Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs.

Osteosarcoma (OS) is a heterogeneous, aggressive malignancy of the bone that disproportionally affects children and adolescents. Therapeutic interventions for OS are limited, which is in part due to the complex tumor microenvironment (TME). As such, we used single-cell RNA sequencing (scRNA-seq) to describe the cellular and molecular composition of the TME in 6 treatment-naïve dogs with spontaneously occurring primary OS. Through analysis of 35,310 cells, we identified 41 transcriptomically distinct cell types including the characterization of follicular helper T cells, mature regulatory dendritic cells (mregDCs), and 8 tumor-associated macrophage (TAM) populations. Cell-cell interaction analysis predicted that mregDCs and TAMs play key roles in modulating T cell mediated immunity. Furthermore, we completed cross-species cell type gene signature homology analysis and found a high degree of similarity between human and canine OS. The data presented here act as a roadmap of canine OS which can be applied to advance translational immuno-oncology research.

Identification and screening of circular RNAs during adipogenic differentiation of ovine preadipocyte by RNA-seq.

Circular RNAs (circRNAs) are a class of non-coding RNAs that play important roles in preadipocyte differentiation and adipogenesis. However, little is known about genome-wide identification, expression profile, and function of circRNAs in sheep. To investigate the role of circRNAs during ovine adipogenic differentiation, the subcutaneous adipose tissue of Tibetan rams was collected in June 2022. Subsequently, the preadipocytes were immediately isolated from collected adipose tissue and then induced to begin differentiation. The adipocytes samples cultured on days 0, 2, and 8 of preadipocytes differentiation were used to perform RNA sequencing (RNA-seq) analysis to construct the expression profiles of circRNAs. Subsequently, the function of differentially expressed circRNAs was investigated by performing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of their parent genes. Finally, a circRNAs-miRNAs-mRNAs network involved in adipogenic differentiation was been analyzed. As a result, a total of 6,449 candidate circRNAs were identified in ovine preadipocytes. Of these circRNAs identified, 63 candidate circRNAs were differentially expressed among the three differentiation stages and their parent genes were mainly enriched in acetyl-CoA metabolic process, positive regulation of lipid biosynthetic process, positive regulation of steroid biosynthetic process, and focal adhesion pathway (P < 0.05). Based on a circRNAs-miRNAs-mRNAs regulatory network constructed, circ_004977, circ_006132 and circ_003788 were found to function as competing endogenous RNAs (ceRNAs) to regulate ovine preadipocyte differentiation and lipid metabolism. The results provide an improved understanding of functions and molecular mechanisms of circRNAs underlying ovine adipogenesis in sheep.

The moderate fat deposition contributes to improve mutton quality, which is associated with the differentiation of preadipocytes. To investigate roles of circular RNAs (circRNAs) in preadipocyte differentiation, we identified circRNAs on days 0, 2, and 8 of preadipocytes differentiation and compared the expression profile of circRNAs at different adipogenic differentiation stages. A total of 6,449 candidate circRNAs were identified, among which 63 candidate circRNAs were differentially expressed among the three differentiation stages. The parent genes of differentially expressed circRNAs were enriched in several biological process and pathways related to lipid metabolism and synthesis. In addition, several circRNAs may regulate ovine preadipocyte differentiation by interacting with microRNAs (miRNAs). The results reveal the potential roles of circRNAs in adipogenic differentiation of sheep.

Combined ATAC-seq, RNA-seq, and GWAS analysis reveals glycogen metabolism regulatory network in Jinjiang oyster ( Crassostrea ariakensis).

