Maternal diet modulates the infant microbiome and intestinal Flt3L necessary for dendritic cell development and immunity to respiratory infection.

Poor maternal diet during pregnancy is a risk factor for severe lower respiratory infections (sLRIs) in the offspring, but the underlying mechanisms remain elusive. Here, we demonstrate that in mice a maternal low-fiber diet (LFD) led to enhanced LRI severity in infants because of delayed plasmacytoid dendritic cell (pDC) recruitment and perturbation of regulatory T cell expansion in the lungs. LFD altered the composition of the maternal milk microbiome and assembling infant gut microbiome. These microbial changes reduced the secretion of the DC growth factor Flt3L by neonatal intestinal epithelial cells and impaired downstream pDC hematopoiesis. Therapy with a propionate-producing bacteria isolated from the milk of high-fiber diet-fed mothers, or supplementation with propionate, conferred protection against sLRI by restoring gut Flt3L expression and pDC hematopoiesis. Our findings identify a microbiome-dependent Flt3L axis in the gut that promotes pDC hematopoiesis in early life and confers disease resistance against sLRIs.

Human milk oligosaccharides: potential therapeutic aids for allergic diseases.

Childhood allergy, including asthma, eczema, and food allergies, is a major global health burden, with prevalence increasing dramatically and novel interventions needed. Emerging research suggests that human milk oligosaccharides (HMOs), complex glycans found in breastmilk, have allergy-protective properties, indicating exciting therapeutic potential. This review evaluates current literature on the role of HMOs in allergy, assesses underlying immunological mechanisms, and discusses future research needed to translate findings into clinical implications. HMOs may mediate allergy risk through multiple structure-specific mechanisms, including microbiome modification, intestinal barrier maturation, immunomodulation, and gene regulation. Findings emphasize the importance of breastfeeding encouragement and HMO-supplemented formula milk for high allergy-risk infants. Although further investigation is necessary to determine the most efficacious structures against varying allergy phenotypes and their long-term efficacy, HMOs may represent a promising complementary tool for childhood allergy prevention.

D-galactose might mediate some of the skeletal muscle hypertrophy-promoting effects of milk-A nutrient to consider for sarcopenia?

Sarcopenia is a process of progressive aging-associated loss of skeletal muscle mass (SMM) recognized as a serious global health issue contributing to frailty and increased all-cause mortality. Exercise and nutritional interventions (particularly intake of dairy products and milk) demonstrate good efficacy, safety, and broad applicability. Here, we propose that at least some of the well-documented favorable effects of milk and milk-derived protein supplements on SMM might be mediated by D-galactose, a monosaccharide present in large quantities in milk in the form of disaccharide lactose (milk sugar). We suggest that ingestion of dairy products results in exposure to D-galactose in concentrations metabolized primarily via the Leloir pathway with the potential to (i) promote anabolic signaling via maintenance of growth factor (e.g., insulin-like growth factor 1 [IGF-1]) receptor mature glycosylation patterns; and (ii) provide extracellular (liver glycogen) and intracellular substrates for short (muscle glycolysis) and long-term (muscle glycogen, intramyocellular lipids) energy availability. Additionally, D-galactose might optimize the metabolic function of skeletal muscles by increasing mitochondrial content and stimulating glucose and fatty acid utilization. The proposed potential of D-galactose to promote the accretion of SMM is discussed in the context of its therapeutic potential in sarcopenia.

Ecoevolutionary processes structure milk microbiomes across the mammalian tree of life.

Milk production is an ancient adaptation that unites all mammals. Milk contains a microbiome that can contribute to offspring health and microbial-immunological development. We generated a comprehensive milk microbiome dataset (16S rRNA gene) for the class Mammalia, representing 47 species from all placental superorders, to determine processes structuring milk microbiomes. We show that across Mammalia, milk exposes offspring to maternal bacterial and archaeal symbionts throughout lactation. Deterministic processes of environmental selection accounted for 20% of milk microbiome assembly processes; milk microbiomes were similar from mammals with the same host superorder (Afrotheria, Laurasiathera, Euarchontoglires, and Xenarthra: 6%), environment (marine captive, marine wild, terrestrial captive, and terrestrial wild: 6%), diet (carnivore, omnivore, herbivore, and insectivore: 5%), and milk nutrient content (sugar, fat, and protein: 3%). We found that diet directly and indirectly impacted milk microbiomes, with indirect effects being mediated by milk sugar content. Stochastic processes, such as ecological drift, accounted for 80% of milk microbiome assembly processes, which was high compared to mammalian gut and mammalian skin microbiomes (69% and 45%, respectively). Even amid high stochasticity and indirect effects, our results of direct dietary effects on milk microbiomes provide support for enteromammary trafficking, representing a mechanism by which bacteria are transferred from the mother's gut to mammary gland and then to offspring postnatally. The microbial species present in milk reflect both selective pressures and stochastic processes at the host level, exemplifying various ecological and evolutionary factors acting on milk microbiomes, which, in turn, set the stage for offspring health and development.

