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Histone lysine demethylase 4A (KDM4A) plays a crucial role in regulating cell proliferation, cell differentiation, development and tumorigenesis. However, little is known about the function of KDM4A in muscle development and regeneration. Here, we found that the conditional ablation of KDM4A in skeletal muscle caused impairment of embryonic and postnatal muscle formation. The loss of KDM4A in satellite cells led to defective muscle regeneration and blocked the proliferation and differentiation of satellite cells. Myogenic differentiation and myotube formation in KDM4A-deficient myoblasts were inhibited. Chromatin immunoprecipitation assay revealed that KDM4A promoted myogenesis by removing the histone methylation mark H3K9me3 at MyoD, MyoG and Myf5 locus. Furthermore, inactivation of KDM4A in myoblasts suppressed myoblast differentiation and accelerated H3K9me3 level. Knockdown of KDM4A in vitro reduced myoblast proliferation through enhancing the expression of the cyclin-dependent kinase inhibitor P21 and decreasing the expression of cell cycle regulator Cyclin D1. Together, our findings identify KDM4A as an important regulator for skeletal muscle development and regeneration, orchestrating myogenic cell proliferation and differentiation.

High-mobility group box 2 (HMGB2) is an abundant, chromatin-associated protein that plays an essential role in the regulation of transcription, cell proliferation, differentiation, and tumorigenesis. However, the underlying mechanism of HMGB2 in adipogenesis remains poorly known. Here, we provide evidence that HMGB2 deficiency in preadipocytes impedes adipogenesis, while overexpression of HMGB2 increases the potential for adipogenic differentiation. Besides, depletion of HMGB2 in vivo caused the decrease in body weight, white adipose tissue (WAT) mass, and adipocyte size. Consistently, the stromal vascular fraction (SVF) of adipose tissue derived from hmgb2 −/− mice presented impaired adipogenesis. When hmgb2 −/− mice were fed with high-fat diet (HFD), the body size, and WAT mass were increased, but at a lower rate. Mechanistically, HMGB2 mediates adipogenesis via enhancing expression of C/EBPβ by binding to its promoter at “GGGTCTCAC” specifically during mitotic clonal expansion (MCE) stage, and exogenous expression of C/EBPβ can rescue adipogenic abilities of preadipocytes in response to HMGB2 inhibition. In general, our findings provide a novel mechanism of HMGB2-C/EBPβ axis in adipogenesis and a potential therapeutic target for obesity.

Triggering receptor expressed on myeloid cells 2 (TREM2) serves as an anti-inflammatory receptor, negatively regulating the innate immune response. TREM2 is mainly expressed on dendritic cells and macrophages, the target cells of porcine reproductive and respiratory syndrome virus (PRRSV). Thus, we investigated the potential role of TREM2 in PRRSV infection in porcine alveolar macrophages (PAMs). We found that there was an increased expression of TREM2 upon PRRSV infection in vitro. TREM2 silencing restrained the replication of PRRSV, whereas TREM2 overexpression facilitated viral replication. The cytoplasmic tail domain of TREM2 interacted with PRRSV Nsp2 to promote infection. TREM2 downregulation led to early activation of PI3K/NF-κB signaling, thus reinforcing the expression of proinflammatory cytokines and type I interferons. Due to the enhanced cytokine expression, a disintegrin and metalloproteinase 17 was activated to promote the cleavage of membrane CD163, which resulted in suppression of infection. Furthermore, exogenous soluble TREM2 (sTREM2)-mediated inhibition of PRRSV attachment might be attributed to its competitive binding to viral envelope proteins. In pigs, following PRRSV challenge in vivo, the expression of TREM2 in lungs and lymph nodes as well as the production of sTREM2 were significantly increased. These novel findings indicate that TREM2 plays a role in regulating PRRSV replication via the inflammatory response. Therefore, our work describes a novel antiviral mechanism against PRRSV infection and suggests that targeting TREM2 could be a new approach in the control of the PRRSV infection.

In-feed antibiotics can influence intestinal microbial structures in born and early-life within a period. However, the impact of antibiotics on gut microbiota during long-term antibiotic-free and antibiotic breeding at porcine-fattening phase have not been studied extensively so far. Here, we conducted a systematic 16S rRNA gene sequencing-based study combined with metagenomic analysis to reveal the variation of diversity and function of gut microbiota between antibiotic-free (treatment group, TG) and antibiotic (a mixture of flavomycin and enramycin, control group, CG) breeding at various stages of fattening pigs. In the present study, Bacteroidetes, Firmicutes, and Proteobacteria phyla were the core microbiomes in fattening pig gut microbiota. The ratio between Firmicutes and Bacteroidetes significantly increased with age (P = 0.03). TG showed significantly higher relative abundance of Proteobacteria and Fibrobacteres phyla than CG. The microbial community can be divided into several notably clustered blocks based on cooperative and competitive correlations. These blocks centered on numerous special genera, which play essential roles in body development and disease prevention. TG showed obviously higher proportions of metabolic pathways related to metabolism, endocrine system, nervous system and excretory system, but pathways included carbohydrate metabolism and immune system diseases in CG. Collectively, this study has comprehensively demonstrated microbial diversities, differences and correlations among gut microbiota, microbial metabolism and gene functions during long-term antibiotic-free breeding. This work provides a novel resource and information with positive implications for pig husbandry production and disease prevention.

