We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.
Rationale: Glial scars present a major obstacle for neuronal regeneration after stroke. Thus, approaches to promote their degradation and inhibit their formation are beneficial for stroke recovery. The interaction of microglia and astrocytes is known to be involved in glial scar formation after stroke; however, how microglia affect glial scar formation remains unclear. Methods: Mice were treated daily with M2 microglial small extracellular vesicles through tail intravenous injections from day 1 to day 7 after middle cerebral artery occlusion. Glial scar, infarct volume, neurological score were detected after ischemia. microRNA and related protein were examined in peri-infarct areas of the brain following ischemia. Results: M2 microglial small extracellular vesicles reduced glial scar formation and promoted recovery after stroke and were enriched in miR-124. Furthermore, M2 microglial small extracellular vesicle treatment decreased the expression of the astrocyte proliferation gene signal transducer and activator of transcription 3, one of the targets of miR-124, and glial fibrillary acidic protein and inhibited astrocyte proliferation both in vitro and in vivo . It also decreased Notch 1 expression and increased Sox2 expression in astrocytes, which suggested that astrocytes had transformed into neuronal progenitor cells. Finally, miR-124 knockdown in M2 microglial small extracellular vesicles blocked their effects on glial scars and stroke recovery. Conclusions: Our results showed, for the first time, that microglia regulate glial scar formation via small extracellular vesicles, indicating that M2 microglial small extracellular vesicles could represent a new therapeutic approach for stroke.
Plant intracellular nucleotide binding leucine-rich repeat (NLR) immune receptors play critical roles in pathogen surveillance. Most plant NLRs characterized so far were found to use a single domain/sensor to recognize pathogen effectors. Here we report that the Sw-5b NLR immune receptor uses two distinct domains to detect the viral movement protein NSm encoded by tospovirus. In addition to its leucine-rich repeat (LRR) domain that has been previously reported, the N-terminal Solanaceae domain (SD) of Sw-5b also interacts with NSm and a conserved 21-amino-acid region of NSm (NSm 21). The specific interaction between Sw-5b SD and NSm is required for releasing the inhibitory effect of coiled-coil domain on the NB-ARC-LRR region. Furthermore, we found that the binding of NSm affects the nucleotide binding activity of the NB-ARC-LRR in vitro, while Sw-5b NB-ARC-LRR is activated only when NSm and NSm 21 levels are high. Interestingly, Sw-5b SD could significantly enhance the ability of the NB-ARC-LRR to detect low levels of NSm effector and facilitate its activation and induction of defense response. An Sw-5b SD mutant that is disrupted in NSm recognition failed to enhance the ability of the NB-ARC-LRR to sense low levels of NSm and NSm 21. Taken together, our results suggest that Sw-5b SD functions as an extra sensor and the NB-ARC-LRR as an activator, and that Sw-5b NLR adopts a two-step recognition mechanism to enhance viral effector perception.
Many studies have shown that oxidative stress is a major cause of cellular injuries in a variety of human diseases including cognitive impairment. Tea polyphenols (TPs), natural plant flavonoids found in tea plant leaves, possess the bioactivity to affect the pathogenesis of several chronic diseases via antioxidant associated mechanisms. However, the possible antioxidant and neuroprotective properties of TPs in the brain of mice housed in constant darkness and in HO-stimulated SH-SY5Y cells are yet to be elucidated. In this study, pretreatment with TPs markedly attenuated HO-elicited cell viability loss and mitochondrial dysfunction, suppressed the induced apoptosis and reduced the elevated levels of intracellular ROS and HO. Additionally, TPs modulate the nuclear translocation of Nrf2 and the TrkB/CREB/BDNF signaling pathway by provoking the PI3K/AKT pathway and thus, they transcriptionally regulate the downstream expression of antioxidant enzymes including HO-1, NQO-1, and BDNF in SH-SY5Y cells. Furthermore, an in vivo study revealed that housing mice in constant darkness, simulating shift work disruption in humans, notably affects the AKT/CREB/BDNF signal pathway and the nuclear translocation of Nrf2 and its downstream phase II detoxification enzymes in brain tissue. Remarkably, TP supplementation through drinking water eliminated these changes. These results suggest that TPs possess protective effects against oxidative stress-triggered cognitive impairment, which might be a potential nutritional preventive strategy for neurodegenerative diseases implicated with oxidative stress in shift workers.
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