DPM-1001 decreased copper levels and ameliorated deficits in a mouse model of Wilson's disease.

The levels of copper, which is an essential element in living organisms, are under tight homeostatic control. Inactivating mutations in ATP7B, a P-type Cu-ATPase that functions in copper excretion, promote aberrant accumulation of the metal, primarily the in liver and brain. This condition underlies Wilson's disease, a severe autosomal recessive disorder characterized by profound hepatic and neurological deficits. Current treatment regimens rely on the use of broad specificity metal chelators as "decoppering" agents; however, there are side effects that limit their effectiveness. Here, we present the characterization of DPM-1001 {methyl 4-[7-hydroxy-10,13-dimethyl-3-({4-[(pyridin-2-ylmethyl)amino]butyl}amino)hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl] pentanoate} as a potent and highly selective chelator of copper that is orally bioavailable. Treatment of cell models, including fibroblasts derived from Wilson's disease patients, eliminated adverse effects associated with copper accumulation. Furthermore, treatment of the toxic milk mouse model of Wilson's disease with DPM-1001 lowered the levels of copper in the liver and brain, removing excess copper by excretion in the feces while ameliorating symptoms associated with the disease. These data suggest that it may be worthwhile to investigate DPM-1001 further as a new therapeutic agent for the treatment of Wilson's disease, with potential for application in other indications associated with elevated copper, including cancer and neurodegenerative diseases.

Exploring Galectin Interactions with Human Milk Oligosaccharides and Blood Group Antigens Identifies BGA6 as a Functional Galectin-4 Ligand.

Galectins (Gals), a family of multifunctional glycan-binding proteins, have been traditionally defined as ß-galactoside binding lectins. However, certain members of this family have shown selective affinity towards specific glycan structures including human milk oligosaccharides (HMOs) and blood group antigens. In this work, we explored the affinity of human galectins (particularly Gal-1, -3, -4, -7 and -12) towards a panel of oligosaccharides including HMOs and blood group antigens using a complementary approach based on both experimental and computational techniques. While prototype Gal-1 and Gal-7 exhibited differential affinity for type I vs. type II Lac/LacNAc residues and recognized fucosylated neutral glycans, chimera-type Gal-3 showed high binding affinity towards poly-LacNAc structures including LNnH and LNnO. Notably, the tandem-repeat human Gal-12 showed preferential recognition of 3-fucosylated glycans, a unique feature among members of the galectin family. Finally, Gal-4 presented a distinctive glycan-binding activity characterized by preferential recognition of specific blood group antigens, also validated by saturation transfer difference nuclear magnetic resonance (STD-NMR) experiments. Particularly, we identified oligosaccharide blood group A type 6 (BGA6) as a biologically relevant Gal-4 ligand, which specifically inhibited IL-6 secretion induced by this lectin on human peripheral blood mononuclear cells. These findings highlight unique determinants underlying specific recognition of HMOs and blood group antigens by human galectins, emphasizing the biological relevance of Gal-4-BGA6 interactions, with critical implications in the development and regulation of inflammatory responses.

Rat hepatocytes secrete free oligosaccharides.

We recently established a method for the isolation of serum-free oligosaccharides, and characterized various features of their structures. However, the precise mechanism for how these glycans are formed still remains unclarified. To further investigate the mechanism responsible for these serum glycans, here, we utilized rat primary hepatocytes to examine whether they are able to secrete free glycans. Our findings indicated that a diverse array of free oligosaccharides such as sialyl/neutral free N-glycans (FNGs), as well as sialyl lactose/LacNAc-type glycans, were secreted into the culture medium by primary hepatocytes. The structural features of these free glycans in the medium were similar to those isolated from the sera of the same rat. Further evidence suggested that an oligosaccharyltransferase is involved in the release of the serum-free N-glycans. Our results indicate that the liver is indeed secreting various types of free glycans directly into the serum.

