Role of LsrR in the regulation of biofilm formation in mammary pathogenic Escherichia coli.

BACKGROUND: Mammary Pathogenic Escherichia coli (MPEC) is an important pathogen that can escape the attack of the host immune system through biofilm formation and proliferate in the mammary gland continuously, resulting in mastitis in cows and causing enormous economic losses. As an effector of AI-2 quorum sensing, LsrR extensively affects the expression levels of hundreds of genes related to multiple biological processes in model E. coli strain. However, the regulatory role of LsrR in MPEC and whether it is involved in pathogenesis has been seldom reported. RESULTS: In this study, the function of LsrR in strain MPEC5, obtained from a milk sample in dairy cows with mastitis, was investigated by performing high-throughput sequencing (RNA-seq) assays. The results revealed that LsrR down-regulated the transcript levels of fimAICDFGH (encoding Type 1 pili), which have been reported to be associated with biofilm formation process. Biofilm assays confirmed that deletion of lsrR resulted in a significant increase in biofilm formation in vitro. In addition, electrophoretic mobility shift assay (EMSA) provided evidence that LsrR protein could directly bind to the promoter regions of fimAICDFGH in a dose-dependent manner. CONCLUSIONS: These results indicate that LsrR protein inhibits the biofilm formation ability of MPEC5 by directly binding to the fimAICDFGH promoter region. This study presents a novel clue for further exploration of the prevention and treatment of MPEC.

Regulatory impacts of PPARGC1A gene expression on milk production and cellular metabolism in buffalo mammary epithelial cells.

The PPARGC1A gene plays a fundamental role in regulating cellular energy metabolism, including adaptive thermogenesis, mitochondrial biogenesis, adipogenesis, gluconeogenesis, and glucose/fatty acid metabolism. In a previous study, our group investigated seven SNPs in Mediterranean buffalo associated with milk production traits, and the current study builds on this research by exploring the regulatory influences of the PPARGC1A gene in buffalo mammary epithelial cells (BuMECs). Our findings revealed that knockdown of PPARGC1A gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis. Additionally, we observed downregulated triglyceride secretion after PPARGC1A knockdown. Furthermore, the critical genes related to milk production, including the STATS, BAD, P53, SREBF1, and XDH genes were upregulated after RNAi, while the FABP3 gene, was downregulated. Moreover, Silencing the PPARGC1A gene led to a significant downregulation of ß-casein synthesis in BuMECs. Our study provides evidence of the importance of the PPARGC1A gene in regulating cell growth, lipid, and protein metabolism in the buffalo mammary gland. In light of our previous research, the current study underscores the potential of this gene for improving milk production efficiency and overall dairy productivity in buffalo populations.

Methods for investigating STAT3 regulation of lysosomal function in mammary epithelial cells.

The transcription factor STAT3 is activated by multiple cytokines and other extrinsic factors. It plays a key role in immune and inflammatory responses and, when dysregulated, in tumourigenesis. STAT3 is also an indispensable mediator of the cell death process that occurs during post-lactational regression of the mammary gland, one of the most dramatic examples of physiological cell death in adult mammals. During this involution of the gland, STAT3 powerfully enhances the lysosomal system to efficiently remove superfluous milk-producing mammary epithelial cells via a lysosomal-mediated programmed cell death pathway. The lysosome is a membrane-enclosed  cytoplasmic organelle that digests and recycles cellular waste, with an important role as a signalling centre that monitors cellular metabolism. Here, we describe key strategies for investigating the role of STAT3 in regulating lysosomal function using a mammary epithelial cell culture model system. These include protocols for lysosome enrichment and enzyme activity assays, in addition to microscopic analyses of the vesicular compartment in cell lines. Collectively, these approaches provide the tools to investigate multiple aspects of lysosome biogenesis and function, and to define both direct and indirect roles for STAT3.

Fused in sarcoma (FUS) inhibits milk production efficiency in mammals.

