DNA Aptamer Confirmation and Utilization for the Cyanotoxin, Cylindrospermopsin

Cyanotoxins are posing an increasing threat to the health of humans and wildlife. Cylindrospermopsin is a cyanotoxin that occurs in warm climates and is harmful when ingested. The toxic effects of CYN can affect multiple organ systems. The effects, coupled with the evidence of a mass contamination of a water supply in Australia, prove that CYN needs to be investigated further. Aptamers have become a desirable method for detection of CYN as a result of an aptamer’s high specificity and the ability to scale up experiments. Aptamers have been designed to bind with a variety of targets, including cyanotoxins. An aptamer for CYN was identified by Elshafey et al. This study aims to confirm the binding of the aptamer to CYN and the selectivity of the aptamer using fluorescent biosensing and circular dichroism. Aptamer affinity capture was used to investigate the possibility of a real world application of the aptamer

D. pulex.

<p><i>D</i>. <i>pulex</i> were exposed to ethylene blue for 15 min, rinsed thoroughly, and imaged using a Maximum Intensity Projection Z Plane Stack (10x, Leica SP5 Scanning Laser Confocal Microscope). The intensity image presented here was used as proof of concept during the characterization of ethylene blue.</p

<i>D</i>. <i>pulex</i> survival with vitamin D₃.

<p><i>D</i>. <i>pulex</i> (N = 12) exposed to acute UV-A in the presence of vitamin D₃. Blue line = 0 mg D₃; Black line = 5 mg D₃; Red line = 10 mg D₃. No reproduction was observed in any individuals during the experimental period. Error = standard error of the mean across 3 trials.</p

Synthetic route for the preparation of fluorescently labeled vitamin D₃.

<p>The carbodiimidazole (CDI) coupling of vitamin D₃ and the fluorescent dye linker.</p

D. pulex.

<p><i>D</i>. <i>pulex</i> were exposed to ethylene blue for 15 min, rinsed thoroughly, and imaged using a Maximum Intensity Projection Z Plane Stack (10x, Leica SP5 Scanning Laser Confocal Microscope). The intensity image presented here was used as proof of concept during the characterization of ethylene blue.</p

UV-Stressed <i>Daphnia pulex</i> Increase Fitness through Uptake of Vitamin D₃

<div><p>Ultraviolet radiation is known to be highly variable in aquatic ecosystems. It has been suggested that UV-exposed organisms may demonstrate enough phenotypic plasticity to maintain the relative fitness of natural populations. Our long-term objective is to determine the potential photoprotective effect of vitamin D₃ on <i>Daphnia pulex</i> exposed to acute or chronic UV radiation. Herein we report our initial findings in this endeavor. <i>D</i>. <i>pulex</i> survival and reproduction (fitness) was monitored for 5 d as a proof of concept study. Significantly higher fitness was observed in the <i>D</i>. <i>pulex</i> with D₃ than those without (most extreme effects observed were 0% survival in the absence of D₃ and 100% with 10 ppm D₃). Vitamin D₃ was isolated from the culture media, the algal food (<i>Pseudokirchneriella</i>), and the <i>D</i>. <i>pulex</i> and quantified using high performance liquid chromatography (HPLC). Vitamin D₃ was fluorescently labeled using a phenothiazinium dye and added to cultures of <i>D</i>. <i>pulex</i>. Images demonstrating the uptake of D₃ into the tissues and carapace of the <i>D</i>. <i>pulex</i> were acquired. Our initial findings suggest a positive role for D₃ in ecosystems as both UV-stressed algae and <i>Daphnia</i> sequester D₃, and <i>D</i>. <i>pulex</i> demonstrate increased fitness in the presence of D₃.</p></div

Tracking vitamin D₃ in <i>D</i>. <i>pulex</i>.

<p>(A) A live <i>D</i>. <i>pulex</i> was placed in a 1:10 solution of ethylene blue, with no vitamin D₃ (“control”) and images were captured 30 min post exposure (10x, Leica SP5 Scanning Laser Confocal Microscope). Note the presence of the dye in the gut tract (red stain). (B). Live <i>D</i>. <i>pulex</i> were placed in a 1:10 solution of ethylene blue linked vitamin D₃, rinsed thoroughly, and images were captured 120 min post exposure (10x). Image was compiled from a 90-min time lapse to capture sequestration of vitamin D₃ (red) into the tissues of the <i>D</i>. <i>pulex</i> from the intestine. The high intensity red “dots” in the image are concentrated dye on the outside of the carapace that was not removed during the rinsing stages. The yellow circle indicates a region where D₃ sequestration was detected.</p