MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that are widely found in organisms and play an important regulatory role in various biological processes, especially immune and inflammatory responses. However, the function of miR-320b in the inflammatory responses of bovine mammary epithelial cells (bMECs) remains to be elucidated. In this study, we examined the miR-320b mimic transduction group (miR-320b_mimic) and negative control mimic transduction group (NC_mimic) of lipopolysaccharide-treated bMECs using data-independent acquisition (DIA) proteomics and untargeted metabolomics. Subsequently, we performed a joint analysis of the sequencing data. Proteomic analysis identified 330 differentially abundant proteins (DAPs) primarily related to PPAR, ferroptosis, arachidonic acid metabolism, IL-17, and complement and coagulation cascades. Metabolome analysis identified 128 and 66 differentially accumulated metabolites (DAMs) in the positive and negative ion mode primarily involved in linoleic acid metabolism, cholesterol metabolism, AMPK, MAPK, and chemokine. Integrated metabolomics and proteomics analysis revealed the co-enrichment of DAPs and DAMs in choline metabolism in cancer, endocrine resistance, glycerophospholipid metabolism, primary bile acid biosynthesis, and the ferroptosis signaling pathways. The results of quantitative real-time PCR (RT-qPCR) showed that compared with the NC_mimic group, mRNA expression levels of COX-2, IL-12 A, iNOS, MAPK1, and MAPK14 genes were significantly down-regulated, and the mRNA expression levels of PPARγ, CEBPα, CEBPβ, FABP4, and LPL genes were significantly up-regulated in the miR-320b_mimic group. These results provide crucial insights into the molecular regulatory functions of miR-320b and offer valuable data for further research on molecular breeding aimed at enhancing mastitis resistance in bovine animals.

Bovine mastitis (BM) is a major source of morbidity among cattle, imposing a significant burden on the dairy industry. Understanding the molecular mechanisms that confer susceptibility to bovine mastitis offers potential for therapeutic targeting, but these mechanisms remain complex and incompletely understood.

This study aims to identify key molecular players and regulatory networks involved in bovine mastitis by integrating multiple expression datasets and applying network analysis.

We integrated data from three BM expression datasets and applied weighted gene co-expression network analysis (WGCNA) to identify specific modules and hub genes related to BM. Within the module that best correlated with mastitis, we constructed a network of mRNA-miRNA and protein-protein interactions.

Our analysis revealed that TNFRSF1A and NCF1 are implicated as hub genes in the host's immune response to BM. Additionally, we identified bta-mir-2881 as a potential regulator of both TNFRSF1A and NCF1. These genes are crucial in regulating the immune response and defence against bacterial infections, highlighting their importance in mastitis susceptibility.

Identifying miRNAs like bta-mir-2881 that may target key genes involved in mastitis susceptibility can shed light on the molecular mechanisms underlying the disease. This provides new targets for BM prevention and treatment, potentially alleviating the significant burden of the disease on the dairy industry.

Mastitis is an inflammatory condition that affects dairy cow's mammary glands. Traditional treatment approaches with antibiotics are increasingly leading to challenging scenarios such as antimicrobial resistance. In order to mitigate the unwanted side effects of antibiotics, alternative strategies such as those that harness the host immune system response, also known as immunotherapy, have been implemented. Immunotherapy approaches to treat bovine mastitis aims to enhance the cow's immune response against pathogens by promoting pathogen clearance, and facilitating tissue repair. Various studies have demonstrated the potential of immunotherapy for reducing the incidence, duration and severity of mastitis. Nevertheless, majority of reported therapies are lacking in specificity hampering their broad application to treat mastitis. Meanwhile, advancements in mastitis immunotherapy hold great promise for the dairy industry, with potential to provide effective and sustainable alternatives to traditional antibiotic-based approaches. This review synthesizes immunotherapy strategies, their current understanding and potential future perspectives. The future perspectives should focus on the development of precision immunotherapies tailored to address individual pathogens/group of pathogens, development of combination therapies to address antimicrobial resistance, and the integration of nano- and omics technologies. By addressing research gaps, the field of mastitis immunotherapy can make significant strides in the control, treatment and prevention of mastitis, ultimately benefiting both animal and human health/welfare, and environment health.