Glycogen serves as the principal energy reserve for metabolic processes in aquatic shellfish and substantially contributes to the flavor and quality of oysters. The Jinjiang oyster ( Crassostrea ariakensis) is an economically and ecologically important species in China. In the present study, RNA sequencing (RNA-seq) and assay for transposase-accessible chromatin using sequencing (ATAC-seq) were performed to investigate gene expression and chromatin accessibility variations in oysters with different glycogen contents. Analysis identified 9 483 differentially expressed genes (DEGs) and 7 215 genes with significantly differential chromatin accessibility (DCAGs) were obtained, with an overlap of 2 600 genes between them. Notably, a significant proportion of these genes were enriched in pathways related to glycogen metabolism, including "Glycogen metabolic process" and "Starch and sucrose metabolism". In addition, genome-wide association study (GWAS) identified 526 single nucleotide polymorphism (SNP) loci associated with glycogen content. These loci corresponded to 241 genes, 63 of which were categorized as both DEGs and DCAGs. This study enriches basic research data and provides insights into the molecular mechanisms underlying the regulation of glycogen metabolism in C. ariakensis.

Motility-related microRNAs identified in pig seminal plasma exosomes by high-throughput small RNA sequencing.

Boar fertility is a key determinant of the production efficiency of the whole pig breeding industry and boar sperm motility is the seminal parameter with the greatest impact on the fecundity of a sow. Exosomes are small, extracellular vesicles found in many body fluids. Seminal plasma exosomes, which are secreted by the epididymis, prostate, seminal vesicles, and testes, contain a large number of miRNAs, the types and levels of which can reflect the physiological state of source cells. It has been shown that the expression profile of seminal plasma exosomal miRNA differs between low-motility semen and normal semen. The aim of this study was to investigate the relationship between semen motility and exosomal miRNA profiles to obtain information that would allow to predict boar fertility, as well as contribute to the understanding of the mechanisms by which exosomal miRNAs regulate semen motility. Three high-motility (semen motility >90 %) and three low-motility (semen motility <80 %) semen samples were collected from Landrace and Yorkshire boars, respectively, and seminal plasma exosomes were extracted by ultracentrifugation. Exosome characterization was performed using transmission electron microscopy, NTA, and Western blot. The expression profiles of exosomal miRNAs associated with semen motility in the two boar breeds were subsequently determined by small RNA sequencing. The results showed that 297 known miRNAs and 295 novel RNAs were co-expressed in the four groups. Notably, six miRNAs (ssc-miR-122-5p, ssc-miR-486, ssc-miR-451, ssc-miR-345-3p, ssc-miR-362, and ssc-miR-500-5p) were found to be differentially expressed in both boar breeds. Enrichment analysis of the target genes of the differentially expressed miRNAs showed that they were mainly involved in biological processes such as regulation of transcription from RNA polymerase II promoter, regulation of gene expression, and intracellular signal transduction and signaling pathways such as the PI3K-Akt, MAPK, and Ras signaling pathways. The six differentially expressed miRNAs identified in this study have significant potential as noninvasive markers of boar semen motility. Meanwhile, the results of the enrichment analysis provide novel insights into the mechanisms underlying the regulation of semen motility.

Characterization of the semen microbiota of healthy stud dogs using 16S RNA sequencing.

The reproductive microbiota of male dogs has never been investigated using culture-independent sequencing techniques. The purpose of the present study was to get seminal knowledge on the microbiota of the ejaculate. Specifically, factors as the fraction of the ejaculate, the sperm quality (normospermia, teratozoospermia), and the living environment were evaluated. The sperm-rich and the prostatic fractions of the ejaculate were collected from healthy stud dogs. Following the sperm analysis, samples from twenty animals (normospermic n = 10 and teratozoospermic n = 10) were stored at - 80 °C until further processing including DNA extraction and 16S rRNA sequencing. Alpha- (Shannon index) and beta- (Bray-Curtis, Unweighted UniFrac) diversities were assessed and compared (PERMANOVA) based on the group of samples (biological samples from the ejaculate and controls), the fraction of the ejaculate (sperm-rich and prostatic fractions), the animal group (normospermia and teratozoospermia), and the living environment of the animal (kennel or pet living in-house). The most abundant bacterial phyla in canine semen samples were Proteobacteria, Firmicutes, and Actinobacteria. Overall, the dominant bacterial family was that of Pasteurellaceae The genus Mycoplasma was never detected. No differences in terms of bacterial composition were found based on the fraction of the ejaculate and based on the animal group (P > 0.05). On the other hand, differences in alpha and beta diversities were highlighted based on the living environment (P = 0.001). Overall, the results of the present study provide preliminary insights on dog semen microbiota, opening a new chapter in the field of canine andrology. Our results suggest that the environment may play a role in influencing the reproductive microbiota of male dogs and that the prostatic fraction of the ejaculate can be used for further research as a representative of the semen microbiota.

