Comparative transcriptional chase analyses of a short-lived study mRNA.

<p>tTA-expressing HeLa cells were engineered to stably express a short-lived derivative human β-globin mRNA (β^ARE). Experiments were conducted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040827#pone-0040827-g003" target="_blank">Fig. 3</a>, including the calculation of mRNA t_1/2 values. (A) Aggregate analysis using the conventional method. Cell aliquots were amended with tet at t₀, and sacrificed at 30-min intervals. Points represent the mean ± S.D. from three replicate experiments. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with tet at 5-min intervals and sacrificed simultaneously after 120 min. Points represent the mean ± S.D. of three independent experiments. (C, D) Analyses of individual replicates. Curves illustrate results from individual replicates that were analyzed in aggregate in panels A and B. Data are presented as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040827#pone-0040827-g003" target="_blank">Fig. 3</a>. (C) Conventional method. (D) Reverse-chase method.</p

Values used in the current report.

<p>Values used in the current report.</p

Experimental schemata for transcriptional chase analyses of mRNA stability.

<p>(A) Conventional chase method. Identical aliquots cultured in doxycycline (dox)-supplemented media (thick line) are sacrificed at defined intervals (arrowheads). A hypothetical 80-hr chase experiment is illustrated. (B) Reverse-chase method. Identical aliquots cultured in dox-free media (thin line) are amended with dox at defined intervals, and sacrificed simultaneously at the conclusion of the experiment.</p

HeLa cell expansion under transcriptional chase conditions.

<p>Data from dox-supplementation experiments in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040827#pone-0040827-g002" target="_blank">Fig. 2</a> (1 µg/mL) was regressed to an exponential function, and expansion factors defined [j = 0.0224 (h) or 0.0004 (min)].</p

Effects of cell doubling time on the apparent t_1/2 value of a study mRNA.

<p>(A) Hypothetical mRNA decay. Cell aliquots, each originally containing 1.0×10⁶ cells, are incubated for defined intervals between 0 and 24 hours. Cells in each aliquot double every 6 hours, in parallel with the total amount of β-actin mRNA. At T = 0, each aliquot contains an equal quantity [1 arbitrary unit (au)] of an infinitely stable β-globin mRNA, encoded by a gene that has been transcriptionally silenced. The decline in the ratio of the globin:actin mRNAs, which erroneously indicates a half-life value of 6 hours for the globin mRNA, fails to account for interval expansion in cell number. These same principles apply to mRNAs with finite stabilities (panel B). (B) True and uncorrected t_1/2 values in cells with different doubling times. Curves illustrate the uncorrected half-life for a test mRNA, following transcriptional silencing of its encoding gene, if the interval expansion in cell number is not considered. The examples utilize an mRNA with a true t_1/2 = 6 h, expressed in cells that are growth arrested (curve <i>a</i>) or display doubling times of 24, 8, 6, or 4 hours (curves <i>b</i> through <i>e</i>, respectively).</p

Comparative transcriptional chase analyses of a long-lived test mRNA.

<p>(A) Aggregate analysis using the conventional method. Cell aliquots were amended with dox at t₀, and sacrificed at defined intervals. Levels of β^WT mRNA were determined by RT-qPCR relative to control dox-indifferent β-actin mRNA, using the ΔΔCt method. Normalized RT-qPCR values for β^WT mRNA were corrected for aliquot-specific cell numbers, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve. (B) Aggregate analysis using the reverse-chase method. Cell aliquots were amended with dox at defined intervals and sacrificed simultaneously at t = 80 h. Normalized values for β^WT mRNA were determined by RT-qPCR, then plotted. Points represent the mean ± S.D. from three replicate experiments. A t_1/2 value was calculated from the exponential decay constant corresponding to the best-fit curve, corrected for an expansion factor describing the growth rate of cultured HeLa cells. (C) Analyses of individual replicates using the conventional method. Normalized values for each of three biological replicates reported in panel A were corrected for the number of cells present in each aliquot at the time of sacrifice, and t_1/2 values calculated. (D) Analyses of individual replicates using the reverse-chase method. Normalized values for each of three biological replicates reported in panel B were directly plotted, and t_1/2 values calculated following correction for interval cell expansion.</p

HeLa cell growth is not affected by doxycycline supplement.

<p>Values are the mean of three independent cultures; error bars indicate ±1 S.E.</p

Prolonging the shelf life of <i>Lumbricus terrestris</i> erythrocruorin for use as a novel blood substitute

Limitations associated with the storage of red blood cells have motivated the development of novel blood substitutes that are able to withstand long-term storage at elevated temperatures. The hemoglobin of the earthworm Lumbricus terrestris (LtEc) is an attractive blood substitute candidate, since it is resistant to oxidation and aggregation during storage. Several factors were investigated to optimize the thermal and oxidative stability of LtEc during storage, including pH, antioxidant supplements, and deoxygenation. A strategy for the reduction of fully oxidized LtEc with antioxidants was also developed. Overall, LtEc was shown to have the highest thermal stability in Ringer’s Modified Lactate solution with 10 mM HEPES at pH 7.0. Deoxygenation of the LtEc was also shown to significantly reduce oxidation of the ferrous heme iron (e.g., þ2+ after 7 d at 37 °C = 75.7%). However, even in cases where oxidation does occur, the addition of 1.8 mM ascorbic acid (AA) was found to reduce 98.3% of the oxidized LtEc (37 μM heme). Most importantly, the oxygen transport properties of LtEc were unaffected by storage at high temperatures or oxidation followed by reduction with AA. These results show that LtEc can be stored at high temperatures (37 °C) without any significant loss of function.</p

Increasing the stability of <i>Lumbricus terrestris</i> erythrocruorin <i>via</i> poly(acrylic acid) conjugation

<p>Since donated red blood cells must be constantly refrigerated, they are often unavailable in remote areas and battlefields. The goal of this study was to synthesize a highly stable blood substitute that does not require refrigeration. Specifically, the extracellular haemoglobin (a.k.a. erythrocruorin, Ec) of the earthworm <i>Lumbricus terrestris</i> erythrocruororin (LtEc) was cross-linked with poly(acrylic acid) (PAA) and ethylene diamine (EDA). PAGE analysis of the LtEc nanoparticles reveals cross-linking between subunits, while dynamic light scattering and scanning electron microscopy show that cross-linking significantly increases the size of the LtEc nanoparticles (164 ± 13.9 nm). Cross-linking also significantly increased the thermal stability of the LtEc nanoparticles by 10 °C (<i>T</i>_m = 72 ± 0.84 °C) relative to native LtEc (<i>T</i>_m = 62 ± 0.6 °C). In addition, while native LtEc rapidly dissociates at pH 9, the LtEc nanoparticles resist subunit dissociation up to pH 10. The oxygen affinity of the LtEc nanoparticles (P₅₀ = 6.85 ± 0.13 mm Hg) is much higher than native LtEc (P₅₀ = 26.67 ± 0.4 mm Hg), but the cooperativity (<i>n</i> = 2.43 ± 0.12) is not affected. Altogether, these results show that cross-linking LtEc with PAA and EDA provides a potential blood substitute with increased stability and oxygen affinity.</p

Presentation1_Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling.pdf

In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.</p