MFG-E8 Ameliorates Nerve Injury-Induced Neuropathic Pain by Regulating Microglial Polarization and Neuroinflammation via Integrin ß3/SOCS3/STAT3 Pathway in Mice.

Spinal microglial polarization plays a crucial role in the pathological processes of neuropathic pain following peripheral nerve injury. Accumulating evidence suggests that milk fat globule epidermal growth factor-8 (MFG-E8) exhibits anti-inflammatory effect and regulates microglial polarization through the integrin ß3 receptor. However, the impact of MFG-E8 on microglial polarization in the context of neuropathic pain has not yet been investigated. In this study, we evaluated the effect of MFG-E8 on pain hypersensitivity and spinal microglial polarization following spared nerve injury (SNI) of the sciatic nerve in mice. We determined the molecular mechanisms underlying the effects of MFG-E8 on pain hypersensitivity and spinal microglial polarization using pain behavior assessment, western blot (WB) analysis, immunofluorescence (IF) staining, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and small interfering RNA (siRNA) transfection. Our findings indicate that SNI significantly increased the levels of MFG-E8 and integrin ß3 expressed in microglia within the spinal cord of mice. Additionally, we observed that intrathecal injection of recombinant human MFG-E8 (rhMFG-E8) alleviated SNI induced-mechanical allodynia and thermal hyperalgesia. Furthermore, the results suggested that rhMFG-E8 facilitated M2 microglial polarization and ameliorated neuroinflammation via integrin ß3/SOCS3/STAT3 pathway in the spinal cord of mice with SNI. Importantly, these effects were negated by integrin ß3 siRNA, or SOCS3 siRNA. These results demonstrate that MFG-E8 ameliorates peripheral nerve injury induced-mechanical allodynia and thermal hyperalgesia by driving M2 microglial polarization and mitigating neuroinflammation mediated by integrin ß3/SOCS3/STAT3 pathway in the spinal cord of mice. MFG-E8 may serve as a promising target for the treatment of neuropathic pain.

Liriodendrin stimulates proliferation and milk protein synthesis of mammary epithelial cells via the PI3K-DDX18 signaling.

Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25 mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35 mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes ß-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling.

ARID1B blocks methionine-stimulated mTOR activation to inhibit milk fat and protein synthesis in and proliferation of mouse mammary epithelial cells.

Met can function through the mTOR signaling pathway, but the molecular mechanism is not fully understood. Here we investigated the role of ARID1B in this regulatory process. ARID1B knockdown promoted milk fat and protein synthesis in and cell proliferation of HC11 cells and increased mTOR mRNA expression and protein phosphorylation, whereas ARID1B gene activation had the opposite effects. ARID1B gene activation totally blocked Met's stimulation on mTOR mRNA expression. ARID1B bound to one region of the mTOR promoter, and Met reduced the binding of ARID1B on this promoter. LY294002 blocked Met-induced reduction of ARID1B mRNA and protein level. Cycloheximide treatment did not affect the decrease of ARID1B by Met. MG132 but not chloroquine restored ARID1B degradation induced by Met. Our data reveal that ARID1B is a key negative regulator of milk fat and protein synthesis in and proliferation of HC11 cells, and blocks Met-stimulated mTOR gene transcription.

MFG-E8 (LACTADHERIN): a novel marker associated with cerebral amyloid angiopathy.

