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Abstract Artemisinin is a component part of current frontline medicines for the treatment of malaria. The aim of this study is to make analogues of artemisinin using microbial transformation and evaluate their in vitro antimalarial activity. A panel of microorganisms were screened for biotransformation of artemisinin (1). The biotransformation products were extracted, purified and isolated using silica gel column chromatography and semi-preparative HPLC. Spectroscopic methods including LC-HRMS, GC–MS, FT-IR, 1D and 2D NMR were used to elucidate the structure of the artemisinin metabolites.1H NMR spectroscopy was further used to study the time-course biotransformation. The antiplasmodial activity (IC50) of the biotransformation products of 1 against intraerythrocytic cultures of Plasmodium falciparum were determined using bioluminescence assays. A filamentous fungus Aspergillus niger CICC 2487 was found to possess the best efficiency to convert artemisinin (1) to a novel derivative, 4-methoxy-9,10-dimethyloctahydrofuro-(3,2-i)-isochromen-11(4H)-one (2) via ring rearrangement and further degradation, along with three known derivatives, compound (3), deoxyartemisinin (4) and 3-hydroxy-deoxyartemisinin (5). Kinetic study of the biotransformation of artemisinin indicated the formation of artemisinin G as a key intermediate which could be hydrolyzed and methylated to form the new compound 2. Our study shows that the anti-plasmodial potency of compounds 2, 3, 4 and 5 were ablated compared to 1, which attributed to the loss of the unique peroxide bridge in artemisinin (1). This is the first report of microbial degradation and ring rearrangement of artemisinin with subsequent hydrolysis and methoxylation by A.niger. Key points • Aspergillus niger CICC 2487 was found to be efficient for biotransformation of artemisinin • A novel and unusual artemisinin derivative was isolated and elucidated • The peroxide bridge in artemisinin is crucial for its high antimalarial potency • The pathway of biotransformation involves the formation of artemisinin G as a key intermediate

Background: In the malaria parasite Plasmodium falciparum the expression of ‘var’ virulence genes is regulated through epigenetic mechanisms. Its sirtuin epigenetic regulators have a direct effect on var gene expression patterns, are increased in a laboratory strain of P. falciparum exposed to heat shock and are positively associated with fever. A Gambia study extended this association to blood lactate and var genes commonly expressed in severe malaria, and between PfSir2A and group B var. A Kenyan study extended this association to between PfSir2A and overall var transcript level. These observations suggest a mechanism through which stress phenotypes in the human host might be sensed via a parasite sirtuin, and virulence gene expression modulated accordingly. Methods: In vitro experiments were conducted using laboratory and recently-laboratory-adapted Kenyan isolates of P. falciparum to follow up the correlative findings of the field study. To investigate a potential cause-and-effect relationship between host stress factors and parasite gene expression, qPCR was used to measure the expression of sirtuins and var genes after highly synchronous cultured parasites had been exposed to 2h or 6h of heat shock at 40°C or elevated lactate.Results: Heat shock was shown to influence the expression of PfSir2B in the trophozoites, whereas exposure to lactate was not. After the ring stages were exposed to heat shock, sirtuins, severe-disease-associated upsA and upsB var genes and var genes in general were not altered. More biological replicate experiments will be needed to confirm our observations.Conclusions: This study demonstrates that heat stress in laboratory and recently-laboratory-adapted isolates of P. falciparum results in a small increase in PfSir2B transcripts in the trophozoite stages only. We could not establish if heat shock can cause an increased expression of sirtuins or var genes in the ring stages. By contrast, the association between hyperlactataemia and sirtuin/var gene expression that was previously observed in vivo appears to be coincidental rather than causative.

