A non-cell-autonomous circadian rhythm of bioluminescence reporter activities in individual duckweed cells.

The circadian clock is responsible for the temporal regulation of various physiological processes in plants. Individual cells contain a circadian oscillator consisting of a clock gene circuit that coordinates physiological rhythms within the plant body in an orderly manner. The coordination of time information has been studied from the perspective of cell-cell local coupling and long-distance communication between tissues based on the view that the behavior of circadian oscillators represents physiological rhythms. Here, we report the cellular circadian rhythm of bioluminescence reporters that are not governed by the clock gene circuit in expressing cells. We detected cellular bioluminescence rhythms with different free-running periods in the same cells using a dual-color bioluminescence monitoring system in duckweed (Lemna minor) transfected with Arabidopsis CIRCADIAN CLOCK ASSOCIATED 1::luciferace+ (AtCCA1::LUC+) and Cauliflower mosaic virus 35S::modified click-beetle red-color luciferase (CaMV35S::PtRLUC) reporters. Co-transfection experiments with the two reporters and a clock gene-overexpressing effector revealed that the AtCCA1::LUC+ rhythm, but not the CaMV35S::PtRLUC rhythm, was altered in cells with a dysfunctional clock gene circuit. This indicated that the AtCCA1::LUC+ rhythm is a direct output of the cellular circadian oscillator, whereas the CaMV35S::PtRLUC rhythm is not. After plasmolysis, the CaMV35S::PtRLUC rhythm disappeared, whereas the AtCCA1::LUC+ rhythm persisted. This suggests that the CaMV35S::PtRLUC bioluminescence has a symplast/apoplast-mediated circadian rhythm generated at the organismal level. The CaMV35S::PtRLUC-type bioluminescence rhythm was also observed when other bioluminescence reporters were expressed. These results reveal that the plant circadian system consists of both cell-autonomous and noncell-autonomous rhythms that are unaffected by cellular oscillators.

Detection of Uncoupled Circadian Rhythms in Individual Cells of Lemna minor using a Dual-Color Bioluminescence Monitoring System.

The plant circadian oscillation system is based on the circadian clock of individual cells. Circadian behavior of cells has been observed by monitoring the circadian reporter activity, such as bioluminescence of AtCCA1::LUC+. To deeply analyze different circadian behaviors in individual cells, we developed the dual-color bioluminescence monitoring system that automatically measured the luminescence of two luciferase reporters simultaneously at a single-cell level. We selected a yellow-green-emitting firefly luciferase (LUC+) and a red-emitting luciferase (PtRLUC) that is a mutant form of Brazilian click beetle ELUC. We used AtCCA1::LUC+ and CaMV35S::PtRLUC. CaMV35S::LUC+ was previously reported as a circadian reporter with a low-amplitude rhythm. These bioluminescent reporters were introduced into the cells of a duckweed, Lemna minor, by particle bombardment. Time series of the bioluminescence of individual cells in a frond were obtained using a dual-color bioluminescence monitoring system with a green-pass- and red-pass filter. Luminescence intensities from the LUC+ and PtRLUC of each cell were calculated from the filtered luminescence intensities. We succeeded in reconstructing the bioluminescence behaviors of AtCCA1::LUC+ and CaMV35S::PtRLUC in the same cells. Under prolonged constant light conditions, AtCCA1::LUC+ showed a robust circadian rhythm in individual cells in an asynchronous state in the frond, as previously reported. By contrast, CaMV35S::PtRLUC stochastically showed circadian rhythms in a synchronous state. These results strongly suggested the uncoupling of cellular behavior between these circadian reporters. This dual-color bioluminescence monitoring system is a powerful tool to analyze various stochastic phenomena accompanying large cell-to-cell variation in gene expression.

Positron emission tomography/computed tomography detection of increased 18F-fluorodeoxyglucose uptake in the cardiac atria of patients with atrial fibrillation.

BACKGROUND: Direct evidence of inflammatory activity in the atria of patients with atrial fibrillation (AF) is scarce. We assessed the capability of positron-emission tomography/computed tomography (PET/CT) to diagnose AF based on fluorodeoxyglucose (FDG) uptake in the atrial wall. METHODS AND RESULTS: Among 8233 patients who underwent FDG-PET/CT as work-up for malignancies, we identified 180 consecutive patients with AF (2.2%). Of those, we selected 137 patients who had fasted >12 h before FDG injection for inclusion in the experimental group (88 men and 49 women; age: 72.7 ±â€¯8.9 years). Controls were 62 age- and sex-matched patients without AF. For visual analysis, we used a 4-point grading system. For quantitative analysis, we used the maximum standard uptake value (SUVmax) in the left (LA) and right atrial (RA) myocardium and the target-to-background ratio (TBR) of SUVmax to blood pool activity. The sensitivity, specificity, and positive-predictive value for detecting AF visually were 54.0%, 95.2%, and 96.1%, respectively; for quantitative analysis, the respective values were 65.7%, 75.8%, and 85.7%. Multivariable analysis of 11 clinical and imaging variables showed significant associations with RA SUVmax (odds ratio [OR]: 14.353, P = 0.026) and LA volume (OR: 1.371, P = 0.0001). The RA TBR was greater in cases with persistent AF than in those with paroxysmal AF (P < 0.0001). Pathological investigation of 4 autopsy hearts confirmed infiltration of extravascular macrophages and lymphocytes in the regions with FDG uptake. CONCLUSIONS: Higher atrial FDG uptake was associated with AF. PET/CT could be a useful tool for detecting local inflammation in the atria with AF.

Estimation of myocardial flow reserve utilizing an ultrafast cardiac SPECT: Comparison with coronary angiography, fractional flow reserve, and the SYNTAX score.

BACKGROUND: Quantitative assessment of myocardial flow reserve (MFR) by single photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI) is challenging but may facilitate evaluation of multi-vessel coronary artery disease (CAD). METHODS: We enrolled 153 patients with suspected or known CAD, referred for pharmacological stress MPI. They underwent a 99mTc-perfusion stress/rest SPECT with an ultrafast cadmium-zinc-telluride (CZT) camera. Dynamic data were acquired and time-activity curves fitted to a 1-tissue compartment analysis with input function. K1 was assigned for stress and rest data. The MFR index (MFRi) was calculated as K1 stress/K1 at-rest. The findings were validated by invasive coronary angiography in 69 consecutive patients. RESULTS: The global MFRi was 1.46 (1.16-1.76), 1.33 (1.12-1.54), and 1.18 (1.01-1.35), for 1-vessel disease (VD), 2-VD, and 3-VD, respectively. In the 3-VD, global MFRi was lower than that in 0-VD (1.63 [1.22-2.04], P<0.0001) and 1-VD (P=0.003). Multivariate logistic regression analysis for 3-VD showed significant associations with smoking history (odds ratio [OR]: 4.4 [0.4-8.4]), left ventricular ejection fraction (OR: 61.6 [57.5-66.0]), and global MFRi (OR: 119.6 [111.5-127.7], P=0.002). A cut-off value of 1.3 yielded 93.3% sensitivity and 75.9% specificity for diagnosing 3-VD. Fractional flow reserve positively correlated with regional MFRi (r=0.62, P=0.008), and the SYNTAX score correlated negatively with global MFRi (r=0.567, P=0.0003). CONCLUSION: We developed and validated a clinically available method for MFR quantification by dynamic 99mTc-perfusion SPECT utilizing a CZT camera, which improves the detectability of multi-vessel CAD.