<i>In vivo</i> evolution of resistance to artesunate + mefloquine.

<p>Artesunate (ATN) and mefloquine (MF) were given together to <i>P. chabaudi</i>-infected mice over many generations. After twenty seven sub-inoculations under increasing ATN + MF exposure, the drug-resistant population was cloned. One of these clones, denoted AS-ATNMF1, was selected from subsequent studies.</p

Drug resistance lineage of <i>Plasmodium chabaudi</i> AS.

<p>Note: the Figure does not depict all drug-resistant clones within the AS lineage. Only those relevant to the present work are represented. Each drug used to select resistant clones is noted inside each arrow and the increasing colour tonality of the arrow represents an approximation of the increase in drug doses during the evolution of resistance. The length of each arrow depicts the approximate relative time for generating the resistance phenotype. The generation of pyrimethamine resistance was a result of a single-step selection, whilst for all other drugs the evolution of resistance resulted from prolonged exposures to small increments in drug concentrations over many generations. The genotypic differences between a particular parasite and its progenitor are noted in brackets. Asterisks depict the clones used in this work.</p

Typical Western blot result depicting MDR1 expression in the artesunate + mefloquine resistant clone AS-ATNMF1.

<p>Hybridization was carried out with α anti Pgh1 antibodies and α anti tubulin antibodies. AS-3CQ and AS-ATN were used as one-<i>mdr1</i> copy controls and AS-15MF was used as the two-<i>mdr1</i> copies control.</p

Oligonucleotide primers and TaqMan probes used to ascertain <i>mdr1</i> copy number and transcription levels.

<p><i>F</i>: forward; <i>R</i>: Reverse; <i>Tp</i>: TaqMan probe.</p

Differences in <i>mdr1</i> genomic DNA, cDNA and protein amounts between the different parasite clones.

<p>Columns represent mean of five independent experiments with corresponding Standard Error bars. Cases were differences in relative <i>mdr1</i> amounts between a particular parasite and its one copy comparator (AS-3CQ or AS-ATN) are statistically significant after Student's t-tests are represented by * (p≤0.01) or ** (0.05≥p≥0.01).</p

Relationships between genomic DNA, transcript and protein amounts of <i>mdr1</i> between the different parasite clones.

<p>Values were determined by dividing the mean N-fold expression between each molecular species.</p

Drug test results.

<p>Each line represents the evolution of parasitaemia in each parasite clone from day 4 post-inoculum (p.i) in the absence of treatment (a), under artesunate + meloquine treatment (b), under mefloquine treatment (c) or under artesunate treatment (d). Each data point is a mean of % parasitaemias ± SE resulting from reads of groups of two and five mice in the untreated and treated groups, respectively.</p