Background Cylicodiscus gabunensis Harms (Family Leguminosae) (CG) is an African medicinal plant used as a treatment of various ailments including malaria, liver diseases, and gastrointestinal disturbances. Its extracts showed potent in vitro antibacterial activity. However, the antibacterial components are unknown. Methods In this study, the stem bark of the CG plant was extracted and its antibacterial property against a panel of Gram-negative and Gram-positive bacterial strains assessed using the disk diffusion assay method. Bioassay-guided fractionation of the bioactive extracts was employed to identify bioactive constituents using both gas and liquid chromatography mass spectrometry. Chemical synthesis was used to make the analogues of gallic acid. Microplate dilution assays and scanning electron microscopy (SEM) were used to evaluate the antibacterial properties and mechanism of action of the active fractions and pure compounds. Results The most bioactive sub-fractions derived from CG comprised of ethyl gallate, gallic acid and polyphenols. Five alkyl/alkenyl gallates were synthesized. A preliminary structure-activity relationship of gallic acid derivatives was obtained using the synthetic analogues and a series of commercially available phenolic compounds. Increasing the length of alkyl chains generally increases the potency of the alkyl gallates. Introducing a double bond with restricted conformations of the C-5 side chain has little effect on the antibacterial property. SEM analysis of the effect of alkyl gallates on Staphylococcus aureus indicates that they appear to interrupt S. aureus bacterial cell wall integrity. Conclusions The results of this research rationalise the ethnobotanical use of C. gabunensis and suggest that gallate derivatives may serve as promising antibacterial agents for the treatment of infectious diseases. Electronic supplementary material The online version of this article (10.1186/s12906-019-2589-2) contains supplementary material, which is available to authorized users.

The use of plant-derived natural products for the treatment of tropical parasitic diseases often has ethnopharmacological origins. As such, plants grown in temperate regions remain largely untested for novel anti-parasitic activities. We describe here a screen of the PhytoQuest Phytopure library, a novel source comprising over 600 purified compounds from temperate zone plants, against in vitro culture systems for Plasmodium falciparum, Leishmania mexicana, Trypanosoma evansi and T. brucei. Initial screen revealed 6, 65, 15 and 18 compounds, respectively, that decreased each parasite’s growth by at least 50% at 1–2 µM concentration. These initial hits were validated in concentration-response assays against the parasite and the human HepG2 cell line, identifying hits with EC50 &lt, 1 μM and a selectivity index of &gt, 10. Two sesquiterpene glycosides were identified against P. falciparum, four sterols against L. mexicana, and five compounds of various scaffolds against T. brucei and T. evansi. An L. mexicana resistant line was generated for the sterol 700022, which was found to have cross-resistance to the anti-leishmanial drug miltefosine as well as to the other leishmanicidal sterols. This study highlights the potential of a temperate plant secondary metabolites as a novel source of natural products against tropical parasitic diseases.

Bisbenzylisoquinoline (BBIQ) alkaloids are a diverse group of natural products that demonstrate a range of biological activities. In this study, the in vitro antiplasmodial activity of three BBIQ alkaloids (cycleanine [compound 1], isochondodendrine [compound 2], and 2′-norcocsuline [compound 3]) isolated from the Triclisia subcordata Oliv. medicinal plant traditionally used for the treatment of malaria in Nigeria are studied alongside two semisynthetic analogues (compounds 4 and 5) of cycleanine., Bisbenzylisoquinoline (BBIQ) alkaloids are a diverse group of natural products that demonstrate a range of biological activities. In this study, the in vitro antiplasmodial activity of three BBIQ alkaloids (cycleanine [compound 1], isochondodendrine [compound 2], and 2′-norcocsuline [compound 3]) isolated from the Triclisia subcordata Oliv. medicinal plant traditionally used for the treatment of malaria in Nigeria are studied alongside two semisynthetic analogues (compounds 4 and 5) of cycleanine. The antiproliferative effects against a chloroquine-resistant Plasmodium falciparum strain were determined using a SYBR green 1 fluorescence assay. The in vivo antimalarial activity of cycleanine is then investigated in suppressive, prophylactic, and curative murine malaria models after infection with a chloroquine-sensitive Plasmodium berghei strain. BBIQ alkaloids (compounds 1 to 5) exerted in vitro antiplasmodial activities with 50% inhibitory concentration (IC50) at low micromolar concentrations and the two semisynthetic cycleanine analogues showed an improved potency and selectivity compared to those of cycleanine. At oral doses of 25 and 50 mg/kg body weight of infected mice, cycleanine suppressed the levels of parasitemia and increased mean survival times significantly compared to those of the control groups. The metabolites and metabolic pathways of cycleanine were also studied using high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry. Twelve novel metabolites were detected in rats after intragastric administration of cycleanine. The metabolic pathways of cycleanine were demonstrated to involve hydroxylation, dehydrogenation, and demethylation. Overall, these in vitro and in vivo results provide a basis for the future evaluation of cycleanine and its analogues as leads for further development.