Breast milk transmission and involvement of mammary glands in tick-borne flavivirus infected mice.

Tick-borne flaviviruses (TBFs) are transmitted to humans through milk and tick bites. Although a case of possible mother-to-child transmission of tick-borne encephalitis virus (TBEV) through breast milk has been reported, this route has not been confirmed in experimental models. Therefore, in this study, using type I interferon receptor-deficient A129 mice infected with Langat virus (LGTV), we aimed to demonstrate the presence of infectious virus in the milk and mammary glands of infected mice. Our results showed viral RNA of LGTV in the pup's stomach milk clots (SMCs) and blood, indicating that the virus can be transmitted from dam to pup through breast milk. In addition, we observed that LGTV infection causes tissue lesions in the mammary gland, and viral particles were present in mammary gland epithelial cells. Furthermore, we found that milk from infected mice could infect adult mice via the intragastric route, which has a milder infection process, longer infection time, and a lower rate of weight loss than other modes of infection. Specifically, we developed a nano-luciferase-LGTV reporter virus system to monitor the dynamics of different infection routes and observed dam-to-pup infection using in vivo bioluminescence imaging. This study provides comprehensive evidence to support breast milk transmission of TBF in mice and has helped provide useful data for studying TBF transmission routes.IMPORTANCETo date, no experimental models have confirmed mother-to-child transmission of tick-borne flavivirus (TBF) through breastfeeding. In this study, we used a mouse model to demonstrate the presence of infectious viruses in mouse breast milk and mammary gland epithelial cells. Our results showed that pups could become infected through the gastrointestinal route by suckling milk, and the infection dynamics could be monitored using a reporter virus system during breastfeeding in vivo. We believe our findings have provided substantial evidence to understand the underlying mechanism of breast milk transmission of TBF in mice, which has important implications for understanding and preventing TBF transmission in humans.

Characterization of highly pathogenic avian influenza virus in retail dairy products in the US.

In March 2024, clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) was detected in dairy cattle in the US, and it was discovered that the virus could be detected in raw milk. Although affected cow's milk is diverted from human consumption and current pasteurization requirements are expected to reduce or eliminate infectious HPAIV from the milk supply, a study was conducted to characterize whether the virus could be detected by quantitative real-time RT-PCR (qrRT-PCR) in pasteurized retail dairy products and, if detected, to determine whether the virus was viable. From 18 April to 22 April 2024, a total of 297 samples of Grade A pasteurized retail milk products (23 product types) were collected from 17 US states that represented products from 132 processors in 38 states. Viral RNA was detected in 60 samples (20.2%), with qrRT-PCR-based quantity estimates (non-infectious) of up to 5.4log1050% egg infectious doses per mL, with a mean and median of 3.0log10/mL and 2.9log10/mL, respectively. Samples that were positive for type A influenza by qrRT-PCR were confirmed to be clade 2.3.4.4 H5 HPAIV by qrRT-PCR. No infectious virus was detected in any of the qrRT-PCR-positive samples in embryonating chicken eggs. Further studies are needed to monitor the milk supply, but these results provide evidence that the infectious virus did not enter the US pasteurized milk supply before control measures for HPAIV were implemented in dairy cattle.IMPORTANCEHighly pathogenic avian influenza virus (HPAIV) infections in US dairy cattle were first confirmed in March 2024. Because the virus could be detected in raw milk, a study was conducted to determine whether it had entered the retail food supply. Pasteurized dairy products were collected from 17 states in April 2024. Viral RNA was detected in one in five samples, but infectious virus was not detected. This provides a snapshot of HPAIV in milk products early in the event and reinforces that with current safety measures, infectious viruses in milk are unlikely to enter the food supply.

Regulatory roles of dopamine D2 receptor in milk protein production and apoptosis in mammary epithelial cells.

