RASGRP1 targeted by H3K27me3 regulates myoblast proliferation and differentiation in mice and pigs.

Skeletal muscle is not only the largest organ in the body that is responsible for locomotion and exercise but also crucial for maintaining the body's energy metabolism and endocrine secretion. The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important histone modifications that participates in muscle development regulation by repressing the transcription of genes. Previous studies indicate that the RASGRP1 gene is regulated by H3K27me3 in embryonic muscle development in pigs, but its function and regulatory role in myogenesis are still unclear. In this study, we verify the crucial role of H3K27me3 in RASGRP1 regulation. The gain/loss function of RASGRP1 in myogenesis regulation is performed using mouse myoblast C2C12 cells and primarily isolated porcine skeletal muscle satellite cells (PSCs). The results of qPCR, western blot analysis, EdU staining, CCK-8 assay and immunofluorescence staining show that overexpression of RASGRP1 promotes cell proliferation and differentiation in both skeletal muscle cell models, while knockdown of RASGRP1 leads to the opposite results. These findings indicate that RASGRP1 plays an important regulatory role in myogenesis in both mice and pigs.

Research on the development and testing methods of physical education and agility training equipment in universities.

Introduction: Because of the problems of insufficient funds and traditional training methods in college sports agile training, an agile training system based on a wireless ad hoc network was developed to evaluate the effect of improving the sensitive quality of ordinary college students. Based on the ESP-MESH network, the lower computer realizes automatic networking between devices and tests the performance of the mesh network. Fourteen male college students received 9 weeks of agility training, with seven students in each of two groups: traditional agility training and agile equipment training. The researchers evaluated the performance of both groups in rapid disguise, body coordination, changing movements, and predictive decision-making. Results: There was no significant difference between the groups before training, but there were significant differences in the four abilities after training (p < 0.01). The experimental group had significant differences in rapid direction change and physical coordination (p < 0.05), and in changing movement and predictive decision-making ability (p < 0.01). Conclusion: Both traditional training and agile equipment training improve the agility quality of college students, and the latter shows better results in certain abilities. However, limited by other physical qualities, the improvement of motor changes and predictive decision-making ability is not as obvious as the other two abilities.

Integrated Analysis of DNA Methylation and Gene Expression in Porcine Placental Development.

Proper placental development is crucial for the conceptus to grow and survive, because the placenta is responsible for transporting nutrients and oxygen from the pregnant female to the developing fetus. However, the processes of placental morphogenesis and fold formation remain to be fully elucidated. In this study, we used whole-genome bisulfite sequencing and RNA sequencing to produce a global map of DNA methylation and gene expression changes in placentas from Tibetan pig fetuses 21, 28, and 35 days post-coitus. Substantial changes in morphology and histological structures at the uterine-placental interface were revealed via hematoxylin-eosin staining. Transcriptome analysis identified 3959 differentially expressed genes (DEGs) and revealed the key transcriptional properties in three stages. The DNA methylation level in the gene promoter was negatively correlated with gene expression. We identified a set of differentially methylated regions associated with placental developmental genes and transcription factors. The decrease in DNA methylation level in the promoter was associated with the transcriptional activation of 699 DEGs that were functionally enriched in cell adhesion and migration, extracellular matrix remodeling, and angiogenesis. Our analysis provides a valuable resource for understanding the mechanisms of DNA methylation in placental development. The methylation status of different genomic regions plays a key role in establishing transcriptional patterns from placental morphogenesis to fold formation.

Comprehensive analysis of pre-mRNA alternative splicing regulated by m6A methylation in pig oxidative and glycolytic skeletal muscles.

BACKGROUND: Different types of skeletal myofibers exhibit distinct physiological and metabolic properties that are associated with meat quality traits in livestock. Alternative splicing (AS) of pre-mRNA can generate multiple transcripts from an individual gene by differential selection of splice sites. N6-methyladenosine (m6A) is the most abundant modification in mRNAs, but its regulation for AS in different muscles remains unknown.  RESULTS: We characterized AS events and m6A methylation pattern in pig oxidative and glycolytic muscles. A tota1 of 1294 differential AS events were identified, and differentially spliced genes were significantly enriched in processes related to different phenotypes between oxidative and glycolytic muscles. We constructed the regulatory network between splicing factors and corresponding differential AS events and identified NOVA1 and KHDRBS2 as key splicing factors. AS event was enriched in m6A-modified genes, and the methylation level was positively correlated with the number of AS events in genes. The dynamic change in m6A enrichment was associated with 115 differentially skipping exon (SE-DAS) events within 92 genes involving in various processes, including muscle contraction and myofibril assembly. We obtained 23.4% SE-DAS events (27/115) regulated by METTL3-meditaed m6A and experimentally validated the aberrant splicing of ZNF280D, PHE4DIP, and NEB. The inhibition of m6A methyltransferase METTL3 could induce the conversion of oxidative fiber to glycolytic fiber in PSCs. CONCLUSION: Our study suggested that m6A modification could contribute to significant difference in phenotypes between oxidative and glycolytic muscles by mediating the regulation of AS. These findings would provide novel insights into mechanisms underlying muscle fiber conversion.

