Expression patterns and functional analysis of porcine lnc-34015.

Long noncoding RNAs (lncRNAs) play important roles in immune regulation in humans and animals. The lnc-34015 was discovered to be critical for the development of muscles, based on the muscle transcriptome of pigs; however, the underlying molecular mechanism requires better understanding. Here, the sequence characteristics of lnc-34015 were analyzed and a competitive endogenous RNA regulatory network of lncRNA was predicted. The developmental expression trend and tissue expression profiles of lnc-34015 were investigated using quantitative polymerase chain reaction. The lnc-34015 sequence is overlapped with introns 11 and 12 of CWF19L1, while CWF19L1, PKD2L1, and CHUK were identified as cis-regulatory genes of lnc-34015. Bioinformatics analyses revealed that lnc-34015 binds to 15 microRNAs (miRNAs), including miR-3646, miR-377-3p, and miR-190b-3p, to regulate downstream gene expression. GO and KEGG enrichment results show that lnc-34015 was mainly involved in cell proliferation, stress response, transcriptional regulation, and alternative splicing. The expression trend of lnc-34015 in muscle was similar to that of target genes and opposite to that of miRNAs. The expression of lnc-34015 was significantly higher in the porcine small intestine and IPEC-J2 cells. Our findings suggest that lnc-34015 regulates CHUK, ZBTB20, and XIAP gene expression by competing with endogenous RNAs to regulate porcine inflammatory responses.

HMG20A Inhibit Adipogenesis by Transcriptional and Epigenetic Regulation of MEF2C Expression.

Obesity and its associated metabolic disease do serious harm to human health. The transcriptional cascade network with transcription factors as the core is the focus of current research on adipogenesis and its mechanism. Previous studies have found that HMG domain protein 20A (HMG20A) is highly expressed in the early stage of adipogenic differentiation of porcine intramuscular fat (IMF), which may be involved in regulating adipogenesis. In this study, HMG20A was found to play a key negative regulatory role in adipogenesis. Gain- and loss-of-function studies revealed that HMG20A inhibited the differentiation of SVF cells and C3H10T1/2 cells into mature adipocytes. RNA-seq was used to screen differentially expressed genes after HMG20A knockdown. qRT-PCR and ChIP-PCR confirmed that MEF2C was the real target of HMG20A, and HMG20A played a negative regulatory role through MEF2C. HMG20A binding protein LSD1 was found to alleviate the inhibitory effect of HMG20A on adipogenesis. Further studies showed that HMG20A could cooperate with LSD1 to increase the H3K4me2 of the MEF2C promoter and then increase the expression of MEF2C. Collectively, these findings highlight a role for HMG20A-dependent transcriptional and epigenetic regulation in adipogenesis.

Electron attachment to solvated dGpdG: effects of stacking on base-centered and phosphate-centered valence-bound radical anions.

To explore the nature of electron attachment to guanine-centered DNA single strands in the presence of a polarizable medium, a theoretical investigation of the DNA oligomer dinucleoside phosphate deoxyguanylyl-3',5'-deoxyguanosine (dGpdG) was performed by using density functional theory. Four different electron-distribution patterns for the radical anions of dGpdG in aqueous solution have been located as local minima on the potential energy surface. The excess electron is found to reside on the proton of the phosphate group (dGp(H-)dG), or on the phosphate group (dGp(.-)dG), or on the nucleobase at the 5' position (dG(.-)pdG), or on the nucleobase at the 3' position (dGpdG(.-)), respectively. These four radical anions are all expected to be electronically viable species under the influence of the polarizable medium. The predicted energetics of the radical anions follows the order dGp(.-)dG>dG(.-)pdG>dGpdG(.-)>dGp(H-)dG. The base-base stacking pattern in DNA single strands seems unaffected by electron attachment. On the contrary, intrastrand H-bonding is greatly influenced by electron attachment, especially in the formation of base-centered radical anions. The intrastrand H-bonding patterns revealed in this study also suggest that intrastrand proton transfer might be possible between successive guanines due to electron attachment to DNA single strands.

Effects of Maternal Factors and Postpartum Environment on Early Colonization of Intestinal Microbiota in Piglets.