Regulatory effects of circular RNA on hypoxia adaptation in chicken embryos.

The Tibetan chicken, a native breed of the Tibetan plateau, is adapted to the high-altitude and hypoxic environment of the plateau. As endogenous molecules, circular RNAs (circRNAs) have been shown to play an important role in the adaptation to hypoxic environments and regulation of angiogenesis. In this study, highland Tibetan and lowland Chahua chicken eggs were incubated in a hypoxic environment and the chorionic allantoic membrane was collected for Ribo-Zero RNA sequencing. A total of 1,414 circRNAs, mostly derived from exons, were identified. Of these, 93 differentially expressed circRNAs were detected between Tibetan and Chahua chickens. Combined with the differentially expressed miRNAs and mRNAs identified in our previous study, we identified four circRNAs (circBRD1, circPRDM2, circPTPRS, and circDENND4C). These circRNAs may act as competing endogenous RNA (ceRNA) to upregulate APOA1 expression by absorbing novel_miR_589, thereby regulating angiogenesis and affecting hypoxia adaptation in chicken embryos. The regulatory circRNAs/novel_miR_589/APOA1 axis provides valuable evidence for a better understanding of the specific functions and molecular mechanisms of circRNAs in plateau hypoxia adaptation in Tibetan chickens.

The chorioallantoic membrane (CAM) is a key respiratory organ involved in early chicken embryo development. It is abundant in blood vessels and plays an important role in gas exchange in the chicken embryo. Hypoxia, a state of insufficient oxygen, can lead to abnormal angiogenesis. Tibetan chickens (TC), owing to their unique genetic background, have adapted to a hypoxic environment and are able to maintain the balance of angiogenesis. In this study, we aimed to identify potential circular RNAs (circRNAs) and their key regulatory networks related to angiogenesis by comparing highland and lowland chickens. Using RNA sequencing, we identified 93 circRNAs that were differentially expressed in the CAM between highland and lowland chickens. The key circRNAs and circRNA-miRNA-mRNA regulatory networks that affect angiogenesis were constructed using bioinformatic methods. This work provides a novel analysis of the molecular mechanisms of hypoxia adaptation in TC.

HOXB8 overexpression induces morphological changes in chicken mandibular skin: an RNA-seq analysis.

The Huiyang beard chicken is a well-known Chinese local breed known for its elongated feathers gathered from both sides of the face (muffs) and below the beak (beard), as well as short wattles (SW). The muff and beard (Mb) mutation is caused by ectopic upregulation of the homeobox B8 (HOXB8) gene in the mandibular skin; and the chi-square test showed a significant correlation between SW and Mb genotypes. However, the underlying molecular mechanisms that regulate Mb and SW variations remain unclear. In this study, we investigated the transcriptomes of the mandibular skin and wattles of chickens with and without the Mb genotype to elucidate the molecular basis of these traits. Our results show that HOXB8 is expressed at significantly higher levels in both the mandibular skin and wattles of Mb chickens than in those of wild-type chickens, indicating that HOXB8 regulates both the Mb and SW phenotypes. Key genes for keratin synthesis were highly expressed in the mandibular skin of Mb chickens, suggesting that HOXB8 may play a role in feather development. In wattles, changes in the expression of extracellular matrix synthesis genes may contribute to SW traits. DNA-binding motif analyses revealed that differentially expressed genes were likely to be directly regulated by HOXB8 binding, indicating that HOXB8 may directly or indirectly regulate feather follicle development and wattle growth. Our study identified both known and novel targets, including several genes not previously implicated in feather development and mesenchymal formation. These findings provide insights into the molecular mechanisms of skin appendage variation in birds and offer potential applications in breeding poultry breeds with unique phenotypes.

