Network visualization of genes involved in skeletal muscle myogenesis in livestock animals.

BACKGROUND: Muscle growth post-birth relies on muscle fiber number and size. Myofibre number, metabolic and contractile capacities are established pre-birth during prenatal myogenesis. The aim of this study was to identify genes involved in skeletal muscle development in cattle, sheep, and pigs - livestock. RESULTS: The cattle analysis showed significant differences in 5043 genes during the 135-280 dpc period. In sheep, 444 genes differed significantly during the 70-120 dpc period. Pigs had 905 significantly different genes for the 63-91 dpc period.The biological processes and KEGG pathway enrichment results in each species individually indicated that DEGs in cattle were significantly enriched in regulation of cell proliferation, cell division, focal adhesion, ECM-receptor interaction, and signaling pathways (PI3K-Akt, PPAR, MAPK, AMPK, Ras, Rap1); in sheep - positive regulation of fibroblast proliferation, negative regulation of endothelial cell proliferation, focal adhesion, ECM-receptor interaction, insulin resistance, and signaling pathways (PI3K-Akt, HIF-1, prolactin, Rap1, PPAR); in pigs - regulation of striated muscle tissue development, collagen fibril organization, positive regulation of insulin secretion, focal adhesion, ECM-receptor interaction, and signaling pathways (PPAR, FoxO, HIF-1, AMPK). Among the DEGs common for studied animal species, 45 common genes were identified. Based on these, a protein-protein interaction network was created and three significant modules critical for skeletal muscle myogenesis were found, with the most significant module A containing four recognized hub genes - EGFR, VEGFA, CDH1, and CAV1. Using the miRWALK and TF2DNA databases, miRNAs (bta-miR-2374 and bta-miR-744) and transcription factors (CEBPB, KLF15, RELA, ZNF143, ZBTB48, and REST) associated with hub genes were detected. Analysis of GO term and KEGG pathways showed that such processes are related to myogenesis and associated with module A: positive regulation of MAP kinase activity, vascular endothelial growth factor receptor, insulin-like growth factor binding, focal adhesion, and signaling pathways (PI3K-Akt, HIF-1, Rap1, Ras, MAPK). CONCLUSIONS: The identified genes, common to the prenatal developmental period of skeletal muscle in livestock, are critical for later muscle development, including its growth by hypertrophy. They regulate valuable economic characteristics. Enhancing and breeding animals according to the recognized genes seems essential for breeders to achieve superior gains in high-quality muscle mass.

Designing of a new multi-epitope vaccine against Leishmania major using Leish-F1 epitopes: An In-silico study.

Cutaneous leishmaniasis (CL) is the most common form of the disease which can cause malignant lesions on the skin. Vaccination for the prevention and treatment of leishmaniasis can be the most effective way to combat this disease. In this study, we designed a novel multi-epitope vaccine against Leishmania major (L. major) using immunoinformatics tools to assess its efficacy in silico. Sequences of Leish-F1 protein (TSA, Leif, and LMSTI1) of L. major were taken from GenBank. The helper T (Th) and cytotoxic T (Tc) epitopes of the protein were predicted. The final multi-epitope consisted of 18 CTL epitopes joined by AAY linker. There were also nine HTL epitopes in the structure of the vaccine construct, joined by GPGPG linker. The profilin adjuvant (the toll-like receptor 11 agonist) was also added into the construct by AAY Linker. There were 613 residues in the structure of the vaccine construct. The multi-epitope vaccine candidate was stable and non-allergic. The data obtained from the binding of final multi-epitope vaccine-TLR11 residues (band lengths and weighted scores) unveiled the ligand and the receptor high score of binding affinity. Moreover, in silico assessment of the vaccine construct cloning achieved its suitable expression in E. coli host. Based on these results, the current multi-epitope vaccine prevents L. major infection in silico, while further confirmatory assessments are required.

Identification of Hub Genes and Target miRNAs Crucial for Milk Production in Holstein Friesian Dairy Cattle.