Intestinal microbiota significantly influences the intake, storage, and utilization of body nutrients, as well as animal growth and development. The establishment of microbiota is affected by many factors, such as delivery and feeding modes, antibiotics, disease, and the surrounding environment. In this study, we selected Chinese indigenous Mashen and Jinfen White pigs as the study subjects. To explore the source and factors affecting the piglet intestinal microbiota, 16S rRNA gene sequencing was performed to analyze the microbial composition of the feces, saliva, vaginal secretions, and colostrum of parturient sows, feces and saliva of newborn piglets, and surrounding environment samples. The results showed that the microbiota of the saliva of sows and piglets is structurally similar to that of the environment and is dominated by the phylum Proteobacteria, including Acinetobacter, Actinomyces, and Pseudomonas. The core genus in the vaginal secretions and colostrum of sows was Pseudomonas. Among the fecal samples, the core bacterial genera in sows before and after delivery were Clostridium sensu_stricto_1 and Christensenellaceae_R-7_group, while in piglets at 1 d of age, Pseudomonas and Escherichia-Shigella were most abundant. These results indicate that microbiota in feces, colostrum, and vaginal secretions of sows more easily colonized piglet intestines through a symbiotic effect. The environmental and salivary microbiota could also affect the early colonization and succession of the intestinal microbiota of piglets to some extent. This study provides a theoretical basis for sow delivery protection and early nursing of piglets and background for the research and development of microbial agents to improve piglet intestinal health.

Integrated Omics Analysis Reveals Alterations in the Intestinal Microbiota and Metabolites of Piglets After Starvation.

Obesity is a serious public health problem. Short-term starvation is an effective way to lose weight but can also cause harm to the body. However, a systematic assessment of the relationship between the intestinal microbiota and metabolites after complete fasting is lacking. Pigs are the best animal models for exploring the mechanisms of human nutrition digestion and absorption, metabolism, and disease treatment. In this study, 16S rRNA sequencing and liquid chromatography-mass spectrometry were used to analyze the changes in the intestinal microbiota and metabolite profiles in piglets under starvation stress. The results show that the microbial composition was changed significantly in the starvation groups compared with the control group (P < 0.05), suggesting that shifts in the microbial composition were induced by starvation stress. Furthermore, differences in the correlation of the intestinal microbiota and metabolites were observed in the different experimental groups. Starvation may disrupt the homeostasis of the intestinal microbiota and metabolite profile and affect the health of piglets. However, piglets can regulate metabolite production to compensate for the effects of short-term starvation. Our results provide a background to explore the mechanism of diet and short-term hunger for intestinal homeostasis.

circRNAome profiling reveals circFgfr2 regulates myogenesis and muscle regeneration via a feedback loop.

BACKGROUND: Circular RNAs (circRNAs) represent a novel class of non-coding RNAs formed by a covalently closed loop and play crucial roles in many biological processes. Several circRNAs associated with myogenesis have been reported. However, the dynamic expression, function, and mechanism of circRNAs during myogenesis and skeletal muscle development are largely unknown. METHODS: Strand-specific RNA-sequencing (RNA-seq) and microarray datasets were used to profile the dynamic circRNAome landscape during skeletal muscle development and myogenic differentiation. Bioinformatics analyses were used to characterize the circRNAome and identify candidate circRNAs associated with myogenesis. Bulk and single-cell RNA-seq were performed to identify the downstream genes and pathways of circFgfr2. The primary myoblast cells, C2C12 cells, and animal model were used to assess the function and mechanism of circFgfr2 in myogenesis and muscle regeneration in vitro or in vivo by RT-qPCR, western blotting, dual-luciferase activity assay, RNA immunoprecipitation, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation. RESULTS: We profiled the dynamic circRNAome in pig skeletal muscle across 27 developmental stages and detected 52 918 high-confidence circRNAs. A total of 2916 of these circRNAs are conserved across human, mouse, and pig, including four circRNAs (circFgfr2, circQrich1, circMettl9, and circCamta1) that were differentially expressed (|log2 fold change| > 1 and adjusted P value < 0.05) in various myogenesis systems. We further focused on a conserved circRNA produced from the fibroblast growth factor receptor 2 (Fgfr2) gene, termed circFgfr2, which was found to inhibit myoblast proliferation and promote differentiation and skeletal muscle regeneration. Mechanistically, circFgfr2 acted as a sponge for miR-133 to regulate the mitogen-activated protein kinase kinase kinase 20 (Map3k20) gene and JNK/MAPK pathway. Importantly, transcription factor Kruppel like factor 4 (Klf4), the downstream target of the JNK/MAPK pathway, directly bound to the promoter of circFgfr2 and affected its expression via an miR-133/Map3k20/JNK/Klf4 auto-regulatory feedback loop. RNA binding protein G3BP stress granule assembly factor 1 (G3bp1) inhibited the biogenesis of circFgfr2. CONCLUSIONS: The present study provides a comprehensive circRNA resource for skeletal muscle study. The functional and mechanistic analysis of circFgfr2 uncovered a circRNA-mediated auto-regulatory feedback loop regulating myogenesis and muscle regeneration, which provides new insight to further understand the regulatory mechanism of circRNAs.