Malaria transmission and endemicity in Africa remains hugely disproportionate compared to the rest of the world. The complex life cycle of P. falciparum (Pf) between the vertebrate human host and the anopheline vector results in differential expression of genes within and between hosts. An in-depth understanding of Pf interaction with various human genes through regulatory elements will pave way for identification of newer tools in the arsenal for malaria control. Therefore, the regulatory elements (REs) involved in the over- or under-expression of various host immune genes hold the key to elucidating alternative control measures that can be applied for disease surveillance, prompt diagnosis and treatment. We carried out an RNAseq analysis to identify differentially expressed genes and network elucidation of non-coding RNAs and target genes associated with immune response in individuals with different clinical outcomes. Raw RNAseq datasets, retrieved for analyses include individuals with severe (Gambia-20), symptomatic (Burkina Faso-15), asymptomatic (Mali-16) malaria as well as uninfected controls (Tanzania-20; Mali-36). Of the total 107 datasets retrieved, we identified 5534 differentially expressed genes (DEGs) among disease and control groups. A peculiar pattern of DEGs was observed, with individuals presenting with severe/symptomatic malaria having the highest and most diverse upregulated genes, while a reverse phenomenon was recorded among asymptomatic and uninfected individuals. In addition, we identified 141 differentially expressed micro RNA (miRNA), of which 78 and 63 were upregulated and downregulated respectively. Interactome analysis revealed a moderate interaction between DEGs and miRNAs. Of all identified miRNA, five were unique (hsa-mir-32, hsa-mir-25, hsa-mir-221, hsa-mir-29 and hsa-mir-148) because of their connectivity to several genes, including hsa-mir-221 connected to 16 genes. Six-hundred and eight differentially expressed long non coding RNA (lncRNA) were also identified, including SLC7A11, LINC01524 among the upregulated ones. Our study provides important insight into host immune genes undergoing differential expression under different malaria conditions. It also identified unique miRNAs and lncRNAs that modify and/or regulate the expression of various immune genes. These regulatory elements we surmise, have the potential to serve a diagnostic purpose in discriminating between individuals with severe/symptomatic malaria and those with asymptomatic infection or uninfected, following further clinical validation from field isolates.

Intramuscular fat (IMF) deposition profoundly influences meat quality and economic value in beef cattle production. Meanwhile, contemporary developments in epigenetics have opened new outlooks for understanding the molecular basics of IMF regulation, and it has become a key area of research for world scholars. Therefore, the aim of this paper was to provide insight and synthesis into the intricate relationship between epigenetic mechanisms and IMF deposition in beef cattle. The methodology involves a thorough analysis of existing literature, including pertinent books, academic journals, and online resources, to provide a comprehensive overview of the role of epigenetic studies in IMF deposition in beef cattle. This review summarizes the contemporary studies in epigenetic mechanisms in IMF regulation, high-resolution epigenomic mapping, single-cell epigenomics, multi-omics integration, epigenome editing approaches, longitudinal studies in cattle growth, environmental epigenetics, machine learning in epigenetics, ethical and regulatory considerations, and translation to industry practices from perspectives of IMF deposition in beef cattle. Moreover, this paper highlights DNA methylation, histone modifications, acetylation, phosphorylation, ubiquitylation, non-coding RNAs, DNA hydroxymethylation, epigenetic readers, writers, and erasers, chromatin immunoprecipitation followed by sequencing, whole genome bisulfite sequencing, epigenome-wide association studies, and their profound impact on the expression of crucial genes governing adipogenesis and lipid metabolism. Nutrition and stress also have significant influences on epigenetic modifications and IMF deposition. The key findings underscore the pivotal role of epigenetic studies in understanding and enhancing IMF deposition in beef cattle, with implications for precision livestock farming and ethical livestock management. In conclusion, this review highlights the crucial significance of epigenetic pathways and environmental factors in affecting IMF deposition in beef cattle, providing insightful information for improving the economics and meat quality of cattle production.

