Efficient TurboID-based proximity labelling method for identifying terminal sialic acid glycosylation in living cells.

TurboID, a proximity labelling method based on mutant biotin ligase, is an efficient new technique for recognizing protein-protein interactions and has been successfully applied to living cells. Sialic acid is typically the terminal monosaccharide attached to many glycoproteins and plays many important roles in many biological processes. However, the lack of enrichment methods for terminal sialic acid glycosylation in vivo hinders the identification and analysis of this glycosylation. Here, we introduce TurboID to identify terminal sialic acid glycosylation in living cells. SpCBM, the carbohydrate-binding domain of sialidase from Streptococcus pneumoniae, is fused with TurboID and overexpressed in HeLa cells. After streptavidin-based purification and detection by mass spectrometry, 31 terminal sialic acid N-glycosylated sites and 1359 putative terminal sialic acid glycosylated proteins are identified, many of which are located in the cytoplasm and nucleus.

Berberine ameliorates neuronal AD-like change via activating Pi3k/PGCε pathway.

IR (insulin resistance) in diabetic brain gave rise to the generation of toxic factor Aß42 and axon collapse which were the marker of AD (Alzheimer's disease)-like lesions in the circumstance of diabetes mellitus. But the underling molecular mechanism was not clear. Chronic HGHI (high glucose and high insulin) exposure accelerates IR has been reported in type II diabetes models. Berberine has been shown to promising effect for IR in vitro and in vivo. This study demonstrates the protective effect and the underlying mechanism of berberine on HGHI-induced IR. HGHI-induced cells were used to mimic the hyperinsulinemia resulting in IR. Berberine was used to uncover the mechanisms for the treatment of hyperinsulinemia in IR model. Morris water maze (MWM), PET imaging, CCK8 assay, ELISA assay, glucose kits, microscopy, and western blot analysis were performed to evaluate the protective effects of berberine. Berberine-improved HGHI-induced IR was correlated with the increase of glucose application in neurons. Meanwhile, the expressions of Pi3K, as well as GLUT3, PKCε, and APP were downregulated in the model, while p-IRS Ser307 was upregulated compared with Normal group. Fortunately, these scenes were reversed by berberine administration. Furthermore, berberine decreased GSK3ß Y216 expressions, inhibited the production of oligomer Aß42 and extended neuronal axon. The monomeric berberine treatment improves IR that may be involved in glucose effective application, rectifying the related proteins of the aberrant insulin pathway. Additionally, it suppressed the generation of Aß42 and ameliorated neuron axon damage. Finally, berberine improves DM (diabetes mellitus)-induced cognitive impairment.

Ferulic acid attenuates oxidative DNA damage and inflammatory responses in microglia induced by benzo(a)pyrene.

Over-activation of microglia disrupts the physiological homeostasis of the brain, and induces inflammatory response and other processes which are implicated in neurodegenerative diseases. Therefore, theoretically, suppression of neuroinflammation would slow the progression of neurodegenerative disease. In this study, we investigated the possible protective effects of Ferulic acid (FA) against benzo(a)pyrene (BaP)-induced microglial activation using BV2 cells as the model system. Exposure of BV2 cells to BaP (10 µM) significantly increased DNA damage and the production of pro-inflammatory mediators, including nitric oxide (NO), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), reactive oxygen species (ROS), malondialdehyde (MDA), and cytokines (interleukins-1ß and -6). On the other hand, when BaP-treated BV2 cells were further incubated with FA (10, 20, 40, or 80 mg/mL) for another 24 h, a significant reduction in BaP-induced DNA damage and the release of multiple pro-inflammatory and cytotoxic factors (including interleukin-1ß, interleukin-6, NO, and ROS) was observed in a dose-dependent manner. Further study revealed that the microglial NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3) signaling pathway was involved in the protective effect of FA. Taken together, these results suggested that FA suppressed BaP-induced toxicity in microglia, and thus may exert neuroprotective effects by inhibiting microglia-mediated pro-inflammatory response.

MiR-28-5p relieves neuropathic pain by targeting Zeb1 in CCI rat models.

MicroRNAs (miRNAs) are recognized as significant regulators of neuropathic pain. Moreover, neuroinflammation can contribute a lot to the progression of neuropathic pain. MiR-28-5p has been reported to be involved in many pathological diseases. However, little is known about the function of miR-28-5p in neuropathic pain development. Our current study was designed to investigate the biological roles of miR-28-5p in neuropathic pain in a rat model established by chronic sciatic nerve injury (CCI). Here, we observed that miR-28-5p was decreased in CCI rats. MiR-28-5p overexpression was able to alleviate neuropathic pain behaviors including mechanical and thermal hyperalgesia. Meanwhile, inflammation-correlated biomarkers such as Cyclooxygenase 2 (Cox-2), interleukin-6 (IL-6), and IL-1ß were greatly promoted in CCI rats and they were inhibited by miR-28-5p upregulation. In addition, zinc finger E-box-binding homeobox 1 (Zeb1) is a kind of transcription factor that is involved in various diseases. Here, in our study, Zeb1 was predicted as a downstream target of miR-28-5p. miR-28-5p can bind with the 3'-untranslated region of Zeb1, which was validated by carrying out dual-luciferase reporter assay. Moreover, we found that Zeb1 was significantly increased in CCI rats and miR-28-5p can modulate Zeb1 expression negatively. Theoverexpression of Zeb1 can disturb neuropathic pain development, which was repressed by the increase of miR-28-5p by upregulating Cox-2, IL-6, and IL-1ß levels. By taking all of these together, it was indicated in our study that miR-28-5p can reduce neuropathic pain progression by targeting Zeb1 in vivo. Our data implied that miR-28-5p/Zeb1 axis can be a novel therapeutic target for neuropathic pain treatment.