Dairy milk production is a quantitative trait that is controlled by many biological and environmental factors. This study employs a network-driven systems approach and clustering algorithm to uncover deeper insights into its genetic associations. We analyzed the GSE33680 dataset from the GEO database to understand the biological importance of milk production through gene expression and modules. In this study, we employed CytoNCA and ClusterONE plugins within Cytoscape for network analysis. Moreover, miRWalk software was utilized to detect miRNAs, and DAVID was employed to identify gene ontology and pathways. The results revealed 140 up-regulated genes and 312 down-regulated genes. In addition, we have identified 91 influential genes and 47 miRNAs that are closely associated with milk production. Through our examination of the network connecting these genes, we have found significant involvement in important biological processes such as calcium ion transit across cell membranes, the BMP signaling pathway, and the regulation of MAPK cascade. The conclusive network analysis further reveals that GAPDH, KDR, CSF1, PYGM, RET, PPP2CA, GUSB, and PRKCA are closely linked to key pathways essential for governing milk production. Various mechanisms can control these genes, making them valuable for breeding programs aiming to enhance selection indexes.

Impact of inclusion non-additive effects on genome-wide association and variance's components in Scottish black sheep.

CONTEXT: It's well-documented that most economic traits have a complex genetic structure that is controlled by additive and non-additive gene actions. Hence, knowledge of the underlying genetic architecture of such complex traits could aid in understanding how these traits respond to the selection in breeding and mating programs. Computing and having estimates of the non-additive effect for economic traits in sheep using genome-wide information can be important because; non-additive genes play an important role in the prediction accuracy of genomic breeding values and the genetic response to the selection. AIM: This study aimed to assess the impact of non-additive effects (dominance and epistasis) on the estimation of genetic parameters for body weight traits in sheep. METHODS: This study used phenotypic and genotypic belonging to 752 Scottish Blackface lambs. Three live weight traits considered in this study were included in body weight at 16, 20, and 24 weeks). Three genetic models including additive (AM), additive + dominance (ADM), and additive + dominance + epistasis (ADEM), were used. KEY RESULTS: The narrow sense heritability for weight at 16 weeks of age (BW16) were 0.39, 0.35, and 0.23, for 20 weeks of age (BW20) were 0.55, 0.54, and 0.42, and finally for 24 weeks of age (BW24) were 0.16, 0.12, and 0.02, using the AM, ADM, and ADEM models, respectively. The additive genetic model significantly outperformed the non-additive genetic model (p < 0.01). The dominance variance of the BW16, BW20, and BW24 accounted for 38, 6, and 30% of the total phenotypic, respectively. Moreover, the epistatic variance accounted for 39, 0.39, and 47% of the total phenotypic variances of these traits, respectively. In addition, our results indicated that the most important SNPs for live weight traits are on chromosomes 3 (three SNPS including s12606.1, OAR3_221188082.1, and OAR3_4106875.1), 8 (OAR8_16468019.1, OAR8_18067475.1, and OAR8_18043643.1), and 19 (OAR19_18010247.1), according to the genome-wide association analysis using additive and non-additive genetic model. CONCLUSIONS: The results emphasized that the non-additive genetic effects play an important role in controlling body weight variation at the age of 16-24 weeks in Scottish Blackface lambs. IMPLICATIONS: It is expected that using a high-density SNP panel and the joint modeling of both additive and non-additive effects can lead to better estimation and prediction of genetic parameters.

A genome-wide association study of morphometric traits in dromedaries.

BACKGROUND: Investigating genomic regions associated with morphometric traits in camels is valuable, because it allows a better understanding of adaptive and productive features to implement a sustainable management and a customised breeding program for dromedaries. OBJECTIVES: With a genome-wide association study (GWAS) including 96 Iranian dromedaries phenotyped for 12 morphometric traits and genotyped-by-sequencing (GBS) with 14,522 SNPs, we aimed at identifying associated candidate genes. METHODS: The association between SNPs and morphometric traits was investigated using a linear mixed model with principal component analysis (PCA) and kinship matrix. RESULTS: With this approach, we detected 59 SNPs located in 37 candidate genes potentially associated to morphometric traits in dromedaries. The top associated SNPs were related to pin width, whither to pin length, height at whither, muzzle girth, and tail length. Interestingly, the results highlight the association between whither height, muzzle circumference, tail length, whither to pin length. The identified candidate genes were associated with growth, body size, and immune system in other species. CONCLUSIONS: We identified three key hub genes in the gene network analysis including ACTB, SOCS1 and ARFGEF1. In the central position of gene network, ACTB was detected as the most important gene related to muscle function. With this initial GWAS using GBS on dromedary camels for morphometric traits, we show that this SNP panel can be effective for genetic evaluation of growth in dromedaries. However, we suggest a higher-density SNP array may greatly improve the reliability of the results.

Identification of Key Genes and Biological Pathways Associated with Skeletal Muscle Maturation and Hypertrophy in Bos taurus, Ovis aries, and Sus scrofa.