Brain accumulation of amyloid-beta (Aß) is a crucial feature in Alzheimer´s disease (AD) and cerebral amyloid angiopathy (CAA), although the pathophysiological relationship between these diseases remains unclear. Numerous proteins are associated with Aß deposited in parenchymal plaques and/or cerebral vessels. We hypothesized that the study of these proteins would increase our understanding of the overlap and biological differences between these two pathologies and may yield new diagnostic tools and specific therapeutic targets. We used a laser capture microdissection approach combined with mass spectrometry in the APP23 transgenic mouse model of cerebral-ß-amyloidosis to specifically identify vascular Aß-associated proteins. We focused on one of the main proteins detected in the Aß-affected cerebrovasculature: MFG-E8 (milk fat globule-EGF factor 8), also known as lactadherin. We first validated the presence of MFG-E8 in mouse and human brains. Immunofluorescence and immunoblotting studies revealed that MFG-E8 brain levels were higher in APP23 mice than in WT mice. Furthermore, MFG-E8 was strongly detected in Aß-positive vessels in human postmortem CAA brains, whereas MFG-E8 was not present in parenchymal Aß deposits. Levels of MFG-E8 were additionally analysed in serum and cerebrospinal fluid (CSF) from patients diagnosed with CAA, patients with AD and control subjects. Whereas no differences were found in MFG-E8 serum levels between groups, MFG-E8 concentration was significantly lower in the CSF of CAA patients compared to controls and AD patients. Finally, in human vascular smooth muscle cells MFG-E8 was protective against the toxic effects of the treatment with the Aß40 peptide containing the Dutch mutation. In summary, our study shows that MFG-E8 is highly associated with CAA pathology and highlights MFG-E8 as a new CSF biomarker that could potentially be used to differentiate cerebrovascular Aß pathology from parenchymal Aß deposition.

UFL1 regulates milk protein and fat synthesis-related gene expression of bovine mammary epithelial cells probably via the mTOR signaling pathway.

UFL1 is an ufmylation (a novel post-translational modification) E3 ligase, mainly located in the endoplasmic reticulum (ER), that has emerged as a significant regulator of several physiological and pathological processes. Yet its physiological function in milk synthesis in bovine mammary epithelial cells (BMECs) remains unknown. In this study, we investigated the effects of UFL1 in milk protein and fat synthesis-related gene expression, with a particular emphasis on the role of UFL1 in LPS-treated BMECs. Results showed that UFL1 depletion significantly reduced the expression of milk protein and fat synthesis-related gene and mTOR phosphorylation in both normal and LPS-treated BMECs. Overexpression of UFL1 enhanced the activation of the mTOR and milk protein and fat synthesis-related gene expression. Collectively, these above results strongly demonstrate that UFL1 could regulate milk protein and fat synthesis-related gene expression of BMECs probably via the mTOR signaling pathway.

Novel Crabtree negative yeast from rumen fluids can improve rumen fermentation and milk quality.

Upgrading the nutritive value of rice straw (RS) is necessary to increase its contribution to enhancing meat and milk production. Present work verified whether novel Crabtree negative yeast inoculant could promote RS utilization, rumen fermentation, and milk quality in tropical crossbred lactating Holstein cows. The new stain of Crabtree negative yeasts (Pichia kudriavzevii KKU20 and Candida tropicalis KKU20) was isolated from the rumen of dairy cattle. This study used 6 multiparous crossbreds between Holstein Frisian × Zebu dairy cows in their mid-lactation period. Dairy cows were randomly allocated to three ensiled RS with various yeast stains including Saccharomyces cerevisiae, P. kudriavzevii KKU20, and C. tropicalis KKU20 according to a 3 × 3 replicated Latin square design. Crabtree-negative yeast (P. kudriavzevii and C. tropicalis) increased the apparent digestibility of dry matter by about 6.9% when compared with Crabtree-positive yeast (S. cerevisiae). Bacterial populations were highest with ensiled RS by C. tropicalis KKU20. Ensiled RS with Crabtree-negative yeasts were significantly increased with total volatile fatty acids, but they did not affect volatile fatty acid profiles. Milk protein precentage was highest at 35.6 g/kg when C. tropicalis was fed, and lowest when applied with S. cerevisiae and P. kudriavzevii KKU20 in ensiled RS at 34.5 and 34.1 g/kg, respectively. Thus, feeding ensiled RS with novel Crabtree negative yeast could improve RS digestion, rumen fermentation, and milk protein content in dairy cows.