Efficient milk production in mammals confers evolutionary advantages by facilitating the transmission of energy from mother to offspring. However, the regulatory mechanism responsible for the gradual establishment of milk production efficiency in mammals, from marsupials to eutherians, remains elusive. Here, we find that mammary gland of the marsupial sugar glider contained milk components during adolescence, and that mammary gland development is less dynamically cyclic compared to that in placental mammals. Furthermore, fused in sarcoma (FUS) is found to be partially responsible for this establishment of low efficiency. In mouse model, FUS inhibit mammary epithelial cell differentiation through the cyclin-dependent kinase inhibitor p57Kip2, leading to lactation failure and pup starvation. Clinically, FUS levels are negatively correlated with milk production in lactating women. Overall, our results shed light on FUS as a negative regulator of milk production, providing a potential mechanism for the establishment of milk production from marsupial to eutherian mammals.

Gross anatomy of vascular supply and drainage of mammary fat pads in mice models.

This work extensively studied the vasculature of mice mammary fat pads (BALB/c and C57BL/6) with special reference to haematogenous drainage routes. Mammary fat pads were five pairs (first cervical, second and third thoracic, fourth abdominal and fifth inguinal), bilaterally symmetrical, extending laterally and continuously with the subcutaneous fascia. The superficial cervical artery and vein primarily accomplished the blood vasculature of the first mammary fat pad, while the lateral thoracic and external thoracic arteries and veins supplied the second and third mammary fat pads. The superficial cervical vein (found parallel to the superficial cervical artery) drained into the external jugular vein. The lateral thoracic artery and external thoracic artery branched almost at the same level as the axillary artery (branch of subclavian artery), the latter being more medial in position. However, in some specimens, the branching of both arteries appeared to be at the same level, and their origins were indistinguishable. The lateral thoracic vein that was parallel to the lateral thoracic artery drained to the axillary vein close to the drainage of the external thoracic vein. The lateral thoracic, superficial caudal epigastric, iliolumbar and external thoracic arteries and veins vascularized the fourth mammary fat pad and displayed anastomosis among themselves. The iliolumbar vein (found parallel to the iliolumbar artery) drained into the inferior vena cava. The superficial caudal epigastric vein (found parallel to the superficial caudal epigastric artery (SCaEA)) drained into the femoral vein. Unlike humans, the internal thoracic artery and vein did not participate in the vasculature of mammary fat pads. The SCaEA and vein supplied blood and drained the fifth mammary fat pad. The anatomical continuity of the fourth and fifth mammary fat pads provided common drainage for both mammary fat pads. The BALB/c and C57BL/6 mice strains studied did not differ in topography and size of mammary fat pads. The vascular supply and drainage of the mammary fat pads also did not differ in the strains studied. Only minor variations could be noted in the small veins draining into the lateral thoracic vein. Lateral tributaries seen in the terminal end of the lateral thoracic vein were absent in the C57BL/6 mice.

In the Murine and Bovine Maternal Mammary Gland Signal Transducer and Activator of Transcription 3 is Activated in Clusters of Epithelial Cells around the Day of Birth.

Signal transducers and activators of transcription (STAT) proteins regulate mammary development. Here we investigate the expression of phosphorylated STAT3 (pSTAT3) in the mouse and cow around the day of birth. We present localised colocation analysis, applicable to other mammary studies requiring identification of spatially congregated events. We demonstrate that pSTAT3-positive events are multifocally clustered in a non-random and statistically significant fashion. Arginase-1 expressing cells, consistent with macrophages, exhibit distinct clustering within the periparturient mammary gland. These findings represent a new facet of mammary STAT3 biology, and point to the presence of mammary sub-microenvironments.

Methionine and Leucine Promote mTOR Gene Transcription and Milk Synthesis in Mammary Epithelial Cells through the eEF1Bα-UBR5-ARID1A Signaling.

