Live-cell imaging of cAMP in Flamindo2/nlsFlamindo2-expressing COS7 cells.

<p>(A) Representative images showing changes in fluorescence intensity (FI) by 50 µM forskolin application in Flamindo2-expressing COS7 cells. (B) Time course of a normalized decrease in FI induced by reagents, which increase intracellular cAMP levels. Each reagent was applied at 2 min (dotted line). Three traces (black and gray lines) from single cells in three independent experiments are shown in all of the graphs. The black traces indicate representative data. (C) Representative images showing changes in FI by 10 mM bicarbonate application in nlsFlamindo2-expressing COS7 cells. (D) Different effects of bicarbonate and forskolin on cAMP dynamics in the nucleus. Bicarbonate or forskolin was applied at 2 min. Three traces (black and gray lines) from single cells in three independent experiments are shown in all of the graphs. The black trace indicates representative data.</p

Schematic representation of the domain structure of Flamindo2.

<p>Citrine is a mutant of yellow fluorescent protein (YFP). cDNAs for yellow fluorescent cAMP indicators, Flamindo and Flamindo2, were created by insertion of DNA fragments encoding the cAMP-binding domain of mEPAC1 (199–358) and N- and C- terminus linker peptides including restriction sites. Nucleus-targeted Flamindo2 (nlsFlamindo2) was created by fusion of NLS (nuclear localization signal; MPKKKRKVEDVDP) to the N-terminus of Flamindo2.</p

Comparison of single FP-based indicators.

<p>I, Intensiometric; R, Ratiometric.</p><p>*Estimated from graphs or descriptions.</p

Spectral characterization of Flamindo2.

<p>(A) Fluorescence spectra of Flamindo2 protein. Excitation and emission spectra were measured in the presence (dotted lines, +cAMP) or absence (solid lines, -cAMP) of 1 mM cAMP. Each fluorescence intensity (FI) was normalized to the peak of FI in the absence of cAMP. The representative excitation/emission spectra data from three independent experiments was shown in the graph. (B) Absorbance spectra of Flamindo2 protein in the presence (dotted line, +cAMP) or absence (solid line, -cAMP) of 1 mM cAMP. The representative absorption spectra data from three independent experiments was shown in the graph. (C) Titration curves of cAMP and cGMP. The peak of FI at each concentration of cAMP (closed circles) or cGMP (closed squares) was normalized to the peak of FI in the absence of cAMP or cGMP. The results are mean ± SD (n = 3) (D) Titration curves against pH. The peak of FI at each pH in the presence (closed circles) or absence (open circles) of 1 mM cAMP was normalized to the peak of FI at pH 9.0 in the absence of cAMP. The results are mean ± SD (n = 3).</p

Dual-color imaging of cAMP and Ca²⁺ in Flamindo2 and R-GECO co-expressing HeLa cells.

<p>(A) Images of changes in fluorescence of Flamindo2 and R-GECO induced by 100 µM noradrenaline. (B) Time course of changes in cAMP (solid line) and Ca²⁺ (dotted line) induced by 100 µM noradrenaline. Noradrenaline was applied at 120 s. Representative trace is shown in the graph.</p

Ca²⁺ dynamics and muscle displacement of the beetle leg muscle under electrical stimulation with multiple pulse trains (100 Hz, 10% duty cycle, 2 V) for 3 s.

<p>Pseudocolor time series images of beetle leg muscle dosed with (A) Fluo-8 (60 µM) and (C) Cell Tracker (60 µM) indicators. ROIs are indicated by yellow region (as also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116655#pone.0116655.s001" target="_blank">S1B Fig.</a>). The color scale is given on the right side of each image (A) or (C) respectively. Fluorescence intensity dynamics of (B) Fluo-8 and (D) Cell Tracker under electrical stimulation, digitized with ImageJ software from the ROI shown in (A) and (C) respectively. The stimulus timing is indicated by grey shading. The Fluo-8 pseudocolor images illustrate the fluorescence intensity dynamics that correspond to the Ca²⁺ dynamics inside the leg muscle: it increased at the start of electrical stimulation, was maintained during the application of the stimulus, and finally slowly decreased after the stimulus stopped. The Cell Tracker pseudocolor images display that the muscle displacement also causes intensity change during electrical stimulation, which slightly affects the Fluo-8 measurement.</p

Oral Dosing of Chemical Indicators for <i>In Vivo</i> Monitoring of Ca²⁺ Dynamics in Insect Muscle

