Project description:Breast milk is the primary source of nutrition for newborns, and rich in immunological components. microRNAs (miRNAs), a well-defined group of non-coding small RNAs, are present in various body fluids (such as breast milk), which are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and regulate target gene expression and recipient cell function. We present the lactation-related miRNA expression profiles in porcine milk exosomes across entire lactation period in pig industry (newborn to 28 days after birth) using deep sequencing technology. We found that the immune-related miRNAs are presented and enriched in breast milk exosomes, and generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs exhibited the higher abundances in the colostrum (newborn to 3 days after birth) than that in the mature milk (7 to 28 days after birth), as well as in the serum of colostrum-feeding piglets compared with the only mature milk-feeding piglets. These immune-related miRNAs-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are prelude to the in-depth investigations of the essential roles of the breast milk in the development of the infant’s immune system. Eight small RNA libraries in porcine breast milk exosomes of six lactigenous stages (0, 3, 7, 14, 21 and 28 days after birth) from three female pigs were sequenced.
Project description:Breast milk is the primary source of nutrition for newborns, and rich in immunological components. microRNAs (miRNAs), a well-defined group of non-coding small RNAs, are present in various body fluids (such as breast milk), which are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and regulate target gene expression and recipient cell function. We present the lactation-related miRNA expression profiles in porcine milk exosomes across entire lactation period in pig industry (newborn to 28 days after birth) using deep sequencing technology. We found that the immune-related miRNAs are presented and enriched in breast milk exosomes, and generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs exhibited the higher abundances in the colostrum (newborn to 3 days after birth) than that in the mature milk (7 to 28 days after birth), as well as in the serum of colostrum-feeding piglets compared with the only mature milk-feeding piglets. These immune-related miRNAs-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are prelude to the in-depth investigations of the essential roles of the breast milk in the development of the infant’s immune system.
Project description:Colostrum and milk have high nutritional value and provide a complete diet for neonates, along with bioactive substances which modulate various functions such as immune defense. Exosomes are membranous vesicles of endocytic origin, recently been considered as major players in cell-cell communication. The mechanisms by which milk components can prime the infant’s active immunity are not entirely clear, and exosomes are suggested to be essential for the infant’s physiological development. We assessed the exosomal proteome profile from milk samples obtained from 10 healthy sows, at day 0 (colostrum), day 7, and 14 post-partum. Exosomes were isolated by ultracentrifugation coupled with size exclusion chromatography, and were characterized by nanoparticle tracking analysis, transmission electron microscopy and Western blotting for exosome markers. Isolated exosomes were in-gel digested and after TMT-labelling of the peptides, they were subjected to LC-MS/MS. Quantitative proteomics analysis revealed different proteome profiles for colostrum exosomes and milk exosomes, and functional analysis highlighted pathways related to immune response, cellular development, and regulation of cellular processes. This study endorses the importance of exosomes as active biocomponents of milk and provides knowledge for future studies exploring their role in regulation of immunity and growth of the newborn.
Project description:The protein profile of bovine milk serum was characterised as milk transitions from colostrum to transition milk over the first 5 days of lactation. Samples were collected from first and third parity cows at days 0, 2, 5 (D0, D2, D5) after calving. Following isolation of the milk serum fraction, label-free quantitative proteomics was carried out following normalisation by total protein concentration. Protein profiles indicated samples clustered by day postpartum, but not by parity. Proteins (n = 471) were identified and relative quantification was performed, with 199 protein groups showing altered abundance by day of lactation (fold change ≥ 2, P < 0.05). Elevated levels of immune proteins, including immunoglobulins and complement proteins were detected in colostrum with levels significantly decreasing by D2. These findings provide an outline of the dynamics of the protein profile of bovine milk and colostrum in early lactation.