Menthol Targeting AMPK Alleviates the Inflammatory Response of Bovine Mammary Epithelial Cells and Restores the Synthesis of Milk Fat and Milk Protein.

Mastitis is one of the most serious diseases that causes losses in the dairy industry, seriously impairing milk production and milk quality, and even affecting human health. Menthol is a cyclic monoterpene compound obtained from the stem and leaves of peppermint, which has a variety of biological activities, including anti-inflammatory and antioxidant activity. The purpose of this study was to investigate the preventive effect of menthol on the lipopolysaccharide-induced inflammatory response in primary bovine mammary gland epithelial cells (BMECs) and its anti-inflammatory mechanism. First, BMECs were isolated and amplified from the udders of Holstein cows by enzymatic hydrolysis. BMECs were treated with menthol (10, 50, 100, 200 µM) for 1h, followed by lipopolysaccharide (5µg/ml) for 12 h. Lipopolysaccharide treatment upregulated the protein levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (INOS) and the mRNA abundance of tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), and interleukin-1ß (IL-1ß), while menthol was able to inhibit this effect. The inhibitory effect of menthol on proinflammatory factors was significantly reduced when autophagy was blocked using 3-Methyladenine (5µg/ml), an inhibitor of autophagy. Furthermore, lipopolysaccharide treatment reduced the expression levels of milk lipids and milk proteins, which were inhibited by menthol. In addition, menthol (200 µM) treatment was able to significantly upregulate the expression level of autophagy-related protein LC3B, downregulate the expression level of P62, promote the expression abundance of autophagy-related gene mRNA, and enhance significantly enhance autophagic flux. Interestingly, treatment of BMECs with menthol (200 µM) promoted the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51 like kinase 1 (ULK1) and increased the nuclear localization of nuclear factor-E2 associated factor 2 (Nrf-2). When the AMPK pathway was blocked using compound C (10µg/ml), an inhibitor of AMPK, autophagy was significantly inhibited. Autophagy levels were significantly decreased after blocking the Nrf-2 pathway using ML385 (5µg/ml), an inhibitor of Nrf-2. Overall, the data suggest that menthol activates the AMPK-ULK1 pathway to initiate the onset of autophagy and maintains the level of autophagy through the AMPK-Nrf-2 pathway. In conclusion, the findings suggest that menthol may alleviate the inflammatory response in BMECs via the AMPK/ULK1/Nrf-2/autophagy pathway.

MFG-E8 alleviates oxygen-glucose deprivation-induced neuronal cell apoptosis by STAT3 regulating the selective polarization of microglia.

Background: Ischemic stroke is a complex pathological process, involving inflammatory reaction, energy metabolism disorder, free radical injury, cell apoptosis and other aspects. Accumulating evidences have revealed that MFG-E8 had a protective effect on multiple organ injuries. However, the comprehensive function and mechanism of MFG-E8 in ischemic brain remain largely unclear.Methods: BV-2 cells were treated with recombinant murine MFG-E8 (rmMFG-E8) or/and Colivelin TFA after exposing for 4 h with oxygen glucose deprivation (OGD). Cell viability and apoptosis were assessed by MTT assay and Flow cytometry. RT-qPCR and Western blot assays were applied to examine the expression levels of MFG-E8, apoptosis-related proteins and M1/M2 polarization markers.Results: Our results demonstrated that OGD significantly inhibited microglial viability and facilitated apoptosis. In addition, we found that OGD downregulated MFG-E8 expression, and MFG-E8 inhibited OGD-induced microglial apoptosis and promoted microglial M2 polarization. In terms of mechanism, we proved that MFG-E8 regulated OGD-induced microglial M1/M2 polarization by inhibiting p-STAT3 and SOCS3 expressions, which was reversed by STAT3 activator (Colivelin TFA). Finally, we verified MFG-E8 alleviated OGD-induced neuronal cell apoptosis by M2 polarization of BV-2 cells.Conclusions: We demonstrated that MFG-E8 reduced neuronal cell apoptosis by enhancing activation of microglia via STAT3 signaling. Therefore, we suggested that MFG-E8 might provide a novel mechanism for ischemic stroke.

