- The unique properties of the EVs protect the cargo against degradation. - We profiled the ncRNAs (non-coding RNA) present in the EVs from seven dairy products - raw whole milk, heat-treated skim milk, homogenized heat-treated skim milk, pasteurized homogenized skim milk, pasteurized heavy whipping cream, sweet cream buttermilk and cultured buttermilk with four replicates each, obtained at different processing steps from a commercial dairy plant. - The most abundant miRNAs were bta-miR-125a and human homolog miR-718 based on the abundance values of read count obtained from the milk samples.bta-miR-125a is involved in host bacterial and viral immune response, and human homolog miR-718 is involved in the regulation of p53, VEGF, and IGF signaling pathways, respectively.. - In addition, our study explored the putative roles of other ncRNAs which included 88 piRNAs (piwi-interacting RNA), 64 antisense RNAs, and 105 lincRNAs (long-intergenic ncRNAs) contained in the bovine exosomes.. - The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. - If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. - To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.. - The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.. - Full list of author information is available at the end of the article. - They regulate around 60% of protein-coding genes in the human genome at the trans- lational level [2]. - These piRNA interact with the piwi protein subfamily of the argonaute family [4].. - Regardless of whether or not dietary ncRNA are absorbed, it is plausible that extracellular vesicles (EVs) contained in milk may confer some benefit to the health of the mam- mary gland, the immune system of the calf pre gut-closure (i.e., the first few days after birth),and the maturation of the neonatal gastrointestinal(GI) tract because of the role of milk in the maturation of the neonate.. - Furthermore, studies have implicated the deg- radation of miRNAs implicit in the pathogenesis of asthma and allergic diseases with different steps of processing extracted from whole bovine milk [19].. - In the following study, we hypothesized that raw milk collected from the bulk tank of a commercial dairy processing plant would contain growth- and immune- modulatory ncRNAs that might confer benefits or other effects to the health of the mammary gland of the cow and the maturation and development of the GI of the calf. - First, we sought to characterize ncRNAs present in raw milk obtained from the bulk tank (unprocessed), meant to be representative of the “average” cow, and to identify putative targets related to growth- and immune-function. - We performed differential expression analysis for all miRNAs present in the samples (Supplemental Data).. - Our results suggest there were a greater number of up- regulated miRNA in the six treated groups compared to the control (raw whole milk) (Fig. - probed miRTarBase to find their experimentally found gene targets. - miRNA-gene targets are not well-annotated in Bos Taurus compared to model organ- isms such as H. - Hence, many of the miRNA were instead found to have putative gene targets H. - Hence for expanding our discovery process of finding miRNAs which are essential for immune and developmental pro- cesses, we also considered the gene targets in the model organisms H. - Out of 52 putative target-genes in Bos Taurus we shortlisted by comparing with miRTaRBase, 42 genes had multiple hits, with the reference list in the PANTHER database (Supplemental Table 2, Fig. - 1 Volcano plots (log 10 ( P -value) versus log 2 (fold regulation)) showing up-regulated (colored in blue) and down-regulated miRNAs(colored in red) for each milk sample compared to control (raw whole milk).miRNAs not differentially regulated in the samples has been not included while plotting for clarity. - 0031347), and regulation of immune system process (GO:0002682), and these genes were overrepresented in the query set compared to the default reference dataset with fold enrichment values of and 5.99, respectively (yellow highlights in Supple- mental Table 2). - These overrepresentation values, based on fold-enrichment in GO analysis, suggest that our dataset had more genes related to the immune-response than to any other biological functions when compared with the reference set in the PANTHER database. - Pathways with miRNA-gene targets include developmental processes. - Pathways with putative miRNA-gene targets include signaling regulation, immune response, and development From our GO association studies described in the previous two sub-sections, we were able to shortlist 27 putative target genes which were associated with GO:. - Based on statistical testing using DESeq2 R package, we fail to reject null hypothesis which suggests there is not much significant change in overall milk miRNA abundance values of all the miRNAs found in the six treatment groups(heat-treated skim milk, homogenized heat-treated skim milk, pasteurized homogenized skim milk, heavy whipping cream, sweet cream buttermilk and cultured buttermilk when compared to raw whole milk (control);although we do report the few number of miRNAs where we observe significant change in miRNA abundances (Table 2, Fig. - processing or doesn’t get reduced because of the processing stages (Figs. - miR-718, one of the most