Differentially expressed transcripts of Tetracapsuloides bryosalmonae (Cnidaria) between carrier and dead-end hosts involved in key biological processes: novel insights from a coupled approach of FACS and RNA sequencing.

Tetracapsuloides bryosalmonae is a malacosporean endoparasite that infects a wide range of salmonids and causes proliferative kidney disease (PKD). Brown trout serves as a carrier host whereas rainbow trout represents a dead-end host. We thus asked if the parasite adapts to the different hosts by changing molecular mechanisms. We used fluorescent activated cell sorting (FACS) to isolate parasites from the kidney of brown trout and rainbow trout following experimental infection with T. bryosalmonae. The sorted parasite cells were then subjected to RNA sequencing. By this approach, we identified 1120 parasite transcripts that were expressed differentially in parasites derived from brown trout and rainbow trout. We found elevated levels of transcripts related to cytoskeleton organisation, cell polarity, peptidyl-serine phosphorylation in parasites sorted from brown trout. In contrast, transcripts related to translation, ribonucleoprotein complex biogenesis and subunit organisation, non-membrane bounded organelle assembly, regulation of protein catabolic process and protein refolding were upregulated in rainbow trout-derived parasites. These findings show distinct molecular adaptations of parasites, which may underlie their distinct outcomes in the two hosts. Moreover, the identification of these differentially expressed transcripts may enable the identification of novel drug targets that may be exploited as treatment against T. bryosalmonae. We here also describe for the first time how FACS based isolation of T. bryosalmonae cells from infected kidney of fish fosters research and allows to define differentially expressed parasite transcripts in carrier and dead-end fish hosts.

RNA-seq reveals the role of miR-223 in alleviating inflammation of bovine mammary epithelial cells.

Bovine mammary epithelial cells (bMECs) are involved in the early defense against the invasion of intramammary pathogens and are essential for the health of bovine mammary gland. MicroRNA (MiRNA) is a key factor that regulates cell state and physiological function. In the present study, the transcriptome profiles of miR-223 inhibitor transfection group (miR-223_Inhibitor) and negative control inhibitor transfection group (NC_Inhibitor) within bMECs were detected via the RNA sequencing (RNA-seq) platform. Based on these experiments, the differentially expressed mRNAs (DE-mRNAs) of the miR-223_Inhibitor transfection group were screened, and the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional analyses of DE-mRNAs were performed. The results revealed that compared with the NC_Inhibitor, 224 differentially expressed genes (DEGs) were identified in the miR-223_Inhibitor, including 184 upregulated and 40 downregulated genes. The functional annotation of the above DEGs indicated that some of these genes are involved in the immune response generated by extracellular substance stimulation, regulation of the activity of cytokines and chemokines, and the immune signaling pathways of NF-κB and TNF. Meanwhile, miR-223_inhibitor upregulated the immune key genes IRF1 and NFκBIA, cytokines IL-6 and IL-24, as well as chemokines CXCL3, CXCL5, and CCR6, triggering a signaling cascade response that exacerbated inflammation in bMECs. These results suggested that miR-223 plays an important role in inhibiting the inflammatory response and maintaining the stability of bMECs, and is a potential target for treating mastitis in dairy cows.

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.

Evaluate the developmental competence of human 8-cell embryos by single-cell RNA sequencing.