UFM1-Specific Ligase 1 Ligating Enzyme 1 Mediates Milk Protein and Fat Synthesis-Related Gene Expression via the JNK Signaling Pathway in Mouse Mammary Epithelial Cells.

Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) has been characterized as a ubiquitin-like (Ubl) protein that affects a range of cellular processes across various pathways. In this study, mouse mammary epithelial cells (HC11 cell line) and UFL1 knockout (KO) mice were used to establish UFL1 knockdown models to explore the influence of UFL1 on milk protein and fat synthesis in the mouse mammary gland and the underlying mechanisms. This is the first study to show UFL1 localization in mouse mammary epithelial cells. UFL1 depletion by transfected UFL1 siRNA (siUFL1) caused aggravated apoptosis. In addition, UFL1 depletion suppressed milk protein synthesis-related protein level in vivo and in vitro. Conversely, ACACA and FASN expressions increased in UFL1-deficient mice. Moreover, UFL1 depletion increased triglyceride synthesis levels and inhibited the p-JNK expression. Importantly, the expression of proteins related to milk protein synthesis was decreased in JNK- and UFL1-deficient cells, whereas proteins related to milk fat synthesis showed the opposite trend, indicating that UFL1 affects milk protein and fat synthesis via the suppression of JNK activation. Overall, our findings indicate that UFL1 plays a key role in mammary milk and fat synthesis via JNK activation.

Post-ruminal supplies of glucose and casein, but not acetate, stimulate milk protein synthesis in dairy cows through differential effects on mammary metabolism.

Amino acids and glucose have been shown to regulate protein synthesis in the mammary gland through their effects on cellular signaling pathways. Acetate might also have an effect on protein synthesis via the AMP-activated kinase signaling pathway, because it is the main energy source for the mammary secretory cell. Thus, the objective of this experiment was to evaluate the effects of casein and energy-yielding nutrients (acetate and glucose), and their combination, on performance and mammary metabolism. Six multiparous Holstein cows, averaging 49 kg of milk/d, were used in a 6 × 6 Latin square design with 14-d periods. Cows were fed to 100% National Research Council requirements for metabolizable protein (MP) and energy (ME) for 9 d, after which they were feed-restricted for 5 d to 85% of their individual ad libitum intake and then abomasally infused with 1 of 6 treatments. Treatments were acetate (A), glucose (G), each at 5% of ad libitum ME intake, casein (C) at 15% of ad libitum MP intake, A + C, G + C, or a saline solution (negative control). Casein infused alone increased milk protein yield numerically, with 25% recovery of the infused casein in milk protein. Glucose infused alone increased milk and milk protein yield and promoted the highest efficiency of nitrogen utilization (37%), with an efficiency of MP use for milk protein of 58%. We discovered no effect of treatment on mammary plasma flow, and the increase in milk protein yield with glucose infusion was brought about by greater mammary AA clearance rate. Infusion of casein and glucose together further increased milk protein yield in an additive fashion, and 47% of the infused casein was recovered in milk protein. Acetate infused alone had no effect on milk protein yield but increased milk fat yield numerically, suggesting that the greater amount of acetate taken up by the mammary gland was used for milk fat synthesis. Infusion of acetate and casein together yielded responses similar to those of casein alone. In conclusion, glucose has a major effect on stimulating milk protein synthesis, and the mammary gland has the ability to increase its supply of nutrients to match its synthetic capacity.

Targeted disruption of TORC1 retards young squab growth by inhibiting the synthesis of crop milk protein in breeding pigeon (Columba livia).

