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Milk fat globule membrane (MFGM) proteins have recently gained increasing attention, due to their significant biological function. However, the glycosylation of proteins in human MFGM during lactation has not been studied in detail. In this study, through mass spectroscopy-based N-glycoproteomics, we analyzed protein glycosylation of human MFGM. A total of 912 N-glycosylation sites on 506 N-glycoproteins were identified in human colostrum and mature milk MFGM. Among them, 220 N-glycoproteins with 304 N-glycosylation sites were differentially expressed in colostrum and mature milk MFGM. Gene Ontology (GO) analysis revealed various biological processes, cellular components, and molecular functions of the differentially expressed N-glycoproteins. Specifically, these glycoproteins were involved in biological processes such as single-organism processes, biological regulation, regulation of biological processes, response to stimulus and localization; were cellular components in organelles, membranes, and the extracellular region; and had different molecular functions such as protein binding, receptor activity, and hydrolase activity. KEGG pathway analysis suggested that the majority of the differentially expressed N-glycoproteins were associated with phagosome, cell adhesion molecule and some disease-related pathways. Our results provide an in-depth understanding of the quantitative changes in N-glycosylation of proteins in human colostrum and mature MFGM, and extend our knowledge of the N-glycoproteome and of the distribution of N-glycosylation sites in human MFGM during lactation, providing insight into the biological functions of the highlighted glycoproteins.

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We investigated the cytoprotective effects of anthocyanins in Aronia melanocarpa against apoptosis induced by Aβ1–42, a key mediator of AD pathophysiology. We measured intracellular calcium with a colorimetric kit, cellular apoptosis with DAPI, intracellular ROS with the fluorescent marker 2,3-dimethoxy-1,4-naphthoquinone, mitochondrial membrane potential with JC-1, and ATP with a colorimetric kit. Gene transcription and protein expression levels of calmodulin, cytochrome c, caspase-9, cleaved caspase-3, Bcl-2, and Bax were analyzed by RT-PCR and Western blotting. The results showed that pretreatment with anthocyanins significantly inhibited Aβ1–42-induced apoptosis, decreased intracellular calcium and ROS, and increased ATP and mitochondrial membrane potential. RT-PCR and Western blotting revealed that anthocyanins upregulated the gene transcription and protein expression of calmodulin and Bcl-2 and downregulated those of cytochrome c, caspase-9, cleaved caspase-3, and Bax. A. melanocarpa anthocyanins protected SH-SY5Y cells against Aβ1–42-induced apoptosis by regulating Ca2+ homeostasis and apoptosis-related genes and inhibiting mitochondrial dysfunction.

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