Abstract: The transition of maternal to zygotic gene expression regulation is critical for human preimplantation embryo development. In recent years, single-cell RNA sequencing (scRNA-seq) had been applied to detect the factors that regulate human oocyte maturation and early embryo development. Here, the evaluation of transcriptomes in single blastomere from the embryo collected from patients by scRNA-seq was performed. There were 20 blastomeres biopsied from 8-cell embryos of seven patients who received more than two ART cycles due to low embryo competence. Meanwhile, ten cells were collected from 8-cell embryos of four patients who received ART treatment due to male or tubal factors. The blastomeres were then evaluated using the previously established scRNA-seq method to determine the associations between their gene expression and developmental competence. The total number of genes detected in 8-cell embryos that failed to form blastocyst including maternal and zygotic mRNAs was reduced. There were 324 differently expressed genes detected among the 8-cell embryos including 65 genes that were significantly suppressed in the 8-cell embryos that failed to form blastocyst. Further analysis found these 8-cell embryos arrested at the cleavage stage due to the dysfunction of the cell cycle, DNA transcription activity, histone methylation, and cell division-related genes such as SMCO-1, ZNF271P,ZNF679, ASF1b, BEX3, DPPA2, and ORC4. The alterations of gene expression detected in human 8-cell embryos are tightly associated with its developmental competence and could be used as targets to enhance embryo development or parameters to predict the embryo's development outcomes. Lay summary: Many females are suffering infertility due to the failure of embryonic development at early stages due to unknown causes. At the very beginning of human embryo development, the embryos start to express its own genes, which should be achieved at 8-cell stage. In current research, we isolated one cell from 8-cell embryos and detected the gene expression at single-cell level. Then the remaining cells of these embryos were cultured to form blastocyst. Meanwhile, the data was analyzed according to the outcomes of embryo development. We detected 324 differently expressed genes between the 8-cell embryos that succeeded and failed to form blastocyst. Our research showed the association between the gene expression and the developmental competence of 8-cell embryos. The findings could be used to predict the embryo quality and potential therapy target to improve the efficiency of assisted reproductive techniques.

Gene expression pattern and molecular mechanisms involved in Shal and Sangsari sheep fertility using RNA-Seq.

Ovulation rate and litter size are the main reproductive traits with high economic value in the sheep breeding industry. In this study, three Shal ewes (multiparous) and three Sangsari ewes (uniparous) at the age of 5 were used. The live weight was between 45 and 50 kg at an extremely body condition score of 3. These breeds are marked seasonal reproduction activity and are often bred in semi-closed breeding systems. Total RNAs were extracted from the ovarian tissues, and RNA sequencing was carried out. The DAVID (Database for Annotation, Visualization and Integrated Discovery) database was then used to annotate genes, and the string database and the Cytoscape software were used to investigate their interactions. Then path-act network analysis and gene-act network analysis were investigated. The results indicated that 19,932 genes were differentially expressed. The 5968 differentially expressed genes were identified in Shal ewe's ovarian tissue compared to Sangsari ewes (FDR < 0.05), of which 2921 genes were up-regulated and 3047 genes were down-regulated. Bioinformatics analysis exhibited that most of the biological processes and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways associated with significant DEGs (Differentially Expressed Genes) in the two studied breeds are associated with oocyte maturation and metabolism. MAPK signalling pathways and Ubiquitin-mediated proteolysis are the most important biological pathways associated with reproductive and fertility traits in the Shal breed. AKT3, MAPK8, MAPK9 and RELA genes are also important genes related to the fertility of multiparous sheep. Analysis of ovarian RNA-seq data identified that most of the differentially expressed genes were involved in various reproductive processes including folliculogenesis, ovulation, ovarian and embryonic development. The MAPK signalling pathway had the most interaction with other pathways, and the AKT3 gene could be a powerful candidate gene in the reproduction and fertility of Shal sheep. These results could pave the way for future efforts to address sheep prolificacy barriers.

Small RNA deep sequencing revealed microRNAs' involvement in modulating cellular senescence and immortalization state.