Knockdown of YY1 Inhibits XIST Expression and Enhances Cloned Pig Embryo Development.

The technique of cloning has wide applications in animal husbandry and human biomedicine. However, the very low developmental efficiency of cloned embryos limits the application of cloning. Ectopic XIST-expression-induced abnormal X chromosome inactivation (XCI) is a primary cause of the low developmental competence of cloned mouse and pig embryos. Knockout or knockdown of XIST improves cloning efficiency in both pigs and mice. The transcription factor Yin yang 1(YY1) plays a critical role in XCI by triggering the transcription of X-inactive specific transcript (XIST) and facilitating the localization of XIST RNA on the X chromosome. This study aimed to investigate whether RNA interference to suppress the expression of YY1 can inhibit erroneous XIST expression, rescue abnormal XCI, and improve the developmental ability of cloned pig embryos. The results showed that YY1 binds to the 5' regulatory region of the porcine XIST gene in pig cells. The microinjection of YY1 siRNA into cloned pig embryos reduced the transcript abundance of XIST and upregulated the mRNA level of X-linked genes at the 4-cell and blastocyst stages. The siRNA-mediated knockdown of YY1 altered the transcriptome and enhanced the in vitro and in vivo developmental efficiency of cloned porcine embryos. These results suggested that YY1 participates in regulating XIST expression and XCI in cloned pig embryos and that the suppression of YY1 expression can increase the developmental rate of cloned pig embryos. The present study established a new method for improving the efficiency of pig cloning.

Soil Nitrogen Content Detection Based on Near-Infrared Spectroscopy.

Traditional soil nitrogen detection methods have the characteristics of being time-consuming and having an environmental pollution effect. We urgently need a rapid, easy-to-operate, and non-polluting soil nitrogen detection technology. In order to quickly measure the nitrogen content in soil, a new method for detecting the nitrogen content in soil is presented by using a near-infrared spectrum technique and random forest regression (RF). Firstly, the experiment took the soil by the Xunsi River in the area of Hubei University of Technology as the research object, and a total of 143 soil samples were collected. Secondly, NIR spectral data from 143 soil samples were acquired, and chemical and physical methods were used to determine the content of nitrogen in the soil. Thirdly, the raw spectral data of soil samples were denoised by preprocessing. Finally, a forecast model for the soil nitrogen content was developed by using the measured values of components and modeling algorithms. The model was optimized by adjusting the changes in the model parameters and Gini coefficient (∆Gini), and the model was compared with the back propagation (BP) and support vector machine (SVM) models. The results show that: the RF model modeling set prediction R2C is 0.921, the RMSEC is 0.115, the test set R2P is 0.83, and the RMSEP is 0.141; the detection of the soil nitrogen content can be realized by using a near-infrared spectrum technique and random forest algorithm, and its prediction accuracy is better than that of the BP and SVM models; using ∆ Gini to optimize the RF modeling data, the spectral information of the soil nitrogen content can be extracted, and the data redundancy can be reduced effectively.

Comparative evaluation of production performances of cloned pigs derived from superior Duroc boars.

Since pig was successfully cloned in 2000, somatic cell nuclear transfer (SCNT) became a promising technique in preserving and expanding the genetics of superior boars. Assessing the safety, growth performance, and reproductive performance of cloned pigs and their progeny is critical for their wide application. In this study, three superior Duroc boars were used to construct 61,736 SCNT-cloned embryos. The semen quality and reproductive performance of the cloned Duroc pigs and the growth performances of their progeny were evaluated. Results showed that the cloned pigs derived from superior boars produced semen with normal quality and exhibited similar reproductive performance as the donor boars, whose progenies showed greater growth performance than those derived from non-cloned pigs under the same feed condition. The results shed light on the application of cloning technology in the conservation and expansion of the genetic resources of Duroc pigs.