Dopamine D2 receptors (D2Rs) play crucial roles in regulating diverse physiological functions of the central nervous system and peripheral organs. D2Rs are also expressed in mammary glands. However, which cell types express D2Rs and whether they are involved in milk production remains unclear. The present findings revealed that D2Rs are expressed in the apical regions of the lateral membranes of mammary epithelial cells (MECs) in lactating mice. We also investigated the effects of the D2R agonist bromocriptine and/or antagonist domperidone on intracellular cAMP levels, milk protein production, and apoptosis in a lactation culture model of MECs that produce major milk components like lactating MECs in vivo. We found that bromocriptine decreased intracellular cAMP levels, whereas domperidone dose-dependently neutralized this effect. Bromocriptine also inhibited casein and lactoferrin production and suppressed activities of STAT5 and glucocorticoid receptors (GRs). Domperidone neutralized the inhibition of casein production as well as STAT5 and GR inactivation induced by bromocriptine. Furthermore, D2R activation by bromocriptine induced apoptosis and inactivated ERK, a signaling molecule responsible for promoting cell proliferation and survival. Domperidone attenuated ERK inactivation and apoptosis induced by bromocriptine. These findings suggest that D2Rs play regulatory roles in milk protein production and apoptosis in MECs.

Targeting PD-L1 in cholangiocarcinoma using nanovesicle-based immunotherapy.

This study demonstrates the potential of using biological nanoparticles to deliver RNA therapeutics targeting programmed death-ligand 1 (PD-L1) as a treatment strategy for cholangiocarcinoma (CCA). RNA therapeutics offer prospects for intracellular immune modulation, but effective clinical translation requires appropriate delivery strategies. Milk-derived nanovesicles were decorated with epithelial cellular adhesion molecule (EpCAM) aptamers and used to deliver PD-L1 small interfering RNA (siRNA) or Cas9 ribonucleoproteins directly to CCA cells. In vitro, nanovesicle treatments reduced PD-L1 expression in CCA cells while increasing degranulation, cytokine release, and tumor cell cytotoxicity when tumor cells were co-cultured with T cells or natural killer cells. Similarly, immunomodulation was observed in multicellular spheroids that mimicked the tumor microenvironment. Combining targeted therapeutic vesicles loaded with siRNA to PD-L1 with gemcitabine effectively reduced tumor burden in an immunocompetent mouse CCA model compared with controls. This proof-of-concept study demonstrates the potential of engineered targeted nanovesicle platforms for delivering therapeutic RNA cargoes to tumors, as well as their use in generating effective targeted immunomodulatory therapies for difficult-to-treat cancers such as CCA.

Cellular and transcriptional diversity over the course of human lactation.

Human breast milk (hBM) is a dynamic fluid that contains millions of cells, but their identities and phenotypic properties are poorly understood. We generated and analyzed single-cell RNA-sequencing (scRNA-seq) data to characterize the transcriptomes of cells from hBM across lactational time from 3 to 632 d postpartum in 15 donors. We found that the majority of cells in hBM are lactocytes, a specialized epithelial subset, and that cell-type frequencies shift over the course of lactation, yielding greater epithelial diversity at later points. Analysis of lactocytes reveals a continuum of cell states characterized by transcriptional changes in hormone-, growth factor-, and milk production-related pathways. Generalized additive models suggest that one subcluster, LC1 epithelial cells, increases as a function of time postpartum, daycare attendance, and the use of hormonal birth control. We identify several subclusters of macrophages in hBM that are enriched for tolerogenic functions, possibly playing a role in protecting the mammary gland during lactation. Our description of the cellular components of breast milk, their association with maternal­infant dyad metadata, and our quantification of alterations at the gene and pathway levels provide a detailed longitudinal picture of hBM cells across lactational time. This work paves the way for future investigations of how a potential division of cellular labor and differential hormone regulation might be leveraged therapeutically to support healthy lactation and potentially aid in milk production.

DNA methylation haplotype block signatures responding to Staphylococcus aureus subclinical mastitis and association with production and health traits.

BACKGROUND: DNA methylation has been documented to play vital roles in diseases and biological processes. In bovine, little is known about the regulatory roles of DNA methylation alterations on production and health traits, including mastitis. RESULTS: Here, we employed whole-genome DNA methylation sequencing to profile the DNA methylation patterns of milk somatic cells from sixteen cows with naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis and ten healthy control cows. We observed abundant DNA methylation alterations, including 3,356,456 differentially methylated cytosines and 153,783 differential methylation haplotype blocks (dMHBs). The DNA methylation in regulatory regions, including promoters, first exons and first introns, showed global significant negative correlations with gene expression status. We identified 6435 dMHBs located in the regulatory regions of differentially expressed genes and significantly correlated with their corresponding genes, revealing their potential effects on transcriptional activities. Genes harboring DNA methylation alterations were significantly enriched in multiple immune- and disease-related pathways, suggesting the involvement of DNA methylation in regulating host responses to S. aureus subclinical mastitis. In addition, we found nine discriminant signatures (differentiates cows with S. aureus subclinical mastitis from healthy cows) representing the majority of the DNA methylation variations related to S. aureus subclinical mastitis. Validation of seven dMHBs in 200 cows indicated significant associations with mammary gland health (SCC and SCS) and milk production performance (milk yield). CONCLUSIONS: In conclusion, our findings revealed abundant DNA methylation alterations in milk somatic cells that may be involved in regulating mammary gland defense against S. aureus infection. Particularly noteworthy is the identification of seven dMHBs showing significant associations with mammary gland health, underscoring their potential as promising epigenetic biomarkers. Overall, our findings on DNA methylation alterations offer novel insights into the regulatory mechanisms of bovine subclinical mastitis, providing further avenues for the development of effective control measures.