Background: In the malaria parasite Plasmodium falciparum the expression of ‘var’ virulence genes is regulated through epigenetic mechanisms. Two deacetylase enzymes of the sirtuin family have been implicated in this epigenetic control in laboratory-adapted parasites. A previous study of var gene expression in parasites isolated directly from Gambian malaria patients found that high expression levels of severe-disease-associated var variants correlated with high expression of the PfSir2A sirtuin, and these expression patterns also correlated with patient phenotypes of fever and hyperlactataemia. Together, the observations suggest a mechanism through which stress phenotypes in the human host might be sensed via a parasite sirtuin, and virulence gene expression modulated accordingly. Methods: In vitro experiments were conducted using recently-laboratory-adapted Kenyan isolates of P. falciparum to follow up the correlative findings of the field study. To investigate a potential cause-and-effect relationship between host stress factors and parasite gene expression, qPCR was used to measure the expression of sirtuins and var genes after cultured parasites had been exposed to 2h or 6h of heat shock at 40°C or elevated lactate at 5mM.Results: Heat shock was shown to influence the expression of both sirtuins and var genes, whereas exposure to lactate was not. Heat shock in the trophozoite stage resulted in modest upregulation of the expression of sirtuins, particularly PfSir2B, by 2-3 fold in all strains tested. Interestingly, when heat shock was applied in ring stages PfSir2A was still upregulated but PfSir2B was downregulated. This correlated with a general upregulation of ring-stage var transcription, and particularly of severe-disease-associated upsA and upsB var genes, but there was no clear pattern in the dominant var gene(s) ultimately expressed by heat-shocked parasites. Conclusions: This study demonstrates for the first time that heat stress in recently-laboratory-adapted isolates of P. falciparum results in altered sirtuin expression – PfSir2B as well as PfSir2A – and also the upregulation of var gene expression. These may be strategies evolved by the parasite to survive heat stress when a human host experiences malarial fevers. By contrast, the association between hyperlactataemia and sirtuin/var gene expression that was previously observed in vivo appears to be coincidental rather than causative.

The achievement of the Sustainable Development Goals (SDGs) demands unprecedented resources and efforts. Remittances as one of the largest development finance flows are an important source of income for millions of households in developing countries and offer tremendous potential to contribute towards the achievement of Agenda 2030. However, the high cost of sending remittances limits their full potential. The global average cost of sending USD 200 is 6.9% of the remittance. SDG 10 C aims to reduce the cost to less than 3% and to eliminate remittance corridors with cost higher than 5% by 2030. Blockchain technology promises to disintermediate banks, transform the financial landscape and drastically reduce the cost of cross-border transactions, yet there is a need for further evidence on this topic. The OECD Development Co-operation Directorate (DCD) has developed this paper to provide an overview of diverse perspectives on the intersection of blockchain technology and remittances by exploring the opportunities and challenges of this technology for reducing the cost of remittances. The paper identifies several limitations, such as data privacy risks, regulatory uncertainty and last-mile delivery, among others, while investigating whether blockchain technology is the solution to reduce the cost of remittances.

Thymoquinone (TQ), 2-isopropyl-5-methyl-1,4-benzoquinone, a natural product isolated from Nigella sativa L., has previously been demonstrated to exhibit antiproliferative activity in vitro against a range of cancers as well as the human malarial parasite Plasmodium falciparum. We describe here the synthesis of a series of analogues of TQ that explore the potential for nitrogen-substitution to this scaffold, or reduction to a hydroquinone scaffold, in increasing the potency of this antiproliferative activity against ovarian cancer cell lines and P. falciparum. In addition, alkyl or halogen-substituted analogues were commercially sourced and tested in parallel. Several TQ analogues with improved potency against ovarian cancer cells and P. falciparum were found, although this increase is suggested to be moderate. Key aspects of the structure activity relationship that could be further explored are highlighted.

