Highly Sensitive and Rapid Characterization of the Development of Synchronized Blood Stage Malaria Parasites Via Magneto-Optical Hemozoin Quantification.

The rotating-crystal magneto-optical diagnostic (RMOD) technique was developed as a sensitive and rapid platform for malaria diagnosis. Herein, we report a detailed in vivo assessment of the synchronized Plasmodium vinckei lentum strain blood-stage infections by the RMOD method and comparing the results to the unsynchronized Plasmodium yoelii 17X-NL (non-lethal) infections. Furthermore, we assess the hemozoin production and clearance dynamics in chloroquine-treated compared to untreated self-resolving infections by RMOD. The findings of the study suggest that the RMOD signal is directly proportional to the hemozoin content and closely follows the actual parasitemia level. The lack of long-term accumulation of hemozoin in peripheral blood implies a dynamic equilibrium between the hemozoin production rate of the parasites and the immune system's clearing mechanism. Using parasites with synchronous blood stage cycle, which resemble human malaria parasite infections with Plasmodium falciparum and Plasmodium vivax, we are demonstrating that the RMOD detects both hemozoin production and clearance rates with high sensitivity and temporal resolution. Thus, RMOD technique offers a quantitative tool to follow the maturation of the malaria parasites even on sub-cycle timescales

Secondary Structure Prediction of Protein Constructs Using Random Incremental Truncation and Vacuum-Ultraviolet CD Spectroscopy

A novel uracil-DNA degrading protein factor (termed UDE) was identified in Drosophila melanogaster with no significant structural and functional homology to other uracil-DNA binding or processing factors. Determination of the 3D structure of UDE is excepted to provide key information on the description of the molecular mechanism of action of UDE catalysis, as well as in general uracil-recognition and nuclease action. Towards this long-term aim, the random library ESPRIT technology was applied to the novel protein UDE to overcome problems in identifying soluble expressing constructs given the absence of precise information on domain content and arrangement. Nine constructs of UDE were chosen to decipher structural and functional relationships. Vacuum ultraviolet circular dichroism (VUVCD) spectroscopy was performed to define the secondary structure content and location within UDE and its truncated variants. The quantitative analysis demonstrated exclusive alpha-helical content for the full-length protein, which is preserved in the truncated constructs. Arrangement of alpha-helical bundles within the truncated protein segments suggested new domain boundaries which differ from the conserved motifs determined by sequence-based alignment of UDE homologues. Here we demonstrate that the combination of ESPRIT and VUVCD spectroscopy provides a new structural description of UDE and confirms that the truncated constructs are useful for further detailed functional studies

Uracil-Containing DNA in Drosophila: Stability, Stage-Specific Accumulation, and Developmental Involvement

Base-excision repair and control of nucleotide pools safe-guard against permanent uracil accumulation in DNA relying on two key enzymes: uracil–DNA glycosylase and dUTPase. Lack of the major uracil–DNA glycosylase UNG gene from the fruit fly genome and dUTPase from fruit fly larvae prompted the hypotheses that i) uracil may accumulate in Drosophila genomic DNA where it may be well tolerated, and ii) this accumulation may affect development. Here we show that i) Drosophila melanogaster tolerates high levels of uracil in DNA; ii) such DNA is correctly interpreted in cell culture and embryo; and iii) under physiological spatio-temporal control, DNA from fruit fly larvae, pupae, and imago contain greatly elevated levels of uracil (200–2,000 uracil/million bases, quantified using a novel real-time PCR–based assay). Uracil is accumulated in genomic DNA of larval tissues during larval development, whereas DNA from imaginal tissues contains much less uracil. Upon pupation and metamorphosis, uracil content in DNA is significantly decreased. We propose that the observed developmental pattern of uracil–DNA is due to the lack of the key repair enzyme UNG from the Drosophila genome together with down-regulation of dUTPase in larval tissues. In agreement, we show that dUTPase silencing increases the uracil content in DNA of imaginal tissues and induces strong lethality at the early pupal stages, indicating that tolerance of highly uracil-substituted DNA is also stage-specific. Silencing of dUTPase perturbs the physiological pattern of uracil–DNA accumulation in Drosophila and leads to a strongly lethal phenotype in early pupal stages. These findings suggest a novel role of uracil-containing DNA in Drosophila development and metamorphosis and present a novel example for developmental effects of dUTPase silencing in multicellular eukaryotes. Importantly, we also show lack of the UNG gene in all available genomes of other Holometabola insects, indicating a potentially general tolerance and developmental role of uracil–DNA in this evolutionary clade