Amino acids are essential for the activation of the mechanistic target of rapamycin (mTOR), but the corresponding molecular mechanism is not yet fully understood. We previously found that Met stimulated eukaryotic elongation factor α (eEF1Bα) nuclear localization in bovine mammary epithelial cells (MECs). Herein, we explored the role and molecular mechanism of eEF1Bα in methionine (Met)- and leucine (Leu)-stimulated mTOR gene transcription and milk synthesis in MECs. eEF1Bα knockdown decreased milk protein and fat synthesis, cell proliferation, and mTOR mRNA expression and phosphorylation, whereas eEF1Bα overexpression had the opposite effects. QE-MS analysis detected that eEF1Bα was phosphorylated at Ser106 in the nucleus and Met and Leu stimulated p-eEF1Bα nuclear localization. eEF1Bα knockdown abrogated the stimulation of Met and Leu by mTOR mRNA expression and phosphorylation, and this regulatory role was dependent on its phosphorylation. Akt knockdown blocked the stimulation of Met and Leu by eEF1Bα and p-eEF1Bα expression. ChIP-PCR detected that p-eEF1Bα bound only to the -548 to -793 nt site in the mTOR promoter, and ChIP-qPCR further detected that Met and Leu stimulated this binding. eEF1Bα mediated Met and Leu' stimulation on mTOR mRNA expression and phosphorylation through inducing AT-rich interaction domain 1A (ARID1A) ubiquitination degradation, and this process depended on eEF1Bα phosphorylation. p-eEF1Bα interacted with ARID1A and ubiquitin protein ligase E3 module N-recognition 5 (UBR5), and UBR5 knockdown rescued the decrease of the ARID1A protein level by eEF1Bα overexpression. Both eEF1Bα and p-eEF1Bα were highly expressed in mouse mammary gland tissues during the lactating period. In summary, we reveal that Met and Leu stimulate mTOR transcriptional activation and milk protein and fat synthesis in MECs through eEF1Bα-UBR5-ARID1A signaling.

TRPS1 maintains luminal progenitors in the mammary gland by repressing SRF/MRTF activity.

The transcription factor TRPS1 is a context-dependent oncogene in breast cancer. In the mammary gland, TRPS1 activity is restricted to the luminal population and is critical during puberty and pregnancy. Its function in the resting state remains however unclear. To evaluate whether it could be a target for cancer therapy, we investigated TRPS1 function in the healthy adult mammary gland using a conditional ubiquitous depletion mouse model where long-term depletion does not affect fitness. Using transcriptomic approaches, flow cytometry and functional assays, we show that TRPS1 activity is essential to maintain a functional luminal progenitor compartment. This requires the repression of both YAP/TAZ and SRF/MRTF activities. TRPS1 represses SRF/MRTF activity indirectly by modulating RhoA activity. Our work uncovers a hitherto undisclosed function of TRPS1 in luminal progenitors intrinsically linked to mechanotransduction in the mammary gland. It may also provide new insights into the oncogenic functions of TRPS1 as luminal progenitors are likely the cells of origin of many breast cancers.

ABCD4 is associated with mammary gland development in mammals.

BACKGROUND: Mammary gland development is a critical process in mammals, crucial for their reproductive success and offspring nourishment. However, the functional roles of key candidate genes associated with teat number, including ABCD4, VRTN, PROX2, and DLST, in this developmental process remain elusive. To address this gap in knowledge, we conducted an in-depth investigation into the dynamic expression patterns, functional implications, and regulatory networks of these candidate genes during mouse mammary gland development. RESULTS: In this study, the spatial and temporal patterns of key genes were characterized in mammary gland development. Using time-series single-cell data, we uncovered differences in the expression of A bcd4, Vrtn, Prox2, and Dlst in cell population of the mammary gland during embryonic and adult stages, while Vrtn was not detected in any cells. We found that only overexpression and knockdown of Abcd4 could inhibit proliferation and promote apoptosis of HC11 mammary epithelial cells, whereas Prox2 and Dlst had no significant effect on these cells. Using RNA-seq and qPCR, further analysis revealed that Abcd4 can induce widespread changes in the expression levels of genes involved in mammary gland development, such as Igfbp3, Ccl5, Tlr2, and Prlr, which were primarily associated with the MAPK, JAK-STAT, and PI3K-AKT pathways by functional enrichment. CONCLUSIONS: These findings revealed ABCD4 as a candidate gene pivotal for regulating mammary gland development and lactation during pregnancy by influencing PRLR expression.

