Exploration of mobile genetic elements in the ruminal microbiome of Nellore cattle.

Metagenomics has made it feasible to elucidate the intricacies of the ruminal microbiome and its role in the differentiation of animal production phenotypes of significance. The search for mobile genetic elements (MGEs) has taken on great importance, as they play a critical role in the transfer of genetic material between organisms. Furthermore, these elements serve a dual purpose by controlling populations through lytic bacteriophages, thereby maintaining ecological equilibrium and driving the evolutionary progress of host microorganisms. In this study, we aimed to identify the association between ruminal bacteria and their MGEs in Nellore cattle using physical chromosomal links through the Hi-C method. Shotgun metagenomic sequencing and the proximity ligation method ProxiMeta were used to analyze DNA, getting 1,713,111,307 bp, which gave rise to 107 metagenome-assembled genomes from rumen samples of four Nellore cows maintained on pasture. Taxonomic analysis revealed that most of the bacterial genomes belonged to the families Lachnospiraceae, Bacteroidaceae, Ruminococcaceae, Saccharofermentanaceae, and Treponemataceae and mostly encoded pathways for central carbon and other carbohydrate metabolisms. A total of 31 associations between host bacteria and MGE were identified, including 17 links to viruses and 14 links to plasmids. Additionally, we found 12 antibiotic resistance genes. To our knowledge, this is the first study in Brazilian cattle that connect MGEs with their microbial hosts. It identifies MGEs present in the rumen of pasture-raised Nellore cattle, offering insights that could advance biotechnology for food digestion and improve ruminant performance in production systems.

Detection of potential functional variants based on systems-biology: the case of feed efficiency in beef cattle.

BACKGROUND: Potential functional variants (PFVs) can be defined as genetic variants responsible for a given phenotype. Ultimately, these are the best DNA markers for animal breeding and selection, especially for polygenic and complex phenotypes. Herein, we described the identification of PFVs for complex phenotypes (in this case, Feed Efficiency in beef cattle) using a systems-biology driven approach based on RNA-seq data from physiologically relevant organs. RESULTS: The systems-biology coupled with deep molecular phenotyping by RNA-seq of liver, muscle, hypothalamus, pituitary, and adrenal glands of animals with high and low feed efficiency (FE) measured by residual feed intake (RFI) identified 2,000,936 uniquely variants. Among them, 9986 variants were significantly associated with FE and only 78 had a high impact on protein expression and were considered as PFVs. A set of 169 significant uniquely variants were expressed in all five organs, however, only 27 variants had a moderate impact and none of them a had high impact on protein expression. These results provide evidence of tissue-specific effects of high-impact PFVs. The PFVs were enriched (FDR < 0.05) for processing and presentation of MHC Class I and II mediated antigens, which are an important part of the adaptive immune response. The experimental validation of these PFVs was demonstrated by the increased prediction accuracy for RFI using the weighted G matrix (ssGBLUP+wG; Acc = 0.10 and b = 0.48) obtained in the ssGWAS in comparison to the unweighted G matrix (ssGBLUP; Acc = 0.29 and b = 1.10). CONCLUSION: Here we identified PFVs for FE in beef cattle using a strategy based on systems-biology and deep molecular phenotyping. This approach has great potential to be used in genetic prediction programs, especially for polygenic phenotypes.

Exploring the Regulatory Potential of Long Non-Coding RNA in Feed Efficiency of Indicine Cattle.

