Plasma interleukin responses as predictors of outcome stratification in patients after major trauma: a prospective observational two centre study.

Background and objectives: There is a need to develop objective risk stratification tools to define efficient care pathways for trauma patients. Biomarker-based point of care testing may strengthen existing clinical tools currently available for this purpose. The dysregulation of pro- and anti-inflammatory cytokines in the pathogenesis of organ failure is well recognised. This study was carried out to evaluate whether blood concentrations of IL-6, IL-10, and IL-6:IL-10 ratios in the early stages of the illness are significantly different in patients with worsening organ function. Materials and methods: In this prospective observational cohort study, plasma concentrations of IL-6 and IL-10 on days 1, 3 and 5 were measured in 91 major trauma patients using a multiplexed cytometric bead array approach. A composite measure of adverse outcome - defined as SOFA ≥ 2 or mortality at 7 days, was the primary outcome. IL-6 and IL-10 concentrations in early samples (days 1, 3 & 5) in patients who developed SOFA ≥ 2 on day 7 were compared against those who did not. Similar composite outcome groups at day 5 and in groups with worsening or improving SOFA scores (ΔSOFA) at days 7 and 5 were undertaken as secondary analyses. Results: Stratification on day 7, 44 (48%) patients showed adverse outcomes. These adverse outcomes associated with significantly greater IL-6 concentrations on days 1 and 5 (Day 1: 47.65 [23.24-78.68] Vs 73.69 [39.93 - 118.07] pg/mL, P = 0.040 and Day 5: 12.85 [5.80-19.51] Vs 28.90 [8.78-74.08] pg/mL; P = 0.0019). Similarly, IL-10 levels were significantly greater in the adverse outcome group on days 3 and 5 (Day 3: 2.54 [1.76-3.19] Vs 3.16 [2.68-4.21] pg/mL; P = 0.044 and Day 5: 2.03 [1.65-2.55] Vs 2.90 [2.00-5.06] pg/mL; P <0.001). IL-6 and IL-10 concentrations were also significantly elevated in the adverse outcome groups at day 3 and day 5 when stratified on day 5 outcomes. Both IL-6 and IL-6:IL-10 were found to be significantly elevated on days 1 and 3 when stratified based on ΔSOFA at day 5. This significance was lost when stratified on day 7 scores. Conclusions: Early IL-6 and IL-10 concentrations are significantly greater in patients who develop worsening organ functions downstream. These differences may provide an alternate biomarker-based approach to strengthen risk stratification in trauma patients.

Evaluation of 4-Aminoquinoline Hydrazone Analogues as Potential Leads for Drug-Resistant Malaria.

The emergence of resistance to first-line antimalarial drugs calls for the development of new therapies for drug-resistant malaria. The efficacy of quinoline-based antimalarial drugs has prompted the development of novel quinolines. A panel of 4-aminoquinoline hydrazone analogues were tested on the multidrug-resistant K1 strain of Plasmodium falciparum: IC50 values after a 48 h cycle ranged from 0.60 to 49 µM, while the 72 h cycle ranged from 0.026 to 0.219 µM. Time-course assays were carried out to define the activity of the lead compounds, which inhibited over 50% growth in 24 h and 90% growth in 72 h. Cytotoxicity assays with HepG2 cells showed IC50 values of 0.87-11.1 µM, whereas in MDBK cells, IC50 values ranged from 1.66 to 11.7 µM. High selectivity indices were observed for the lead compounds screened at 72 h on P. falciparum. Analyses of stage specificity revealed that the ring stages of the parasite life cycle were most affected. Based on antimalarial efficacy and in vitro safety profiles, lead compound 4-(2-benzylidenehydrazinyl)-6-methoxy-2-methylquinoline 2 was progressed to drug combination studies for the detection of synergism, with a combinatory index of 0.599 at IC90 for the combination with artemether, indicating a synergistic antimalarial activity. Compound 2 was screened on different strains of P. falciparum (3D7, Dd2), which maintained similar activity to K1, suggesting no cross-resistance between multidrug resistance and sensitive parasite strains. In vivo analysis with 2 showed the suppression of parasitaemia with P. yoelii NL (non-lethal)-treated mice (20 mg/kg and 5 mg/kg).