Comparative Evaluation of the Ileum Microbiota Composition in Piglets at Different Growth Stages.

Intestinal microbiota can affect the intake, storage, and absorption of nutrients in the body, thereby greatly impacting the growth and development of animals. In addition to diet, the breed and growth stages of pigs could also affect changes in the intestinal microbiota. However, research on the developmental changes in the ileum microbiota of piglets remains unclear. In this study, the ileum microbiota of Jinfen White and Mashen piglets at different developmental stages were investigated using 16S rRNA sequencing. Physiologically, the villus height of the ileum decreased, and the crypt depth increased during the development of the two pig breeds. Additionally, the serum antioxidant factors in the Jinfen White piglets were significantly higher than in the Mashen piglets at the end of the nursing stage. A total of 690 operational taxonomic units (OTUs) belonging to 21 phyla and 286 genera were identified, of which Firmicutes and Proteobacteria were the dominant phyla during the development of both the Jinfen White and Mashen piglets, accounting for ∼90% of all OTUs. Further research revealed differences in dominant bacteria between the two breeds. With increasing age, the ileum microbial diversity increased, and in both the pig breeds, the proportion of Firmicutes increased, whereas the proportion of Proteobacteria decreased. Additionally, different samples were characterized by specific genera, and different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were predicted at certain developmental stages. Finally, the correlation between the ileum microbiota and physiological features was analyzed, and it was suggested that the host and environmental factors play important roles in the formation of the microbial community structure in piglets. In summary, we delineated the structure, function, and differences in ileum microbiota between Jinfen White and Mashen piglets during different growth stages. This study helps to understand the development of the intestinal microbiota in local and hybrid pig breeds.

Identification of Ovarian Circular RNAs and Differential Expression Analysis between MeiShan and Large White Pigs.

MeiShan and Large White pigs differ in their female fecundity. However, the mechanisms behind the gene expression and regulation that cause these differences remain unclear. In this study, we profiled circRNAs and identified 5,879 circRNAs from the ovaries of MeiShan and Large White pigs. Eighty-five circRNAs were differentially expressed between the two pig breeds. Of these, 37 were up-regulated and 48 were down-regulated in MeiShan pigs. Gene ontology enrichment analysis suggested that the differentially expressed circRNA were involved in the hormone-mediated signaling pathway. We verified that circSCIN and its parent gene, scinderin (SCIN), were differentially expressed by reverse transcription and quantitative PCR (RT-qPCR). Luciferase assays demonstrated that circSCIN can target and sponge miR-133 and miR-148a/b. The identification of differentially expressed circRNAs (DECs) and their regulatory functions increased our understanding of the differences in reproductive efficiency between MeiShan and Large White pigs.

The possibility of the decomposition of 2'-deoxyribose moiety of thymidine induced by the low energy electron attachment.

To evaluate the possibility of the decomposition of 2-deoxyribose moiety of thymidine induced by low energy electrons (LEE) attachment, the transition states and the energy barriers of the bond breaking processes of the ribose of the nucleoside have been studied theoretically by applying the density functional theory with the double zeta basis sets (DZP++). The energy barriers for the breakage of the C-C bonds (C(1')-C(2'), C(2')-C(3'), C(3')-C(4'), and C(4')-C(5')) of the ribose group of the radical anion of thymidine are found to be high (ca. 42-57 kcal/mol). The total energies of the C-C bond-broken products are significantly higher than that of the radical anion dT(*-). The decomposition of dT(*-) through the C-C bond rupture is unlikely to take place. The rupture of the C(1')-O(4') bond of dT(*-) needs an activation energy as low as 10.4 kcal/mol. However, the reversed reaction (C(1')-O(4') bond formation) needs the activation energy low as 0.3 kcal/mol. Therefore, the intermediate product LM1(C1')-(O4') is unlikely to be stable and the C(1')-O(4') bond-broken is not favored. The activation energy of the C(4')-O(4') bond rupture process amounts to 20.5 kcal/mol. The total energy of the C(4')-O(4') bond broken product is about 6.5 kcal/mol lower than that of the reactant dT(*-). The subsequent N1-glycosidic bond breaking process is found to have a very low energy barrier. Therefore, the LEE-induced base release through the C(4')-O(4') bond rupture might be a possible pathway.

