Ultrasound modification on milk fat globule membrane and soy lecithin to improve the physicochemical properties, microstructure and stability of mimicking human milk fat emulsions.

Starting from the consideration of the structure of human milk fat globule (MFG), this study aimed to investigate the effects of ultrasonic treatment on milk fat globule membrane (MFGM) and soy lecithin (SL) complexes and their role in mimicking human MFG emulsions. Ultrasonic power significantly affected the structure of the MFGM-SL complex, further promoting the unfolding of the molecular structure of the protein, and then increased solubility and surface hydrophobicity. Furthermore, the microstructure of mimicking MFG emulsions without sonication was unevenly distributed, and the average droplet diameter was large. After ultrasonic treatment, the droplets of the emulsion were more uniformly dispersed, the particle size was smaller, and the emulsification properties and stability were improved to varying degrees. Especially when the ultrasonic power was 300 W, the mimicking MFG emulsion had the highest encapsulation rate and emulsion activity index and emulsion stability index were increased by 60.88 % and 117.74 %, respectively. From the microstructure, it was observed that the spherical droplets of the mimicking MFG emulsion after appropriate ultrasonic treatment remain well separated without obvious flocculation. This study can provide a reference for the screening of milk fat globules mimicking membrane materials and the further utilization and development of ultrasound in infant formula.

Perilipin membrane integration determines lipid droplet heterogeneity in differentiating adipocytes.

The storage of fat within lipid droplets (LDs) of adipocytes is critical for whole-body health. Acute fatty acid (FA) uptake by differentiating adipocytes leads to the formation of at least two LD classes marked by distinct perilipins (PLINs). How this LD heterogeneity arises is an important yet unresolved cell biological problem. Here, we show that an unconventional integral membrane segment (iMS) targets the adipocyte specific LD surface factor PLIN1 to the endoplasmic reticulum (ER) and facilitates high-affinity binding to the first LD class. The other PLINs remain largely excluded from these LDs until FA influx recruits them to a second LD population. Preventing ER targeting turns PLIN1 into a soluble, cytoplasmic LD protein, reduces its LD affinity, and switches its LD class specificity. Conversely, moving the iMS to PLIN2 leads to ER insertion and formation of a separate LD class. Our results shed light on how differences in organelle targeting and disparities in lipid affinity of LD surface factors contribute to formation of LD heterogeneity.

Construction of nanoparticles from blueberry anthocyanins-lecithin/gum Arabic improves lipid droplet accumulation and gut microbiota disturbance in HFD-induced obese mice.

The digestive instability of anthocyanins (ACNs) limits their application in food nutrition, especially precision nutrition. Blueberry ACNs-loaded nanoparticles (Lipo/GA-ACNs NPs) were prepared using gum arabic (GA) as the delivery carrier and liposomal vesicles (Lipo) prepared from soy lecithin as the targeting scaffold. The average particle size of the NPs was 99.4 nm, and the polydispersion index (PDI) was 0.46. The results showed that the presence of the Lipo-GA matrix enhanced the NPs' in vitro stability and antioxidant activity. In addition, the in vitro biocompatibility, uptake ability, lipid-lowering activity, and free-radical scavenging ability were improved to a certain extent. In a high-fat diet (HFD)-induced obese mouse model, oral administration of ACNs-LNP (LNP, liver-targeted nanoparticle) showed better effects on body weight, liver injury, and lipid droplet accumulation in the liver than ACNs. In addition, ACNs-LNP also played a role in regulating HFD-induced gut microbiota imbalance. These results provide a promising ACNs delivery strategy with the potential to be developed into a functional food that targets the liver to prevent fatty liver.

Phosphoproteomics Revealed Differentially Expressed Sites and Function of the Bovine Milk Fat Globule Membrane in Colostrum and Mature Milk.