Mastitis caused by multiple factors remains one of the most common and costly disease of the dairy industry. Multi-omics approaches enable the comprehensive investigation of the complex interactions between multiple layers of information to provide a more holistic view of disease pathogenesis. Therefore, this study investigated the genomic and epigenomic signatures and the possible regulatory mechanisms underlying subclinical mastitis by integrating RNA sequencing data (mRNA and lncRNA), small RNA sequencing data (miRNA) and DNA methylation sequencing data of milk somatic cells from 10 healthy cows and 20 cows with naturally occurring subclinical mastitis caused by Staphylococcus aureus or Staphylococcus chromogenes.

Functional investigation of the data sets through gene set analysis uncovered 3458 biological process GO terms and 170 KEGG pathways with altered activities during subclinical mastitis, provided further insights into subclinical mastitis and revealed the involvement of multi-omics signatures in the altered immune responses and impaired mammary gland productivity during subclinical mastitis. The abundant genomic and epigenomic signatures with significant alterations related to subclinical mastitis were observed, including 30,846, 2552, 1276 and 57 differential methylation haplotype blocks (dMHBs), differentially expressed genes (DEGs), lncRNAs (DELs) and miRNAs (DEMs), respectively. Next, 5 factors presenting the principal variation of differential multi-omics signatures were identified. The important roles of Factor 1 (DEG, DEM and DEL) and Factor 2 (dMHB and DEM), in the regulation of immune defense and impaired mammary gland functions during subclinical mastitis were revealed. Each of the omics within Factors 1 and 2 explained about 20% of the source of variation in subclinical mastitis. Also, networks of important functional gene sets with the involvement of multi-omics signatures were demonstrated, which contributed to a comprehensive view of the possible regulatory mechanisms underlying subclinical mastitis. Furthermore, multi-omics integration enabled the association of the epigenomic regulatory factors (dMHBs, DELs and DEMs) of altered genes in important pathways, such as 'Staphylococcus aureus infection pathway' and 'natural killer cell mediated cytotoxicity pathway', etc., which provides further insights into mastitis regulatory mechanisms. Moreover, few multi-omics signatures (14 dMHBs, 25 DEGs, 18 DELs and 5 DEMs) were identified as candidate discriminant signatures with capacity of distinguishing subclinical mastitis cows from healthy cows.

The integration of genomic and epigenomic data by multi-omics approaches in this study provided a better understanding of the molecular mechanisms underlying subclinical mastitis and identified multi-omics candidate discriminant signatures for subclinical mastitis, which may ultimately lead to the development of more effective mastitis control and management strategies.

Bovine mastitis is one of the most serious and costly disease affecting dairy cattle production. The present study explored the inflammatory response and autoprotective mechanism of a novel specific high expression BMNCR (bovine mastitis related long non-coding RNA) in S. aureus induced mastitis by miR-145/CBFB axis in dairy cows from the perspective of molecular genetics. In bovine mammary epithelial cells, we preformed loss of function experiments to detect changes in cytokine, proliferation and apoptosis by qRT-PCR, western blot, flow cytometry and EdU staining. The results demonstrated that BMNCR significantly increased cell apoptosis, and inhibited cell proliferation. However, the secretion of IL-1α, IL-2, IL-6, IL-8 and IL-12 were enhanced after knock-down BMNCR. Bioinformatics analysis demonstrated that BMNCR could target 8 miRNAs, in-depth analyses indicated that BMNCR acts as a molecular sponge for bta-miR-145 and CBFB was one of 23 target gene of bta-miR-145 . The results of the present study demonstrated that the role of BMNCR in S. aureus induced mastitis can be mediated by sponge bta-miR-145 activating CBFB expression. BMNCR could be a potential target for mastitis diagnosis and therapy, which may enrich the theoretical research of therapeutic intervention, and further increase milk yield and improve milk quality.