Long non-coding RNA (lncRNA) can regulate several aspects of gene expression, being associated with complex phenotypes in humans and livestock species. In taurine beef cattle, recent evidence points to the involvement of lncRNA in feed efficiency (FE), a proxy for increased productivity and sustainability. Here, we hypothesized specific regulatory roles of lncRNA in FE of indicine cattle. Using RNA-Seq data from the liver, muscle, hypothalamus, pituitary gland and adrenal gland from Nellore bulls with divergent FE, we submitted new transcripts to a series of filters to confidently predict lncRNA. Then, we identified lncRNA that were differentially expressed (DE) and/or key regulators of FE. Finally, we explored lncRNA genomic location and interactions with miRNA and mRNA to infer potential function. We were able to identify 126 relevant lncRNA for FE in Bos indicus, some with high homology to previously identified lncRNA in Bos taurus and some possible specific regulators of FE in indicine cattle. Moreover, lncRNA identified here were linked to previously described mechanisms related to FE in hypothalamus-pituitary-adrenal axis and are expected to help elucidate this complex phenotype. This study contributes to expanding the catalogue of lncRNA, particularly in indicine cattle, and identifies candidates for further studies in animal selection and management.

An epigenetic screening determines BET proteins as targets to suppress self-renewal and tumorigenicity in canine mammary cancer cells.

Targeting self-renewal and tumorigenicity has been proposed as a potential strategy against cancer stem cells (CSCs). Epigenetic proteins are key modulators of gene expression and cancer development contributing to regulation and maintenance of self-renewal and tumorigenicity. Here, we have screened a small-molecule epigenetic inhibitor library using 3D in vitro models in order to determine potential epigenetic targets associated with self-renewal and tumorigenicity in Canine Mammary Cancer (CMC) cells. We identified inhibition of BET proteins as a promising strategy to inhibit CMC colonies and tumorspheres formation. Low doses of (+)-JQ1 were able to downregulate important genes associated to self-renewal pathways such as WNT, NOTCH, Hedgehog, PI3K/AKT/mTOR, EGF receptor and FGF receptor in CMC tumorspheres. In addition, we observed downregulation of ZEB2, a transcription factor important for the maintenance of self-renewal in canine mammary cancer cells. Furthermore, low doses of (+)-JQ1 were not cytotoxic in CMC cells cultured in 2D in vitro models but induced G2/M cell cycle arrest accompanied by upregulation of G2/M checkpoint-associated genes including BTG2 and CCNG2. Our work indicates the BET inhibition as a new strategy for canine mammary cancers by modulating the self-renewal phenotype in tumorigenic cells such as CSCs.

Identification of two molecular subtypes in canine mast cell tumours through gene expression profiling.

Mast cell tumours (MCTs) are common neoplasms in dogs and are usually regarded as potentially malignant. Several studies have attempted to identify biomarkers to better predict biological behaviours for this tumour. The aim of this study was to identify pathways connected to clinical and histopathological malignancies, shorter survival times, and poor prognoses associated with MCTs. We performed genome-wide gene expression analyses on tissues obtained from 15 dogs with single MCTs, and identified two distinct tumour subtypes-high-risk and low-risk-associated with differences in histological grades, survival times, Ki67 indices, and occurrence of death due the disease. Comparative analyses of RNA sequence profiles revealed 71 genes that were differentially expressed between high- and low-risk MCTs. In addition to these analyses, we also examined gene co-expression networks to explore the biological functions of the identified genes. The network construction revealed 63 gene modules, of which 4 were significantly associated with the more aggressive tumour group. Two of the gene modules positively correlated with high-risk MCTs were also associated with cell proliferation and extracellular matrix-related terms. At the top of the extracellular matrix module category, genes with functions directly related to those of cancer-associated fibroblasts (CAFs) were identified. Immunohistochemical analyses also revealed a greater number of CAFs in high-risk MCTs. This study provides a method for the molecular characterisation of canine MCTs into two distinct subtypes. Our data indicate that proliferation pathways are significantly involved in malignant tumour behaviours, which are known to be relevant for the induction and maintenance of MCTs. Finally, animals presenting high-risk MCTs overexpress genes associated with the extracellular matrix that can be robustly linked to CAF functions. We suggest that CAFs in the MCT stroma contribute to cancer progression.

Systems Biology Reveals NR2F6 and TGFB1 as Key Regulators of Feed Efficiency in Beef Cattle.