Highly Sensitive and Rapid Characterization of the Development of Synchronized Blood Stage Malaria Parasites Via Magneto-Optical Hemozoin Quantification

The rotating-crystal magneto-optical diagnostic (RMOD) technique was developed as a sensitive and rapid platform for malaria diagnosis. Herein, we report a detailed in vivo assessment of the synchronized Plasmodium vinckei lentum strain blood-stage infections by the RMOD method and comparing the results to the unsynchronized Plasmodium yoelii 17X-NL (non-lethal) infections. Furthermore, we assess the hemozoin production and clearance dynamics in chloroquine-treated compared to untreated self-resolving infections by RMOD. The findings of the study suggest that the RMOD signal is directly proportional to the hemozoin content and closely follows the actual parasitemia level. The lack of long-term accumulation of hemozoin in peripheral blood implies a dynamic equilibrium between the hemozoin production rate of the parasites and the immune system’s clearing mechanism. Using parasites with synchronous blood stage cycle, which resemble human malaria parasite infections with Plasmodium falciparum and Plasmodium vivax, we are demonstrating that the RMOD detects both hemozoin production and clearance rates with high sensitivity and temporal resolution. Thus, RMOD technique offers a quantitative tool to follow the maturation of the malaria parasites even on sub-cycle timescales

The results of analytical gel filtration and the determination of the oligomeric status of each truncated fragments.

<p>The results of analytical gel filtration and the determination of the oligomeric status of each truncated fragments.</p

Alignment and scale-up of selected UDE truncated fragments.

<p><b>(A)</b> The restricted nine UDE truncated fragments from the identified protein clusters that were chosen for scale-up. Arrows show the expression compatible boundaries compared to the previously designated conserved motifs determined by the alignment of UDE homologues sequences. <b>(B)</b> The optimized expression of the nine UDE constructs in <i>E</i>. <i>coli</i> BL21 cells before (-) and after (+) IPTG induction. <b>(C)</b> Purification of recombinant UDE constructs by Ni²⁺-affinity chromathography. Gel slice images show the supernatant (termed as “Sup” on the figure) of cell lysis and the 300 mM imidazole elution (termed as “Elu” on the figure) fractions for each constructs. Note that the entire purification process can be followed on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.s001" target="_blank">S1 Fig</a> that shows the whole SDS-PAGE gels, not only the supernatant and imidazole elution samples.</p

Secondary structure content determination of UDE and its fragments using VUVCD and SELCON3 program.

<p><b>(A)</b> Vacuum-ultraviolet circular dichroism (Δε) spectra of the UDE protein and its nine truncated fragments measured over the wavelength region of λ = 170–255 nm. The spectra are sorted into two panels for better visibility and spectra of UDE (red) is shown in both panels for reference <b>(B)</b> Decomposition of the CD spectra of UDE and its selected fragments using six secondary structure components; regular/distorted α-helix (rH/dH), regular/distorted β-strand (rS/dS), turn (T), and disordered structure (D). Upper panel: CD spectrum of UDE as measured and as fitted using the six components [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref022" target="_blank">22</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref024" target="_blank">24</a>]. Spectra of the components are also plotted with magnitudes proportional to their ratios in the full-length protein. Lower Panel: Difference spectra corresponding to UDE-NA2 and NG3-NA3 together with the fittings based on the spectra of the six basic components.</p

Screening expression level and solubility of UDE truncation libraries.

<p><b>(A)</b> Size fractionation of UDE fragments generated by unidirectional truncation on agarose gel. In the lanes next to the DNA ladders is the vector with total length UDE gene at higher position while the empty vector is at a lower position. N1–N3 and C1–C3 marked samples show by the exonuclease III truncation generated UDE constructs. <b>(B)</b> Assessment of UDE sublibraries size and diversity by PCR screen. <b>(C)</b> Separation of purified protein fractions on Ni²⁺-NTA resin from N-terminal (upper panels) and C-terminal (bottom panels) libraries on SDS-PAGE.</p

Unidirectional truncation of UDE gene.

<p><b>(A)</b> The location of designed starting points along the UDE sequence indicating the predicted disordered segments and the conserved motifs. <b>(B)</b> The truncated UDE gene fragments generated by N-terminal truncation were fused in-frame with the biotin acceptor peptide and out-of-frame with hexahistidine tag, while fragments produced by C-terminal truncation were fused in-frame with hexahistidine tag and out-of-frame with BAP.</p