High-throughput analysis of the transcriptional patterns of sexual genes in malaria.

BACKGROUND: Plasmodium falciparum (Pf) is the leading protozoan causing malaria, the most devastating parasitic disease. To ensure transmission, a small subset of Pf parasites differentiate into the sexual forms (gametocytes). Since the abundance of these essential parasitic forms is extremely low within the human host, little is currently known about the molecular regulation of their sexual differentiation, highlighting the need to develop tools to investigate Pf gene expression during this fundamental mechanism. METHODS: We developed a high-throughput quantitative Reverse-Transcription PCR (RT-qPCR) platform to robustly monitor Pf transcriptional patterns, in particular, systematically profiling the transcriptional pattern of a large panel of gametocyte-related genes (GRG). Initially, we evaluated the technical performance of the systematic RT-qPCR platform to ensure it complies with the accepted quality standards for: (i) RNA extraction, (ii) cDNA synthesis and (iii) evaluation of gene expression through RT-qPCR. We then used this approach to monitor alterations in gene expression of a panel of GRG upon treatment with gametocytogenesis regulators. RESULTS: We thoroughly elucidated GRG expression profiles under treatment with the antimalarial drug dihydroartemisinin (DHA) or the metabolite choline over the course of a Pf blood cycle (48 h). We demonstrate that both significantly alter the expression pattern of PfAP2-G, the gametocytogenesis master regulator. However, they also markedly modify the developmental rate of the parasites and thus might bias the mRNA expression. Additionally, we screened the effect of the metabolites lactate and kynurenic acid, abundant in severe malaria, as potential regulators of gametocytogenesis. CONCLUSIONS: Our data demonstrate that the high-throughput RT-qPCR method enables studying the immediate transcriptional response initiating gametocytogenesis of the parasites from a very low volume of malaria-infected RBC samples. The obtained data expand the current knowledge of the initial alterations in mRNA profiles of GRG upon treatment with reported regulators. In addition, using this method emphasizes that asexual parasite stage composition is a crucial element that must be considered when interpreting changes in GRG expression by RT-qPCR, specifically when screening for novel compounds that could regulate Pf sexual differentiation.

Malaria parasites both repress host CXCL10 and use it as a cue for growth acceleration.

Pathogens are thought to use host molecular cues to control when to initiate life-cycle transitions, but these signals are mostly unknown, particularly for the parasitic disease malaria caused by Plasmodium falciparum. The chemokine CXCL10 is present at high levels in fatal cases of cerebral malaria patients, but is reduced in patients who survive and do not have complications. Here we show a Pf 'decision-sensing-system' controlled by CXCL10 concentration. High CXCL10 expression prompts P. falciparum to initiate a survival strategy via growth acceleration. Remarkably, P. falciparum inhibits CXCL10 synthesis in monocytes by disrupting the association of host ribosomes with CXCL10 transcripts. The underlying inhibition cascade involves RNA cargo delivery into monocytes that triggers RIG-I, which leads to HUR1 binding to an AU-rich domain of the CXCL10 3'UTR. These data indicate that when the parasite can no longer keep CXCL10 at low levels, it can exploit the chemokine as a cue to shift tactics and escape.

Bioproduction of 2-phenylethanol in a biphasic ionic liquid aqueous system.

2-Phenylethanol (PEA) is a commercial flavor and fragrance compound, with a rose-like odor, used in the cosmetics and food industries. Saccharomyces cerevisiae strains produce PEA in a growth-associated manner but are prone to product inhibition, resulting in low production yields. The aim of this study was to use immiscible ionic liquids (ILs) in a biphasic system to enhance the PEA concentration by means of in situ product removal (ISPR). Nine ILs were tested for their influence on growing yeast cells, and five of them were found to be biocompatible. A correlation between the IL structure and the effect on yeast growth was investigated. [Tf(2)N] anions were found to be the most biocompatible in comparison to [PF(6)] and [BF(4)], and the pyridinium and ammonium cations were slightly preferable than the imidazolium cation. Furthermore, the longer the alkyl side chain on the imidazolium ring, the less it is biocompatible, with major significance above six carbons. The five biocompatible ILs were tested for PEA recovery capability by determining their distribution coefficients (K(D)), with the highest value of 17.6 obtained for BMIM[Tf(2)N]. Finally, ILs were tested for their efficiency as ISPR solvents under stress conditions of a high product concentration. A 3-5-fold increase in the total PEA concentration produced by the cells was obtained with MPPyr[Tf(2)N], OMA[Tf(2)N], and BMIM[Tf(2)N], demonstrating the potential of ILs for enhancing productivity in bioprocesses using growing cells.

Improvement of natural isolates of Saccharomyces cerevisiae strains for synthesis of a chiral building block using classic genetics.

The asymmetric bio-reduction of 4-chloro-acetoacetic-acid-ethyl-ester to the pharmaceutical building block (S)-4-chloro-3-hydroxybutanoate-ethyl-ester requires the utilization of an enantioselective robust biocatalyst. Some of the natural Saccharomyces cerevisiae strains, isolated from Mount Carmel National Park in Israel, were characterized as resistant to environmental stress. Nevertheless, these strains showed relatively low enantiomeric-excess (ee), while a laboratory strain, Y103, exhibited a selectivity of 98% ee. The enantioselective lab strain was crossed with the multi-stress resistant environmental isolate (93% ee) followed by backcross with Y103, to subsequently obtain a haploid offspring of backcross-1, exhibiting both high multi-stress resistance and high enantioselectivity (98% ee). Introducing osmotic (1 M NaCl), oxidative (0.6 mM H(2)O(2)) and thermal stress (44 degrees C) to growing cultures of the enantioselective parent, resulted in a decrease of 24-32% in specific activity, while the enantioselectivity of the stress-resistant parent decreased by 4-12% ee. Unlike its original parental strains, the new strain maintained constant specific activity and enantioselectivity when introduced to the various stress factors. This work shows that the classic introgression method, can serve as a viable approach for creating a robust enantioselective biocatalyst, designed for industrial production of chiral compounds.