Selected Derivatives of Erythromycin B-In Silico and Anti-Malarial Studies.

Erythromycin A is an established anti-bacterial agent against Gram-positive bacteria, but it is unstable to acid. This led to an evaluation of erythromycin B and its derivatives because these have improved acid stability. These compounds were investigated for their anti-malarial activities, by their in silico molecular docking into segments of the exit tunnel of the apicoplast ribosome from Plasmodium falciparum. This is believed to be the target of the erythromycin A derivative, azithromycin, which has mild anti-malarial activity. The erythromycin B derivatives were evaluated on the multi-drug (chloroquine, pyrimethamine, and sulfadoxine)-resistant strain K1 of P. falciparum for asexual growth inhibition on asynchronous culture. The erythromycin B derivatives were identified as active in vitro inhibitors of asexual growth of P. falciparum with low micro-molar IC50 values after a 72 h cycle. 5-Desosaminyl erythronolide B ethyl succinate showed low IC50 of 68.6 µM, d-erythromycin B 86.8 µM, and erythromycin B 9-oxime 146.0 µM on the multi-drug-resistant K1 of P. falciparum. Based on the molecular docking, it seems that a small number of favourable interactions or the presence of unfavourable interactions of investigated derivatives of erythromycin B with in silico constructed segment from the exit tunnel from the apicoplast of P. falciparum is the reason for their weak in vitro anti-malarial activities.

Improved biopharmaceutical attributes of lumefantrine using choline mimicking drug delivery system: preclinical investigation on NK-65 P.berghei murine model.

BACKGROUND: Lumefantrine (LMF) is first-line antimalarial drug, possesses activity against almost all human malarial parasites, but the in vivo activity of this molecule gets thwarted due to its low and inconsistent oral bioavailability (i.e. 4-12%) owing to poor biopharmaceutical attributes. METHODS: Lumefantrine phospholipid complex (LMF-PC) was prepared by rota-evaporation method following job's plot technique for the selection of apt stoichiometric ratios. Docking studies were carried out to determine the possible interaction(s) of LMF with phosphatidylcholine analogue. Comparative in vitro physiochemical, solid-state characterization, MTT assay, dose-response on P. falciparum, in vivo efficacy studies including pharmacokinetic and chemosuppression on NK-65 P. berghei infected mice were carried out. RESULTS: Aqueous solubility was distinctly improved (i.e. 345 times) with phospholipid complex of LMF. Cytotoxicity studies on Hela and fibroblast cell lines demonstrated safety of LMF-PC with selectivity indices of 4395 and 5139, respectively. IC50 value was reduced almost 2.5 folds. Significant enhancement in Cmax (3.3-folds) and AUC (2.7-folds) of rat plasma levels indicated notable pharmacokinetic superiority of LMF-PC over LMF suspension. Differential leukocytic count and cytokine assay delineated plausible immunoregulatory role of LMF-PC with nearly 98% chemosuppression and over 30 days of post-survival. CONCLUSION: Superior antimalarial efficacy and survival time with full recovery of infected mice revealed through histopathological studies.

Lead Optimization of Dehydroemetine for Repositioned Use in Malaria.