Non-coding RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), together with transcription factors, are critical pre-, co-, and post-transcriptional regulators. In addition to their criteria as ideal biomarkers, they have great potential in disease prognosis, diagnosis, and treatment of complex diseases. Investigation of regulatory mechanisms in the context of bovine mastitis, as most common and economic disease in the dairy industry, to identify elements influencing the expression of candidate genes as key regulators of the mammary immune response is not yet fully understood. Transcriptome profiles (50 RNA-Seq and 50 miRNA-Seq samples) of bovine monocytes induced by Str. uberis were used for co-expression module detection and preservation analysis using the weighted gene co-expression network analysis (WGCNA) approach. Assigned mi-, lnc-, and m-modules used to construct the integrated regulatory networks and miRNA-lncRNA-mRNA regulatory sub-networks. Remarkably, we have identified 18 miRNAs, five lncRNAs, and seven TFs as key regulators of str. uberis-induced mastitis. Most of the genes introduced here, mainly involved in immune response, inflammation, and apoptosis, were new to mastitis. These findings may help to further elucidate the underlying mechanisms of bovine mastitis, and the discovered genes may serve as signatures for early diagnosis and treatment of the disease.

Mastitis is known as intramammary inflammation, which has a multifactorial complex phenotype. However, the underlying molecular pathogenesis of mastitis remains poorly understood. In this study, we utilized a combination of RNA-seq and miRNA-seq techniques, along with computational systems biology approaches, to gain a deeper understanding of the molecular interactome involved in mastitis. We retrieved and processed one hundred transcriptomic libraries, consisting of 50 RNA-seq and 50 matched miRNA-seq data, obtained from milk-isolated monocytes of Holstein-Friesian cows, both infected with Streptococcus uberis and non-infected controls. Using the weighted gene co-expression network analysis (WGCNA) approach, we constructed co-expressed RNA-seq-based and miRNA-seq-based modules separately. Module-trait relationship analysis was then performed on the RNA-seq-based modules to identify highly-correlated modules associated with clinical traits of mastitis. Functional enrichment analysis was conducted to understand the functional behavior of these modules. Additionally, we assigned the RNA-seq-based modules to the miRNA-seq-based modules and constructed an integrated regulatory network based on the modules of interest. To enhance the reliability of our findings, we conducted further analyses, including hub RNA detection, protein-protein interaction (PPI) network construction, screening of hub-hub RNAs, and target prediction analysis on the detected modules. We identified a total of 17 RNA-seq-based modules and 3 miRNA-seq-based modules. Among the significant highly-correlated RNA-seq-based modules, six modules showed strong associations with clinical characteristics of mastitis. Functional enrichment analysis revealed that the turquoise module was directly related to inflammation persistence and mastitis development. Furthermore, module assignment analysis demonstrated that the blue miRNA-seq-based module post-transcriptionally regulates the turquoise RNA-seq-based module. We also identified a set of different RNAs, including hub-hub genes, hub-hub TFs (transcription factors), hub-hub lncRNAs (long non-coding RNAs), and hub miRNAs within the modules of interest, indicating their central role in the molecular interactome underlying the pathogenic mechanisms of S. uberis infection. This study provides a comprehensive insight into the molecular crosstalk between immunoregulatory mRNAs, miRNAs, and lncRNAs during S. uberis infection. These findings offer valuable directions for the development of molecular diagnosis and biological therapies for mastitis.

The aim of this study was to compare the cargos of miRNA in exosomes isolated from the milk of healthy (H) cows, cows at risk of mastitis (ARM), and cows with subclinical mastitis (SCM). Based on the number of somatic cells and the percentage of polymorphonuclear cells, 10 cows were assigned to group H, 11 to group ARM, and 11 to group SCM. After isolating exosomes in milk by isoelectric precipitation and ultracentrifugation, the extracted RNA was sequenced to 50 bp long single reads, and these were mapped against Btau_5.0.1. The resulting 225 miRNAs were uploaded to the miRNet suite, and target genes for Bos taurus were identified based on the miRTarBase and miRanda databases. The list of differentially expressed target genes resulting from the comparisons of the three groups was enriched using the Function Explorer of the Kyoto Encyclopedia of Genes and Genomes. A total of 38, 18, and 12 miRNAs were differentially expressed (DE, p < 0.05) in the comparisons of H vs. ARM, ARM vs. SCM, and H vs. SCM, respectively. Only 1 DE miRNA was shared among the three groups (bta-mir-221), 1 DE miRNA in the H vs. SCM comparison, 9 DE miRNAs in the ARM vs. SCM comparison, and 21 DE miRNAs in the H vs. ARM comparison. A comparison of the enriched pathways of target genes from the H, SCM, and ARM samples showed that 19 pathways were differentially expressed in the three groups, while 56 were expressed in the H vs. SCM comparison and 57 in the H vs. ARM comparison. Analyzing milk exosome miRNA cargos can be considered as a promising approach to study the complex molecular machinery set in motion in response to mastitis in dairy cows.