One type of large and intricate post-translational modification of milk proteins that has significant biological implications is phosphorylation. The characterization of phosphoproteins found in the bovine milk fat globule membrane (MFGM) is still mostly unknown. Here, label-free phosphoproteomics was used to identify 94 phosphorylation sites from 54 MFGM phosphoproteins in bovine colostrum (BC) and 136 phosphorylation sites from 91 MFGM phosphoproteins in bovine mature milk (BM). αs1-Casein and ß-casein were the most phosphorylated proteins in bovine colostrum. In bovine mature milk, perilipin-2 was the protein with the greatest number of phosphorylation sites. The results show that bovine colostrum MFGM phosphoproteins were mainly involved in immune function, whereas bovine mature MFGM phosphoproteins were mainly involved in metabolic function. Plasminogen and osteopontin were the most strongly interacting proteins in colostrum, whereas perilipin-2 was the most strongly interacting protein in bovine mature milk. This work demonstrates the unique alterations in the phosphorylation manner of the bovine MFGM protein during lactation and further expands our knowledge of the site characteristics of bovine MFGM phosphoproteins. This result confirms the value of MFGM as a reference ingredient for infant formula during different stages.

Ultrasound-induced structural changes of different milk fat globule membrane protein-phospholipids complexes and their effects on physicochemical and functional properties of emulsions.

Ultrasonic technology is a non-isothermal processing technology that can be used to modify the physicochemical properties of food ingredients. This study investigated the effects of ultrasonic time (5 min, 10 min, 15 min) and power (150 W,300 W,500 W) on the structural properties of three types of phospholipids composed of different fatty acids (milk fat globule membrane phospholipid (MPL), egg yolk lecithin (EYL), soybean lecithin (SL)) and milk fat globule membrane protein (MFGMP). We found that the ultrasound treatment changed the conformation of the protein, and the emulsions prepared by the pretreatment showed better emulsification and stability, the lipid droplets were also more evenly distributed. Meanwhile, the flocculation phenomenon of the lipid droplets was significantly improved compared with the non-ultrasonic emulsions. Compared with the three complexes, it was found that ultrasound had the most significant effect on the properties of MPL-MFGMP, and its emulsion state was the most stable. When the ultrasonic condition was 300 W, the particle size of the emulsion decreased significantly (from 441.50 ±â€¯4.79 nm to 321.77 ±â€¯9.91 nm) at 15 min, and the physical stability constants KE decreased from 14.49 ±â€¯0.702 % to 9.4 ±â€¯0.261 %. It can be seen that proper ultrasonic pretreatment can effectively improve the stability of the system. At the same time, the emulsification performance of the emulsion had also been significantly improved. While the accumulation phenomenon occurred when the ultrasonic power was 150 W and 500 W. These results showed that ultrasonic pretreatment had great potential to improve the properties of emulsions, and this study would provide a theoretical basis for the application of emulsifier in the emulsions.

Comparative Site-Specific O-Glycosylation Analysis of the Milk Fat Globule Membrane Proteome in Donkey Colostrum and Mature Milk.

Donkey milk fat globule membrane (MFGM) proteins are a class of membrane-bound secreted proteins with broad-spectrum biofunctional activities; however, their site-specific O-glycosylation landscapes have not been systematically mapped. In this study, an in-depth MFGM O-glycoproteome profile of donkey milk during lactation was constructed based on an intact glycopeptide-centered, label-free glycoproteomics pipeline, with 2137 site-specific O-glycans from 1121 MFGM glycoproteins and 619 site-specific O-glycans from 217 MFGM glycoproteins identified in donkey colostrum and donkey mature milk, respectively. As lactation progressed, the number of site-specific O-glycans from three glycoproteins significantly increased, whereas that of 11 site-specific O-glycans from five glycoproteins significantly decreased. Furthermore, donkey MFGM O-glycoproteins with core-1 and core-2 core structures and Lewis and sialylated branch structures may be involved in regulating apoptosis. The findings of this study reveal the differences in the composition of donkey MFGM O-glycoproteins and their site-specific O-glycosylation modification dynamic change rules during lactation, providing a molecular basis for understanding the complexity and biological functions of donkey MFGM protein O-glycosylation.

Fabrication of curcumin-loaded oleogels using camellia oil bodies and gum arabic/chitosan coatings for controlled release applications.