Drug repositioning offers an effective alternative to de novo drug design to tackle the urgent need for novel antimalarial treatments. The antiamoebic compound emetine dihydrochloride has been identified as a potent in vitro inhibitor of the multidrug-resistant strain K1 of Plasmodium falciparum (50% inhibitory concentration [IC50], 47 nM ± 2.1 nM [mean ± standard deviation]). Dehydroemetine, a synthetic analogue of emetine dihydrochloride, has been reported to have less-cardiotoxic effects than emetine. The structures of two diastereomers of dehydroemetine were modeled on the published emetine binding site on the cryo-electron microscopy (cryo-EM) structure with PDB code 3J7A (P. falciparum 80S ribosome in complex with emetine), and it was found that (-)-R,S-dehydroemetine mimicked the bound pose of emetine more closely than did (-)-S,S-dehydroisoemetine. (-)-R,S-dehydroemetine (IC50 71.03 ± 6.1 nM) was also found to be highly potent against the multidrug-resistant K1 strain of P. falciparum compared with (-)-S,S-dehydroisoemetine (IC50, 2.07 ± 0.26 µM), which loses its potency due to the change of configuration at C-1'. In addition to its effect on the asexual erythrocytic stages of P. falciparum, the compound exhibited gametocidal properties with no cross-resistance against any of the multidrug-resistant strains tested. Drug interaction studies showed (-)-R,S-dehydroemetine to have synergistic antimalarial activity with atovaquone and proguanil. Emetine dihydrochloride and (-)-R,S-dehydroemetine failed to show any inhibition of the hERG potassium channel and displayed activity affecting the mitochondrial membrane potential, indicating a possible multimodal mechanism of action.

Investigating antimalarial drug interactions of emetine dihydrochloride hydrate using CalcuSyn-based interactivity calculations.

The widespread introduction of artemisinin-based combination therapy has contributed to recent reductions in malaria mortality. Combination therapies have a range of advantages, including synergism, toxicity reduction, and delaying the onset of resistance acquisition. Unfortunately, antimalarial combination therapy is limited by the depleting repertoire of effective drugs with distinct target pathways. To fast-track antimalarial drug discovery, we have previously employed drug-repositioning to identify the anti-amoebic drug, emetine dihydrochloride hydrate, as a potential candidate for repositioned use against malaria. Despite its 1000-fold increase in in vitro antimalarial potency (ED50 47 nM) compared with its anti-amoebic potency (ED50 26-32 uM), practical use of the compound has been limited by dose-dependent toxicity (emesis and cardiotoxicity). Identification of a synergistic partner drug would present an opportunity for dose-reduction, thus increasing the therapeutic window. The lack of reliable and standardised methodology to enable the in vitro definition of synergistic potential for antimalarials is a major drawback. Here we use isobologram and combination-index data generated by CalcuSyn software analyses (Biosoft v2.1) to define drug interactivity in an objective, automated manner. The method, based on the median effect principle proposed by Chou and Talalay, was initially validated for antimalarial application using the known synergistic combination (atovaquone-proguanil). The combination was used to further understand the relationship between SYBR Green viability and cytocidal versus cytostatic effects of drugs at higher levels of inhibition. We report here the use of the optimised Chou Talalay method to define synergistic antimalarial drug interactivity between emetine dihydrochloride hydrate and atovaquone. The novel findings present a potential route to harness the nanomolar antimalarial efficacy of this affordable natural product.

Bio-inspired artemether-loaded human serum albumin nanoparticles for effective control of malaria-infected erythrocytes.

AIM: The intra-erythrocytic development of the malarial parasite is dependent on active uptake of nutrients, including human serum albumin (HSA), into parasitized red blood cells (pRBCs). We have designed HSA-based nanoparticles as a potential drug-delivery option for antimalarials. METHODS: Artemether-loaded nanoparticles (AANs) were designed and antimalarial activity evaluated in vitro/in vivo using Plasmodium falciparum/Plasmodium berghei species, respectively. RESULTS: Selective internalization of AAN into Plasmodium-infected RBCs in preference to healthy erythrocytes was observed using confocal imaging. In vitro studies showed 50% dose reduction for AAN as compared with drug-only controls to achieve IC50 levels of inhibition. The nanoparticles exhibited twofold higher peak drug concentrations in RBCs with antimalarial activity at 50% of therapeutic doses in P. bergei infected mice. CONCLUSION: Novel HSA-based nanoparticles offer safe and effective approach for selective targeting of antimalarial drugs.

"Omics"-Informed Drug and Biomarker Discovery: Opportunities, Challenges and Future Perspectives.