Project description:Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA sequencing technology to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. We find that transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during the same postpartum time frame could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding M-NM-2-casein (CSN2) and a-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, PTPRF may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors to sub-optimal lactation in humans. Milk fat mRNA profiles were generated from Day 2 and mature milk samples obtained from lactating mothers
Project description:Human milk fat globules, by enveloping cell contents during their secretion into milk, are a rich source of mammary cell RNA. Here, we pair this non-invasive mRNA source with RNA sequencing technology to probe the milk fat layer transcriptome during three stages of lactation: colostral, transitional, and mature milk production. We find that transcriptional profiles cluster not by postpartum day, but by milk Na:K ratio, indicating that women sampled during the same postpartum time frame could be at markedly different stages of gene expression. Each stage of lactation is characterized by a dynamic range (105-fold) in transcript abundances not previously observed with microarray technology. We discovered that transcripts for isoferritins and cathepsins are strikingly abundant during colostrum production, highlighting the potential importance of these proteins for neonatal health. Two transcripts, encoding β-casein (CSN2) and a-lactalbumin (LALBA), make up 45% of the total pool of mRNA in mature lactation. Genes significantly expressed across all stages of lactation are associated with making, modifying, transporting, and packaging milk proteins. Stage-specific transcripts are associated with immune defense during the colostral stage, up-regulation of the machinery needed for milk protein synthesis during the transitional stage, and the production of lipids during mature lactation. We observed strong modulation of key genes involved in lactose synthesis and insulin signaling. In particular, PTPRF may serve as a biomarker linking insulin resistance with insufficient milk supply. This study provides the methodology and reference data set to enable future targeted research on the physiological contributors to sub-optimal lactation in humans.
Project description:We monitored longitudinal changes in bovine milk IgG in samples from four cows at 9 time points in between 0.5-28 days following calving. We used peptide-centric LC-MS/MS on proteolytic digests of whole bovine milk, resulting in the combined identification of 212 individual bovine milk protein sequences, with IgG making up >50% of the protein content of every 0.5 d colostrum sample, which reduced to ≤3% in mature milk. In parallel, we analysed IgG captured from the bovine milk samples to characterise its N-glycosylation, using dedicated methods for bottom-up glycoproteomics employing product ion-triggered hybrid fragmentation. The bovine milk IgG N-glycosylation profile was revealed to be very heterogeneous, consisting of >40 glycoforms. Furthermore, these N-glycosylation profiles changed substantially over the period of lactation, but consistently across the four individual cows. We identified NeuAc sialylation as the key abundant characteristic of bovine colostrum IgG, significantly decreasing in the first days of lactation, and barely detectable in mature bovine milk IgG. We also report, for the first time to our knowledge, the identification of subtype IgG3 in bovine milk, alongside the better-documented IgG1 and IgG2. The detailed molecular characteristics we describe of the bovine milk IgG, and their dynamic changes during lactation, are important not only for the fundamental understanding of the calf’s immune development, but also for understanding bovine milk and its bioactive components in the context of human nutrition.
Project description:α-Lactalbumin is an abundant protein present in the milk of most mammals, and is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to one month post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e., Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation primarily with N-acetylneuraminic acid (NeuAc) and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) antennae were characteristic biochemical features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform profile revealed lactational dynamics; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc antennae per glycan, at the expense of LacdiNAc antennae. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.
Project description:Milk is an easily digestible source of nutrients and bioactive factors, and its composition reflects the neonate's needs and changes from colostrum to transitional and mature milk. Our objective was to measure milk fat, lactose, total carbohydrate, and protein content in parallel with global proteome of homogenate milk samples to characterize changes across the three phases of swine lactation. Milk samples were collected from multiparous sows on postnatal day 0 (D0: colostrum), 3 (D3: early transitional), 7 (D7: late transitional) and 14 (D14: mature). Liquid chromatography tandem mass spectrometry proteomic analysis of homogenate D0, D3, and D14 milk samples (n=6) identified 772 proteins corresponding to 501 non-redundant protein-coding genes, of which 207 proteins were high confidence (detected in n=3 sows/day). Of the high confidence proteins, 81 were common to all days. Among the proteins that changed between the days was the decrease in multiple apolipoproteins and the milk fat globule membrane protein XDH. There were variable changes in complement factors, where as 14-3-3 proteins (YWHAQ,YWHAE) increased across the days. Our data provide a good characterization of milk proteome changes that likely reflect mammary function as well as the neonate's phase-specific developmental needs. These data, may be useful in developing approaches to enhance the health and welfare of swine.