Genome-wide profiling of Sus scrofa circular RNAs across nine organs and three developmental stages.

The spatio-temporal expression patterns of Circular RNA (circRNA) across organs and developmental stages are critical for its function and evolution analysis. However, they remain largely unclear in mammals. Here, we comprehensively analysed circRNAs in nine organs and three skeletal muscles of Guizhou miniature pig (S. scrofa), a widely used biomedical model animal. We identified 5,934 circRNAs and analysed their molecular properties, sequence conservation, spatio-temporal expression pattern, potential function, and interaction with miRNAs. S. scrofa circRNAs show modest sequence conservation with human and mouse circRNAs, are flanked by long introns, exhibit low abundance, and are expressed dynamically in a spatio-temporally specific manner. S. scrofa circRNAs show the greatest abundance and complexity in the testis. Notably, 31% of circRNAs harbour well-conserved canonical miRNA seed matches, suggesting that some circRNAs act as miRNAs sponges. We identified 149 circRNAs potentially associated with muscle growth and found that their host genes were significantly involved in muscle development, contraction, chromatin modification, cation homeostasis, and ATP hydrolysis-coupled proton transport; moreover, this set of genes was markedly enriched in genes involved in tight junctions and the calcium signalling pathway. Finally, we constructed the first public S. scrofa circRNA database, allowing researchers to query comprehensive annotation, expression, and regulatory networks of circRNAs.

Comparative analysis of DNA methylome and transcriptome of skeletal muscle in lean-, obese-, and mini-type pigs.

DNA methylation plays a pivotal role in biological processes by affecting gene expression. However, how DNA methylation mediates phenotype difference of skeletal muscle between lean-, obese-, and mini-type pigs remains unclear. We systematically carried out comparative analysis of skeletal muscle by integrating analysis of genome-wide DNA methylation, mRNA, lncRNA and miRNA profiles in three different pig breeds (obese-type Tongcheng, lean-type Landrace, and mini-type Wuzhishan pigs). We found that the differentially methylated genes (DMGs) were significantly associated with lipid metabolism, oxidative stress and muscle development. Among the identified DMGs, 253 genes were related to body-size and obesity. A set of lncRNAs and mRNAs including UCP3, FHL1, ANK1, HDAC4, and HDAC5 exhibited inversely changed DNA methylation and expression level; these genes were associated with oxidation reduction, fatty acid metabolism and cell proliferation. Gene regulatory networks involved in phenotypic variation of skeletal muscle were related to lipid metabolism, cellular movement, skeletal muscle development, and the p38 MAPK signaling pathway. DNA methylation potentially influences the propensity for obesity and body size by affecting gene expression in skeletal muscle. Our findings provide an abundant information of epigenome and transcriptome that will be useful for animal breeding and biomedical research.

Recent progress on the applications of multifunctional glyconanoparticles.

Glyconanoparticles are very useful tools for proteomic and glycomics research. They mainly contain glycosylated gold nanoparticles, glycosylated quantum dots, and magnetic glyconanoparticles. This review summarized the glyconanoparticle progress on biolabeling, in vitro or in vivo imaging, biosensing, targeted drug delivery, and other biomedical applications in recent years. The core of glyconanoparticle applications is to study the carbohydrate-mediated interactions, which opens the new field in glycobiology.

Microsolvation pattern of the hydrated radical anion of uracil: U-(H2O)n (n = 3-5).

The microsolvation patterns of the uracil radical anion in water clusters U-(H2O)n with n ranging from 3 to 5 were investigated by the density functional theory approach. The electron detachment energies (VDE) of the stable anionic complexes with different numbers of hydration water are predicted. The linear dependence of the VDE value of the most stable anionic complexes with respect to the hydration number suggests the importance of the clustered waters in the microsolvation of the radical anion of the nucleobases. The formation of the water clusters is found to be necessary in the most stable conformers of the tri-, tetra-, and pentahydrated radical anion of uracil. The microsolvation pattern with three or more well-separated hydration water molecules in the first hydration layer is less stable than the arrangement with the waters in tight clusters. The charge transfer between the anionic uracil and the hydration water is high. Good agreement between the experimental and the theoretical vertical detachment energy yield in this study further demonstrates the practicability of the B3LYP/DZP++ approach in the study of radical anions of the DNA subunits.