This study has explored the potential of plant-derived oil bodies (OBs)-based oleogels as novel drug delivery systems for in vitro release under simulated physiological conditions. To obtain stable OBs-based oleogels, gum arabic (GA) and chitosan (CH) were coated onto the curcumin-loaded OBs using an electrostatic deposition technique, followed by 2,3,4-trihydroxybenzaldehyde (TB) induced Schiff-base cross-linking. Microstructural analyses indicated successful encapsulation of curcumin into the hydrophobic domain of the OBs through a pH-driven method combined with ultrasound treatment. The curcumin encapsulation efficiency of OBs increased up to 83.65 % and 92.18 % when GA and GA-CH coatings were applied, respectively, compared to uncoated OBs (63.47 %). In addition, GA-CH coatings retained the structural integrity of oleogel droplets with superior oil-holding capacity (99.07 %), while TB addition induced interconnected 3D-network structures with excellent gel strength (≥4.8 × 105 Pa) and thermal stability (≥80 °C). GA-CH coated oleogels appeared to provide the best protection for loaded bioactive against UV irradiation and high temperature-induced degradation during long-term storage. The combination of biopolymer coatings and TB-induced Schiff-base cross-linking synergistically hindered the simulated gastric degradability of oleogels, releasing only 23.35 %, 12.46 % and 7.19 % of curcumin by GA, GA-CH and GA-CH-TB stabilized oleogels, respectively, while also resulting in sustained release effects during intestinal conditions.

Prepared Radix Polygoni Multiflori and emodin alleviate lipid droplet accumulation in nonalcoholic fatty liver disease through MAPK signaling pathway inhibition.

As one of the most common liver diseases, nonalcoholic fatty liver disease (NAFLD) affects almost one-quarter of the world's population. Although the prevalence of NAFLD is continuously rising, effective medical treatments are still inadequate. Radix Polygoni Multiflori (RPM) is a traditional Chinese herbal medicine. As a processed product of RPM, prepared Radix Polygoni Multiflori (PRPM) has been reported to have antioxidant and anti-inflammatory effects. This study investigated whether PRPM treatment could significantly improve NAFLD. We used recent literature, the Herb database and the SwissADME database to isolate the active compounds of PRPM. The OMIM, DisGeNET and GeneCards databases were used to isolate NAFLD-related target genes, and GO functional enrichment and KEGG pathway enrichment analyses were conducted. Moreover, PRPM treatment in NAFLD model mice was evaluated. The results indicate that the target genes are mainly enriched in the AMPK and de novo lipogenesis signaling pathways and that PRPM treatment improves NAFLD disease in model mice. Here, we found the potential benefits of PRPM against NAFLD and demonstrated in vivo and in vitro that PRPM and its ingredient emodin downregulate phosphorylated P38/P38, phosphorylated ERK1/2 and genes related to de novo adipogenesis signaling pathways and reduce lipid droplet accumulation. In conclusion, our findings revealed a novel therapeutic role for PRPM in the treatment of NAFLD and metabolic inflammation.

The role of membrane components on the oleosome lubrication properties.

HYPOTHESIS: Oleosomes are natural oil droplets with a unique phospholipid/protein membrane, abundant in plant seeds, from which they can be extracted and used in emulsion-based materials, such as foods, cosmetics and pharmaceutics. The lubrication properties of such materials are essential, on one hand, due to the importance of the in-mouth creaminess for the consumed products or the importance of spreading the topical creams. Therefore, here, we will evaluate the lubrication properties of oleosomes, and how these properties are affected by the components at the oleosome membrane. EXPERIMENT: Oleosomes were extracted, and their oral lubricating properties were evaluated using tribology. To understand the influence of the oil droplet membrane composition, reconstituted oleosomes were also studied, with membranes that differed in protein/lecithin ratio. Additionally, whey protein- and lecithin-stabilised emulsions were used as reference samples. Confocal laser scattering microscopy was used to study the samples visually before and after tribological analysis. FINDINGS: Oleosomes followed a ball-bearing mechanism, which was probably related to their high physical stability due to the presence of membrane proteins. When the membrane protein concentration at the surface was reduced, the droplet stability weakened, leading to plating-out lubrication. Following our results, we elucidated the oleosome lubrication mechanism and showed their possible control by changing the membrane composition.