The pharmaceutical industry faces unsustainable program failure despite significant increases in investment. Dwindling discovery pipelines, rapidly expanding R&D budgets and increasing regulatory control, predict significant gaps in the future drug markets. The cumulative duration of discovery from concept to commercialisation is unacceptably lengthy, and adds to the deepening crisis. Existing animal models predicting clinical translations are simplistic, highly reductionist and, therefore, not fit for purpose. The catastrophic consequences of ever-increasing attrition rates are most likely to be felt in the developing world, where resistance acquisition by killer diseases like malaria, tuberculosis and HIV have paced far ahead of new drug discovery. The coming of age of Omics-based applications makes available a formidable technological resource to further expand our knowledge of the complexities of human disease. The standardisation, analysis and comprehensive collation of the "data-heavy" outputs of these sciences are indeed challenging. A renewed focus on increasing reproducibility by understanding inherent biological, methodological, technical and analytical variables is crucial if reliable and useful inferences with potential for translation are to be achieved. The individual Omics sciences-genomics, transcriptomics, proteomics and metabolomics-have the singular advantage of being complimentary for cross validation, and together could potentially enable a much-needed systems biology perspective of the perturbations underlying disease processes. If current adverse trends are to be reversed, it is imperative that a shift in the R&D focus from speed to quality is achieved. In this review, we discuss the potential implications of recent Omics-based advances for the drug development process.

Drug repositioning as a route to anti-malarial drug discovery: preliminary investigation of the in vitro anti-malarial efficacy of emetine dihydrochloride hydrate.

BACKGROUND: Drug repurposing or repositioning refers to the usage of existing drugs in diseases other than those it was originally used for. For diseases like malaria, where there is an urgent need for active drug candidates, the strategy offers a route to significantly shorten the traditional drug development pipelines. Preliminary high-throughput screens on patent expired drug libraries have recently been carried out for Plasmodium falciparum. This study reports the systematic and objective further interrogation of selected compounds reported in these studies, to enable their repositioning as novel stand-alone anti-malarials or as combinatorial partners. METHODS: SYBR Green flow cytometry and micro-titre plate assays optimized in the laboratory were used to monitor drug susceptibility of in vitro cultures of P. falciparum K1 parasite strains. Previously described fixed-ratio methods were adopted to investigate drug interactions. RESULTS: Emetine dihydrochloride hydrate, an anti-protozoal drug previously used for intestinal and tissue amoebiasis was shown to have potent inhibitory properties (IC50 doses of ~ 47 nM) in the multidrug resistant K1 strain of P. falciparum. The sum 50% fractional inhibitory concentration (∑FIC50, 90) of the interaction of emetine dihydrochloride hydrate and dihydroartemisinin against the K1 strains of P. falciparum ranged from 0.88-1.48. CONCLUSION: The results warrant further investigation of emetine dihydrochloride hydrate as a potential stand-alone anti-malarial option. The interaction between the drug and the current front line dihydroartemisinin ranged from additive to mildly antagonistic in the fixed drug ratios tested.

Impact of pre-analytical factors on the proteomic analysis of formalin-fixed paraffin-embedded tissue.

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a tremendous potential resource for biomarker discovery, with large numbers of samples in hospital pathology departments and links to clinical information. However, the cross-linking of proteins and nucleic acids by formalin fixation has hampered analysis and proteomic studies have been restricted to using frozen tissue, which is more limited in availability as it needs to be collected specifically for research. This means that rare disease subtypes cannot be studied easily. Recently, improved extraction techniques have enabled analysis of FFPE tissue by a number of proteomic techniques. As with all clinical samples, pre-analytical factors are likely to impact on the results obtained, although overlooked in many studies. The aim of this review is to discuss the various pre-analytical factors, which include warm and cold ischaemic time, size of sample, fixation duration and temperature, tissue processing conditions, length of storage of archival tissue and storage conditions, and to review the studies that have considered these factors in more detail. In those areas where investigations are few or non-existent, illustrative examples of the possible importance of specific factors have been drawn from studies using frozen tissue or from immunohistochemical studies of FFPE tissue.

Initial development and validation of a novel extraction method for quantitative mining of the formalin-fixed, paraffin-embedded tissue proteome for biomarker investigations.