Unlike rodent cells, spontaneous immortalization of avian cells and human cells is a very rare event. According to patent publications and current literature, there are no more than 4 spontaneously immortalized chicken embryo fibroblast (CEF) cell lines established up to date. One of those cell lines is ADOL (Avian Disease and Oncology Laboratory) ZS-1 cell line, which was established by continuous passaging of the CEFs derived from the specific pathogen free (SPF) 0.TVB*S1 (commonly known as rapid feathering susceptible or RFS) genetic line of chickens. The RFS genetic line of chickens was developed and has been maintained on the SPF chicken farm of USDA-ARS facility, ADOL, in East Lansing, Michigan, which is known as one of a few lines of chickens that are free of any known avian endogenous virus genes. To explore potential roles that epigenetic factors may play in modulating cellular senescence processes and spontaneous immortalization state, total RNAs extracted from samples of the RFS primary CEFs, RFS CEFs reached the 21st passage, and the ZS-1 cells were subjected to small RNA sequencing. Collectively, a total of 531 miRNAs was identified in the 3 types of samples. In contrast to the primary CEF samples, 50 miRNAs were identified with significantly differential expression only in the 21st passage samples; a different subset of 63 differentially expressed miRNAs was identified only in the ZS-1 samples; the majority of differentially expressed miRNAs identified in both the 21st passage CEF and the ZS-1 samples were more or less directionally consistent. Gene Ontology analysis results suggested that the epigenetic factor, miRNAs, plays a role in modulating the cellular senescence and spontaneous immortalization processes through various bioprocesses and key pathways including ErbB and MAPK signaling pathways. These findings provided the experimental and bioinformatic evidence for a better understanding on the epigenetic factor of miRNAs in association with cellular senescence and spontaneous immortalization process in avian cells.

Small RNA sequencing and profiling of serum-derived exosomes from African swine fever virus-infected pigs.

African swine fever (ASF) virus (ASFV) is responsible for one of the most severe swine diseases worldwide, with a morbidity rate of up to 100%; no vaccines or antiviral medicines are available against the virus. Exosomal miRNAs from individual cells can regulate the immune response to infectious diseases. In this study, pigs were infected with an ASFV Pig/HN/07 strain that was classified as acute form, and exosomal miRNA expression in the serum of infected pigs was analyzed using small RNA sequencing (small RNA-seq). Twenty-seven differentially expressed (DE) miRNAs were identified in the ASFV-infected pigs compared to that in the uninfected controls. Of these, 10 were upregulated and 17 were downregulated in the infected pigs. All DE miRNAs were analyzed using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the DE miRNAs were found to be highly involved in T-cell receptor signaling, cGMP-PKG signaling, Toll-like receptor, MAPK signaling, and mTOR signaling pathways. Furthermore, the Cytoscape network analysis identified the network of interactions between DE miRNAs and target genes. Finally, the transcription levels of four miRNA genes (ssc-miR-24-3p, ssc-miR-130b-3p, ssc-let-7a, and ssc-let-7c) were examined using quantitative real-time PCR (qRT-PCR) and were found to be consistent with the small RNA-seq data. These DE miRNAs were associated with cellular genes involved in the pathways related to immune response, virus-host interactions, and several viral genes. Overall, our findings provide an important reference and improve our understanding of ASF pathogenesis and the immune or protective responses during an acute infection in the host.

African swine fever is a viral disease caused by African swine fever virus (ASFV) which induces a big threat to the pig industry in the world. To date, there are no vaccines or antiviral medicines against the ASFV. Therefore, it is important to improve the understanding of the pathogenesis of ASFV and host­pathogen interaction using miRNA that may regulate genes related to the immune system. This study aimed to investigate the differentially expressed (DE) miRNA in serum-derived exosomes from African swine fever virus infected pigs. We successfully infected pigs with an ASFV Pig/HN/07 strain and identified the DE miRNAs in serum-derived exosomes using small RNA sequencing. Our results showed that total of 27 miRNAs were differentially expressed in serum-derived exosomes from ASFV-infected pigs. We analyzed the small RNA sequencing results using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and found that most DE miRNA may regulate the expression of genes related with the immune response pathway (T-cell receptor signaling pathway, cGMP-PKG signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, etc.).

Identification of differentially expressed long non-coding RNAs and messenger RNAs involved with muscle development in Dazu black goats through RNA sequencing.