Pectin polysaccharide contribution to oleosome extraction after wet milling of rapeseed.

Oleosomes are lipid composites providing energy storage in oilseeds. They possess a unique structure, comprised of a triglyceride core stabilized by a phospholipid-protein membrane, and they have shown potential to be used as ingredients in several food applications. Intact oleosomes are extracted by an aqueous process which includes soaking, milling, and gravitational separation. However, the details of the complexes formed between oleosomes, proteins and pectin polysaccharides during this extraction are not known. It was hypothesized that pectins play an important role during the oleosome separation, and different proteins will be complexed on the surface of the oleosomes, depending on the pH of extraction. Rapeseed extracts were treated with and without pectinase (Pectinex Ultra SP-L) and extracted at pH 5.7 or 8.5, as this will affect electrostatic complexation. Acidic conditions led to co-extraction of storage proteins, structured as dense oleosome emulsions, stabilized by a network of proteins and polysaccharides. Pectinase intensified this effect, highlighting pectic polysaccharides' role in bridging interactions among proteins and oleosomes under acidic conditions. The presence of this dense interstitial layer around the oleosomes protected them from coalescence during extraction. Conversely, under alkaline conditions, the extraction process yielded more purified oleosomes characterized by a larger particle size, most likely due to coalescence. Nevertheless, pectinase addition at pH 8.5 mitigated coalescence tendencies. These results contribute to a better understanding of the details of the colloidal complexes formed during extraction and can be used to modulate the composition of the extracted fractions, with significant consequences not only for yields and purity but also for the functional properties of the ingredients produced.

Identification of a ß-Carboline Alkaloid from Chemoselectively Derived Vanilla Bean Extract and Its Prevention of Lipid Droplet Accumulation in Human Hepatocytes (HepG2).

Targeting bioactive compounds to prevent lipid droplet accumulation in the liver, we explored an antioxidative extract from vanilla bean (Vainilla planifolia) after chemo-selective derivatization through heating and acid modification. The chemical analysis of vanilla bean extract through chemoselective derivatization resulted in the identification of sixteen compounds (34-50) using LC-MS/MS analysis. A ß-carboline alkaloid with a piperidine C-ring and a vanillin moiety at C-1 (34) was identified by molecular networking and diagnostic fragmentation filtering approaches. ß-carboline alkaloid 34 exhibited significant inhibitory activity of lipid droplet accumulation (LDAI) in oleic acid-loaded hepatocellular carcinoma HepG2 cells. The LDAI activity was associated with both activation of lipolysis and suppression of lipogenesis in the cells. The study indicates that crude plant extracts, following chemoselective derivatization, may contain bioactive compounds that could be beneficial in preventing hepatosteatosis and could serve as a source of lead compounds for drug development. This approach may be useful to investigate other mixtures of natural products and food resources.

Placental extract suppresses lipid droplet accumulation by autophagy during the differentiation of adipose-derived mesenchymal stromal/stem cells into mature adipocytes.

OBJECTIVE: Placental extract, which contains various bioactive compounds, has been used as traditional medicine. Many studies have demonstrated additional applications of placental extract and provided a scientific basis for the broad spectrum of its effects. We have previously reported that porcine placental extract (PPE) strongly suppresses adipogenesis in a 3T3-L1 preadipocyte cell line, inhibiting differentiation. This study aimed to examine the effect of PPE on the accumulation of lipid droplets (LD) in adipose-derived mesenchymal stromal/stem cells (ASC). RESULTS: The study findings revealed that PPE decreased the size of LD during the differentiation of ASC into mature adipocytes. RT-qPCR analysis revealed that PPE increased the gene expression of lysosomal acid lipase A (Lipa), a lipolysis-related gene, in ASC-differentiated adipocytes. However, no differences were noted in the adipocyte differentiation markers (Pparg, Cebpa, and Adipoq), or the adipogenesis-related genes (Dgat1, Dgat2, Fasn, Soat1, and Soat2). In addition, PPE promoted autophagosome formation, which was partially co-localized with the LD, indicating that PPE accelerated the degradation of LD by inducing autophagy (termed lipophagy) during the differentiation of ASC into mature adipocytes. These results suggest that the use of PPE may be a potential novel treatment for regulating adipogenesis for the treatment of obesity.