Annotated formalin-fixed, paraffin-embedded (FFPE) tissue archives constitute a valuable resource for retrospective biomarker discovery. However, proteomic exploration of archival tissue is impeded by extensive formalin-induced covalent cross-linking. Robust methodology enabling proteomic profiling of archival resources is urgently needed. Recent work is beginning to support the feasibility of biomarker discovery in archival tissues, but further developments in extraction methods which are compatible with quantitative approaches are urgently needed. We report a cost-effective extraction methodology permitting quantitative proteomic analyses of small amounts of FFPE tissue for biomarker investigation. This surfactant/heat-based approach results in effective and reproducible protein extraction in FFPE tissue blocks. In combination with a liquid chromatography-mass spectrometry-based label-free quantitative proteomics methodology, the protocol enables the robust representative and quantitative analyses of the archival proteome. Preliminary validation studies in renal cancer tissues have identified typically 250-300 proteins per 500 ng of tissue with 1D LC-MS/MS with comparable extraction in FFPE and fresh frozen tissue blocks and preservation of tumor/normal differential expression patterns (205 proteins, r = 0.682; p < 10(-15)). The initial methodology presented here provides a quantitative approach for assessing the potential suitability of the vast FFPE tissue archives as an alternate resource for biomarker discovery and will allow exploration of methods to increase depth of coverage and investigate the impact of preanalytical factors.

Development and validation of a novel protein extraction methodology for quantitation of protein expression in formalin-fixed paraffin-embedded tissues using western blotting.

The development of efficient formaldehyde cross-link reversal strategies will make the vast diagnostic tissue archives of pathology departments amenable to prospective and retrospective translational research, particularly in biomarker-driven proteomic investigations. Heat-induced antigen retrieval strategies (HIARs) have achieved varying degrees of cross-link reversal, potentially enabling archival tissue usage for proteomic applications outside its current remit of immunohistochemistry (IHC). While most successes achieved so far have been based on retrieving tryptic peptide fragments using shot-gun proteomic approaches, attempts at extracting full-length, non-degraded, immunoreactive proteins from archival tissue have proved challenging. We have developed a novel heat-induced antigen retrieval strategy using SDS-containing Laemmli buffer for efficient intact protein recovery from formalin-fixed tissues for subsequent analysis by western blotting. Protocol optimization and comparison of extraction efficacies with frozen tissues and current leader methodology is presented. Quantitative validation of methodology was carried out in a cohort of matched tumour/normal, frozen/FFPE renal tissue samples from 10 patients, probed by western blotting for a selected panel of seven proteins known to be differentially expressed in renal cancer. Our data show that the protocol enables efficient extraction of non-degraded, full-length, immunoreactive protein, with tumour versus normal differential expression profiles for a majority of the panel of proteins tested being comparable to matched frozen tissue controls (rank correlation, r = 0.7292, p < 1.825e-09). However, the variability observed in extraction efficacies for some membrane proteins emphasizes the need for cautious interpretation of quantitative data from this subset of proteins. The method provides a viable, cost-effective quantitative option for the validation of potential biomarker panels through a range of clinical samples from existing diagnostic archives, provided that validation of the method is first carried out for the specific proteins under study.

Mining the archival formalin-fixed paraffin-embedded tissue proteome: opportunities and challenges.

The significant potential of tissue-based proteomic biomarker studies can be restricted by difficulties in accessing samples in optimal fresh-frozen form. While archival formalin-fixed tissue collections with attached clinical and outcome data represent a valuable alternate resource, the use of formalin as a fixative which induces protein cross-linking, has generally been assumed to render them unsuitable for proteomic studies. However, this view has been challenged recently with the publication of several papers accomplishing variable degrees of heat-induced reversal of cross-links. Although still in its infancy and requiring the quantitative optimisation of several critical parameters, formalin-fixed tissue proteomics holds promise as a powerful tool for biomarker-driven translational research. Here, we critically review the current status of research in the field, highlighting challenges which need to be addressed for robust quantitative application of protocols to ensure confident high impact inferences can be made.

Microscale solution isoelectric focusing as an effective strategy enabling containment of hemeoglobin-derived products for high-resolution gel-based analysis of the Plasmodium falciparum proteome.

