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Background and Purpose Blood-stage Plasmodium parasites cause morbidity and mortality from malaria. Parasite resistance to drugs makes development of new chemotherapies an urgency. Aminoacyl-tRNA synthetases have been validated as antimalarial drug targets. We explored long-term effects of borrelidin and mupirocin in lethal P. yoelii murine malaria. Experimental Approach Long-term (up to 340 days) immunological responses to borrelidin or mupirocin were measured after an initial 4 day suppressive test. Prophylaxis and cure were evaluated and the inhibitory effect on the parasites analysed. Key Results Borrelidin protected against lethal malaria at 0.25 mg·kg−1·day−1. Antimalarial activity of borrelidin correlated with accumulation of trophozoites in peripheral blood. All infected mice treated with borrelidin survived and subsequently developed immunity protecting them from re-infection on further challenges, 75 and 340 days after the initial infection. This long-term immunity in borrelidin-treated mice resulted in negligible parasitaemia after re-infections and marked increases in total serum levels of antiparasite IgGs with augmented avidity. Long-term memory IgGs mainly reacted against high and low molecular weight parasite antigens. Immunofluorescence microscopy showed that circulating IgGs bound predominantly to late intracellular stage parasites, mainly schizonts. Conclusions and Implications Low borrelidin doses protected mice from lethal malaria infections and induced protective immune responses after treatment. Development of combination therapies with borrelidin and selective modifications of the borrelidin molecule to specifically inhibit plasmodial threonyl tRNA synthetase should improve therapeutic strategies for malaria.

Most drugs against malaria that are available or under development target a single process of the parasite infective cycle, favouring the appearance of resistant mutants which are easily spread in areas under chemotherapeutic treatments. Maslinic acid (MA) is a low toxic natural pentacyclic triterpene for which a wide variety of biological and therapeutic activities have been reported. Previous work revealed that Plasmodium falciparum erythrocytic cultures were inhibited by MA, which was able to hinder the maturation from ring to schizont stage and, as a consequence, prevent the release of merozoites and the subsequent invasion. We show here that MA effectively inhibits the proteolytic processing of the merozoite surface protein complex, probably by inhibition of PfSUB1. In addition, MA was also found to inhibit metalloproteases of the M16 family by a non-chelating mechanism, suggesting the possible hindrance of plasmodial metalloproteases belonging to that family, such as falcilysin and apicoplast peptide-processing proteases. Finally, in silico target screening was used to search for other potential binding targets that may have remained undetected. Among the targets identified, the method recovered two for which experimental activity could be confirmed, and suggested several putative new targets to which MA could have affinity. One of these unreported targets, phospholipase A2, was shown to be partially inhibited by MA. These results suggest that MA may behave as a multi-targeted drug against the intra-erythrocytic cycle of Plasmodium, providing a new tool to investigate the synergistic effect of inhibiting several unrelated processes with a single compound, a new concept in antimalarial research.

This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated polymerase chain reaction amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high-throughput analyses.

Nna1 has some sequence similarity to metallocarboxypeptidases, but the biochemical characterization of Nna1 has not previously been reported. In this work we performed a detailed genomic scan and found >100 Nna1 homologues in bacteria, Protista, and Animalia, including several paralogs in most eukaryotic species. Phylogenetic analysis of the Nna1-like sequences demonstrates a major divergence between Nna1-like peptidases and the previously known metallocarboxypeptidases subfamilies: M14A, M14B, and M14C. Conformational modeling of representative Nna1-like proteins from a variety of species indicates an unusually open active site, a property that might facilitate its action on a wide variety of peptide and protein substrates. To test this, we expressed a recombinant form of one of the Nna1-like peptidases from Caenorhabditis elegans and demonstrated that this protein is a fully functional metallocarboxypeptidase that cleaves a range of C-terminal amino acids from synthetic peptides. The enzymatic activity is activated by ATP/ADP and salt-inactivated, and is preferentially inhibited by Z-Glu-Tyr dipeptide, which is without precedent in metallocarboxypeptidases and resembles tubulin carboxypeptidase functioning; this hypothesis is strongly reinforced by the results depicted in Kalinina et al. published as accompanying paper in this journal. Our findings demonstrate that the M14 family of metallocarboxypeptidases is more complex and diverse than expected, and that Nna1-like peptidases are functional variants of such enzymes, representing a novel subfamily (we propose the name M14D) that contributes substantially to such diversity.

The bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (G6PD-6PGL) found in Plasmodium falciparum has unique structural and functional characteristics restricted to this genus. This study was designed to examine the effects of RNA-mediated PfG6PD-6PGL gene silencing in cultures of P. falciparum on the expression of parasite antioxidant defense genes at the transcription level. The highest degree of G6PD-6PGL silencing achieved was 86% at the mRNA level, with a recovery to almost normal levels within 24 h, indicating only transient diminished expression of the PfG6PD-6PGL gene. PfG6PD-6PGL silencing caused arrest of the trophozoite stage and enhanced gametocyte formation. In addition, an immediate transcriptional response was shown by thioredoxin reductase suggesting that P. falciparum G6PD-6PGL plays a physiological role in the specific response of the parasite to intracellullar oxidative stress. P. falciparum transfection with an empty DNA vector also promoted intracellular stress, as determined by mRNA up-regulation of antioxidant genes. Collectively, our findings point to an important role for this enzyme in the parasite's infection cycle. The different characteristics of G6PD-6PGL with respect to its homologue in the host make it an ideal target for therapeutic strategies.

Background In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. Methods We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. Results Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the ‘transcription index’ (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. Conclusions The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels. Copyright © 2006 John Wiley & Sons, Ltd.