Integrated analysis of genomics and transcriptomics revealed the genetic basis for goaty flavor formation in goat milk.

Goat milk exhibits a robust and distinctive "goaty" flavor. However, the underlying genetic basis of goaty flavor remains elusive and requires further elucidation at the genomic level. Through comparative genomics analysis, we identified divergent signatures of certain proteins in goat, sheep, and cow. MMUT has undergone a goat-specific mutation in the B12 binding domain. We observed the goat FASN exhibits nonsynonymous mutations in the acyltransferase domain. Structural variations in these key proteins may enhance the capacity for synthesizing goaty flavor compounds in goat. Integrated omics analysis revealed the catabolism of branched-chain amino acids contributed to the goat milk flavor. Furthermore, we uncovered a regulatory mechanism in which the transcription factor ZNF281 suppresses the expression of the ECHDC1 gene may play a pivotal role in the accumulation of flavor substances in goat milk. These findings provide insights into the genetic basis underlying the formation of goaty flavor in goat milk. STATEMENT OF SIGNIFICANCE: Branched-chain fatty acids (BCFAs) play a crucial role in generating the distinctive "goaty" flavor of goat milk. Whether there is an underlying genetic basis associated with goaty flavor is unknown. To begin deciphering mechanisms of goat milk flavor development, we collected transcriptomic data from mammary tissue of goat, sheep, cow, and buffalo at peak lactation for cross-species transcriptome analysis and downloaded nine publicly available genomes for comparative genomic analysis. Our data indicate that the catabolic pathway of branched-chain amino acids (BCAAs) is under positive selection in the goat genome, and most genes involved in this pathway exhibit significantly higher expression levels in goat mammary tissue compared to other species, which contributes to the development of flavor in goat milk. Furthermore, we have elucidated the regulatory mechanism by which the transcription factor ZNF281 suppresses ECHDC1 gene expression, thereby exerting an important influence on the accumulation of flavor compounds in goat milk. These findings provide insights into the genetic mechanisms underlying flavor formation in goat milk and suggest further research to manipulate the flavor of animal products.

Association of breast milk-derived arachidonic acid-induced infant gut dysbiosis with the onset of atopic dermatitis.

OBJECTIVE: The specific breast milk-derived metabolites that mediate host-microbiota interactions and contribute to the onset of atopic dermatitis (AD) remain unknown and require further investigation. DESIGN: We enrolled 250 mother-infant pairs and collected 978 longitudinal faecal samples from infants from birth to 6 months of age, along with 243 maternal faecal samples for metagenomics. Concurrently, 239 corresponding breast milk samples were analysed for metabolomics. Animal and cellular experiments were conducted to validate the bioinformatics findings. RESULTS: The clinical findings suggested that a decrease in daily breastfeeding duration was associated with a reduced incidence of AD. This observation inspired us to investigate the effects of breast milk-derived fatty acids. We found that high concentrations of arachidonic acid (AA), but not eicosapentaenoic acid (EPA) or docosahexaenoic acid, induced gut dysbiosis in infants. Further investigation revealed that four specific bacteria degraded mannan into mannose, consequently enhancing the mannan-dependent biosynthesis of O-antigen and lipopolysaccharide. Correlation analysis confirmed that in infants with AD, the abundance of Escherichia coli under high AA concentrations was positively correlated with some microbial pathways (eg, 'GDP-mannose-derived O-antigen and lipopolysaccharide biosynthesis'). These findings are consistent with those of the animal studies. Additionally, AA, but not EPA, disrupted the ratio of CD4/CD8 cells, increased skin lesion area and enhanced the proportion of peripheral Th2 cells. It also promoted IgE secretion and the biosynthesis of prostaglandins and leukotrienes in BALB/c mice fed AA following ovalbumin immunostimulation. Moreover, AA significantly increased IL-4 secretion in HaCaT cells costimulated with TNF-α and INF-γ. CONCLUSIONS: This study demonstrates that AA is intimately linked to the onset of AD via gut dysbiosis.

