Cyanide treatment alters Pi levels in different cell types of <i>C</i>. <i>elegans</i>.

<p>(A) 24 h adult hermaphrodites were imaged prior to and after treatment with 10 mM sodium cyanide to study the effect of cyanide on cytosolic Pi levels. Plotted values are mean FRET/cpVenus with standard deviation measured from 11 independent worms. P values were obtained from Student’s T test. (B) Graph shows the effect of cyanide on fluorescence intensity of purified sensor protein. cpVenus emission was measured with purified sensor protein in silicone well isolators prior to and after treatment with 10 mM sodium cyanide. Plotted data represent mean cpVenus intensity from 8 independent wells. (C) Plot shows the effect of cyanide on FRET/cpVenus ratios of <i>C</i>. <i>elegans</i> intestinal cells. The FRET/cpVenus ratio was calculated from multiple ROIs in the intestine of a worm after treating it with 10 mM sodium cyanide. The linear relationship between FRET and cpVenus shows that the ratio FRET/cpVenus is unaffected by cyanide. FRET Excitation-445 nm: Emission- 542/27 nm, cpVenus Excitation- 515 nm: Emission 542/27 nm.</p

cpFLIPPi-6.4m can report rapid changes in Pi-induced FRET <i>in vivo</i>.

<p>(A) Image showing spread of the injected fluid along the intestine, visualized by injecting propidium iodide. (B) Rapid decrease in FRET/eCFP ratio after Pi injection into the intestinal cells. Bars represent mean FRET/eCFP ratio taken from ROIs 50–100 μm from the puncture. FRET Excitation-445 nm: Emission- 542/27 nm, eCFP Excitation- 445 nm: Emission 483/32 nm. Scale bar is 100 microns.</p

<i>In vivo</i> Pi response models generated from the <i>in vitro</i> Pi binding curves.

<p> The black lines represent the experimentally generated dose dependent Pi response curve of purified cpFLIPPi-6.4m protein plotting Pi concentration against either (A) FRET/eCFP or (B) FRET/cpVenus. The curves follow the binding equation: R-R_o = (R_max-R_o)*L)/(K_d+L). R is the FRET ratio; R_o is the FRET ratio with no ligand (Pi); R_max is the FRET ratio at ligand saturation; L is ligand (Pi); K_d is the apparent dissociation constant. From the FRET/eCFP curve, R_o is 1.7, R_max is 1.1. From the FRET/cpVenus curve, R_o is 1.0, R_max is .88. The apparent K_d for both curves is 6.4 mM. The magenta lines represent the model of <i>in vivo</i> Pi dependent response curves generated by substituting the R_{o and} R_max for (A) FRET/eCFP or (B) FRET/cpVenus tail values measured from <i>in vivo</i> L1 larva and old adult animals. The K_d was kept the same as the <i>in vitro</i> curve. The asterisk represents the K_d. The capped lines represent the Pi range between 0.25x and 4x of the K_d.</p

Nutrient starvation alters Pi levels in different cell types of <i>C</i>. <i>elegans</i>.

<p>24 h adult hermaphrodites were held in NGM plates without bacteria and imaged to compare FRET ratios with the control group, maintained in NGM plates with bacteria. Plotted values are mean FRET/eCFP with standard deviation measured from 13 independent worms. FRET Excitation-445 nm: Emission- 542/27 nm, eCFP Excitation- 445 nm: Emission 483/32 nm. P values were obtained from Student’s T test.</p

Pi levels differ during development and in different cell types.