Background Skeletal muscle development is a multistep process whose understanding is central in a broad range of fields and applications, from the potential medical value to human society, to its economic value associated with improvement of agricultural animals. Skeletal muscle initiates in the somites, with muscle precursor cells generated in the dermomyotome and dermomyotome-derived myotome before muscle differentiation ensues, a developmentally regulated process that is well characterized in model organisms. However, the regulation of skeletal muscle ontogeny during embryonic development remains poorly defined in farm animals, for instance in pig. Here, we profiled gene expression and chromatin accessibility in developing pig somites and myotomes at single-cell resolution. Results We identified myogenic cells and other cell types and constructed a differentiation trajectory of pig skeletal muscle ontogeny. Along this trajectory, the dynamic changes in gene expression and chromatin accessibility coincided with the activities of distinct cell type-specific transcription factors. Some novel genes upregulated along the differentiation trajectory showed higher expression levels in muscular dystrophy mice than that in healthy mice, suggesting their involvement in myogenesis. Integrative analysis of chromatin accessibility, gene expression data, and in vitro experiments identified EGR1 and RHOB as critical regulators of pig embryonic myogenesis. Conclusions Collectively, our results enhance our understanding of the molecular and cellular dynamics in pig embryonic myogenesis and offer a high-quality resource for the further study of pig skeletal muscle development and human muscle disease.

Gas‐phase mechanisms provide important oxidant and gaseous precursors for secondary aerosol formation. Different gas‐phase mechanisms may lead to different predictions of gases, aerosols, and aerosol direct and indirect effects. In this study, WRF/Chem‐MADRID simulations are conducted over the continental United States for July 2001, with three different gas‐phase mechanisms, a default one (i.e., CBM‐Z) and two newly implemented ones (i.e., CB05 and SAPRC‐99). Simulation results are evaluated against available surface observations, satellite data, and reanalysis data. The model with these three gas‐phase mechanisms gives similar predictions of most meteorological variables in terms of spatial distribution and statistics, but large differences exist in shortwave radiation and temperature and relative humidity at 2 m at individual sites under cloudy conditions, indicating the importance of aerosol semi‐direct and indirect effects on these variables. Large biases exist in the simulated wind speed at 10 m, cloud water path, cloud optical thickness, and precipitation, due to uncertainties in current cloud microphysics and surface layer parameterizations. Simulations with all three gas‐phase mechanisms well reproduce surface concentrations of O3, CO, NO2, and PM2.5, and column NO2. Larger biases exist in the surface concentrations of nitrate and organic matter (OM) and in the spatial distribution of column CO, tropospheric ozone residual, and aerosol optical depth, due to uncertainties in primary OM emissions, limitations in model representations of chemical transport, and radiative processes. Different gas‐phase mechanisms lead to different predictions of mass concentrations of O3 (up to 5 ppb), PM2.5 (up to 0.5 μg m−3), secondary inorganic PM2.5 species (up to 1.1 μg m−3), organic PM (up to 1.8 μg m−3), and number concentration of PM2.5 (up to 2 × 104 cm−3). Differences in aerosol mass and number concentrations further lead to sizeable differences in simulated cloud condensation nuclei (CCN) and cloud droplet number concentration (CDNC) due to the feedback mechanisms among H2SO4 vapor, PM2.5 number, CCN, and CDNC through gas‐phase chemistry, new particle formation via homogeneous nucleation, aerosol growth, and aerosol activation by cloud droplets. This study illustrates the important impact of gas‐phase mechanisms on chemical and aerosol predictions, their subsequent effects on meteorological predictions, and a need for an accurate representation of such feedbacks through various atmospheric processes in the model. The online‐coupled models that simulate feedbacks between meteorological variables and chemical species may provide more accurate representations of the real atmosphere for regulatory applications and can be applied to simulate chemistry‐climate feedbacks over a longer period of time. Key Points Gas‐phase mechanisms in a fully coupled meteorology‐chemistry model Different gas‐phase predictions cause different aerosol/cloud predictions Aerosol feedbacks should be considered in future regulatory modeling