Walnut (Juglans regia L.) kernel oil bodies recovered by aqueous extraction for utilization as ingredient in food emulsions: Exploration of their microstructure, composition and the effects of homogenization, pH, and salt ions on their physical stability.

Natural oil-in-water emulsions containing plant oil bodies (OBs), also called oleosomes, rich in health-promoting omega-3 polyunsaturated fatty acids (ω3 PUFA) are of increasing interest for food applications. In this study, we focused on walnut kernel OBs (WK-OBs) and explored their microstructure, composition and physical stability in ionic environments as well as the impact of homogenization. A green process involving aqueous extraction by grinding of WK allowed the co-extraction of OBs and proteins, and centrifugation was used to recover the WK-OBs. Confocal laser scanning microscopy images showed the spherical shape of WK-OBs with an oil core envelopped by a layer of phospholipids (0.16 % of lipids) and embedded proteins. Their mean diameter was 5.1 ± 0.3 µm. The WK-OBs contained 70.1 % PUFA with 57.8 % ω6 linoleic acid and 12.3 % ω3 α-linolenic acid representing 68 % and 11.6 % of the total fatty acids in the sn-2 position of the triacylglycerols (TAG), respectively. Trilinolein was the main TAG (23.1 %). The WK-OBs also contained sterols (1223 ± 33 mg/kg lipids; 86 % ß-sitosterol), carotenoids (0.62 ± 0.01 mg/kg lipids; 49.2 % ß-carotene), and tocopherols (322.7 ± 7.7 mg/kg lipids; 89 % γ-tocopherol), confirming their interest as health-promoting ingredients. The decrease in the size of WK-OBs under high-pressure homogenization avoided phase separation upon storage. The anionic WK-OB surface at neutral pH was affected by stressful ionic environments (pH, NaCl, CaCl2), that induced aggregation of WK-OBs and decreased the physical stability of the emulsions. Emulsions containing WK-OBs are promising to diversify the market of the ω3-rich plant-based food products and beverages.

Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186.

IMPORTANCE: Since the global market for sterols and vitamin D are grown with a high compound annual growth rate, a sustainable source of these compounds is required to keep up with the increasing demand. Thraustochytrid is a marine oleaginous microorganism that can synthesize several sterols, which are stored as SE in lipid droplets. DGAT2C is an unconventional SE synthase specific to thraustochytrids. Although the primary structure of DGAT2C shows high similarities with that of DGAT, DGAT2C utilizes sterol as an acceptor substrate instead of diacylglycerol. In this study, we examined more detailed enzymatic properties, intracellular localization, and structure-activity relationship of DGAT2C. Furthermore, we successfully developed a method to increase sterol and provitamin D3 productivity of thraustochytrid by more than threefold in the process of elucidating the function of the DGAT2C-specific N-terminal region. Our findings could lead to sustainable sterol and vitamin D production using thraustochytrid.

Effects of choline deficiency and supplementation on lipid droplet accumulation in bovine primary liver cells in vitro.

