMP in the form of organic matter synthesis and energy metabolism. This study obtained lncRNAs and target genes affecting IMP-specific deposition in Jingyuan chickens based on transcriptome analysis, which deepened our insight into the role for lncRNAs in chicken meat quality.

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.

PKM2 promotes myoblast growth and inosine monophosphate-specific deposition in Jingyuan chicken.

Inosine monophosphate (IMP) is widely regarded as an important indicator for evaluating the flavour of poultry meat. However, little is known about the molecular mechanisms affecting the specific deposition of IMP. In this study, we functionally verified PKM2 (Pyruvate kinase M2), a candidate gene related to IMP synthesis, in order to reveal the important role of PKM2 in meat flavour and muscle development of Jingyuan chickens. The results showed that the IMP content in breast muscle of Jingyuan chickens was negatively correlated with PKM2 mRNA expression (r = -0.1710), while the IMP content in leg muscle was significantly positively correlated with PKM2 mRNA expression (r = 0.7350) (P < 0.05). During myogenesis, PKM2 promoted the proliferation rate of myoblasts and the expression of proliferation marker genes, inhibited the apoptosis rate and the expression of apoptosis marker genes, and decreased the expression of differentiation marker genes. Up-regulation of PKM2 enhanced the expression of key genes in the purine metabolic pathway and the de novo synthesis pathway of IMP, and suppressed the expression of key genes in the salvage pathway. ELISA assays showed that PKM2 decreased IMP and hypoxanthine (HX) contents, while adenosine triphosphate (ATP) and uric acid (UA) contents were clearly elevated. In summary, these studies revealed that PKM2 regulates myogenesis and specific deposition of IMP, which can be used to improve the quality of Jingyuan chicken meat.

Identification of key differentially methylated genes in regulating muscle development and intramuscular fat deposition in chickens.

Muscle development and intramuscular fat (IMF) deposition are intricate physiological processes characterized by multiple gene expressions and interactions. In this research, the phenotypic variations in the breast muscle of Jingyuan chickens were examined at three different time points: 42, 126, and 180 days old. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were performed to identify differentially methylated genes (DMGs) responsible for regulating muscle development and IMF deposition. The findings indicate a significant increase in breast muscle weight (BMW), myofiber diameter, and cross-sectional area, as well as IMF content, in correlation with the progressive number of growing days in Jingyuan chickens. The findings also revealed that 380 hypo-methylated and 253 hyper-methylated DMGs were identified between the three groups of breast muscle. Module gene and DMG association analysis identified m6A methylation-mediated multiple DMGs associated with muscle development and fat metabolism. In vitro cell modeling analysis reveals stage-specific differences in the expression of CUBN, MEGF10, BOP1, and BMPR2 during the differentiation of myoblasts and intramuscular preadipocytes. Cycloleucine treatment significantly inhibited the expression levels of CUBN, BOP1, and BMPR2, and promoted the expression of MEGF10. These results suggest that m6A methylation-mediated CUBN, MEGF10, BOP1, and BMPR2 can serve as potential candidate genes for regulating muscle development and IMF deposition, and provide an important theoretical basis for further investigation of the functional mechanism of m6A modification involved in adipogenesis.

Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.).