Puerarin Delays Mammary Gland Aging by Regulating Gut Microbiota and Inhibiting the p38MAPK Signaling Pathway.

Mammary gland aging is one of the most important problems faced by humans and animals. How to delay mammary gland aging is particularly important. Puerarin is a kind of isoflavone substance extracted from Pueraria lobata, which has anti-inflammatory, antioxidant, and other pharmacological effects. However, the role of puerarin in delaying lipopolysaccharide (LPS)-induced mammary gland aging and its underlying mechanism remains unclear. On the one hand, we found that puerarin could significantly downregulate the expression of senescence-associated secretory phenotype (SASP) and age-related indicators (SA-ß-gal, p53, p21, p16) in mammary glands of mice. In addition, puerarin mainly inhibited the p38MAPK signaling pathway to repair mitochondrial damage and delay mammary gland aging. On the other hand, puerarin could also delay the cellular senescence of mice mammary epithelial cells (mMECs) by targeting gut microbiota and promoting the secretion of gut microbiota metabolites. In conclusion, puerarin could not only directly act on the mMECs but also regulate the gut microbiota, thus, playing a role in delaying the aging of the mammary gland. Based on the above findings, we have discovered a new pathway for puerarin to delay mammary gland aging.

Heme oxygenase activates calcium release from the endoplasmic reticulum of bovine mammary epithelial cells to promote TFEB entry into the nucleus to reduce the intracellular load of Mycoplasma bovis.

Heme oxygenase HO-1 (HMOX) regulates cellular inflammation and apoptosis, but its role in regulation of autophagy in Mycoplasma bovis infection is unknown. The objective was to determine how the HO-1/CO- Protein kinase RNA-like endoplasmic reticulum kinase (PERK)-Ca2+- transcription factor EB (TFEB) signaling axis induces autophagy and regulates clearance of M. bovis by bovine mammary epithelial cells (bMECs). M. bovis inhibited autophagy and lysosomal biogenesis in bMECs and suppressed HO-1 protein and expression of related proteins, namely nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (keap1). Activation of HO-1 and its production of carbon monoxide (CO) were required for induction of autophagy and clearance of intracellular M. bovis. Furthermore, when HO-1 was deficient, CO sustained cellular autophagy. HO-1 activation increased intracellular calcium (Ca2+) and cytosolic localization activity of TFEB via PERK. Knockdown of PERK or chelation of intracellular Ca2+ inhibited HO-1-induced M. bovis autophagy and clearance. M. bovis infection affected nuclear localization of lysosomal TFEB in the MiT/TFE transcription factor subfamily, whereas activation of HO-1 mediated dephosphorylation and intranuclear localization of TFEB, promoting autophagy, lysosomal biogenesis and autophagic clearance of M. bovis. Nuclear translocation of TFEB in HO-1 was critical to induce M. bovis transport and survival of infected bMECs. Furthermore, the HO-1/CO-PERK-Ca2+-TFEB signaling axis induced autophagy and M. bovis clearance, providing a viable approach to treat persistent M. bovis infections.

Development and Validation of Matrix of Chemistry, Manufacturing, and Control (MoCMC) System for Intramammary Drug Products (IMM).

PURPOSE: Products formulated for intramammary (IMM) infusion are intended for the delivery of therapeutic moieties directly into the udder through the teat canal to maximize drug exposure at the targeted clinical site, the mammary gland, with little to no systemic drug exposure. Currently, to our knowledge, there has been no in-vitro matrix system available to differentiate between IMM formulations. Our goal is to develop A custom tailored in-vitro "Matrix of Chemistry, Manufacturing and Control" (MoCMC) System to be a promising future tool for identifying inequivalent IMM formulations. MoCMC can detect inter and intra batch variabilities, thereby identifying potential generics versus brand product similarities or differences with a single numeric value and a specific & distinctive fingerprint. METHODS: The FDA-approved IMM formulation, SPECTRAMASTⓇ LC, was selected as the reference product for the MoCMC. Twelve in-house test formulations containing ceftiofur hydrochloride were formulated and characterized. The MoCMC was developed to include six input parameters and three output parameters. The MoCMC system was used to evaluate and compare SPECTRAMASTⓇ LC with its in-house formulations. RESULTS: Based on the MoCMC generated parameters, the distinctive fingerprints of MoCMC for each IMM formulations, and the statistical analyses of MCI and PPI values, in-house formulations, F-01 and F-02 showed consistency while the rest of in-house formulations (F-03-F-12) were significantly different as compared to SPECTRAMASTⓇ LC. CONCLUSION: This research showed that the MoCMC approach can be used as a tool for intra batch variabilities, generics versus brand products comparisons, post-approval formulations changes, manufacturing changes, and formulation variabilities.