Improved breast milk proteome coverage by DIA based LC-MS/MS method.

The breast milk composition includes a multitude of bioactive factors such as viable cells, lipids and proteins. Measuring the levels of specific proteins in breast milk plasma can be challenging because of the large dynamic range of protein concentrations and the presence of interfering substances. Therefore, most proteomic studies of breast milk have been able to identify under 1000 proteins. Optimised procedures and the latest separation technologies used in milk proteome research could lead to more precise knowledge of breast milk proteome. This study (n = 53) utilizes three different protein quantification methods, including direct DIA, library-based DIA method and a hybrid method combining direct DIA and library-based DIA. On average we identified 2400 proteins by hybrid method. By applying these methods, we quantified body mass index (BMI) associated variation in breast milk proteomes. There were 210 significantly different proteins when comparing the breast milk proteome of obese and overweight mothers. In addition, we analysed a small cohort (n = 5, randomly selected from 53 samples) by high field asymmetric waveform ion mobility spectrometry (FAIMS). FAIMS coupled with the Orbitrap Fusion Lumos mass spectrometer, which led to 41.7% higher number of protein identifications compared to Q Exactive HF mass spectrometer.

Intestinal absorption of sphingosine: new insights on generated ceramide species using stable isotope tracing in vitro.

Dietary sphingomyelin (SM) has been reported to favorably modulate postprandial lipemia. Mechanisms underlying these beneficial effects on cardiovascular risk markers are not fully elucidated. Rodent studies showed that tritiated SM was hydrolyzed in the intestinal lumen into ceramides (Cer) and further to sphingosine (SPH) and fatty acids (FA) that were absorbed by the intestine. Our objective was to investigate the uptake and metabolism of SPH and/or tricosanoic acid (C23:0), the main FA of milk SM, as well as lipid secretion in Caco-2/TC7 cells cultured on semipermeable inserts. Mixed micelles (MM) consisting of different digested lipids and taurocholate were prepared without or with SPH, SPH and C23:0 (SPH+C23:0), or C23:0. Triglycerides (TG) were quantified in the basolateral medium, and sphingolipids were analyzed by tandem mass spectrometry. TG secretion increased 11-fold in all MM-incubated cells compared with lipid-free medium. Apical supply of SPH-enriched MM led to increased concentrations of total Cer in cells, and coaddition of C23:0 in SPH-enriched MM led to a preferential increase of C23:0 Cer and C23:0 SM. Complementary experiments using deuterated SPH demonstrated that SPH-d9 was partly converted to sphingosine-1-phosphate-d9, Cer-d9, and SM-d9 within cells incubated with SPH-enriched MM. A few Cer-d9 (2% of added SPH-d9) was recovered in the basolateral medium of (MM+SPH)-incubated cells, especially C23:0 Cer-d9 in (MM+SPH+C23:0)-enriched cells. In conclusion, present results indicate that MM enriched with (SPH+C23:0), such as found in postprandial micelles formed after milk SM ingestion, directly impacts sphingolipid endogenous metabolism in enterocytes, resulting in the secretion of TG-rich particles enriched with C23:0 Cer.

Delineating Bovine Milk Derived Microvesicles from Exosomes Using Proteomics.

In the work presented herein, a simple serial-pelleting purification strategy combined with a mass spectrometry-based proteomics analysis was developed as a means of discerning differences in extracellular vesicle (EV) populations found in bovine milk samples. A sequence of ultracentrifugation speeds was used to generate changes in the abundances of EV populations, allowing for the identification of associated proteins. A metric was developed to determine the relative abundances of proteins in large EVs (>200 nm) and small EVs (<200 nm). Of the 476 proteins consistently found in this study, 340 are associated with vesicular components. Of these, 156 were heavily enriched in large EVs, 155 shared between large and small EVs, and 29 heavily enriched in small EVs. Additionally, out of 68 proteins annotated as exosome proteins, 32 were enriched in large EVs, 27 shared between large and small EVs, 5 enriched in small EVs, and 7 were found to be nonvesicular contaminant proteins. The top correlated proteins in the small EV group were predominantly membrane-bound proteins, whereas the top correlated proteins in the large EV group were mostly cytosolic enzymes for molecular processing. This method provides a means of assessing the origins of vesicle components and provides new potential marker proteins within discrete vesicle populations.