BACKGROUND: Eukaryotic mitogen-activated protein kinase (MAPK/MPK) signaling cascades transduce and amplify environmental signals via three types of reversibly phosphorylated kinases to activate defense gene expression. Canola (oilseed rape, Brassica napus) is a major crop in temperate regions. Identification and characterization of MAPK and MAPK kinases (MAPKK/MKK) of canola will help to elucidate their role in responses to abiotic and biotic stresses. RESULTS: We describe the identification and analysis of seven MKK (BnaMKK) and 12 MPK (BnaMPK) members from canola. Sequence alignments and phylogenetic analyses of the predicted amino acid sequences of BnaMKKs and BnaMPKs classified them into four different groups. We also examined the subcellular localization of four and two members of BnaMKK and BnaMPK gene families, respectively, using green fluorescent protein (GFP) and, found GFP signals in both nuclei and cytoplasm. Furthermore, we identified several interesting interaction pairs through yeast two-hybrid (Y2H) analysis of interactions between BnaMKKs and BnaMPKs, as well as BnaMPK and BnaWRKYs. We defined contiguous signaling modules including BnaMKK9-BnaMPK1/2-BnaWRKY53, BnaMKK2/4/5-BnaMPK3/6-BnaWRKY20/26 and BnaMKK9-BnaMPK5/9/19/20. Of these, several interactions had not been previously described in any species. Selected interactions were validated in vivo by a bimolecular fluorescence complementation (BiFC) assay. Transcriptional responses of a subset of canola MKK and MPK genes to stimuli including fungal pathogens, hormones and abiotic stress treatments were analyzed through real-time RT-PCR and we identified a few of BnaMKKs and BnaMPKs responding to salicylic acid (SA), oxalic acid (OA), Sclerotinia sclerotiorum or other stress conditions. Comparisons of expression patterns of putative orthologs in canola and Arabidopsis showed that transcript expression patterns were generally conserved, with some differences suggestive of sub-functionalization. CONCLUSIONS: We identified seven MKK and 12 MPK genes from canola and examined their phylogenetic relationships, transcript expression patterns, subcellular localization, and protein-protein interactions. Not all expression patterns and interactions were conserved between canola and Arabidopsis, highlighting the limitations of drawing inferences about crops from model species. The data presented here provide the first systematic description of MKK-MPK-WRKY signaling modules in canola and will further improve our understanding of defense responses in general and provide a basis for future crop improvement.

A novel energy harvester based on dual vibrating mechanisms with self-actuation.

This paper introduced a novel energy harvester with a tunnel and drop-shaped bluffbody for self-actuation and wind speed sensing. The harvester exhibits dual vibrating mechanisms of vortex-induced vibration (VIV) and galloping. Theoretical and numerical analyses were conducted to study the energy conversion relationship and fluid field of the harvester, and the conclusions were verified by controlled variable experiments. The optimal design values of inlet angle I 40°, polyvinylidene fluoride (PVDF) angle P 10°, and exit angle E 10° were demonstrated with the highest output of 10.42 Vp-p at the wind speed of 18 m/s. The output voltage of the PVDF energy harvester has a reliable relationship with the wind speed as a function of wind speed sensor, which could be applied for meteorological information collection and fluid flow rate monitoring with further study conducted underwater.

Transcriptome and co-expression network analysis reveals the molecular mechanism of inosine monophosphate-specific deposition in chicken muscle.

The inosine monophosphate (IMP) content in chicken meat is closely related to muscle quality and is an important factor affecting meat flavor. However, the molecular regulatory mechanisms underlying the IMP-specific deposition in muscle remain unclear. This study performed transcriptome analysis of muscle tissues from different parts, feeding methods, sexes, and breeds of 180-day-old Jingyuan chickens, combined with differential expression and weighted gene co-expression network analysis (WGCNA), to identify the functional genes that regulate IMP deposition. Out of the four comparison groups, 1,775, 409, 102, and 60 differentially expressed genes (DEGs) were identified, of which PDHA2, ACSS2, PGAM1, GAPDH, PGM1, GPI, and TPI1 may be involved in the anabolic process of muscle IMP in the form of energy metabolism or amino acid metabolism. WGCNA identified 11 biofunctional modules associated with IMP deposition. The brown, midnight blue, red, and yellow modules were strongly correlated with IMP and cooking loss (p < 0.05). Functional enrichment analysis showed that glycolysis/gluconeogenesis, arginine and proline metabolism, and pyruvate metabolism, regulated by PYCR1, SMOX, and ACSS2, were necessary for muscle IMP-specific deposition. In addition, combined analyses of DEGs and four WGCNA modules identified TGIF1 and THBS1 as potential candidate genes affecting IMP deposition in muscle. This study explored the functional genes that regulate muscle development and IMP synthesis from multiple perspectives, providing an important theoretical basis for improving the meat quality and molecular breeding of Jingyuan chickens.