The aim of the current study was to identify the major genes and pathways involved in the process of hypertrophy and skeletal muscle maturation that is common for Bos taurus, Ovis aries, and Sus scrofa species. Gene expression profiles related to Bos taurus, Ovis aries, and Sus scrofa muscle, with accession numbers GSE44030, GSE23563, and GSE38518, respectively, were downloaded from the GEO database. Differentially expressed genes (DEGs) were screened out using the Limma package of R software. Genes with Fold Change > 2 and an adjusted p-value < 0.05 were identified as significantly different between two treatments in each species. Subsequently, gene ontology and pathway enrichment analyses were performed. Moreover, hub genes were detected by creating a protein−protein interaction network (PPI). The results of the analysis in Bos taurus showed that in the period of 280 dpc−3-months old, a total of 1839 genes showed a significant difference. In Ovis aries, however, during the period of 135dpc−2-months old, a total of 486 genes were significantly different. Additionally, in the 91 dpc−adult period, a total of 2949 genes were significantly different in Sus scrofa. The results of the KEGG pathway enrichment analysis and GO function annotation in each species separately revealed that in Bos taurus, DEGs were mainly enriched through skeletal muscle fiber development and skeletal muscle contraction, and the positive regulation of fibroblast proliferation, positive regulation of skeletal muscle fiber development, PPAR signaling pathway, and HIF-1 signaling pathway. In Ovis aries, DEGs were mainly enriched through regulating cell growth, skeletal muscle fiber development, the positive regulation of fibroblast proliferation, skeletal muscle cell differentiation, and the PI3K-Akt signaling, HIF-1 signaling, and Rap1 signaling pathways. In Sus scrofa, DEGs were mainly enriched through regulating striated muscle tissue development, the negative regulation of fibroblast proliferation and myoblast differentiation, and the HIF-1 signaling, AMPK signaling, and PI3K-Akt signaling pathways. Using a Venn diagram, 36 common DEGs were identified between Bos taurus, Ovis aries, and Sus scrofa. A biological pathways analysis of 36 common DEGs in Bos taurus, Ovis aries, and Sus scrofa allowed for the identification of common pathways/biological processes, such as myoblast differentiation, the regulation of muscle cell differentiation, and positive regulation of skeletal muscle fiber development, that orchestrated the development and maturation of skeletal muscle. As a result, hub genes were identified, including PPARGC1A, MYOD1, EPAS1, IGF2, CXCR4, and APOA1, in all examined species. This study provided a better understanding of the relationships between genes and their biological pathways in the skeletal muscle maturation process.

Identification of Candidate Genes for Pigmentation in Camels Using Genotyping-by-Sequencing.

The coat color of dromedary is usually uniform and varies from black to white, although dark- to light-brown colors are the most common phenotypes. This project was designed to gain knowledge on novel color-related variants using genotyping-by-sequencing (GBS). The association between the SNPs and coat color was tested using MLM (mixed linear models) with kinship matrix. Three GWAS models including white color vs. non-white color, black vs. non-black color, and light-brown vs. dark-brown color were performed. There were no distinct genetic clusters detected based on the color phenotypes. However, admixture occurred among all individuals of the four different coat color groups. We identified nine significant SNPs associated with white color after Bonferroni correction, located close to ANKRD26, GNB1, TSPYL4, TEKT5, DEXI, CIITA, TVP23B, CLEC16A, TMPRSS13, FXYD6, MPZL3, ANKRD26, HFM1, CDC7, TGFBR3, and HACE1 genes in neighboring flanking regions. The 13 significant SNPs associated with black color and the candidate genes were: CAPN7, CHRM4, CIITA, CLEC16A, COL4A4, COL6A6, CREB3L1, DEXI, DGKZ, DGKZ, EAF1, HDLBP, INPP5F, MCMBP, MDK, SEC23IP, SNAI1, TBX15, TEKT5, TMEM189, trpS, TSPYL4, TVP23B, and UBE2V1. The SNAI1 gene interacted with MCIR, ASIP and KIT genes. These genes play a key role in the melanin biosynthetic and pigmentation biological process and melanogenesis biological pathway. Further research using a larger sample size and pedigree data will allow confirmation of associated SNPs and the identified candidate genes.

Gene-Set Enrichment Analysis for Identifying Genes and Biological Activities Associated with Growth Traits in Dromedaries.