<div><p>This paper proposes a remarkably facile staining protocol to visually investigate dynamic physiological events in insect tissues. We attempted to monitor Ca²⁺ dynamics during contraction of electrically stimulated living muscle. Advances in circuit miniaturization and insect neuromuscular physiology have enabled the hybridization of living insects and man-made electronic components, such as microcomputers, the result of which has been often referred as a Living Machine, Biohybrid, or Cyborg Insect. In order for Cyborg Insects to be of practical use, electrical stimulation parameters need to be optimized to induce desired muscle response (motor action) and minimize the damage in the muscle due to the electrical stimuli. Staining tissues and organs as well as measuring the dynamics of chemicals of interest in muscle should be conducted to quantitatively and systematically evaluate the effect of various stimulation parameters on the muscle response. However, existing staining processes require invasive surgery and/or arduous procedures using genetically encoded sensors. In this study, we developed a non-invasive and remarkably facile method for staining, in which chemical indicators can be orally administered (oral dosing). A chemical Ca²⁺ indicator was orally introduced into an insect of interest via food containing the chemical indicator and the indicator diffused from the insect digestion system to the target muscle tissue. We found that there was a positive relationship between the fluorescence intensity of the indicator and the frequency of electrical stimulation which indicates the orally dosed indicator successfully monitored Ca²⁺ dynamics in the muscle tissue. This oral dosing method has a potential to globally stain tissues including neurons, and investigating various physiological events in insects.</p></div

Effect of various electrical stimulation frequencies on Ca²⁺ dynamics and muscle displacement in beetle leg muscle.

<p>Images of beetle leg muscle that was dosed with (A) Fluo-8 (60 µM) and (C) Cell Tracker (60 µM), with the yellow selection indicating the ROI that was used for analysis (as also shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116655#pone.0116655.s001" target="_blank">S1B Fig</a>.). The color scale is given at the bottom left corner of the image. Representative time courses showing the fluorescence intensity dynamics of (B) Fluo-8, and (D) Cell Tracker under various electrical stimulations of multiple pulse trains (50 Hz, 10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz; 10% duty cycle; 2 V) observed from the ROI that are displayed in (A) and (C) respectively. All electrical stimulations were applied for 3 seconds periods with a 27 seconds resting period in between stimulations. The stimulus timing is indicated by grey shading. (E) Relative change in fluorescence intensity ((Δ<i>F</i>/<i>F</i>₀)×100%) for Fluo-8 (blue) and Cell Tracker (red) under varying electrical stimulation frequencies (50 Hz, 10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz; 10% duty cycle; 2 V). The small numbers next to each plot indicate the order of stimulation; i.e., first from 50 Hz followed by varying frequency pulses (10 Hz, 1 Hz, 100 Hz, 1 Hz, and 50 Hz). The Fluo-8 intensity plot shows that Ca²⁺ dynamics are dependent on electrical stimulation frequency, whereas the Cell Tracker plot shows that the muscle displacement contributes a small amount.</p

Effects of <i>Lactobacillus paracasei</i> MCC1849 on total IgA production in mice.

<p>(A) Mice were treated with or without MCC1849 for 5 weeks. Total IgA concentrations in homogenized small intestine and serum samples were determined via ELISA. (B) The proportions of IgA⁺ B220⁺ cells and IgA⁺ plasmablasts (IgA⁺ B220^- cells) in PPs were analyzed via FCM. (C) Gene expression related to the differentiation of IgA⁺ cells was measured via real-time RT-PCR analysis. The level of gene expression was normalized to that of GAPDH mRNA expression in the control group. Data are shown as the mean ± SD. N = 16. *p<0.05, **p<0.01 compared to the control.</p

Effects of <i>Lactobacillus paracasei</i> MCC1849 on total IgA and OVA-specific IgA production in OVA-immunized mice.

<p>(A, B) Mice were treated with or without MCC1849 for 5 weeks. All mice were orally immunized on days 14, 21, and 28 with OVA and cholera toxin. On day 35, mice were euthanized and dissected. Data show the total IgA and OVA-specific IgA concentrations in homogenized small intestine, small-intestine lavage fluid, homogenized colon, colon contents, serum and lung samples. AU: arbitrary unit. Data are shown as the mean ± SD. N = 14. Data are representative three independent experiments. *p<0.05, **p<0.01 compared to the control.</p