Bovine mastitis is one of the most troublesome and costly problems in the modern dairy industry, which is not only difficult to monitor, but can also cause economic losses while having significant implications on public health. However, efficacious preventative methods and therapy are still lacking. Moreover, new drugs and therapeutic targets are in increasing demand due to antibiotic restrictions. In recent years, noncoding RNAs have gained popularity as a topic in pathological and genetic studies. Meanwhile, there is growing evidence that they play a role in regulating various biological processes and developing novel treatment platforms. In light of this, this review focuses on two types of noncoding RNAs, micro RNAs and circular RNAs, and summarizes their characterizations, relationships, potential applications as selection markers, diagnostic or treatment targets and potential applications in RNA-based therapy, in order to shed new light on further research.

MicroRNAs (miRNAs) as post-transcriptionally regulators of gene expression have been shown to be critical regulators to fine-tuning immune responses, besides their criteria for being an ideal biomarker. The regulatory role of miRNAs in responses to most mastitis-causing pathogens is not well understood. Gram-positive Streptococcus uberis (Str. uberis), the leading pathogen in dairy herds, cause both clinical and subclinical infections. In this study, a system biology approach was used to better understand the main post-transcriptional regulatory functions and elements of bovine mammary gland response to Str. uberis infection. Publicly available miRNA-Seq data containing 50 milk samples of the ten dairy cows (five controls and five infected) were retrieved for this current research. Functional enrichment analysis of predicted targets revealed that highly confident responsive miRNAs (4 up- and 19 downregulated) mainly regulate genes involved in the regulation of transcription, apoptotic process, regulation of cell adhesion, and pro-inflammatory signaling pathways. Time series analysis showed that six gene clusters significantly differed in comparisons between Str. uberis-induced samples with controls. Additionally, other bioinformatic analysis, including upstream network analysis, showed essential genes, including TP53 and TGFB1 and some small molecules, including glucose, curcumin, and LPS, commonly regulate most of the downregulated miRNAs. Upregulated miRNAs are commonly controlled by the most important genes, including IL1B, NEAT1, DICER1 enzyme and small molecules including estradiol, tamoxifen, estrogen, LPS, and epigallocatechin. Our study used results of next-generation sequencing to reveal key miRNAs as the main regulator of gene expression responses to a Gram-positive bacterial infection. Furthermore, by gene regulatory network (GRN) analysis, we can introduce the common upregulator transcription factor of these miRNAs. Such milk-based miRNA signature(s) would facilitate risk stratification for large-scale prevention programs and provide an opportunity for early diagnosis and therapeutic intervention.

Non-coding RNAs (ncRNAs) are an abundant class of RNA with varying nucleotide lengths. They have been shown to have great potential in eutherians/human disease diagnosis and treatments and are now gaining more importance for the improvement of diseases in livestock. To date, thousands of ncRNAs have been discovered in the bovine genome and the continuous advancement in deep sequencing technologies and various bioinformatics tools has enabled the elucidation of their roles in bovine health. Among farm animals' diseases, mastitis, a common inflammatory disease in cattle, has caused devastating economic losses to dairy farmers over the last few decades. Here, we summarize the biology of bovine mastitis and comprehensively discuss the roles of ncRNAs in different types of mastitis infection. Based on our findings and relevant literature, we highlighted various evidence of ncRNA roles in mastitis. Different approaches (in vivo versus in vitro) for exploring ncRNA roles in mastitis are emphasized. More particularly, the potential applications of emerging genome editing technologies, as well as integrated omics platforms for ncRNA studies and implications for mastitis are presented.