<p>(A) Pi levels in the intestine of <i>C</i>. <i>elegans</i> at different developmental stages. Plotted data points represent mean FRET/eCFP ratios of the intestine of individual animals. Shown for each developmental stage is the population FRET/eCFP ratio mean and standard deviation values. (B) Intestinal FRET/eCFP ratio of a representative sample is depicted as a ratio image of FRET/eCFP. Mean FRET/eCFP values of 8 individual animals are plotted in the graph after imaging the same region of the intestine sequentially under 10x and 40x magnification to determine the effect of different image capture settings on the FRET/eCFP ratios. The difference between the mean FRET/eCFP ratios was used as an additive correction factor for all subsequent experiments carried out using the 40x objective. (C) Differential interference contrast micrographs and FRET/eCFP ratio images showing variation in FRET ratios in the pharyngeal muscle, head neurons, tail neurons and intestine of a representative animal. (D) Mean FRET/eCFP ratio values are plotted in the graph as separate distributions for individual cell types of 16 animals. Each animal was given a numerical identifier. FRET Excitation-445 nm: Emission- 542/27 nm, eCFP Excitation- 445 nm: Emission 483/32 nm. Scale bar is 50 micron. P values were obtained from Student’s T test.</p

The FRET and eCFP ratio of cpFLIPPi-6.4m between intestinal cells of <i>C</i>. <i>elegans</i> is similar.

<p>(A) eCFP and FRET emission channels, respectively showing donor and non-corrected FRET emission after the donor is excited with the 445nm laser. Inset shows schematic representation of cpFLIPPi-6.4m tethered to the cytosolic face of the cell membrane. Binding of ligand (Pi) alters the orientation or distance between the donor and acceptor and decreases FRET. FRET Excitation-445 nm: Emission- 542/27 nm, eCFP Excitation- 445 nm: Emission 483/32 nm. Scale bar is 100 microns. (B) Pseudo-colored FRET ratio image was created by dividing FRET pixel intensity values by eCFP pixel intensity values. Color bar shows the range of FRET ratios in the intestine. (C) Plot of non-corrected FRET and eCFP mean intensity values obtained from arbitrary user defines ROIs. The red slope line was calculated as a linear regression (least-squares method) of the data points. Slope of the graph represents mean FRET/eCFP ratio of the intestine of a single animal. (D) The upper panel shows eCFP (top) and FRET (bottom) emission images of a 24 hr adult hermaphrodite with differential intestinal cpFLIPPi-6.4m expression. Scale bar is 100 microns. The red boxes between the images show the regions of interest (ROI) used to quantify the eCFP and uncorrected FRET emission intensities. The ROIs were superimposed on the intestinal images and used to calculate the average pixel intensity. The lower panel shows the eCFP intensities (in arbitrary units) plotted against the uncorrected FRET emission intensities for the animal in the upper panel. The red slope line was calculated as a linear regression (least-squares method) of the data points.</p

Cyanide causes expulsion of intestinal Pi.

<p>Temporal profile of Pi expulsion from the intestine after cyanide treatment. Protein solution in the silicone well was measured at 1 min intervals up to 5 min after treating the suspended worms with 10 mM sodium cyanide. The maximum decrease in FRET/eCFP ratio is observed after 2 min then remains stable. The control group was treated with extra sensor protein solution instead of 10 mM sodium cyanide. Data shows mean and standard error values of measurements taken from three individual wells.</p

Environmental Risks of Nano Zerovalent Iron for Arsenate Remediation: Impacts on Cytosolic Levels of Inorganic Phosphate and MgATP^2– in <i>Arabidopsis thaliana</i>

The use of nano zerovalent iron (nZVI) for arsenate (As­(V)) remediation has proven effective, but full-scale injection of nZVI into the subsurface has aroused serious concerns for associated environmental risks. This study evaluated the efficacy of nZVI treatment for arsenate remediation and its potential hazards to plants using Arabidopsis thaliana grown in a hydroponic system. Biosensors for inorganic phosphate (Pi) and MgATP^2– were used to monitor <i>in vivo</i> Pi and MgATP^2– levels in plant cells. The results showed that nZVI could remove As­(V) from growth media, decrease As uptake by plants, and mitigate As­(V) toxicity to plants. However, excess nZVI could cause Pi starvation in plants leading to detrimental effects on plant growth. Due to the competitive adsorption of As­(V) and Pi on nZVI, removing As­(V) via nZVI treatment at an upstream site could relieve downstream plants from As­(V) toxicity and Pi deprivation, in which case 100 mg/L of nZVI was the optimal dosage for remediation of As­(V) at a concentration around 16.13 mg/L