This study was conducted to explore the regulatory role of the target of rapamycin complex 1 (TORC1) signaling pathway in crop milk synthesis in breeding pigeons (Columba livia). Three groups of breeding pigeons in the lactation period (n = 30 pairs/group) were respectively injected with rapamycin (RAPA, a specific inhibitor of the target of rapamycin complex) at doses of 0 (vehicle, control), 0.6, or 1.2 mg/kg body weight (BW)/day via the wing vein for 7 days. The average daily feed intake (ADFI) and BW of the breeding pigeons and the BW of young squabs were respectively recorded throughout the experimental period. The breeding pigeons were sacrificed to collect their crop tissues, crop milk, and serum on the eighth day of the experiment. The results showed that neither 0.6 nor 1.2 mg/kg BW RAPA injection affected BW loss or ADFI in breeding pigeons (P > 0.05), while crop thickness and crop relative weight were significantly decreased (P < 0.05) in the 1.2 mg/kg BW rapamycin-injected group. Simultaneously, RAPA (especially at 1.2 mg/kg BW) decreased the crude protein, αs1-casein, αs2-casein, ß-casein, and amino acid contents (Asp, Thr, Ser, Glu, Gly, Ala, Cys, Val, Met, Ile, Leu, Tyr, Lys, His, Arg, and Pro) of crop milk (P < 0.05) and the concentrations of albumin, total protein, and uric acid in the serum of breeding pigeons (P < 0.05). Additionally, the expression of TORC1 pathway-related proteins (TORC1, S6K1, S6, 4EBP1, and eIF4E) was downregulated in the crop tissues of breeding pigeons by 0.6 or 1.2 mg/kg BW/day RAPA injection (P < 0.05). Accordingly, the average daily gain (ADG) of young squabs declined, and the mortality rate increased significantly (P < 0.05). Together, the results showed that RAPA reduced protein and amino acid levels in the crop milk of breeding pigeons and retarded young squab growth, suggesting a crucial role of TORC1 in crop milk synthesis in breeding pigeons.

High levels of fatty acids inhibit ß-casein synthesis through suppression of the JAK2/STAT5 and mTOR signaling pathways in mammary epithelial cells of cows with clinical ketosis.

Ketosis is a metabolic disease of dairy cows often characterized by high concentrations of ketone bodies and fatty acids, but low milk protein and milk production. The Janus kinase 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5) and the mechanistic target of rapamycin (mTOR) signaling pathways are central for the regulation of milk protein synthesis. The effect of high levels of fatty acids on these pathways and ß-casein synthesis are unknown in dairy cows with clinical ketosis. Mammary gland tissue and blood samples were collected from healthy (n = 15) and clinically-ketotic (n = 15) cows. In addition, bovine mammary epithelial cells (BMEC) were treated with fatty acids, methionine (Met) or prolactin (PRL), respectively. In vivo, the serum concentration of fatty acids was greater (P > 0.05) and the percentage of milk protein (P > 0.05) was lower in cows with clinical ketosis. The JAK2-STAT5 and mTOR signaling pathways were inhibited and the abundance of ß-casein was lower in mammary tissue of cows with clinical ketosis (P > 0.05). In vitro, high levels of fatty acids inhibited the JAK2-STAT5 and mTOR signaling pathways (P > 0.05) and further decreased the ß-casein synthesis (P > 0.05) in BMEC. Methionine or PRL treatment, as positive regulators, activated the JAK2-STAT5 and mTOR signaling pathways to increase the ß-casein synthesis. Importantly, the high concentration of fatty acids attenuated the positive effect of Met or PRL on mTOR, JAK2-STAT5 pathways and the abundance of ß-casein (P > 0.05). Overall, these data indicate that the high concentrations of fatty acids that reach the mammary cells during clinical ketosis inhibit mTOR and JAK2-STAT5 signaling pathways, and further suppress ß-casein synthesis.

Thermally-Induced Lactosylation of Whey Proteins: Identification and Synthesis of Lactosylated ß-lactoglobulin Epitope.