This study aimed to explore the genetic basis of muscle development in goats. The transcriptome dataset for differentially expressed lncRNAs (DELs) and differentially expressed genes (DEGs) of goat muscle at different developmental stages were obtained using RNA-Seq. A total of 447,806,481 and 587,559,465 clean reads in the longissimus dorsi muscle of Dazu black goats between 75d embryonic stage and 1d after birth were generated through Illumina paired-end sequencing, and their mapping rates were 89.82 and 90.99%, respectively. Moreover, 4517 DEGs and 648 DELs were identified, and 4784 lncRNA-mRNA targeting relationships were predicted. Gene function annotation results showed that 4101 DEGs were significantly enriched to 1098 GO terms, and 2014 DEGs were significantly enriched to 40 KEGG pathways, including many GO terms and pathways related to muscle development, such as cell differentiation and Wnt signaling pathway. Then, 10 DELs and 20 DEGs were randomly selected for RT-qPCR verification, and the agreement rate between the verification and RNA-Seq results was 90%, indicating the high reliability of the RNA-Seq data analysis. In conclusion, this study obtained several mRNAs and lncRNAs related to the muscle development of Dazu black goats and identified several targeted regulatory pairs of lncRNA-mRNA. This study may serve as a reference to understand the genetic basis and molecular mechanism of muscle development in goats.

Integrating transcriptome and metabolome to identify key genes regulating important muscular flavour precursors in sheep.

Flavour precursors are the basis of meat flavour, and their metabolism is regulated by a variety of enzymes. Thus, it is of great significance to identify the key genes related to meat flavour precursors. In this study, the difference in flavour precursors and transcriptome between Hu sheep and Dorper with different intramuscular fat (IMF) content were investigated using widely targeted metabolomics and RNA-sequencing technologies. Then, the key genes regulating the metabolism of vital precursors were explored by integrating transcriptome and metabolome. Consequently, 594 metabolites were detected in sheep longissimus dorsi, and 76 differentially abundant metabolites (DAMs) were identified between Hu sheep and Dorper. No DAMs were observed between distinct IMF content groups within each breed. A total of 10 lysophospholipids (LPs), including four lysophospholipid ethanolamines and six lysophospholipid cholines, were identified as the main differential precursors between Hu sheep and Dorper. Furthermore, the weighted gene coexpression network analysis uncovered three differentially coexpression modules that were significantly associated with the content of differential LPs in Dorper. From the three modules, GLB1, PLD3, LPCAT2, DGKE, ACOT7, and CH25H genes were identified as key genes regulating the metabolism of LPs. This work provides an insight into understanding the difference in flavour between different sheep breeds, as well as a basis for further exploring the regulatory mechanism of key genes on LPs.

Whole-transcriptome RNA sequencing reveals the global molecular responses and circRNA/lncRNA-miRNA-mRNA ceRNA regulatory network in chicken fat deposition.

Fat deposition is a vital factor affecting the economics of poultry production. Numerous studies on fat deposition have been done. However, the molecular regulatory mechanism is still unclear. In the present study, the whole-transcriptome RNA sequencing in abdominal fat, back skin, and liver both high- and low-abdominal fat groups was used to uncover the competitive endogenous RNA (ceRNA) regulation network related to chicken fat deposition. The results showed that differentially expressed (DE) genes in abdominal fat, back skin, liver were 1207(784 mRNAs, 330 lncRNAs, 41 circRNAs, 52 miRNAs), 860 (607 mRNAs, 166 lncRNAs, 26 circRNAs, 61 miRNAs), and 923 (501 mRNAs, 262 lncRNAs, 15 circRNAs, 145 miRNAs), respectively. The ceRNA regulatory network analysis indicated that the fatty acid metabolic process, monocarboxylic acid metabolic process, carboxylic acid metabolic process, glycerolipid metabolism, fatty acid metabolism, and peroxisome proliferator-activated receptor (PPAR) signaling pathway took part in chicken fat deposition. Meanwhile, we scan the important genes, FADS2, HSD17B12, ELOVL5, AKR1E2, DGKQ, GPAM, PLIN2, which were regulated by gga-miR-460b-5p, gga-miR-199-5p, gga-miR-7470-3p, gga-miR-6595-5p, gga-miR-101-2-5p. While these miRNAs were competitive combined by lncRNAs including MSTRG.18043, MSTRG.7738, MSTRG.21310, MSTRG.19577, and circRNAs including novel_circ_PTPN2, novel_circ_CTNNA1, novel_circ_PTPRD. This finding provides new insights into the regulatory mechanism of mRNA, miRNA, lncRNA, and circRNA in chicken fat deposition.

