Mu-opioid receptor (MOR) signaling regulates multiple neuronal pathways, including those involved in pain, reward, and respiration. To advance the understanding of MORsr oles in pain modulation, there is an eed for highthroughput screening methods of opioids in vitro and highresolution mapping of opioids in the brain. To fill this need, we designed and characterized ag enetically encoded fluorescent reporter,c alled Single-chain Protein-based Opioid Transmission Indicator Tool for MOR (M-SPOTIT). M-SPOTIT represents an ew and unique mechanism for fluorescent reporter design and can detect MOR activation, leaving ap ersistent green fluorescence mark for image analysis.M -SPOTIT showed an opioid-dependent signal to noise ratio (S/ N) up to 12.5 and was able to detect as fast as a3 0-second opioid exposure in HEK293T cell culture.A dditionally,i t showed an opioid-dependent S/N up to 4.6 in neuronal culture and detected fentanyl with an EC 50 of 15 nM. M-SPOTIT will potentially be useful for high-throughput detection of opioids in cell cultures and cellular-resolution detection of opioids in vivo.M-SPOTITsnovel mechanism can be used as aplatform to design other G-protein-coupled receptor-based sensors.
The mu-opioid receptor (MOR) regulates the neuronal pathways involved in pain, reward, and respiration. To increase our understanding of MOR’s roles in these pathways, there is a need to detect...
Modular fluorescent sensor motifs are needed to design fluorescent sensors for detecting various cellular processes and functional molecules. Here, we took advantage of the versatility of a new sensor motif to design a series of sensors called SPOTon. SPOTon sensors integrate the signal from either opioids, protein−protein interactions, or protease activities to generate persistent green fluorescence. We demonstrate that SPOTon can be engineered with temporal gating to allow detection of these cellular events during a user-defined time window, providing temporal information about cellular processes and functional molecule release. These SPOTon sensors all show a high signal-to-noise ratio, up to 38 for chemical gated opioid detection, 147 for chemical gated protein−protein interaction detection, and 85 for protease activity detection.
Adolescent depression is a serious problem affecting 10.7% of all teens and 29.9% of high school students; 17% of high school students have contemplated suicide. Yet, depression in teens is often unrecognized. This article relays the tragic death of a 17-year-old, along with symptoms of depression and suicide in adolescents; DSM-5 criteria for depression; treatments including protective factors, psychotherapy, and medications; and imparts interventions for addressing this huge but silent crisis.
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