Growth is an important heritable economic trait for dromedaries and necessary for planning a successful breeding program. Until now, genome-wide association studies (GWAS) and QTL-mapping have identified significant single nucleotide polymorphisms (SNPs) associated with growth in domestic animals, but in dromedaries, the number of studies is very low. This project aimed to find biological themes affecting growth in dromedaries. In the first step, 99 candidate SNPs were chosen from a previously established set of SNPs associated with body weight, gain, and birth weight in Iranian dromedaries. Next, 0.5 kb upstream and downstream of each candidate SNP were selected from NCBI (assembly accession: GCA_000803125.3). The annotation of fragments with candidate SNPs regarding the reference genome was retrieved using the Blast2GO tool. Candidate SNPs associated with growth were mapped to 22 genes, and 25 significant biological themes were identified to be related to growth in dromedaries. The main biological functions included calcium ion binding, protein binding, DNA-binding transcription factor activity, protein kinase activity, tropomyosin binding, myosin complex, actin-binding, ATP binding, receptor signaling pathway via JAK-STAT, and cytokine activity. EFCAB5, MTIF2, MYO3A, TBX15, IFNL3, PREX1, and TMOD3 genes are candidates for improving growth in camel breeding programs.

Computational study of zebrafish immune-targeted microarray data for prediction of preventive drug candidates.

Viral hemorrhagic septicemia virus (VHSV) is a rhabdovirus reported to cause economic loss in fish farms. Because of the lack of adequate preventative treatments, the identification of multipath genes involved in VHS infection might be an alternative to explore the possibility of using drugs for the seasonal prevention of this fish disease. We propose labeling a category of drug molecules by further classification and interpretation of the Drug Gene Interaction Database using gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment scores. The study investigated disease networks of up-and down-regulated genes to find those with high interaction as substantial genes in pathways among the different disease networks. We prioritized these genes based on their relationship to those associated with VHS infection in the context of human protein-protein interaction networks and disease pathways. Among the 29 genes as potential drug targets, nine were selected as promising druggable genes (ERBB2, FGFR3, ITGA2B, MAP2K1, NGF, NTRK1, PDGFRA, SCN2B, and SERPINC1). PDGFRA is the most important druggable up-and down-regulated gene and is considered an important gene in the IMATINIB pathway. This study findings indicate a promising approach for drug target prediction for VHS treatment, which might be useful for disease therapeutics.

Exploring membrane proteins of Leishmania major to design a new multi-epitope vaccine using immunoinformatics approach.

Leishmaniasis is one of the major global endemic diseases. Among all the different forms of the disease, cutaneous Leishmaniasis has the highest prevalence worldwide. Treatment with current drugs has not had a significant effect on the improvement of the disease. An attempt to replace an appropriate vaccine that can stimulate host cellular immunity and induce the response of Major histocompatibility complex I (MHCI) and Major histocompatibility complex II (MHCII) against Leishmania is essential. Vaccine production remains a challenge despite the use of different antigens for vaccination against Leishmania major. Hence, we were used the immunoinformatics approach to design a new multi-epitope vaccine against L. major using immunogenic outer membrane proteins. Helper T-lymphocyte (HTL) and Cytotoxic T lymphocyte (CTL) epitopes were predicted and for final confirmation of the selected epitopes, docking analysis, and molecular dynamics simulation was performed. Then, GDGDG linker and profilin adjuvant were added to enhance the immunity of vaccines. The designed vaccine was evaluated in terms of molecular weight, PI, immunogenicity, and allergenicity. Moreover, the secondary and three-dimensional structure of the final construct was identified. In silico cloning approach was carried out to improve expression of the vaccine construct. Finally, molecular docking, followed by molecular dynamic was performed to determine the interaction between multi-epitope vaccine and TLR11. We hope that the designed vaccine can be a good candidate for the development of cutaneous leishmaniasis vaccine. but its effectiveness should be assessed in vivo.

Transcriptomic analysis on the promoter regions discover gene networks involving mastitis in cattle.

Mastitis is one of the costliest diseases in dairy farms caused by infection of different microorganisms such as Escherichia coli, Streptococcus uberis and Staphylococcus aureus. Promoters are significantly involved in regulating gene expression and shedding light on the mechanisms of transcriptional regulation in physiological and immunological processes of the infections. Exploiting regulatory elements such as transcription factor binding sites (TFBSs modules) on the promoter region could reveal co-regulated genes, which allow screating regulatory models and executing a cross-sectional analysis on several databases. In this study, the promoter regions of 11 genes associated with contagious mastitis including CCL4, CXCL8, STAT3, IKBKB, MAPK14, NFKBIA, NFKB1, TNF, IL18, IL6, and HCK were investigated to predict the activating regulatory modules on promoters and to discover the key related transcription factors. By exploring the promoter regions, 228 genes were discovered comprising the same transcription factors modules. Out of 228 genes, 36 were validated using five microarray datasets. The promoter research of these genes revealed that as many as 7 down-regulated and 12 up-regulated genes are predictable in the network. The genes whose functions were associated with the initial gene list (11 genes), were identified by DAVID queries with TFBSs models implying that the approach provides a clear image of the underlying regulatory mechanism of gene expression profile and offers a novel approach in designing gene networks in cattle.