The high temperatures used in the production of milk may induce modifications in proteins structure. Due to occurrence of the Maillard reaction, lactose binds lysine residues in proteins, affecting the nutritional value. Milk is also an important source of allergenic proteins (i.e., caseins, ß-lactoglobulin and α-lactalbumin). Thus, this modification may also affect the allergenicity of these proteins. Focusing on milk whey proteins, a screening on different Ultra High Temperatures (UHT) and pasteurized milk samples was performed to identify lactosylation sites, in particular in protein known epitopes, and to verify the correlation between lactosylation and the harshness of the treatment. Whey proteins were extracted from milk samples after caseins precipitations at pH 4.6 and, after chymotryptic and tryptic in solution digestion, peptides were analysed by UPLC-MS and LTQ-Orbitrap. Results show the presence of lactosylated lysine residues in several known epitopes. Then, a ß-lactoglobulin epitope was selected and synthesized by solid phase synthesis followed by in solution lactosylation, obtaining high reaction yields and purities. The synthesis of lactosylated allergenic epitopes, described here for the first time, is a useful tool for further studies on the technological impacts on food allergenicity.

Immunological and Clinicopathological Significance of MFG-E8 Expression in Patients with Oral Squamous Cell Carcinoma.

Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein secreted by the activated macrophages and acts as a bridge between apoptotic cells and phagocytes. Aside from macrophages, a variety of malignant cells also express MFG-E8. The objective of this study is to elucidate the clinical relevance and significance of MFG-E8 in the tumor microenvironment (TME) of patients with oral squamous cell carcinoma (OSCC). We investigated MFG-E8 expression in 74 patients with OSCC by immunohistochemistry and evaluated the relationship between MFG-E8 expression and various clinicopathological factors including immune cell infiltration. MFG-E8 expression was detected in 34 of 74 (45.9%) patients with OSCC and a significant correlation was observed with levels of infiltrating T cells, macrophages, and immunosuppressive M2 macrophages. Furthermore, MFG-E8 expression was also associated with clinical stage, lymphatic/vascular invasion, and Ki-67+ tumor cells but not with survival. Our results suggest that MFG-E8 may play an important role in shaping the immune suppressive network in TME as well as tumor progression.

Proteomic changes in the milk of water buffaloes (Bubalus bubalis) with subclinical mastitis due to intramammary infection by Staphylococcus aureus and by non-aureus staphylococci.

Subclinical mastitis by Staphylococcus aureus (SAU) and by non-aureus staphylococci (NAS) is a major issue in the water buffalo. To understand its impact on milk, 6 quarter samples with >3,000,000 cells/mL (3 SAU-positive and 3 NAS-positive) and 6 culture-negative quarter samples with <50,000 cells/mL were investigated by shotgun proteomics and label-free quantitation. A total of 1530 proteins were identified, of which 152 were significantly changed. SAU was more impacting, with 162 vs 127 differential proteins and higher abundance changes (P < 0.0005). The 119 increased proteins had mostly structural (n = 43, 28.29%) or innate immune defence functions (n = 39, 25.66%) and included vimentin, cathelicidins, histones, S100 and neutrophil granule proteins, haptoglobin, and lysozyme. The 33 decreased proteins were mainly involved in lipid metabolism (n = 13, 59.10%) and included butyrophilin, xanthine dehydrogenase/oxidase, and lipid biosynthetic enzymes. The same biological processes were significantly affected also upon STRING analysis. Cathelicidins were the most increased family, as confirmed by western immunoblotting, with a stronger reactivity in SAU mastitis. S100A8 and haptoglobin were also validated by western immunoblotting. In conclusion, we generated a detailed buffalo milk protein dataset and defined the changes occurring in SAU and NAS mastitis, with potential for improving detection (ProteomeXchange identifier PXD012355).

Heat stress negatively affects the transcriptome related to overall metabolism and milk protein synthesis in mammary tissue of midlactating dairy cows.

