Proteins can be modified with eight homogenous ubiquitin chains linked by an isopeptide bond between the Cterminus of one ubiquitin and an amine from one of the seven lysines or the N-terminal methionine of the next ubiquitin.These topologically distinct ubiquitin chains signal for many essential cellular functions, such as protein degradation, cell cycle progression, DNA repair, and signal transduction.The lysine 48 (K48)-linked ubiquitin chain is one of the most abundant chains and a major proteasome-targeting signal in cells.Despite recent advancements in imaging linkage-specific polyubiquitin chains, no tool is available for imaging K48 chains in live cells.Here we report on a ubiquitination-induced fluorescence complementation (UiFC) assay for detecting K48 ubiquitin chains in vitro and in live cells.For this assay, two nonfluorescent fragments of a fluorescent protein were fused to the ubiquitininteracting motifs (UIMs) of epsin1 protein.Upon simultaneous binding to a ubiquitin chain, the nonfluorescent fragments of the two fusion proteins are brought in close proximity to reconstitute fluorescence.When used in vitro, UiFC preferentially detected K48 ubiquitin chains with excellent signal-to-noise ratio.Time-lapse imaging revealed that UiFC is capable of monitoring increases in polyubiquitination induced by treatment with proteasome inhibitor, by agents that induce stress, and during mitophagy in live cells.
Introduction This study was conducted to evaluate whether a non-immersive virtual reality (VR)-based intervention can enhance lower extremity movement in patients with cerebral infarction and whether it has greater short-term and long-term effectiveness than conventional therapies (CTs). Materials and methods This was a single-blinded, randomized clinical controlled trial. Forty-four patients with subacute cerebral infarction were randomly allocated to the VR or CT group. All intervention sessions were delivered in the inpatient unit for 3 weeks. Outcomes were measured before (baseline) and after the interventions and at 3-month, 6-month and 1-year follow-ups. The outcomes included clinical assessments of movement and balance function using the Fugl–Meyer Assessment of Lower Extremity (FMA-LE) and Berg Balance Scale (BBS), and gait parameters in the sagittal plane. Results In the VR group, the walking speed after intervention, at 3-month, 6-month, and 1-year follow-ups were significantly greater than baseline ( p = 0.01, <0.001, 0.007, and <0.001, respectively). Compared with baseline, BBS scores after intervention, at 3-month, 6-month, and 1-year follow-ups were significantly greater in both the VR group ( p = 0.006, 0.002, <0.001, and <0.001, respectively) and CT group ( p = <0.001, 0.002, 0.001, and <0.001, respectively), while FMA-LE scores after intervention, at 3-month, 6-month, and 1-year follow-ups were significant increased in the VR group ( p = 0.03, <0.001, 0.003, and <0.001, respectively), and at 3-month, 6-month, and 1-year follow-ups in the CT group ( p = 0.02, 0.004 and <0.001, respectively). In the VR group, the maximum knee joint angle in the sagittal plane enhanced significantly at 6-month follow-up from that at baseline ( p = 0.04). Conclusion The effectiveness of the non-immersive VR-based intervention in our study was observed after the intervention and at the follow-ups, but it was not significantly different from that of CTs. In sum, our results suggest that non-immersive VR-based interventions may thus be a valuable addition to conventional physical therapies to enhance treatment efficacy. Clinical trial registration http://www.chictr.org.cn/showproj.aspx?proj=10541 , ChiCTR-IOC-15006064.
Abstract: Melatonin not only plays a major role in the regulation of circadian rhythms, but is also involved in antioxidative defense and immunomodulation. Circulating melatonin levels are derived primarily from the pineal gland while other sources of melatonin have also been reported. Here, we show for the first time that astrocytes from the rat cortex and glioma C6 cell line synthesize melatonin in vitro. In addition, we show the presence of serotonin, the precursor of melatonin and the two key enzymes in the pathway of melatonin synthesis, i.e. N ‐acetyltransferase and hydroxyndole‐ O ‐methyltransferase in the cultured rat cortical astrocytes. Release of melatonin into the culture medium showed no diurnal changes. These point to astrocytes as a local source of melatonin in the rat brain. Its exact physiological function remains a topic for future studies.
Mesencephalic astrocyte-derived neurotrophic factor (MANF), a 20 kDa secreted protein, was originally derived from a rat mesencephalic type-1 astrocyte cell line. MANF belongs to a novel evolutionally conserved family of neurotrophic factors along with conserved dopamine neurotrophic factor. In recent years, ever-increasing evidence has shown that both of them play a remarkable protective role against various injuries to neurons in vivo or in vitro. However, the characteristics of MANF expression in the different types of glial cells, especially in astrocytes, remain unclear.The model of focal cerebral ischemia was induced by rat middle cerebral artery occlusion. Double-labeled immunofluorescent staining was used to identify the types of neural cells expressing MANF. Primarily cultured glial cells were used to detect the response of glial cells to endoplasmic reticulum stress stimulation. Propidium iodide staining was used to determine dead cells. Reverse transcription PCR and western blotting were used to detect the levels of mRNA and proteins.We found that MANF was predominantly expressed in neurons in both normal and ischemic cortex. Despite its name, MANF was poorly expressed in glial cells, including astrocytes, in normal brain tissue. However, the expression of MANF was upregulated in the glial cells under focal cerebral ischemia, including the astrocytes. This expression was also induced by several endoplasmic reticulum stress inducers and nutrient deprivation in cultured primary glial cells. The most interesting phenomenon observed in this study was the pattern of MANF expression in the microglia. The expression of MANF was closely associated with the morphology and state of microglia, accompanied by the upregulation of BIP/Grp78.These results indicate that MANF expression was upregulated in the activated glial cells, which may contribute to the mechanism of ischemia-induced neural injury.
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