Long non-coding RNA Gm10561 promotes myogenesis by sponging miR-432.

Skeletal myogenesis is a highly ordered process finely regulated by various factors. Long non-coding RNAs play an important regulatory role in myogenesis via multiple mechanisms. In this study, we identified the lncRNA Gm10561, which was upregulated during myogenic differentiation and is highly expressed in skeletal muscle. Knockdown of Gm10561 inhibited the proliferation and differentiation of C2C12 myoblasts in vitro and muscle growth in vivo. Overexpression of Gm10561 promoted the proliferation and differentiation of both C2C12 myoblasts and porcine muscle satellite cells. Notably, lncRNA Gm10561 is localized in the cytoplasm and competitively bound to miR-432, which directly targets MEF2C and E2F3. It was confirmed that lncRNA Gm10561 regulates the proliferation and differentiation of myoblasts by acting as a sponge of miR-432 to modulate MEF2C and E2F3 expression. Thus, the lncRNA-Gm10561-miR-432-MEF2C/E2F3 axis plays an important role in myogenesis.

Comprehensive Analysis of Long Noncoding RNA Modified by m6A Methylation in Oxidative and Glycolytic Skeletal Muscles.

N6-methyladenosine (m6A) is the most common modification in eukaryotic RNAs. Accumulating evidence shows m6A methylation plays vital roles in various biological processes, including muscle and fat differentiation. However, there is a lack of research on lncRNAs' m6A modification in regulating pig muscle-fiber-type conversion. In this study, we identified novel and differentially expressed lncRNAs in oxidative and glycolytic skeletal muscles through RNA-seq, and further reported the m6A-methylation patterns of lncRNAs via MeRIP-seq. We found that most lncRNAs have one m6A peak, and the m6A peaks were preferentially enriched in the last exon of the lncRNAs. Interestingly, we found that lncRNAs' m6A levels were positively correlated with their expression homeostasis and levels. Furthermore, we performed conjoint analysis of MeRIP-seq and RNA-seq data and obtained 305 differentially expressed and differentially m6A-modified lncRNAs (dme-lncRNAs). Through QTL enrichment analysis of dme-lncRNAs and PPI analysis for their cis-genes, we finally identified seven key m6A-modified lncRNAs that may play a potential role in muscle-fiber-type conversion. Notably, inhibition of one of the key lncRNAs, MSTRG.14200.1, delayed satellite cell differentiation and stimulated fast-to-slow muscle-fiber conversion. Our study comprehensively analyzed m6A modifications on lncRNAs in oxidative and glycolytic skeletal muscles and provided new targets for the study of pig muscle-fiber-type conversion.

ITGB6 inhibits the proliferation of porcine skeletal muscle satellite cells.

The formation of embryonic muscle fibers determines the amount of postnatal muscles and is regulated by a variety of signaling pathways and transcription factors. Previously, by using chromatin immunoprecipitation-sequencing and RNA-Seq techniques, we identified a large number of genes that are regulated by H3K27me3 in porcine embryonic skeletal muscles. Among these genes, we found that ITGB6 is regulated by H3K27me3. However, its function in muscle development is unknown. In this study, we first verified that ITGB6 was differentially regulated by H3K27me3 and that its expression levels were upregulated in porcine skeletal muscles at embryonic Days 33, 65, and 90. Then, we performed gain- or loss-of-function studies on porcine skeletal muscle satellite cells to study the role of ITGB6 in porcine skeletal muscle development. The proliferation of porcine skeletal muscle satellite cells was studied through real-time polymerase chain reaction, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, Western blot, and flow cytometry analyses. We found that the ITGB6 gene was regulated by H3K27me3 during muscle development and had an inhibitory effect on the proliferation of porcine skeletal muscle satellite cells.

Neuronatin gene expression levels affect foetal growth and development by regulating glucose transport in porcine placenta.

Imprinted genes play important regulatory roles in the growth and development of placentas and foetuses during pregnancy. In a previous study, we found that the imprinted gene Neuronatin (NNAT) is involved in foetal development; NNAT expression was significantly lower in the placentas of piglets that died neonatally compared to the placentas of surviving piglets. However, the function and mechanism of NNAT in regulating porcine placental development is still unknown. In this study, we collected the placentas of high- and low-weight foetuses at gestational day (GD 65, 90), (n = 4-5 litters/GD) to investigate the role of NNAT in regulating foetal growth and development. We found that the mRNA and protein levels of NNAT were significantly higher in the placentas of high-weight than low-weight foetuses. We then overexpressed NNAT in porcine placental trophoblast cell lines (pTr2) and demonstrated that NNAT activated the PI3K-AKT pathway, and further promoted the expression of glucose transporter 1 (GLUT1) and increased cellular calcium ion levels, which improved glucose transport in placental trophoblast cells in vitro. To conclude, our study suggests that NNAT expression impacts porcine foetal development by regulating placental glucose transport.

