No abstract

No abstract

These results have implications for the prevention of youth suicide, suggesting that mental health status and attitudes towards suicide could be important targets for prevention and intervention for children and adolescents at risk of suicidal ideation.

Summary Acceptor‐sensitised 3‐cube fluorescence resonance energy transfer (FRET) imaging (also termed as E‐FRET imaging) is a popular fluorescence intensity‐based FRET quantification method. Here, an automated E‐FRET microscope with user‐friendly interfaces was set up for dynamical online quantitative live‐cell FRET imaging. This microscope reduces the time of a quantitative E‐FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E‐FRET imaging of the cells can be performed automatically by clicking ‘Capture’ button on interfaces. E‐FRET imaging was performed on the microscope for living cells expressing different FRET tandem constructs. Dynamical E‐FRET imaging on the microscope for live cells coexpressing CFP‐Bax and YFP‐Bax treated by staurosporine (STS) revealed three Bax redistribution stages: Bax translocation from cytosol to mitochondria within 10 min, membrane insertion with conformational change on mitochondrial membrane within about 30 min, and subsequent oligomerisation within about 10 min. Because of excellent user‐friendly interface and stability, the automated E‐FRET microscope is a convenient tool for quantitative FRET imaging of living cell. Lay Description Acceptor‐sensitised 3‐cube fluorescence resonance energy transfer (FRET) imaging (also termed as E‐FRET) is a popular fluorescence intensity‐based FRET quantification methods. E‐FRET measurements are currently performed manually, and a complete FRET measurement takes about 12 s. E‐FRET measurement necessitates not only a skilled operator and specialised equipment but also expertise in the interpretation of FRET signals, a considerable challenge in the application of FRET technology in living cells. Furthermore, manual E‐FRET microscope is hard to perform dynamical quantitative FRET measurement, the ever‐increasing applications in mapping the biochemical signal transduction within cells. Here, an automated E‐FRET microscope with user‐friendly interfaces was set up for dynamical online quantitative live‐cell FRET imaging. This microscope reduces the time of a quantitative E‐FRET imaging from 12 to 3 s. After locating cells, calibration of the microscope and E‐FRET imaging of the cells can be performed automatically by clicking ‘Capture’ button on interfaces. Because of excellent user‐friendly interface and stability, the automated E‐FRET microscope is a convenient tool for quantitative FRET imaging of living cell.

Heavy metal residues and chemical contaminators considered as relevant sources of aquatic environmental pollutants have a generally immunosuppressive effect on aquatic organisms, depressing metabolic activities and immune response. Glutamine: fructose-6-phosphate aminotransferase (GFAT, EC2.6.1.16) is the first, and rate-limiting, enzyme in the hexosamine biosynthetic pathway, and is involved in the regulation of chitin biosynthesis and glycosylation of proteins. We have isolated and characterized GFAT from the white shrimp Litopenaeus vannamei. Amino acid sequence similarity of the Lv-GFAT (L.vannamei-GFAT) was highest to GFATs isolated from insects and mammals (83 % similarity to that of Haemaphysalis longicornis). The open-reading frame of the Lv-GFAT codes for a protein of 41.6 kDa with a calculated isoelectric point of 5.03. RT-PCR assays showed that endogenous Lv-GFAT mRNA is most strongly expressed in the intestine. Further analysis of Lv-GFAT gene expression in hepatopancreas by quantitative real-time PCR demonstrated that Lv-GFAT transcript levels increased when the shrimp were exposed to alkaline pH (9.3) and cadmium stress, but the time when its mRNA expression level peaked differed under these stresses. We also first expressed the recombinant protein of GFAT from shrimps in Escherichia coli. Western blot analyses confirmed that the Lv-GFAT protein was strongly expressed in the hepatopancreas after exposure to the LC-Cd stress. These results suggest that Lv-GFAT expression is stimulated by alkaline pH and cadmium stress and that it may play important roles in resistance of shrimp to environmental stresses.