Systems biology approaches are used as strategy to uncover tissue-specific perturbations and regulatory genes related to complex phenotypes. We applied this approach to study feed efficiency (FE) in beef cattle, an important trait both economically and environmentally. Poly-A selected RNA of five tissues (adrenal gland, hypothalamus, liver, skeletal muscle and pituitary) of eighteen young bulls, selected for high and low FE, were sequenced (Illumina HiSeq 2500, 100 bp, pared-end). From the 17,354 expressed genes considering all tissues, 1,335 were prioritized by five selection categories (differentially expressed, harboring SNPs associated with FE, tissue-specific, secreted in plasma and key regulators) and used for network construction. NR2F6 and TGFB1 were identified and validated by motif discovery as key regulators of hepatic inflammatory response and muscle tissue development, respectively, two biological processes demonstrated to be associated with FE. Moreover, we indicated potential biomarkers of FE, which are related to hormonal control of metabolism and sexual maturity. By using robust methodologies and validation strategies, we confirmed the main biological processes related to FE in Bos indicus and indicated candidate genes as regulators or biomarkers of superior animals.

Liver proteomics unravel the metabolic pathways related to Feed Efficiency in beef cattle.

Improving nutrient utilization efficiency is essential for livestock, given the current scenario of increasing demand for animal protein and sustainable resource use. In this context, understanding the biology of feed efficiency (FE) in beef cattle allows the development of markers for identification and selection of best animals for animal production. Thus, 98 young Nellore bulls were evaluated for FE and at the end of the experiment liver samples from six High Feed Efficient (HFE) and six Low Feed Efficient (LFE) animals were collected for protein extraction, digestion and analysis by HPLC-MS/MS. Data were analyzed for differential abundant proteins (DAPs), protein networks, and functional enrichment. Serum endotoxin was also quantified. We found 42 DAPs and 3 protein networks significantly related to FE. The main pathways associated with FE were: microbial metabolism; biosynthesis of fatty acids, amino acids and vitamins; glycolysis/gluconeogenesis; xenobiotic metabolism and; antigen processing and presentation. Serum endotoxins were significantly higher in LFE animals supporting the results. Therefore, the findings presented here confirmed the altered hepatic metabolism and pronounced hepatic inflammation in LFE animals supporting that the increased bacterial load is at least in part responsible for the hepatic lesions and inflammation in LFE animals.

RNA-Seq transcriptome analysis shows anti-tumor actions of melatonin in a breast cancer xenograft model.

Melatonin is a pleiotropic anti-cancer molecule that controls cancer growth by multiple mechanisms. RNA-Seq can potentially evaluate therapeutic response and its use in xenograft tumor models can differentiate the changes that occur specifically in tumor cells or in the tumor microenvironment (TME). Melatonin actions were evaluated in a xenograft model of triple-negative breast cancer. Balb/c nude mice bearing MDA-MB-231 tumors were treated with melatonin or vehicle. RNA-Seq was performed on the Illumina HiSeq. 2500 and data were mapped against human and mouse genomes separately to differentiate species-specific expression. Differentially expressed (DE) genes were identified and Weighted Gene Co-expression Network Analysis (WGCNA) was used to detect clusters of highly co-expressed genes. Melatonin treatment reduced tumor growth (p < 0.01). 57 DE genes were identified in murine cells, which represented the TME, and were mainly involved in immune response. The WGCNA detected co-expressed genes in tumor cells and TME, which were related to the immune system among other biological processes. The upregulation of two genes (Tnfaip8l2 and Il1f6) by melatonin was validated in the TME, these genes play important roles in the immune system. Taken together, the transcriptomic data suggests that melatonin anti-tumor actions occur through modulation of TME in this xenograft tumor model.

Liver transcriptomic networks reveal main biological processes associated with feed efficiency in beef cattle.