Rumen-protected choline (RPC) supplementation in the periparturient period has in some instances prevented and alleviated fatty liver disease in dairy cows. Mechanistically, however, it is unclear how choline prevents the accumulation of lipid droplets (LD) in liver cells. In this study, primary liver cells isolated from liver tissue obtained via puncture biopsy from 3 nonpregnant mid-lactation multiparous Holstein cows (∼160 d postpartum) were used. Analyses of LD via oil red O staining, protein abundance via Western blotting, and phospholipid content and composition measured by thin-layer chromatography and HPLC/mass spectrometry were performed in liver cells cultured in choline-deficient medium containing 150 µmol/L linoleic acid for 24 h. In a subsequent experiment, lipophagy was assessed in liver cells cultured with 30, 60, or 90 µmol/L choline-chloride. All data were analyzed statistically using SPSS 20.0 via t-tests or one-way ANOVA. Compared with liver cells cultured in Dulbecco's Modified Eagle Medium alone, choline deficiency increased the average diameter of LD (1.59 vs. 2.10 µm), decreased the proportion of small LD (<2 µm) from 75.3% to 56.6%, and increased the proportion of large LD (>4 µm) from 5.6% to 15.0%. In addition, the speed of LD fusion was enhanced by the absence of choline. Among phospholipid species, the phosphatidylcholine (PC) content of liver cells decreased by 34.5%. Seventeen species of PC (PC [18:2_22:6], PC [15:0_16:1], PC [14:0_20:4], and so on) and 6 species of lysophosphatidylcholine (LPC; LPC [15:0/0:0]), PC (22:2/0:0), LPC (20:2/0:0), and so on] were decreased, while PC (14:1_16:1) and LPC (0:0/20:1) were increased. Choline deficiency increased the triglyceride (TAG) content (0.57 vs. 0.39 µmol/mg) in liver cells and increased the protein abundance of sterol regulatory element binding protein 1, sterol regulatory element binding protein cleavage activation protein, and fatty acid synthase by 23.5%, 17%, and 36.1%, respectively. Upon re-supplementation with choline, the phenotype of LD (TAG content, size, proportion, and phospholipid profile) was reversed, and the ratio of autophagy marker LC3II/LC3I protein was significantly upregulated in a dose-dependent manner. Overall, at least in vitro in mid-lactation cows, these data demonstrated that PC synthesis is necessary for normal LD formation, and both rely on choline availability. According to the limitation of the source of liver cells used, further work should be conducted to ascertain that these effects are applicable to liver cells from postpartum cows, the physiological stage where the use of RPC has been implemented for the prevention and treatment of fatty liver.

New Perspectives on Chinese Medicine in Treating Hepatic Fibrosis: Lipid Droplets in Hepatic Stellate Cells.

Hepatic fibrosis (HF) is a wound healing response featuring excessive deposition of the extracellular matrix (ECM) and activation of hepatic stellate cells (HSCs) that occurs during chronic liver injury. As an initial stage of various liver diseases, HF is a reversible pathological process that, if left unchecked, can escalate into cirrhosis, liver failure, and liver cancer. HF is a life-threatening disease presenting morbidity and mortality challenges to healthcare systems worldwide. There is no specific and effective anti-HF therapy, and the toxic side effects of the available drugs also impose a heavy financial burden on patients. Therefore, it is significant to study the pathogenesis of HF and explore effective prevention and treatment measures. Formerly called adipocytes, or fat storage cells, HSCs regulate liver growth, immunity, and inflammation, as well as energy and nutrient homeostasis. HSCs in a quiescent state do not proliferate and store abundant lipid droplets (LDs). Catabolism of LDs is characteristic of the activation of HSCs and morphological transdifferentiation of cells into contractile and proliferative myofibroblasts, resulting in the deposition of ECM and the development of HF. Recent studies have revealed that various Chinese medicines (e.g., Artemisia annua, turmeric, Scutellaria baicalensis Georgi, etc.) are able to effectively reduce the degradation of LDs in HSCs. Therefore, this study takes the modification of LDs in HSCs as an entry point to elaborate on the process of Chinese medicine intervening in the loss of LDs in HSCs and the mechanism of action for the treatment of HF.

Coptidis rhizoma and evodiae fructus against lipid droplet deposition in nonalcoholic fatty liver disease-related liver cancer by AKT.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease in the world. NAFLD has become one of the major factors contributing to hepatocellular carcinoma (HCC) development. However, there are no clear targets and therapeutic drugs for NAFLD-related liver cancer. This study explored the active compounds, target and mechanism of coptidis rhizoma and evodiae fructus in the treatment of NAFLD-related liver cancer based on the network pharmacology and experimental verification. There were 455 intersection targets of NAFLD-related liver cancer, and 65 drug-disease common targets. AKT1 has the highest degree, indicating that it may be a key target of coptidis rhizoma and evodiae fructus in the treatment of NAFLD-related liver cancer. The expression level of AKT1 was high in high-risk group, and the overall survival rate was lower than that in low-risk group. After oleic acid induction, p-AKT expression and lipid droplet deposition were promoted in HepG2 cells. Quercetin and resveratrol reduced lipid droplet deposition in vivo. Moreover, quercetin inhibited p-AKT expression, resveratrol both reduced the expression of p-AKT and AKT. The overall findings suggested that quercetin inhibited AKT in the treatment of NAFLD-related liver cancer.

