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

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

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

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

Spatial distribution of the secondary structure components along UDE protein.

<p>The location of α-helical segments <b>(A)</b> in the full-length (355 aa) UDE protein and <b>(B)</b> in its nine truncated fragments were determined from the CD spectra and the amino acid sequence using a neural network algorithm [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref025" target="_blank">25</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156238#pone.0156238.ref026" target="_blank">26</a>]. The α-helical segments and β-strands are displayed in blue and red, respectively, while both turns and disordered parts appear in yellow. <b>(C)</b> Our final estimate for the secondary structure of UDE obtained as an average of the structure of UDE proposed in panel <b>(A)</b> and the structure of the fragments shown in panel <b>(B)</b> except for CA7. <b>(D)</b> The native structure of the N-terminal end of the full-length UDE was investigated using the evaluation of the CD spectrum of the N- terminal as the difference between the CD spectra of UDE and NA2 according to (Δε1UDE×N1UDE−Δε1NA2×N1NA2)/(N1UDE—N1NA2), where Δε is the molar ellipticity and N is the number of amino acids for UDE and NA2. The same subtraction method was performed with the highly overlapping fragments NG3 and NA3.</p

Using the hemozoin conversion rates reported in the literature for the different parasite stages (rings: 3–5%, trophozoites: 15–20%, and schizonts: 50–70%) [20]–[22], [29], [30] and the stage distribution of the parasites (Fig. 1), we estimated the hemozoin content of culture A (ring stage culture) and culture B (schizont stage culture).

<p>The lower and upper values of the hemozoin content correspond to the lower and upper values of the conversion rates quoted above. Note that the cultures have different parasite densities. We also estimated the hemozoin concentration of the two cultures based on MO signal using the conversion factor c_HZ = 1 ng/<i>µ</i>L →  = 1.4% between the hemozoin concentration and the low-frequency (∼1 Hz) MO signal previously determined for artificial hemozoin crystals suspended in blood <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096981#pone.0096981-Butykai1" target="_blank">[15]</a>.</p

Magneto-optical (MO) detection of parasitemia in synchronized <i>Plasmodium falciparum</i> cultures.

<p>Panel A: Red and blue curves show the frequency dependent MO signal for samples from the ring and schizont stage cultures, respectively, with various levels of parasite density given in <i>µ</i>L⁻¹ units on the right of the respective curves. The green curves shows the signal from uninfected reference samples. Data plotted with triangles and diamonds are the residual signal from freshly hemolyzed uninfected blood and water, respectively. The frequency scale corresponds to the rotation speed of the magnetic field. Panel B: Red and blue squares in panel B are the MO signal values measured at 20 Hz – indicated by a vertical solid line in panel A – for the dilution series prepared from the original ring and schizont stage cultures, respectively. Solid and open squares correspond to the duplicate samples labeled as samples #1 and samples #2. Triangles indicate the results obtained by remeasuring samples #1 with 24 h delay. The solid lines following the trend of the MO signal at higher parasite densities for ring (red line) and schizont (blue line) samples are guides for the eye. For ring and schizont stage samples with parasite densities lower than 10 parasites/<i>µ</i>L and 1 parasites/<i>µ</i>L, respectively, the MO signal does not further decrease. The green horizontal line shows the residual MO signal of uninfected blood, which is the mean detection limit of our method. The 95% confidence levels of this mean detection limit for the ring and schizont stage samples are indicated by red and blue dashed lines, respectively. Correspondingly, for ring and schizont stage samples with parasite density higher than 40 parasites/<i>µ</i>L and 10 parasites/<i>µ</i>L, respectively, the diagnosis is positive with a confidence of at least 95%. The background signal for freshly hemolyzed uninfected blood and water are also shown by dark and light grey lines. All these horizontal indicators are also shown in panel A for reference. The upper horizontal scale shows the corresponding levels of parasitemia.</p

Distribution of parasite life cycle stages in the two <i>Plasmodium falciparum</i> cultures used in the present study.

<p>Panel A: The <i>ring stage culture</i> contained early rings, late rings and some early trophozoites of the first generation after synchronization. The <i>schizont stage culture</i> was on the verge of the first and second life cycles where most of the schizont stages have already turned to early ring stages of the second generation following invasion. Therefore, the ring stage culture contained only the hemozoin present in the parasites up to the early trophozoite stage, while the schizont stage culture had the entire hemozoin content formed during one generation of parasites with the largest portion produced by schizonts. Panel B: Light microscopy images of Giemsa stained thin blood films containing infected red blood cells with parasites in different stages of maturity (taken from these two cultures). In both panels the labels ER, LR, ET, LT, ES and LS correspond to early-ring, late-ring, early-trophozoite, late-trophozoite, early-schizont and late-schizont stages, respectively.</p