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: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.
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: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:The molecular processes underlying human milk production and the effects of mastitic infection are largely unknown because of limitations in obtaining tissue samples. Determination of gene expression in normal lactating women would be a significant step towards understanding why some women display poor lactation outcomes. Here we demonstrate the utility of RNA obtained directly from human milk cells to detect mammary epithelial cell (MEC)-specific gene expression. Milk cell RNA was collected from 5 time points (24 hours pre-partum during the colostrum period, mid lactation, two involution, and during a bout of mastitis) in addition to an involution series comprising three time points. Gene expression profiles were determined by use of human Affymetrix arrays. Milk cells collected during milk production showed that the most highly expressed genes were involved in milk synthesis (eg. CEL, OLAH, FOLR1, BTN1A1, ARG2), while milk cells collected during involution showed a significant down regulation of milk synthesis genes and activation of involution associated genes (eg. STAT3, NF-kB, IRF5, IRF7). Milk cells collected during mastitic infection revealed regulation of a unique set of genes specific to this disease state, whilst maintaining regulation of milk synthesis genes. Use of conventional epithelial cell markers was used to determine the population of MECâ??s within each sample. This paper is the first to describe the milk cell transcriptome across the human lactation cycle and during mastitic infection, providing valuable insight into gene expression of the human mammary gland. Human milk sampling throughout lactation cycle and during mastitic infection.
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:Studies of normal human mammary gland development and function have mostly relied on cell culture, limited surgical specimens, and rodent models. Although RNA extracted from human milk has been used to assay the mammary transcriptome non-invasively, the transcriptome derived from the milk fat layer has not been compared with the mammary-derived transcriptome nor have sources of RNA been quantified in milk. In this study the effects of milk collection and processing on RNA quality and origin were assessed in humans and rhesus macaques. Total RNA in milk was quantitated in acridine orange-stained milk using an automated whole slide scanner and custom-built Globulator software. Total RNA extracted from milk fat, cells in milk, and mammary biopsies of lactating rhesus macaques were compared using RNA sequencing and analysis. Compared with human milk, milk from macaques contained similar amounts of RNA-containing cytoplasmic crescents, but more cells. Total RNA extracted from milk fractions was also evaluated for factors that affect RNA quality. Degradation of RNA extracted from human milk fat was positively correlated with geographic distance from collection site, storage time, and sample type. There were no differences in RNA degradation in macaque milk collected after 10 min or 4 hr accumulation, suggesting that degradation of RNA extracted from milk fat may not occur in the mammary gland. Using RNA-Seq, RNA extracted from macaque milk fat and cells in milk more accurately represented RNA from mammary epithelial cells (cells that produce milk) than did RNA from mammary tissue. Mammary epithelium-specific transcripts were more abundant in macaque milk fat whereas adipose or stroma-specific transcripts were more abundant in mammary tissue. Functional analyses confirmed the validity of milk as a source of RNA from mammary epithelial cells. Analysis of highly abundant putative microRNAs in macaque milk fat revealed a potentially novel non-coding RNA species that is conserved in humans. RNA extracted from the milk fat during lactation accurately portrayed the RNA profile of milk-producing mammary epithelial cells. However, this sample type clearly requires protocols that minimize RNA degradation. Transcript profiles from milk cells, milk fat, and mammary tissue from 6 lactating rhesus macaques at 30 and 90 days lactation; 34 samples run in triplicate
Project description:The molecular processes underlying human milk production and the effects of mastitic infection are largely unknown because of limitations in obtaining tissue samples. Determination of gene expression in normal lactating women would be a significant step towards understanding why some women display poor lactation outcomes. Here we demonstrate the utility of RNA obtained directly from human milk cells to detect mammary epithelial cell (MEC)-specific gene expression. Milk cell RNA was collected from 5 time points (24 hours pre-partum during the colostrum period, mid lactation, two involution, and during a bout of mastitis) in addition to an involution series comprising three time points. Gene expression profiles were determined by use of human Affymetrix arrays. Milk cells collected during milk production showed that the most highly expressed genes were involved in milk synthesis (eg. CEL, OLAH, FOLR1, BTN1A1, ARG2), while milk cells collected during involution showed a significant down regulation of milk synthesis genes and activation of involution associated genes (eg. STAT3, NF-kB, IRF5, IRF7). Milk cells collected during mastitic infection revealed regulation of a unique set of genes specific to this disease state, whilst maintaining regulation of milk synthesis genes. Use of conventional epithelial cell markers was used to determine the population of MEC’s within each sample. This paper is the first to describe the milk cell transcriptome across the human lactation cycle and during mastitic infection, providing valuable insight into gene expression of the human mammary gland.
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.