RNA-seq reveals insights into molecular mechanisms of metabolic restoration via tryptophan supplementation in low birth weight piglet model.

Low birth weight (LBW) is associated with metabolic disorders in early life. While dietary l-tryptophan (Trp) can ameliorate postprandial plasma triglycerides (TG) disposal in LBW piglets, the genetic and biological basis underlying Trp-caused alterations in lipid metabolism is poorly understood. In this study, we collected 24 liver samples from 1-mo-old LBW and normal birth weight (NBW) piglets supplemented with different concentrations of dietary Trp (NBW with 0% Trp, N0; LBW with 0% Trp, L0; LBW with 0.4% Trp, L4; LBW with 0.8% Trp, L8; N = 6 in each group.) and conducted systematic, transcriptome-wide analysis using RNA sequencing (RNA-seq). We identified 39 differentially expressed genes (DEG) between N0 and L0, and genes within "increased dose effect" clusters based on dose-series expression profile analysis, enriched in fatty acid response of gene ontology (GO) biological process (BP). We then identified RNA-binding proteins including SRSF1, DAZAP1, PUM2, PCBP3, IGF2BP2, and IGF2BP3 significantly (P < 0.05) enriched in alternative splicing events (ASE) in comparison with L0 as control. There were significant positive and negative relationships between candidate genes from co-expression networks (including PID1, ANKRD44, RUSC1, and CYP2J34) and postprandial plasma TG concentration. Further, we determined whether these candidate hub genes were also significantly associated with metabolic and cardiovascular traits in humans via human phenome-wide association study (Phe-WAS), and analysis of mammalian orthologs suggests a functional conservation between human and pig. Our work demonstrates that transcriptomic changes during dietary Trp supplementation in LBW piglets. We detected candidate genes and related BP that may play roles on lipid metabolism restoration. These findings will help to better understand the amino acid support in LBW metabolic complications.

Low birth weight (LBW) has been associated with higher rate of mortality and morbidity and the development of metabolic complications, leaving burdens on livestock production and human health care. The feasibility of LBW metabolic restoration via postnatal nutrition compensation has been verified and the role of one of essential amino acids, l-tryptophan (Trp), on rescuing lipid metabolism in LBW was determined, while the underlying molecular mechanism and key gene regulation is little known. Our study was conducted to identify the unique molecular mechanisms between LBW and normal birth weight (NBW), and to identify the metabolic restoration related genes and biological processes after dietary Trp supplementation in LBW piglet model. We found that differentially expressed genes (DEG) between LBW and NBW were related to fatty acid response based on gene ontology enrichment analysis, and LBW piglets supplemented with Trp showed lower postprandial plasma triglycerides (TG) level as NBW, with similar expression feature of lipid metabolism related genes.

RNA-Seq analysis of duck embryo fibroblast cells gene expression during duck Tembusu virus infection.

Duck Tembusu virus (DTMUV), a member of the family Flaviviridae and an economically important pathogen with a broad host range, leads to markedly decreased egg production. However, the molecular mechanism underlying the host-DTMUV interaction remains unclear. Here, we performed high-throughput RNA sequencing (RNA-Seq) to study the dynamic changes in host gene expression at 12, 24, 36, 48 and 60 h post-infection (hpi) in duck embryo fibroblasts (DEF) infected with DTMUV. A total of 3129 differentially expressed genes (DEG) were identified after DTMUV infection. Gene Ontology (GO) category and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these DEG were associated with multiple biological functions, including signal transduction, host immunity, virus infection, cell apoptosis, cell proliferation, and pathogenicity-related and metabolic process signaling pathways. This study analyzed viral infection and host immunity induced by DTMUV infection from a novel perspective, and the results provide valuable information regarding the mechanisms underlying host-DTMUV interactions, which will prove useful for the future development of antiviral drugs or vaccines for poultry, thus benefiting the entire poultry industry.