Chemerin Stimulates the Secretory Activity of BME-UV1 Bovine Mammary Epithelial Cells.

Adipose tissue is an active endocrine gland, synthesizing and secreting multiple signaling molecules termed adipokines. Following the detection of adipokines and their receptors in the mammary tissue of various species, it is indicated that adipokines play a role in the development of the mammary gland. The aim of the present study was to determine the concentration-dependent influence of three adipokines, leptin, adiponectin, and chemerin, on the viability, apoptosis, and secretory activity of BME-UV1 bovine mammary epithelial cells. The study confirmed that BME-UV1 cells contain the leptin receptor (Ob-R) protein, and express transcripts of adiponectin (ADIPOR1 and ADIPOR2) and chemerin (CMLKR1 and GPR1) receptors. Regardless of the administered dose, none of the three tested adipokines had an effect on the viability of BME-UV1 cells, and the number of apoptotic cells remained unchanged. However, chemerin (100 ng/mL) stimulated BME-UV1 cells to synthesize and secrete αS1-casein, the major protein component of milk. These results indicate that chemerin may be a potent regulator of the bovine mammary epithelial cells' functional differentiation, contributing, along with the major systemic hormones and local growth factors, to the development of the bovine mammary gland.

Staphylococcus aureus promotes its intracellular survival by inhibiting Rab11-Rab11FIP4-mediated vesicle trafficking.

Mastitis in dairy cows is mainly caused by bacteria, in which Staphylococcus aureus appears frequently. Epithelial cells, as a major physical barrier of mammary gland, play an important role in preventing mastitis in dairy cows. Our previous study reported that Rab11fip4 (an effector of Rab11) was significantly changed in response to stimulation by S. aureus. So, in this study, the role of Rab11A in phagocytosis of bovine mammary epithelial cells (MAC-T) against S. aureus was evaluated. First, changes of Rab11A and Rab11fip4 were analyzed in response to S. aureus by immunofluorescence and western blotting. Subsequently, the effects of Rab11A and Rab11fip4 on proliferation of S. aureus, as well as formation and function of late endosomes (LEs) and lysosomes (LYSs) were investigated. The results showed that, after infection, Rab11A and Rab11fip4 were recruited to phagosomes containing S. aureus. Rab11A promoted bacterial clearance and rescues the destruction of LEs and LYSs by S. aureus, whereas Rab11fip4 did the opposite. These findings provide new insights into phagocytosis and control of S. aureus in host cells, thus lay the foundation to elucidate the pathogenesis of S. aureus in bovine mastitis.

MiR-103-5p deficiency suppresses lipid accumulation via upregulating PLSCR4 and its host gene PANK3 in goat mammary epithelial cells.

Lipids are intimately related to the unique flavor and nutritional values of goat milk. MicroRNAs (miRNA) participate in the regulation of various biological functions, including the synthesis and degradation of lipids. Several studies have shown that miR-103 is involved in the regulation of lipid metabolism, however, the molecular mechanism by which miR-103 regulates lipid metabolism in goat mammary gland is poorly understood. In this study, miR-103 was knocked out in goat mammary epithelial cells (GMECs) by CRISPR/Cas9, and the accumulation of lipid droplets, triglycerides, and cholesterol in the cells was suppressed subsequently. Overexpression or knockdown of miR-103-5p and miR-103-3p in GMECs revealed that it was miR-103-5p that promoted lipid accumulation but not miR-103-3p. In addition, Pantothenate Kinase 3 (PANK3), the host gene of miR-103, and Phospholipid Scramblase 4 (PLSCR4) were identified as the target genes of miR-103-5p by dual fluorescein and miRNA pulldown. Furthermore, we identified that cellular lipid levels were negatively regulated by PANK3 and PLSCR4. Lastly, in miR-103 knockout GMECs, the knockdown of PANK and PLSCR4 rescued the lipid accumulation. These findings suggest that miR-103-5p promotes lipid accumulation by targeting PLSCR4 and the host gene PANK3 in GMECs, providing new insights for the regulation of goat milk lipids via miRNAs.

