A combination of new screening assays for prioritization of transmission-blocking antimalarials reveals distinct dynamics of marketed and experimental drugs.

OBJECTIVES: The development of drugs to reduce malaria transmission is an important part of malaria eradication plans. We set out to develop and validate a combination of new screening assays for prioritization of transmission-blocking molecules. METHODS: We developed high-throughput assays for screening compounds against gametocytes, the parasite stages responsible for onward transmission to mosquitoes. An existing gametocyte parasitic lactate dehydrogenase (pLDH) assay was adapted for use in 384-well plates, and a novel homogeneous immunoassay to monitor the functional transition of female gametocytes into gametes was developed. A collection of 48 marketed and experimental antimalarials was screened and subsequently tested for impact on sporogony in Anopheles mosquitoes, to directly quantify the transmission-blocking properties of antimalarials in relation to their effects on gametocyte pLDH activity or gametogenesis. RESULTS AND CONCLUSIONS: The novel screening assays revealed distinct stage-specific kinetics and dynamics of drug effects. Peroxides showed the most potent transmission-blocking effects, with an intermediate speed of action and IC50 values that were 20-40-fold higher than the IC50s against the asexual stages causing clinical malaria. Finally, the novel synthetic peroxide OZ439 appeared to be a promising drug candidate as it exerted gametocytocidal and transmission-blocking effects at clinically relevant concentrations.

Least absolute regression network analysis of the murine osteoblast differentiation network.

MOTIVATION: We propose a reverse engineering scheme to discover genetic regulation from genome-wide transcription data that monitors the dynamic transcriptional response after a change in cellular environment. The interaction network is estimated by solving a linear model using simultaneous shrinking of the least absolute weights and the prediction error. RESULTS: The proposed scheme has been applied to the murine C2C12 cell-line stimulated to undergo osteoblast differentiation. Results show that our method discovers genetic interactions that display significant enrichment of co-citation in literature. More detailed study showed that the inferred network exhibits properties and hypotheses that are consistent with current biological knowledge.

Regulation of Notch signaling genes during BMP2-induced differentiation of osteoblast precursor cells.

The bone morphogenetic protein (BMP)-induced Smad signal transduction pathway is an important positive regulator of osteoblast differentiation. BMP and other members of the transforming growth factor-beta (TGF-beta) family have distinct effects on osteoblast differentiation, depending on cell type and cell differentiation status. In C2C12 mesenchymal cells, BMP-induced osteoblast differentiation can be blocked by TGF-beta. In a search for key regulators of osteoblast differentiation we have used microarray analysis to identify genes which are differentially regulated by BMP2 and TGF-beta. Within the first 24 h following the onset of differentiation, 61 BMP2-regulated genes were identified of which the BMP2 effect was counteracted by TGF-beta. The majority of these differentially expressed transcripts are related to signal transduction. Notably, our data show that three Notch signal transduction pathway genes, Lfng, Hey1, and Hes1, are differentially regulated by BMP2 and TGF-beta. This suggests that these genes might function as the focal point for interaction of Smad and Notch signaling during osteoblast differentiation.

Isolation and functional characterization of two distinct sexual-stage-specific promoters of the human malaria parasite Plasmodium falciparum.

Transmission of malaria depends on the successful development of the sexual stages of the parasite within the midgut of the mosquito vector. The differentiation process leading to the production of the sexual stages is delineated by several developmental switches. Arresting the progression through this sexual differentiation pathway would effectively block the spread of the disease. The successful development of such transmission-blocking agents is hampered by the lack of a detailed understanding of the program of gene expression that governs sexual differentiation of the parasite. Here we describe the isolation and functional characterization of the Plasmodium falciparum pfs16 and pfs25 promoters, whose activation marks the developmental switches executed during the sexual differentiation process. We have studied the differential activation of the pfs16 and pfs25 promoters during intraerythrocytic development by transfection of P. falciparum and during gametogenesis and early sporogonic development by transfection of the related malarial parasite P. gallinaceum. Our data indicate that the promoter of the pfs16 gene is activated at the onset of gametocytogenesis, while the activity of the pfs25 promoter is induced following the transition to the mosquito vector. Both promoters have unusual DNA compositions and are extremely A/T rich. We have identified the regions in the pfs16 and pfs25 promoters that are essential for high transcriptional activity. Furthermore, we have identified a DNA-binding protein, termed PAF-1, which activates pfs25 transcription in the mosquito midgut. The data presented here shed the first light on the details of processes of gene regulation in the important human pathogen P. falciparum.

Distinct frequency-distributions of homopolymeric DNA tracts in different genomes.

The unusual base composition of the genome of the human malaria parasite Plasmodium falciparum prompted us to systematically investigate the occurrence of homopolymeric DNA tracts in the P. falciparum genome and, for comparison, in the genomes of Homo sapiens , Saccharomyces cerevisiae , Caenorhabditis elegans , Arabidopsis thaliana , Escherichia coli and Mycobacterium tuberculosis. Comparison of theobserved frequencies with the frequencies as expected for random DNA revealed that homopolymeric (dA:dT) tracts occur well above chance in the eukaryotic genome. In the majority of these genomes, (dA:dT) tract overrepresentation proved to be an exponential function of the tract length. (dG:dC) tract overrepresentation was absent or less pronounced in both prokaryotic and eukaryotic genomes. On the basis of our results, we propose that homopolymeric (dA:dT) tracts are expanded via replication slippage. This slippage-mediated expansion does not operate on tracts with lengths below a critical threshold of 7-10 bp.

Developmentally regulated expression of pfs16, a marker for sexual differentiation of the human malaria parasite Plasmodium falciparum.

Sexual differentiation is essential for the transmission of Plasmodium to mosquitoes and therefore, for the spread of malaria. The molecular mechanisms underlying sexual differentiation are poorly understood but may be elucidated by a detailed study of the regulation of expression of sexual stage specific genes. In the present work we describe the differential expression of the gene encoding the sexual stage specific protein, Pfs16. We have conducted a comparative analysis of pfs16 promoter activity, RNA levels and the rate of de novo protein synthesis during development of Plasmodium falciparum. Furthermore, we have determined the pattern of expression of pfs16 transcripts at the single cell level by in situ hybridisation. We show that the expression of pfs16 is induced immediately following the invasion of a red blood cell in sexually committed ring stage parasites and continues throughout gametocytogenesis and in macrogametes. The expression of pfs16 is regulated at the level of transcription initiation and modulated by a post-transcriptional process. These results demonstrate that the expression of the pfs16 gene is the earliest event in the sexual differentiation process of P. falciparum described to date.

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression.

Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate. A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer. Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine. The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose. Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response. The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate.