Project description:Tissue repair after spinal cord injury (SCI) requires mobilization of immune and glial cells to form a protective barrier that seals the wound and facilitates debris clearing, inflammatory containment, and matrix compaction. This process involves corralling, wherein phagocytic immune cells become confined to the necrotic core surrounded by an astrocytic border. Here, we elucidate a temporally distinct gene signature in injury-activated microglia/macrophages (IAM), which engages axon guidance pathways. Plexin-B2 is upregulated in IAM, which is required for motosensory recovery after SCI. Plexin-B2 deletion in myeloid cells impairs corralling, leading to diffuse tissue damage, inflammatory spillover, and hampered axon regeneration. Corralling begins early and requires Plexin-B2 in both microglia and macrophages. Mechanistically, Plexin-B2 promotes microglia motility, steers IAM away from colliding cells, and facilitates matrix compaction. Our data thus establish Plexin-B2 as an important link that integrates biochemical cues and physical interactions of IAM with the injury microenvironment during wound healing.
Project description:Neatly every proteomic protocol has a Cysteine reduction and alkylation step in it preceding the trypsin digestion. It needs to be done in order to remove the s-s bonds and prevent proteins from folding, thus, provide better access for trypsin. Among various reagents used for alkylation, iodoacetamide (IAM) seems to be the most common. Although, it has been shown that IAM can cause some undesired side reactions [Chernobrovkin et al., 2015; Muller et al., 2017]. In this work, we have compared the effect of four alkylating agents: iodoacetamide (IAM), 4-vinylpyridine (4-VP), chloroacetamide (CAM) and S-Methyl methanethiosulfonate (MMTS). In addition, we tried to check whether post-treatment with DTT (p-red) is reasonable in the case of irreversible alkylation.
Project description:Neatly every proteomic protocol has a Cysteine reduction and alkylation step in it preceding the trypsin digestion. It needs to be done in order to remove the s-s bonds and prevent proteins from folding, thus, provide better access for trypsin. Among various reagents used for alkylation, iodoacetamide (IAM) seems to be the most common. Although, it has been shown that IAM can cause some undesired side reactions [Chernobrovkin et al., 2015; Muller et al., 2017]. In this work, we have compared the effect of four alkylating agents: iodoacetamide (IAM), 4-vinylpyridine (4-VP), chloroacetamide (CAM) and S-Methyl methanethiosulfonate (MMTS). In addition, we tried to check whether post-treatment with DTT (p-red) is reasonable in the case of irreversible alkylation.