Polyphenols and Lactation: Molecular Evidence to Support the Use of Botanical Galactagogues.

Botanicals and herbal supplements contain a diverse array of polyphenols that may affect mammary gland function and promote galactagogue activity. This scoping review is conducted to identify scientific literature elucidating how polyphenols affect mammary gland biology and cellular mechanisms critical for lactation. A literature search of PubMed and Medline reviews relevant studies in dairy animals, rodent models, and cultured mammary epithelial cells that are published from January 2010 until July 2023, to ascertain effects of polyphenols on mechanisms regulating milk production and composition. The PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Review) strategy is applied and 80 studies on polyphenols and their implications on milk production and composition are included in this review. Limited information delineating effects of polyphenols on the molecular pathways that affect lactation are found, although available information suggests modulation of Stat5 signaling/differentiation, Stat3 signaling/remodeling, mTOR and insulin signaling/energy production, and nuclear factor kappa beta (NFκß) signaling/oxidative stress and inflammation may play roles. A profound lack of mechanistic information underscores the critical need for further research to understand the impact of botanical supplements and polyphenols on milk production and composition in humans to establish maternal nutritional guidelines to support lactation and breastfeeding goals.

Exploring the inhibitory impact of Mongolian medicinal He-Zi-3 soup on mammary gland hyperplasia in rats induced by estrogen and progestogen.

ETHNOPHARMACOLOGICAL RELEVANCE: Mammary gland hyperplasia, a prevalent benign breast condition, often serves as a precursor to various other breast diseases. He-Zi-3 soup (HZ-3), a traditional Mongolian remedy, is utilized for treating this condition. AIM OF THE STUDY: To explore the effect and underlying mechanism of HZ-3, a Mongolian medicinal preparation, on mammary gland hyperplasia. MATERIALS AND METHODS: This study aimed to assess the impact of different doses of HZ-3 in a rat model of mammary hyperplasia. The active components within HZ-3 drug serum were identified and analyzed through network pharmacology and target prediction. To elucidate the underlying mechanism of HZ-3 in addressing mammary hyperplasia, we conducted a series of investigations on estradiol-induced mammary hyperplasia in model rates. Assessments included measurements of papilla width and height, hematoxylin and eosin staining, Masson staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot, and immunohistochemistry. RESULTS: Our investigation revealed the identification of 21 compounds, primarily terpenoids, through serum medicinal chemistry screening. Utilizing network pharmacological analysis, we observed predominant regulation through the estrogen pathway, closely associated with key genes including esr1,esr2, ncoa1, krt 19, ctsd, ebag 9, and bcl-2. Assessments encompassing nipple height and width, histological examination, immunohistochemical analysis, and serum hormone levels via enzyme-linked immunosorbent assay demonstrated the inhibitory effect of HZ-3 on mammary hyperplasia in rat models. RT-qPCR and Western blot analyses corroborated these findings, affirming the suppression of mammary hyperplasia by HZ-3 through the activation of estrogen pathway signaling.

Uncoupling protein 1-driven Cre (Ucp1-Cre) is expressed in the epithelial cells of mammary glands and various non-adipose tissues.