Mining key circRNA-associated-ceRNA networks for milk fat metabolism in cows with varying milk fat percentages.

BACKGROUND: Cow milk fat is an essential indicator for evaluating and measuring milk quality and cow performance. Growing research has identified the molecular functions of circular RNAs (circRNAs) necessary for mammary gland development and lactation in mammals. METHOD: The present study analyzed circRNA expression profiling data in mammary epithelial cells (MECs) from cows with highly variable milk fat percentage (MFP) using differential expression analysis and weighted gene co-expression network analysis (WGCNA). RESULTS: A total of 309 differentially expressed circRNAs (DE-circRNAs) were identified in the high and low MFP groups. WGCNA analysis revealed that the pink module was significantly associated with MFP (r = - 0.85, P = 0.007). Parental genes of circRNAs in this module were enriched mainly in lipid metabolism-related signaling pathways, such as focal adhesion, ECM-receptor interaction, adherens junction and AMPK. Finally, six DE-circRNAs were screened from the pink module: circ_0010571, circ_0007797, circ_0002746, circ_0003052, circ_0004319, and circ_0012840. Among them, circ_0002746, circ_0003052, circ_0004319, and circ_0012840 had circular structures and were highly expressed in mammary tissues. Subcellular localization revealed that these four DE-circRNAs may play a regulatory role in the mammary glands of dairy cows, mainly as competitive endogenous RNAs (ceRNAs). Seven hub target genes (GNB1, GNG2, PLCB1, PLCG1, ATP6V0C, NDUFS4, and PIGH) were obtained by constructing the regulatory network of their ceRNAs and then analyzed by CytoHubba and MCODE plugins in Cytoscape. Functional enrichment analysis revealed that these genes are crucial and most probable ceRNA regulators in milk fat metabolism. CONCLUSIONS: Our study identified several vital circRNAs and ceRNAs affecting milk fat synthesis, providing new research ideas and a theoretical basis for cow lactation, milk quality, and breed improvement.

Genomic dissection of the correlation between milk yield and various health traits using functional and evolutionary information about imputed sequence variants of 34,497 German Holstein cows.

BACKGROUND: Over the last decades, it was subject of many studies to investigate the genomic connection of milk production and health traits in dairy cattle. Thereby, incorporating functional information in genomic analyses has been shown to improve the understanding of biological and molecular mechanisms shaping complex traits and the accuracies of genomic prediction, especially in small populations and across-breed settings. Still, little is known about the contribution of different functional and evolutionary genome partitioning subsets to milk production and dairy health. Thus, we performed a uni- and a bivariate analysis of milk yield (MY) and eight health traits using a set of ~34,497 German Holstein cows with 50K chip genotypes and ~17 million imputed sequence variants divided into 27 subsets depending on their functional and evolutionary annotation. In the bivariate analysis, eight trait-combinations were observed that contrasted MY with each health trait. Two genomic relationship matrices (GRM) were included, one consisting of the 50K chip variants and one consisting of each set of subset variants, to obtain subset heritabilities and genetic correlations. In addition, 50K chip heritabilities and genetic correlations were estimated applying merely the 50K GRM. RESULTS: In general, 50K chip heritabilities were larger than the subset heritabilities. The largest heritabilities were found for MY, which was 0.4358 for the 50K and 0.2757 for the subset heritabilities. Whereas all 50K genetic correlations were negative, subset genetic correlations were both, positive and negative (ranging from -0.9324 between MY and mastitis to 0.6662 between MY and digital dermatitis). The subsets containing variants which were annotated as noncoding related, splice sites, untranslated regions, metabolic quantitative trait loci, and young variants ranked highest in terms of their contribution to the traits` genetic variance. We were able to show that linkage disequilibrium between subset variants and adjacent variants did not cause these subsets` high effect. CONCLUSION: Our results confirm the connection of milk production and health traits in dairy cattle via the animals` metabolic state. In addition, they highlight the potential of including functional information in genomic analyses, which helps to dissect the extent and direction of the observed traits` connection in more detail.