Investigation of systemic lupus erythematosus (SLE) with integrating transcriptomics and genome wide association information.

Systemic lupus erythematous (SEL) is a heterogeneous, systemic autoimmune disorder which is defined by its autoantibody pattern. Transcriptomic data analysis has shown pathways and immune system responses associated with SLE. Eight up-regulated genes (SOCE, MMP9, CXCL8, JUN, IL1B, NFKBIA, TNF and FOS) have been examined with four interactions among different pathways. These genes are associated with SNPs which have been identified through two datasets from SLE genome-wide association studies (GWAS). In this investigation, the GWAS results were integrated with pathway analysis of transcriptomes and several genes were detected with known SLE-related variations (TYK2, C5, SH2B, IRF5, IL2RA, STAT4, FCGR2A, IL7R, LYN, HLA-DRB and TNFAIP3). Pathway-based analysis on the Wikipathway Human Collection allowed the identification of prioritized variants in the relevant pathways, such as thymic stromal lymphopoietin (TSLP) signaling pathway linked to LYN, IL7R, STAT4 and rs7574865. Analysis of existing transcriptomes and GWAS data identified eight up-regulated candidate genes with more than four relationships among the different pathways associated with SNPs to pinpoint the relevant loci linked to SLE. The results of this investigation have expanded the number of candidate genes related to SLE and have highlighted possible pathways and GWAS-based methods for gene detection. Identification of the fundamental genes would assist in revealing the mechanisms responsible for SLE.

Identification of Biological Pathways Contributing to Marbling in Skeletal Muscle to Improve Beef Cattle Breeding.

Red meat is an important dietary source that provides part of the nutritional requirements. Intramuscular fat, known as marbling, is located throughout skeletal muscle. Marbling is a trait of major economic relevance that positively influences sensory quality aspects. The aim of the present study was to identify and better understand biological pathways defining marbling in beef cattle. Pathway analysis was performed in PathVisio with publicly available transcriptomic data from semitendinosus muscle of well-marbled and lean-marbled beef. Moreover, for Bos taurus we created a gene identifier mapping database with bridgeDb and a pathway collection in WikiPathways. The regulation of marbling is possibly the result of the interplay between signaling pathways in muscle, fat, and intramuscular connective tissue. Pathway analysis revealed 17 pathways that were significantly different between well-marbled and lean-marbled beef. The MAPK signaling pathway was enriched, and the signaling pathways that play a role in tissue development were also affected. Interestingly, pathways related to immune response and insulin signaling were enriched.

A lentiviral vaccine expressing KMP11-HASPB fusion protein increases immune response to Leishmania major in BALB/C.

Hydrophilic acylated surface protein B (HASPB) is an immunogenic Leishmania-specific protein that antibodies are produced against it in the sera of Leishmania-infected individuals. Kinetoplastid membrane protein 11 (KMP11) is another Leishmania antigen and considered as the suitable candidate for vaccine development Leishmaniasis. It is a highly conserved surface protein expressed in both promastigotes and amastigotes. In this study, KMP11 and HASPB coding sequences were cloned into a pCDH-cGFP lentiviral vector as a fusion protein to be used as a DNA vaccine against L. major. The KMP11-HASPB fusion protein was successfully expressed as evidenced by RT-PCR and Western blot assays. The effect of the vaccine was determined by evaluating the level of IFN-γ, IL-10, IgG1, and IgG2a performed using ELISA as well as determining the parasite load after challenge with L. major in vaccinated mice. The results revealed that IFN-γ, IL-10, IgG1, and IgG2a significantly increased after vaccination using KMP11-HASPB-expressing lentiviruses in BALB/c mice. It is noteworthy that the level of IFN-γ and IgG2a was higher than that of IL-10 and IgG1, respectively, which indicates the activation Th1 cells, macrophages, and cellular immunity. Moreover, the parasite load in the spleen and lymph node of vaccinated mice after challenge was significantly lower than that of controls.