OBJECTIVE: Uncoupling protein 1 (UCP1), a mitochondrial protein responsible for nonshivering thermogenesis in adipose tissue, serves as a distinct marker for thermogenic brown and beige adipocytes. Ucp1-Cre mice are thus widely used to genetically manipulate these thermogenic adipocytes. However, evidence suggests that UCP1 may also be expressed in non-adipocyte cell types. In this study, we investigated the presence of UCP1 expression in different mouse tissues that have not been previously reported. METHODS: We employed Ucp1-Cre mice crossed with Cre-inducible transgenic reporter Nuclear tagging and Translating Ribosome Affinity Purification (NuTRAP) mice to investigate Ucp1-Cre expression in various tissues of adult female mice and developing embryos. Tamoxifen-inducible Ucp1-CreERT2 mice crossed with NuTRAP mice were used to assess active Ucp1 expression in adult mice. Immunostaining, RNA analysis, and single-cell/nucleus RNA-seq (sc/snRNA-seq) data analysis were performed to determine the expression of endogenous UCP1 and Ucp1-Cre-driven reporter expression. We also investigated the impact of UCP1 deficiency on mammary gland development and function using Ucp1-knockout (KO) mice. RESULTS: Ucp1-Cre expression was observed in the mammary glands within the inguinal white adipose tissue of female Ucp1-Cre; NuTRAP mice. Ucp1-Cre was activated during embryonic development in various tissues, including mammary glands, as well as in the brain, kidneys, eyes, and ears, specifically in epithelial cells in these organs. However, Ucp1-CreERT2 showed no or only partial activation in these tissues of adult mice, indicating the potential for low or transient expression of endogenous Ucp1. While sc/snRNA-seq data suggest potential expression of UCP1 in mammary epithelial cells in adult mice and humans, Ucp1-KO female mice displayed normal mammary gland development and function. CONCLUSIONS: Our findings reveal widespread Ucp1-Cre expression in various non-adipose tissue types, starting during early development. These results highlight the importance of exercising caution when interpreting data and devising experiments involving Ucp1-Cre mice.

The Experimental and In Silico-Based Evaluation of NRF2 Modulators, Sulforaphane and Brusatol, on the Transcriptome of Immortalized Bovine Mammary Alveolar Cells.

Changes during the production cycle of dairy cattle can leave these animals susceptible to oxidative stress and reduced antioxidant health. In particular, the periparturient period, when dairy cows must rapidly adapt to the sudden metabolic demands of lactation, is a period when the production of damaging free radicals can overwhelm the natural antioxidant systems, potentially leading to tissue damage and reduced milk production. Central to the protection against free radical damage and antioxidant defense is the transcription factor NRF2, which activates an array of genes associated with antioxidant functions and cell survival. The objective of this study was to evaluate the effect that two natural NRF2 modulators, the NRF2 agonist sulforaphane (SFN) and the antagonist brusatol (BRU), have on the transcriptome of immortalized bovine mammary alveolar cells (MACT) using both the RT-qPCR of putative NRF2 target genes, as well as RNA sequencing approaches. The treatment of cells with SFN resulted in the activation of many putative NRF2 target genes and the upregulation of genes associated with pathways involved in cell survival, metabolism, and antioxidant function while suppressing the expression of genes related to cellular senescence and DNA repair. In contrast, the treatment of cells with BRU resulted in the upregulation of genes associated with inflammation, cellular stress, and apoptosis while suppressing the transcription of genes involved in various metabolic processes. The analysis also revealed several novel putative NRF2 target genes in bovine. In conclusion, these data indicate that the treatment of cells with SFN and BRU may be effective at modulating the NRF2 transcriptional network, but additional effects associated with cellular stress and metabolism may complicate the effectiveness of these compounds to improve antioxidant health in dairy cattle via nutrigenomic approaches.

Droplet-based proteomics reveals CD36 as a marker for progenitors in mammary basal epithelium.

Deep proteomic profiling of rare cell populations has been constrained by sample input requirements. Here, we present DROPPS (droplet-based one-pot preparation for proteomic samples), an accessible low-input platform that generates high-fidelity proteomic profiles of 100-2,500 cells. By applying DROPPS within the mammary epithelium, we elucidated the connection between mitochondrial activity and clonogenicity, identifying CD36 as a marker of progenitor capacity in the basal cell compartment. We anticipate that DROPPS will accelerate biology-driven proteomic research for a multitude of rare cell populations.