Inadequate dry matter intake only partially accounts for the decrease in milk protein synthesis during heat stress (HS) in dairy cows. Our hypothesis is that reduced milk protein synthesis during HS in dairy cows is also caused by biological changes within the mammary gland. The objective of this study was to assess the hypothesis via RNA-Seq analysis of mammary tissue. Herein, four dairy cows were used in a crossover design where HS was induced for 9 days in environmental chambers. There was a 30-day washout between periods. Mammary tissue was collected via biopsy at the end of each environmental period (HS or pair-fed and thermal neutral) for transcriptomic analysis. RNA-Seq analysis revealed HS affected >2,777 genes (false discovery rate-adjusted P value < 0.05) in mammary tissue. Expression of main milk protein-encoding genes and several key genes related to regulation of protein synthesis and amino acid and glucose transport were downregulated by HS. Bioinformatics analysis revealed an overall decrease of mammary tissue metabolic activity by HS (especially carbohydrate and lipid metabolism) and an increase in immune activation and inflammation. Network analysis revealed a major role of TNF, IFNG, S100A8, S100A9, and IGF-1 in inducing/controlling the inflammatory response, with a central role of NF-κB in the process of immunoactivation. The same analysis indicated an overall inhibition of PPARγ. Collectively, these data suggest HS directly controls milk protein synthesis via reducing the transcription of metabolic-related genes and increasing inflammation-related genes.

Meta-analysis of mammary RNA seq datasets reveals the molecular understanding of bovine lactation biology.

A better understanding of the biology of lactation, both in terms of gene expression and the identification of candidate genes for the production of milk and its components, is made possible by recent advances in RNA seq technology. The purpose of this study was to understand the synthesis of milk components and the molecular pathways involved, as well as to identify candidate genes for milk production traits within whole mammary transcriptomic datasets. We performed a meta-analysis of publically available RNA seq transcriptome datasets of mammary tissue/milk somatic cells. In total, 11 562 genes were commonly identified from all RNA seq based mammary gland transcriptomes. Functional annotation of commonly expressed genes revealed the molecular processes that contribute to the synthesis of fats, proteins, and lactose in mammary secretory cells and the molecular pathways responsible for milk synthesis. In addition, we identified several candidate genes responsible for milk production traits and constructed a gene regulatory network for RNA seq data. In conclusion, this study provides a basic understanding of the lactation biology of cows at the gene expression level.

Short-term lactation and mammary metabolism responses in lactating goats to graded removal of methionine from an intravenously infused complete amino acid mixture.

To investigate the possible pathways of Met deficiency to depress milk protein synthesis, 4 lactating goats fitted with jugular vein, mammary vein, and carotid artery catheters and transonic blood flow detectors on the external pudic artery were used in a 4 × 4 Latin square experiment. Goats were fasted for 24 h followed by a 9-h intravenous infusion of an AA mixture plus glucose. Milk yield was recorded and samples were taken in h 2 to 8 of the infusion period, and mammary biopsy was performed in the last hour. Treatments were graded removal of Met from the infused AA mixture to achieve Met content in the infusate of 100 (complete), 60, 30, or 0% of that in casein. Graded Met removal decreased yield of milk, milk protein, and lactose linearly and tended to decrease yield of milk fat linearly. Milk protein yield decreased to 82, 78, and 69% that of complete mixture infusion, respectively, when the 60, 30, and 0% Met infusate was infused. Circulating Met decreased linearly with graded Met removal. Arterial and venous Met decreased to 36 and 23% that of complete mixture infusion, respectively, when all Met was removed out of the mixture. Concomitant with the decreased circulating concentration was a similar increase in mammary Met affinity as reflected by the linearly increased mammary Met clearance rate. The increased affinity plus the linearly increased mammary blood flow totally offset the negative effect of decreased circulating Met concentration on mammary Met uptake. The overall result was similar mammary Met uptakes across treatments ranging from 285.9 to 339.5 µmol/h. Mammary uptakes of the other AA measured were generally not affected by treatments except for a linearly decreased Thr uptake and a trend of linearly increased Glu uptake. Consistent with the behavior of an AA mainly catabolized in the liver and mainly used for protein synthesis in peripheral tissues, mammary uptake to milk output ratios of Met measured in the present study ranged from 1.25 to 1.49 and was not affected by treatments. For the other AA measured, the ratio of Thr was linearly decreased and that of Glu was linearly increased by graded Met removal. Graded Met removal linearly elevated circulating urea N and glucose concentrations, indicating enhanced whole-body catabolism of AA and hepatic gluconeogenesis. Treatments had no significant effects on circulating insulin, growth hormone, and the other hormones and metabolites measured. Phosphorylation status of eIF4E binding protein 1 tended to decrease linearly and that of p70S6k was linearly decreased by graded Met removal, indicating depressed signal in the intracellular mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. In conclusion, results of the present study indicated that the mTORC1 pathway and whole-body AA catabolism rather than mammary uptake appeared the drivers for changes in milk protein synthesis in response to varying Met supply.