abundant miRNAs had the highest abundance in homogenized heat-treated skim milk samples, however, its’ abundance was considerably decreased in down- stream processing (Fig. - Eighty-eight piRNA were present in the samples for which annotations of 37 piRNA were derived from the pirBASE database [26] (Supplemental Table 6a, b). - Apart from miRNA and piRNA, 305 other types of RNA tran- scripts were found in the samples. - Two hundred forty-six tran- scripts were identified in the Ensembl database. - These transcripts included some of the mapped protein-coding regions corresponding to genes responsible for neuron develop- ment, myeloid cell development, vesicle transport, neural differentiation, and others (Supplemental Table 7).. - We found that some of the transcripts in EVs correspond to protein-coding regions (based on probing Ensemble Table 1 4 gene targets of immune and growth-related functional miRNAs found in the milk samples which are connected to most number of KEGG pathways. - miRNA name Gene name Name of the KEGG pathway. - 4 Comparison of log2(abundance values) of Human homolog miRNAs related to immune response and growth related to immune response and growth in the differently treated milk samples and raw whole milk. - database), for genes that participate in development (included in Supplemental Table 4).The milk-derived EVs in this study contain several miRNA with putative targets that are related to immune response and devel- opment. - It is possible that defatting step caused pelleting of larger EVs and enrich- ment of smaller EVs and/or that processing steps prefer- entially destroy larger EVs, amplifying the proportion of the cargo of smaller EVs. - 1), likely be- cause of the presence of lactic acid bacterial culture miRNA [31], or because of other effects of culturing (e.g., pH, bacterial enzymes). - The effects of culturing and the presence of ncRNAs and their putative gene targets related to growth and development warrant further investigation.. - Each silo, or batch, was estimated to be the composite of about 30,000 dairy cows which are almost exclusively of the Holstein breed. - The cooled sour cream was churned, worked, and drained in the same fashion as the sweet cream butter- milk to produce cultured buttermilk.. - The cream layer was removed, and 2 mL of the de-fatted milk was passed through a sterile cellulose ester 0.80 μm syringe filter (Millex-AA syringe filter, EMD Millipore, Billerica, MA, USA) directly onto the exoRNeasy midi prep column. - The pipeline involves normaliz- ing UMI data with DESeq2 method [34], within the pipeline and then computing the fold-change and fold- regulation values for each of the ncRNA entry. - It uses a scaling factor to place the UMI counts across all of the samples into the same scale. - Each sample’s scaling factor is calculated as the median of the ratios of observed counts to the geometric mean of each corre- sponding miRNA across all samples.. - Fold-change is the normalized miRNA expression in each test sample divided by the normalized miRNA expression in the control sample (raw whole milk). - Fold-change values less than one suggests decreased miRNA expression, and the fold-regulation is calculated as the negative inverse of the fold-change. - contrast operation to compare raw whole milk (control) with each of the milk samples. - Computational scanning of miRNA to find associated gene-targets. - We found the experimentally annotated gene targets for miRNA dataset by comparing against the miRTarBase database [35].miRTarBase is a comprehensive database comprising of the experimentally validated miRNA- target interactions. - The gene ontology (GO) annotation of gene targets was carried out via PANTHER database.. - The built-in GO enrichment tool in the database utilizes the binomial test with Bonferroni correction to identify the overrepresentation and underrepresentation (fold-en- richment values) of GO annotations in the query gene data set compared to the frequency of occurrence of GO annotations in the reference species-specific gene dataset employed by the tool. - If the frequency of occurrence of GO annotations is higher in the query dataset compared to the in-built reference dataset, then the GO annotation is overrepresented in the query list. - If the frequency of occurrence of GO annotations is lower in the query dataset compared to the reference, then it is implied the GO annotation is underrepresented in the query dataset relative to the inbuilt reference dataset in the tool.. - We also identified other ncRNA present in the milk samples based on the mapping done by the QIAseq miRNA primary analysis pipeline (QIAGEN, Valencia, CA, USA). - The secondary annotation of piRNAs in the samples was determined using the pirBASE database [23]. - The online version contains supplementary material available at https://doi.. - org/10.1186/s w.. - musculus found in milk samples and their experimentally annotated gene-targets on probing miRTarBase.. - Gene ontology (GO) IDs over-represented in list of gene targets (specific to B. - taurus ) for which miRNAs have been found in the milk samples.. - Gene ontology (GO) IDs over-represented in list of gene targets (specific to H. - sapiens ) for which miRNAs have been found in the milk samples.. - 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