MicroRNA-19a regulates milk fat metabolism by targeting SYT1 in bovine mammary epithelial cells.

MicroRNAs (miRNAs) are important post-transcriptional factors involved in the regulation of gene expression and play crucial roles in biological processes related to milk fat metabolism. Our previous study revealed that miR-19a expression was significantly higher in the mammary epithelial cells of high-milk fat cows than in those of low-milk fat cows. However, the precise molecular mechanisms underlying these differences remain unclear. In this study, we found a high expression of miR-19a in the mammary tissues of dairy cows. The regulatory effects of miR-19a on bovine mammary epithelial cells (BMECs) were analyzed using cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays, which demonstrated that miR-19a significantly inhibited BMEC proliferation. Transfection of the miR-19a mimic into BMECs significantly upregulated the expression of milk fat marker genes LPL, SCAP, and SREBP1, promoting triglyceride (TG) synthesis and lipid droplet formation, whereas the miR-19a inhibitor exhibited the opposite function. TargetScan and miRWalk predictions revealed that synaptotagmin 1 (SYT1) is a target gene of miR-19a. A dual luciferase reporter gene assay, RT-qPCR, and western blot analyses revealed that miR-19a directly targets the 3'-untranslated region (UTR) of SYT1 and negatively regulates SYT1 expression. Functional validation revealed that overexpression of SYT1 in BMECs significantly downregulated the expression of LPL, SCAP, and SREBP1, and inhibited TG synthesis and lipid droplet formation. Conversely, the knockdown of SYT1 had the opposite effect. Altogether, miR-19a plays a crucial role in regulating the proliferation and differentiation of BMECs and regulates biological processes related to TG synthesis and lipid droplet formation by suppressing SYT1 expression. These findings provide a strong foundation for further research on the functional mechanisms underlying milk fat metabolism in dairy cows.

Regulatory role of N6-methyladenosine in intramuscular fat deposition in chicken.

Intramuscular fat (IMF) has a pivotal influence on meat quality, with its deposition being a multifaceted physiological interaction of several regulatory factors. N6-methyladenosine (m6A), the preeminent epigenetic alteration among eukaryotic RNA modifications, holds a crucial role in moderating post-transcriptional gene expression. However, there is a dearth of comprehensive understanding regarding the functional machinery of m6A modification in the context of IMF deposition in poultry. Our current study entails an analysis of the disparities in IMF within the breast and leg of 180-day-old Jingyuan chickens. We implemented methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to delve into the distribution of m6A and its putative regulatory frameworks on IMF deposition in chickens. The findings demonstrated a markedly higher IMF content in leg relative to breast (P < 0.01). Furthermore, the expression of METTL14, WTAP, FTO, and ALKBH5 was significantly diminished in comparison to that of breast (P < 0.01). The m6A peaks in the breast and leg primarily populated 3'untranslated regions (3'UTR) and coding sequence (CDS) regions. The leg, when juxtaposed with the breast, manifested 176 differentially methylated genes (DMGs), including 151 hyper-methylated DMGs and 25 hypo-methylated DMGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a pronounced enrichment of DMGs in the biosynthesis of amino acids, peroxisome, Fatty acid biosynthesis, fatty acid elongation, and cell adhesion molecules (CAMs) pathways. Key DMGs, namely ECH1, BCAT1, and CYP1B1 were implicated in the regulation of muscle lipid anabolism. Our study offers substantial insight and forms a robust foundation for further exploration of the functional mechanisms of m6A modification in modulating IMF deposition. This holds profound theoretical importance for improving and leveraging meat quality in indigenous chicken breeds.

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.