Copper phosphide decorated g-C3N4 catalysts for highly efficient photocatalytic H2 evolution.

Designing functional heterojunctions to enhance photocatalytic hydrogen evolution is still a key challenge in the field of efficient solar energy utilization. Copper phosphides become an ideal material to serve as the cocatalysts during photocatalytic hydrogen evolution by virtue of the lower prices. In this study, we synthesized graphitic carbon nitride (g-C3N4) based catalysts loaded with copper phosphide (Cu3P, Cu97P3), which exhibit superior performance in photocatalytic H2 evolution. Ultraviolet (UV)-visible spectroscopy illustrated that the absorption of light strengthened after the loading of copper phosphide, and the time-resolved transient photoluminescence (PL) spectra showed that the separation and transfer of the photoexcited carriers greatly improved. Moreover, both copper phosphide/g-C3N4 photocatalysts exhibited a relatively high H2 evolution rate: Cu3P/g-C3N4 (maximum 343 µmol h-1 g-1), Cu97P3/g-C3N4 (162.9 µmol h-1 g-1) while copper phosphide themself exhibit no photocatalytic activity. Thus, the copper phosphides (Cu3P, Cu97P3) work as a cocatalyst during photocatalytic H2 evolution. The cycling experiments illustrated that both copper phosphide/g-C3N4 photocatalysts perform excellent stability in the photocatalytic H2 evolution. It is worth noting that while the NaH2PO2 was heated in the tube furnace for phosphorization to obtain Cu3P, the excessive PH3 could pass through the solution of CuSO4 to obtain Cu97P3 at the same time, which significantly improved the utilization of PH3 and reduced the risk of toxicity. This work could provide new strategies to design photocatalysts decorated with copper phosphide for highly efficient visible-light-driven hydrogen evolution.

Genome-Wide Analysis of H3K27me3 in Porcine Embryonic Muscle Development.

The trimethylation of histone H3 lysine 27 (H3K27me3) is one of the most important chromatin modifications, which is generally presented as a repressive mark in various biological processes. However, the dynamic and global-scale distribution of H3K27me3 during porcine embryonic muscle development remains unclear. Here, our study provided a comprehensive genome-wide view of H3K27me3 and analyzed the matching transcriptome in the skeletal muscles on days 33, 65, and 90 post-coitus from Duroc fetuses. Transcriptome analysis identified 4,124 differentially expressed genes (DEGs) and revealed the key transcriptional properties in three stages. We found that the global H3K27me3 levels continually increased during embryonic development, and the H3K27me3 level was negatively correlated with gene expression. The loss of H3K27me3 in the promoter was associated with the transcriptional activation of 856 DEGs in various processes, including skeletal muscle development, calcium signaling, and multiple metabolic pathways. We also identified for the first time that H3K27me3 could enrich in the promoter of genes, such as DES, MYL1, TNNC1, and KLF5, to negatively regulate gene expression in porcine satellite cells (PSCs). The loss of H3K27me3 could promote muscle cell differentiation. Taken together, this study provided the first genome-wide landscape of H3K27me3 in porcine embryonic muscle development. It revealed the complex and broad function of H3K27me3 in the regulation of embryonic muscle development from skeletal muscle morphogenesis to myofiber maturation.

Defective ultra-thin two-dimensional g-C3N4 photocatalyst for enhanced photocatalytic H2 evolution activity.

The two-dimensional semiconductor photocatalytic material has excellent photocatalytic H2 evolution activity. In order to further improve the hydrogen production activity of g-C3N4, this study improved the preparation process of g-C3N4 and obtained a new photocatalyst (name H-CN) with a higher absorption range, larger specific surface area, and faster hydrogen production activity. Compared with the originally prepared g-C3N4, the H-CN absorption range has been improved, and the utilization of visible light has reached 650 nm. When the doping amount of Pt cocatalyst was 1.0 wt%, the H-CN demonstrates excellent photocatalytic hydrogen production activity, with a hydrogen production rate of 4.3 mmol h-1·g-1, which was 7.0 times higher than that pure 1.0 wt% Pt/g-C3N4. The fluorescence spectroscopy of H-CN showed better separation of carriers and longer lifetime. This study has guiding significance for the preparation of subsequent ultra-thin nanosheet photocatalysts and the establishment of high-efficiency photocatalytic systems.

