Bioluminescence (BL) imaging, which utilizes light emitted through the enzymatic reaction of luciferase oxidizing its substrate luciferin, enables sensitive and noninvasive monitoring of life phenomena. Herein, we developed a series of caged furimazine (FMZ) derivatives by introducing a protective group at the C-3 position and a hydroxy group at the C-6 phenyl ring to realize long-term live-cell BL imaging based on the NanoLuc (NLuc)/ NanoKAZ (NKAZ)−FMZ system. The membrane permeability and cytotoxicity of the substrates were evaluated and related to their hydrophobicity. Among the series, the derivative with the bulkiest protective group (adamantanecarbonyl group) and a hydroxy substituent (named Ad-FMZ-OH) showed significantly prolonged and constant BL signal in cells expressing NLuc compared to the native FMZ substrate. This derivative enabled continuous BL imaging at the single-cell level for 24 h. Furthermore, we applied Ad-FMZ-OH to BL imaging of myocyte fusion and succeeded in the consecutive and sensitive monitoring at a single-cell level over a day. In summary, NLuc/NKAZ-caged FMZ derivatives have the potential to be applied to live-cell BL imaging of various life phenomena that require long-term observation.
A quantitative PCR (qPCR) assay using SYBR Green I was developed based on the published sequence of the gtxA gene from Gallibacterium anatis. This method produced reliable specificity, sensitivity, and repeatability. The detection rate of Gallibacterium in 181 clinical samples was 36.5% (66/181) by qPCR, which was superior to the detection rate of Gallibacterium-specific PCR (0/181) and an isolation and identification assay (18.2% or 33/181). No association was found between the prevalence of Gallibacterium and the age of the chickens. Gallibacterium infection was detected in one 4-day-old chicken, showing that infection can occur much earlier than the previously stated fourth week of life. Tissue sample analysis showed that Gallibacterium is mainly located in the trachea and ovaries, based on results from three groups of chicken with different health statuses. Furthermore, a titer analysis suggested that Gallibacterium loads in different organs may correlate with different clinical manifestations of disease. Thus, the qPCR assay developed in the present study is useful for identification and quantitative analysis of gtxA-containing Gallibacterium in various tissue samples from birds and for the assessment of the pathogenic mechanisms of Gallibacterium.
Myogenesis-promoting chemicals are an important source of new pharmaceuticals for the treatment of skeletal muscle atrophy that impairs quality of life. This report presents a robust and quantitative bioluminescence-based assay for screening myogenesis-promoting compounds in chemical libraries. The assay system consists of two stable C2C12 myoblast cell lines, each of which expresses either an N-terminal or a C-terminal split luciferase fragment fused to a naturally split DnaE intein as an indicator for cell fusion. Cell fusion during myogenesis induces bioluminescence in the cytosol because of the reconstitution of luciferases. The luminescence intensity quantitatively represents the progress in the cell fusion and therefore indicates the extent of myogenesis. We applied this assay system to a high-throughput screening of myogenesis-promoting compouns in 1191 pharmacologically proven bioactive small molecules, which revealed two chemical compounds as myogenesis-promoting compounds: Imatinib and Doxazosin mesylate. The assay system enabled a robust and quantitative evaluation of the extent of myogenesis through simple luminescence measurements, and is expected to be widely applicable for high-throughput screening of cell fusion-promoting and inhibiting molecules.
Glucose transporter 4 (GLUT4) is an insulin-regulated glucose transporter, which is vital for blood glucose homeostasis. To clarify the physiological roles of GLUT4, quantitative measurement of GLUT4 exocytosis is indispensable. Herein, we show a rapid detection system for GLUT4 on the cell surface using spontaneous split-luciferase reconstitution. Upon insulin-induced GLUT4 exocytosis, GLUT4 was exposed outside, where luciferase is reconstituted and emitted luminescence. Pretreatment with inhibitors reduced the insulin-induced signal elevation. The results indicate that the developed method is applicable to high-throughput analysis on GLUT4 trafficking, which will greatly accelerate comprehensive research on the physiological roles of GLUT4.
Fluorescence capillary analysis (FCA) realizes trace-level analysis of micro-volume samples; it is easy to operate, extremely low in analytical cost and can significantly lessen environmental pollution from analytical chemistry waste.
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