BACKGROUND: The selection of beef cattle for feed efficiency (FE) traits is very important not only for productive and economic efficiency but also for reduced environmental impact of livestock. Considering that FE is multifactorial and expensive to measure, the aim of this study was to identify biological functions and regulatory genes associated with this phenotype. RESULTS: Eight genes were differentially expressed between high and low feed efficient animals (HFE and LFE, respectively). Co-expression analyses identified 34 gene modules of which 4 were strongly associated with FE traits. They were mainly enriched for inflammatory response or inflammation-related terms. We also identified 463 differentially co-expressed genes which were functionally enriched for immune response and lipid metabolism. A total of 8 key regulators of gene expression profiles affecting FE were found. The LFE animals had higher feed intake and increased subcutaneous and visceral fat deposition. In addition, LFE animals showed higher levels of serum cholesterol and liver injury biomarker GGT. Histopathology of the liver showed higher percentage of periportal inflammation with mononuclear infiltrate. CONCLUSION: Liver transcriptomic network analysis coupled with other results demonstrated that LFE animals present altered lipid metabolism and increased hepatic periportal lesions associated with an inflammatory response composed mainly by mononuclear cells. We are now focusing to identify the causes of increased liver lesions in LFE animals.

Genome-wide association analysis of feed intake and residual feed intake in Nellore cattle.

BACKGROUND: Feed intake plays an important economic role in beef cattle, and is related with feed efficiency, weight gain and carcass traits. However, the phenotypes collected for dry matter intake and feed efficiency are scarce when compared with other measures such as weight gain and carcass traits. The use of genomic information can improve the power of inference of studies on these measures, identifying genomic regions that affect these phenotypes. This work performed the genome-wide association study (GWAS) for dry matter intake (DMI) and residual feed intake (RFI) of 720 Nellore cattle (Bos taurus indicus). RESULTS: In general, no genomic region extremely associated with both phenotypic traits was observed, as expected for the variables that have their regulation controlled by many genes. Three SNPs surpassed the threshold for the Bonferroni multiple test for DMI and two SNPs for RFI. These markers are located on chromosomes 4, 8, 14 and 21 in regions near genes regulating appetite and ion transport and close to important QTL as previously reported to RFI and DMI, thus corroborating the literature that points these two processes as important in the physiological regulation of intake and feed efficiency. CONCLUSIONS: This study showed the first GWAS of DMI to identify genomic regions associated with feed intake and efficiency in Nellore cattle. Some genes and QTLs previously described for DMI and RFI, in other subspecies (Bos taurus taurus), that influences these phenotypes are confirmed in this study.

Bovine NR1I3 gene polymorphisms and its association with feed efficiency traits in Nellore cattle.

The Nuclear receptor 1 family I member 3 (NR1I3), also known as the Constitutive Androstane Receptor (CAR), was initially characterized as a key regulator of xenobiotic metabolism. However, recent biochemical and structural data suggest that NR1I3 is activated in response to metabolic and nutritional stress in a ligand-independent manner. Thus, we prospected the Bovine NR1I3 gene for polymorphisms and studied their association with feed efficiency traits in Nellore cattle. First, 155 purebred Nellore bulls were individually measured for Residual Feed Intake (RFI) and the 25 best (High Feed Efficiency group, HFE) and the 25 worst animals (Low Feed Efficiency group, LFE) were selected for DNA extraction. The entire Bovine NR1I3 gene was amplified and polymorphisms were identified by sequencing. Then, one SNP different between HFE and LFE groups was genotyped in all the 155 animals and in another 288 animals totalizing 443 Nellore bulls genotyped for association of NR1I3 SNPs with feed efficiency traits. We found 24 SNPs in the NR1I3 gene and choose a statistically different SNP between HFE and LFE groups for further analysis. Genotyping of the 155 animals showed a significant association within SNP and RFI (p = 0.04), Residual Intake and BW Gain (p = 0.04) and Dry Matter Intake (p = 0.01). This SNP is located in the 5'flanking promoter region of NR1I3 gene and different alleles alter the binding site for predicted transcriptional factors as HNF4alpha, CREM and c-MYB, leading us to conclude that NR1I3 expression and regulation might be important to feed efficiency.