Assessment of the Growth and Reproductive Performance of Cloned Pietrain Boars.

How to maximize the use of the genetic merits of the high-ranking boars (also called superior ones) is a considerable question in the pig breeding industry, considering the money and time spent on selection. Somatic cell nuclear transfer (SCNT) is one of the potential ways to answer the question, which can be applied to produce clones with genetic resources of superior boar for the production of commercial pigs. For practical application, it is essential to investigate whether the clones and their progeny keep behaving better than the "normal boars", considering that in vitro culture and transfer manipulation would cause a series of harmful effects to the development of clones. In this study, 59,061 cloned embryos were transferred into 250 recipient sows to produce the clones of superior Pietrain boars. The growth performance of 12 clones and 36 non-clones and the semen quality of 19 clones and 28 non-clones were compared. The reproductive performance of 21 clones and 25 non-clones were also tested. Furthermore, we made a comparison in the growth performance between 466 progeny of the clones and 822 progeny of the non-clones. Our results showed that no significant difference in semen quality and reproductive performance was observed between the clones and the non-clones, although the clones grew slower and exhibited smaller body size than the non-clones. The F1 progeny of the clones showed a greater growth rate than the non-clones. Our results demonstrated through the large animal population showed that SCNT manipulation resulted in a low growth rate and small body size, but the clones could normally produce F1 progeny with excellent growth traits to bring more economic benefits. Therefore, SCNT could be effective in enlarging the merit genetics of the superior boars and increasing the economic benefits in pig reproduction and breeding.

Identification and Expression Pattern of EZH2 in Pig Developing Fetuses.

The proper methylation status of histones is essential for appropriate cell lineage and organogenesis. EZH2, a methyltransferase catalyzing H3K27me3, has been abundantly studied in human and mouse embryonic development. The pig is an increasing important animal model for molecular study and pharmaceutical research. However, the transcript variant and temporal expression pattern of EZH2 in the middle and late porcine fetus are still unknown. Here, we identified the coding sequence of the EZH2 gene and characterized its expression pattern in fetal tissues of Duroc pigs at 65- and 90-day postcoitus (dpc). Our results showed that the coding sequence of EZH2 was 2241 bp, encoding 746 amino acids. There were 9 amino acid insertions and an amino acid substitution in this transcript compared with the validated reference sequence in NCBI. EZH2 was ubiquitously expressed in the fetal tissues of two time points with different expression levels. These results validated a different transcript in pigs and characterized its expression profile in fetal tissues of different gestation stages, which indicated that EZH2 played important roles during porcine embryonic development.

Fault diagnosis in a current sensor and its application to fault-tolerant control for an air supply subsystem of a 50 kW-Grade fuel cell engine.

The safety, reliability and stability of air supply subsystems are still problems for the commercial applications of fuel cells; therefore, engine fault diagnosis and fault-tolerant control are essential to protect the fuel cell stack. In this study, a fault diagnosis and fault-tolerant control method based on artificial neural networks (ANNs) has been proposed. The offline ANN modification model was trained with a Levenberg-Marquardt (LM) algorithm based on other sensors' signals relevant to the current sensor of a 50 kW-grade fuel cell engine test bench. The output current was predicted via the ANN identification model according to other relevant sensors and compared with the sampled current sensor signal. The faults in the current sensor were detected immediately once the difference exceeded the given threshold value, and the invalid signals of the current sensor were substituted with the predictive output value of the ANN identification model. Finally, the reconstructed current sensor signals were sent back to a fuel cell controller unit (FCU) to adjust the air flow and rotate speeds of the air compressor. Experimental results show that the typical faults in the current sensor can be diagnosed and distinguished within 0.5 s when the threshold value is 15 A. The invalid signal of current sensor can be reconstructed within 0.1 s. Which ensures that the air compressor operate normally and avoids oxygen starvation. The proposed method can protect the fuel cell stack and enhance the fault-tolerant performance of air supply subsystem used in the fuel cell engine, and it is promising to be utilized in the fault diagnosis and fault-tolerant control of various fuel cell engines and multiple sensor systems.