Label-free quantitative proteomic analysis of milk fat globule membrane proteins in porcine colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are nutritional components with various biological functions. This study aimed to analyze and compare MFGM proteins in porcine colostrum (PC) and porcine mature milk (PM), via label-free quantitative proteomics. In total, 3917 and 3966 MFGM proteins were identified in PC and PM milk, respectively. A total of 3807 common MFGM proteins were found in both groups, including 303 significant differentially expressed MFGM proteins. Gene Ontology (GO) analysis revealed that the differentially expressed MFGM proteins were mainly related to the cellular process, cell, and binding. The dominant pathway of the differentially expressed MFGM proteins was related to the phagosome according to Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. These results reveal crucial insights into the functional diversity of MFGM proteins in porcine milk during lactation and provide theoretical guidance for the development of MFGM proteins in the future.

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment.

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing dysfunction caused by free fatty acids (FAs). The liver, given its critical role in the body's fat metabolism, is persistently threatened by the intracellular accumulation of LDs in the form of both microvesicular and macrovesicular hepatic steatosis. The histologic characterization of LDs is typically based on lipid-soluble diazo dyes, such as Oil Red O (ORO) staining, but a number of disadvantages consistently hamper the use of this analysis with liver specimens. More recently, lipophilic fluorophores 493/503 have become popular for visualizing and locating LDs due to their rapid uptake and accumulation into the neutral lipid droplet core. Even though most applications are well-described in cell cultures, there is less evidence demonstrating the reliable use of lipophilic fluorophore probes as an LD imaging tool in tissue samples. Herein, we propose an optimized boron dipyrromethene (BODIPY) 493/503-based protocol for the evaluation of LDs in liver specimens from an animal model of high-fat diet (HFD)-induced hepatic steatosis. This protocol covers liver sample preparation, tissue sectioning, BODIPY 493/503 staining, image acquisition, and data analysis. We demonstrate an increased number, intensity, area ratio, and diameter of hepatic LDs upon HFD feeding. Using orthogonal projections and 3D reconstructions, it was possible to observe the full content of neutral lipids in the LD core, which appeared as nearly spherical droplets. Moreover, with the fluorophore BODIPY 493/503, we were able to distinguish microvesicles (1 µm < d ≤ 3 µm), intermediate vesicles (3 µm < d ≤ 9 µm), and macrovesicles (d > 9 µm), allowing the successful discrimination of microvesicular and macrovesicular steatosis. Overall, this BODIPY 493/503 fluorescence-based protocol is a reliable and simple tool for hepatic LD characterization and may represent a complementary approach to the classical histological protocols.

Characterization and comparison site-specific N-glycosylation profiling of milk fat globule membrane proteome in donkey and human colostrum and mature milk.

Milk fat globule membrane (MFGM) proteins are highly glycosylated and involved in various biological processes within the body. However, information on site-specific N-glycosylation of MFGM glycoproteins in donkey and human milk remains limited. This study aimed to map the most comprehensive site-specific N-glycosylation fingerprinting of donkey and human MFGM glycoproteins using a site-specific glycoproteomics strategy. We identified 1,360, 457, 2,617, and 986 site-specific N-glycans from 296, 77, 214, and 196 N-glycoproteins in donkey colostrum (DC), donkey mature milk (DM), human colostrum (HC), and human mature milk (HM), respectively. Bioinformatics was used to describe the structure-activity relationships of DC, DM, HC, and HM MFGM N-glycoproteins. The results revealed differences in the molecular composition of donkey and human MFGM N-glycoproteins and the dynamic changes to site-specific N-glycosylation of donkey and human MFGM glycoproteins during lactation, deepening our understanding of the composition of donkey and human MFGM N-glycoproteins and their potential physiological roles.