Taurine Promotes Milk Synthesis via the GPR87-PI3K-SETD1A Signaling in BMECs.

Taurine, a ß-aminosulfonic acid, exerts many cellular physiological functions. It is still unknown whether taurine can regulate milk synthesis in the mammary gland. Therefore, in this study we investigated the effects and mechanism of taurine on milk synthesis in mammary epithelial cells (MECs). Bovine MECs (BMECs) cultured in FBS-free OPTI-MEMImedium were treated with taurine (0, 0.08, 0.16, 0.24, 0.32, and 0.4 mM). Taurine treatment led to increased milk protein and fat synthesis, mTOR phosphorylation, and SREBP-1c protein expression, in a dose-dependent manner, with an apparent maximum at 0.24 mM. Gene function study approaches revealed that the GPR87-PI3K-SETD1A signaling was required for taurine to increase the mTOR and SREBP-1c mRNA levels. Taurine stimulated GPR87 expression and cell membrane localization in a dose dependent manner, suggesting a sensing mechanism of GPR87 to extracellular taurine. Collectively, these data demonstrate that taurine promotes milk synthesis via the GPR87-PI3K-SETD1A signaling.

MFG-E8 overexpression is associated with poor prognosis in breast cancer patients.

BACKGROUND: MFG-E8(Milk fat globule-EGF factor 8), a secreted glycoprotein, plays an exceptional role in various diseases. MFG-E8 overexpression is found in a variety of cancers. However, it remains unclear whether MFG-E8 overexpression is associated with the clinicopathological characteristics and prognosis of human breast cancer. MATERIALS AND METHODS: In this study, we detected the expression and localization of MFG-E8 protein in breast cancer and cancer-adjacent tissues using immunohistochemical staining, Western blot analysis and immunofluorescence. We analyzed the association between MFG-E8 expression and clinical characteristics and outcomes of breast cancer patients with different HR and HER2 statuses. RESULTS: Our results confirmed that MFG-E8 expression increased significantly in breast cancer compared with cancer-adjacent tissues by immunohistochemical staining (P < 0.001). Similarly, the Western blot results further confirmed the increased expression of MFG-E8 in breast cancer compared with cancer-adjacent tissues (P = 0.001). Immunofluorescence staining showed that MFG-E8 was mainly localized in the cytoplasm and membrane of tumor cells, consistent with the immunohistochemical staining results. The high expression levels of MFG-E8 showed a greater association with lymph node metastasis, TNM stage and histological grade (P < 0.001). Moreover, high MFG-E8 expression was related to a shortened overall survival (OS) (P < 0.001) and disease-free survival (DFS) (P < 0.001). Bioinformatics analysis with a Kaplan-Meier plotter also demonstrated a strong association of MFG-E8 mRNA overexpression with a short OS and DFS compared with low MFG-E8 expression (P = 0.040, P = 0.005). CONCLUSIONS: Our findings indicate that MFG-E8 may be a potential marker for poor prognosis and survival in breast cancer.