The high hemeozoin (beta-hemeatin) content of Plasmodium falciparum lysates imposes severe limitations on the analysis of the malarial proteome, in particular compromising the loading capacities of two-dimensional gels. Here we report on the adaptation of a recently developed solution-phase isoelectric focusing-based fractionation technique as a prefractionation strategy for efficient containment of hemeoglobin-derived products and complexity reduction, to facilitate the high-resolution gel-based quantitative analysis of plasmodial lysates.

Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum.

Antifolate drugs that target the biosynthesis and processing of essential folate cofactors are widely used for treatment of chloroquine-resistant falciparum malaria. Salvage of pre-formed folate can strongly compromise the efficacy of these drugs in vitro and the availability of folate from the human host in natural infections also influences therapeutic outcomes. To investigate how different parasite lines respond to the presence of exogenous folate, we measured the effect of the latter on the susceptibility of parasites to sulfa-drug blockage of folate biosynthesis, utilising the parents and 22 progeny of the HB3-Dd2 genetic cross of Plasmodium falciparum, together with selected unrelated lines. Complete linkage of the folate utilisation phenotype was observed to a DNA sequence of 48.6 kb lying between nucleotide positions 738,489 and 787,058 of chromosome 4 and encompassing the dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene locus. Examination of the putative ORFs on this fragment upstream (3) and downstream (4) of dhfr-ts revealed no plausible candidate genes for folate processing. Similarly, a marked heterogeneity in the 5'-UTR regions of Dd2 and HB3, manifest as a directly repeated 256 bp sequence in the former, also did not correlate with the folate utilisation phenotype nor apparently influence levels of dhfr-ts transcripts or protein products. By contrast, the nature of the coding sequence of the dhfr domain appeared to play a direct role, with the single mutant (S108N) HB3-type utilising folic acid much less efficiently than other allelic variants. We also compared the processing of exogenous folic acid, folinic acid and p-aminobenzoic acid (pABA) in metabolic labelling studies of HB3 and Dd2. These support the view that DHFR is likely to have a low-level folate reductase activity as well as its normal function of reducing dihydrofolate to tetrahydrofolate, and that a significant hurdle in the utilisation of exogenous folic acid is the initial reduction of fully oxidised folic acid to dihydrofolate, an activity that the single mutant enzyme found in HB3 is postulated to perform particularly poorly. This would mirror earlier studies indicating that the DHFR activity of HB3 is also compromised relative to other variants.

Translational up-regulation of antifolate drug targets in the human malaria parasite Plasmodium falciparum upon challenge with inhibitors.

The thymidylate cycle in Plasmodium falciparum is essential for cell growth and replication, and dihydrofolate reductase (DHFR), a key enzyme in this cycle, is the target of important antimalarial drugs such as pyrimethamine and cycloguanil. Following previous work, where we found no evidence of upregulation of the dhfr-ts gene upon challenge with pyrimethamine, we investigated the expression at the protein level of the bifunctional gene product, which also carries thymidylate synthase (TS) activity. Challenge of parasite cultures with fluoro-substituted bases that are specific TS inhibitors at levels close to the IC(50) resulted in five to seven-fold increases in enzyme level, as monitored by both DHFR and TS activities, while pyrimethamine and another DHFR-binding inhibitor, WR99210, induced smaller but still significant increases of approximately three-fold. However, when parasites were challenged with tetracycline, an antimalarial not directed at the folate pathway, although an increase was consistently seen above untreated controls, this was at a level of approximately 1.8-fold. These increases reflect enhanced synthesis of the DHFR-TS enzyme, rather than liberation of a latent activity, as they were completely abolished if cultures were pre-incubated with cycloheximide to block de novo protein synthesis. Moreover, none of the above antimalarial drugs was found to significantly alter absolute levels of the dhfr-ts mRNA under the conditions of challenge used. We conclude that, in common with mammalian systems, where a similar phenomenon has been reported, malaria parasites are able to significantly relieve translational constraint when faced with antifolate drug challenge. The data indicate that there is a specific component in addition to a low-level non-specific increment, and that binding to the TS domain of the DHFR-TS protein appears to be better able to relieve this constraint than binding to the DHFR domain.