Background In the pig production industry, artificial insemination (AI) plays an important role in enlarging the beneficial impact of elite boars. Understanding the genetic architecture and detecting genetic markers associated with semen traits can help in improving genetic selection for such traits and accelerate genetic progress. In this study, we utilized a weighted single-step genome-wide association study (wssGWAS) procedure to detect genetic regions and further candidate genes associated with semen traits in a Duroc boar population. Overall, the full pedigree consists of 5284 pigs (12 generations), of which 2693 boars have semen data (143,113 ejaculations) and 1733 pigs were genotyped with 50 K single nucleotide polymorphism (SNP) array. Results Results show that the most significant genetic regions (0.4 Mb windows) explained approximately 2%~ 6% of the total genetic variances for the studied traits. Totally, the identified significant windows (windows explaining more than 1% of total genetic variances) explained 28.29, 35.31, 41.98, and 20.60% of genetic variances (not phenotypic variance) for number of sperm cells, sperm motility, sperm progressive motility, and total morphological abnormalities, respectively. Several genes that have been previously reported to be associated with mammal spermiogenesis, testes functioning, and male fertility were detected and treated as candidate genes for the traits of interest: Number of sperm cells, TDRD5 , QSOX1 , BLK , TIMP3 , THRA , CSF3 , and ZPBP1 ; Sperm motility, PPP2R2B , NEK2 , NDRG , ADAM7 , SKP2 , and RNASET2 ; Sperm progressive motility, SH2B1 , BLK , LAMB1 , VPS4A , SPAG9 , LCN2, and DNM1 ; Total morphological abnormalities, GHR , SELENOP , SLC16A5 , SLC9A3R1 , and DNAI2 . Conclusions In conclusion, candidate genes associated with Duroc boars’ semen traits, including the number of sperm cells, sperm motility, sperm progressive motility, and total morphological abnormalities, were identified using wssGWAS. KEGG and GO enrichment analysis indicate that the identified candidate genes were enriched in biological processes and functional terms may be involved into spermiogenesis, testes functioning, and male fertility.

Zinc finger protein 422 (Zfp422) is a widely expressed zinc finger protein that serves as a transcriptional factor to regulate downstream gene expression, but until now, little is known about its roles in myogenesis. We found here that Zfp422 plays a critical role in skeletal muscle development and regeneration. It highly expresses in mouse skeletal muscle during embryonic development. Specific knockout of Zfp422 in skeletal muscle impaired embryonic muscle formation. Satellite cell-specific Zfp422 deletion severely inhibited muscle regeneration. Myoblast differentiation and myotube formation were suppressed in Zfp422-deleted C2C12 cells, isolated primary myoblasts, and satellite cells. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) revealed that Zfp422 regulated ephrin type-A receptor 7 (EphA7) expression by binding an upstream 169-bp DNA sequence, which was proved to be an enhancer of EphA7. Knocking EphA7 down in C2C12 cells or deleting Zfp422 in myoblasts will inhibit cell apoptosis which is required for myoblast differentiation. These results indicate that Zfp422 is essential for skeletal muscle differentiation and fusion, through regulating EphA7 expression to maintain proper apoptosis.

Recently, three-dimensional (3D) imaging techniques have been used to detect viral invasion and the appearance of specialized structures established in virus-infected cells. These methods have had a positive impact in the field of virology and helped to further our knowledge of how viruses invade cells. Nearly all positive-strand RNA viruses propagate their viral genomes in part through intracellular membranes. Porcine reproductive and respiratory syndrome virus (PRRSV), an Arterivirus, accumulates viral RNA that forms replication complexes (RCs) in infected cells. In this study, using immunofluorescence and electron microscopy (EM), we dissected PRRSV-induced membrane structures in infected cells and determined the correlations between PRRSV particles and vesicles stimulated by PRRSV to understand the structural and dynamic aspects of PRRSV infection. We identified the appropriate time point by determining the 50% tissue culture infectious dose (TCID50) and using qRT-PCR and Western blotting. The co-localization of viruses and organelles was determined by immunofluorescence and immune-electron microscopy (IEM). The ultrastructure of cells infected by PRRSV was observed using EM and electron tomography (ET). In our study, we found that PRRSV dsRNA was located at the endoplasmic reticulum (ER) and autophagosomes; in addition, the N protein was located at the mitochondria, ER and autophagosomes. Vesicles induced by PRRSV appeared at 16 hours post-infection (h.p.i.) and increased in size with time during the infection period. In addition, our findings demonstrated that the virus vesicles originated from the ER, and these two organelle structures connected with each other to form a reticulovesicular network (RVN) that provided a site for virus replication and assembly. Our results revealed that membrane vesicles induced by PRRSV were derived from the ER. The vesicles may provide a location for PRRSV replication and assembly.

Background Autophagy is an essential process in eukaryotic cells in which autophagosomes form to deliver cellular organelles and long-lived proteins to lysosomes for degradation. Many studies have recently identified the regulatory mechanisms involved in the interaction between viral infection and autophagy. Methods LC3 turnover and the proteins in the endoplasmic reticulum (ER) stress pathway were investigated using western blot analysis. The formation and degradation of autophagosomes were detected using immunofluorescence staining. Results Autophagy was activated by porcine reproductive and respiratory syndrome virus (PRRSV) NSP3, NSP5 and NSP9, which are two transmembrane proteins and an RNA-dependent RNA polymerase, respectively. The formation of autophagosomes was induced by NSP3 and NSP5 and developed from the ER; the fusion of these autophagosomes with lysosomes was limited. Although NSP3 and NSP5 are ER transmembrane proteins, these proteins did not activate the ER stress signaling pathways. In addition, the cytoplasmic domain of NSP3 plays a pivotal role in activating autophagy. Conclusions The data presented in this study reveal an important relationship between PRRSV NSPs and autophagy and provide new insights that improve our understanding of the involvement of PRRSV NSPs in the autophagy process.