Quantitative proteomics of the human malaria parasite Plasmodium falciparum and its application to studies of development and inhibition.

The ability to measure accurately comparative levels of protein expression after drug challenge, metabolic stress, developmental programming or other perturbation represents one of the most important goals in post-genomics malaria research. We describe here a simple and robust quantitative methodology that is ideally suited to in vitro experiments designed to study changes in the proteome of the most important of the human parasites, the lethal species Plasmodium falciparum. The metabolic labelling technique we have developed uses parasite uptake of heavy isotope-containing isoleucine during normal growth followed by two-dimensional separation of individual proteins and mass spectrometry. The method is applicable to essentially each of the approximately 5300 proteins of P. falciparum predicted from the completed genome sequence, permitting facile identification and accurate comparative quantification of labelled peptides from any of these proteins synthesized by in vitro cultures subjected to different stimuli. We demonstrate its application to the study of cell cycle changes, where we observe divergent patterns of protein and reported transcript levels indicative of modulation at the translational level. Our data also provide evidence for significant levels of post-translational modification in the parasite, and we measure differences among variants of phosphoethanolamine N-methyltransferase and actin-I across the cell cycle. We have also monitored parasite responses to equipotent doses of the clinical antimalarial inhibitors pyrimethamine and tetracycline and observed differential effects for a number of proteins unrelated to likely targets of these drugs.

Transcriptional analysis of genes encoding enzymes of the folate pathway in the human malaria parasite Plasmodium falciparum.

Folate metabolism in Plasmodium falciparum is essential for cell growth and replication, and the target of important antimalarial agents. The pathway comprises a series of enzymes that convert GTP to derivatives of tetrahydrofolate, which are cofactors in one-carbon transfer reactions. We investigated the expression of five of the genes encoding these enzymes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using a threshold detection technique. We followed changes in mRNA levels as parasites progress through the erythrocytic cell cycle and examined this process in two cloned lines of diverse origins, as well as under stress conditions, induced by either removal of important metabolites or challenge by folate enzyme inhibitors. Although conventionally regarded as performing housekeeping functions, these genes show disparate levels of and changes in expression through the cell cycle, but respond quite uniformly to folate pathway-specific stress factors, with no evidence of feedback at the transcriptional level. Overall, the two genes involved in the thymidylate cycle (encoding dihy-drofolate reductase-thymidylate synthase, dhfr-ts, and serine hydroxymethyltransferase, shmt) gave the most abundant transcripts. However, only the latter showed major variation across the cell cycle, with a peak around the time of onset of DNA replication, possibly indicative of a regulatory function.

The FAR proteins of filarial nematodes: secretion, glycosylation and lipid binding characteristics.

The FAR proteins of nematodes are small ( approximately 20 kDa), helix-rich, fatty acid and retinol-binding (FAR) proteins that appear to be confined to nematodes. We have carried out a comparative sequence and biochemical analysis of selected FAR proteins often species of filarial parasites (from the genera Onchocerca, Brugia, Wuchereria, Loa, Acanthocheilonema and Litomosoides). The sequences fall into two main groups corresponding broadly to the onchocercal and lymphatic filariasis parasites, and only those with unsheathed microfilariae were found to produce glycosylated FAR proteins. The proteins were released into culture medium by all the species and developmental stages investigated. Recombinant forms of two of these proteins (Ov-FAR-1 from O. volvulus and Bm-FAR-1 from B. malayi) were compared for ligand binding in fluorescence-based assays. Both were found to bind all-trans-retinol, (dansylamino) undecanoic acid (DAUDA), and oleic acid by competition. Both produced an identical, and dramatic, blue-shift in the fluorescence emission of DAUDA (from 541 to approximately 483 nm), indicative of similarity in the binding site environments of the two proteins. These findings indicate that there is strong conservation of the biochemical activities of the FAR proteins between the different parasite species, although they appear to have different post-translational modifications which may relate to the biology of the larvae.