Ethnopharmacological relevance and aim\ud A decoction of the bark of Cylicodiscus gabunensis Harms is used as a traditional medicine in the treatment of malaria in Nigeria. This study aims to validate the antimalarial potency of this decoction in vitro against Plasmodium falciparum and define potential bioactive constituents within the C. gabunensis bark.\ud \ud Materials and methods\ud A bioassay-guided separation and fractionation protocol was applied to C. gabunensis extracts, exploiting the use of a Malaria Sybr Green I Fluorescence assay method to monitor antiproliferative effects on parasites as well as define 50% inhibition concentrations. Spectroscopic techniques, including GC-MS, TOF LC-MS and 1H NMR were used to identify phytochemicals present in bioactive fractions. Analogues of gallic acid were synthesized de novo to support the demonstration of the antimalarial action of phenolic acids identified in C. gabunensis bark. In vitro cytotoxicity of plant extracts, fractions and gallate analogues was evaluated against the HepG2 cell line.\ud \ud Results\ud The antimalarial activity of ethanolic extracts of C. gabunensis bark was confirmed in vitro, with evidence for phenolic acids, primarily gallic acid and close analogues such as ethyl gallate, likely providing this effect. Further fractionation produced the most potent fraction with a 50% inhibitory concentration of 4.7 µg/ml. Spectroscopic analysis, including 1H NMR, LC-MS and GC-MS analysis of this fraction and its acid hydrolyzed products, indicated the presence of conjugates of gallic acid with oligosaccharides. The extracts/fractions and synthetic alkyl and alkenyl gallates showed moderate selectivity against P. falciparum.\ud \ud Conclusions\ud These results support the use of the bark of C. gabunensis as a traditional medicine in the treatment of human malaria, with phenolic acid oligosaccharide complexes evident in the most bioactive fractions.

Objectives Rapid rate-of-kill (RoK) is a key parameter in the target candidate profile 1 (TCP1) for the next-generation antimalarial drugs for uncomplicated malaria, termed Single Encounter Radical Cure and Prophylaxis (SERCaP). TCP1 aims to rapidly eliminate the initial parasite burden, ideally as fast as artesunate, but minimally as fast as chloroquine. Here we explore whether the relative RoK of the Medicine for Malaria Venture (MMV) Malaria Box compounds is linked to their mode of action (MoA) and identify scaffolds of medicinal chemistry interest. Methods We used a bioluminescence relative RoK (BRRoK) assay over 6 and 48 h, with exposure to equipotent IC50 concentrations, to compare the cytocidal effects of Malaria Box compounds with those of benchmark antimalarials. Results BRRoK assay data demonstrate the following relative RoKs, from fast to slow: inhibitors of PfATP4>parasite haemoglobin catabolism>dihydrofolate reductase-thymidylate synthase (DHFR-TS)>dihydroorotate dehydrogenase (DHODH)>bc1 complex. Core-scaffold clustering analyses revealed intrinsic rapid cytocidal action for diamino-glycerols and 2-(aminomethyl)phenol, but slow action for 2-phenylbenz-imidazoles, 8-hydroxyquinolines and triazolopyrimidines. Conclusions This study provides proof of principle that a compound’s RoK is related to its MoA and that the target’s intrinsic RoK is also modified by factors affecting a drug’s access to it. Our findings highlight that as we use medicinal chemistry to improve potency, we can also improve the RoK for some scaffolds. Our BRRoK assay provides the necessary throughput for drug discovery and a critical decision-making tool to support development campaigns. Finally, two scaffolds, diamino-glycerols and 2-phenylbenzimidazoles, exhibit fast cytocidal action, inviting medicinal chemistry improvements towards TCP1 candidates.

G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum , which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs.

Biofilm formation on surfaces is an important research topic in ship tribology and medical implants. In this study, dopamine and two types of synthetic peptides were designed and attached to 304 stainless steel surfaces, aiming to inhibit the formation of biofilms. A combinatory surface modification procedure was applied in which dopamine was used as a coupling agent, allowing a strong binding ability with the two peptides. X-ray photoelectron spectroscopy (XPS), elemental analysis, contact angle measurement and surface roughness test were used to evaluate the efficiency of the peptide modification. An antibiofilm assay against Staphylococcus aureus was conducted to validate the antibiofilm capacity of the peptide-modified stainless steel samples. XPS analysis confirmed that the optimal dopamine concentration was 40 µg ml −1 in the coupling reaction. Element analysis showed that dopamine and the peptides had bound to the steel surfaces. The robustness assay of the modified surface demonstrated that most peptide molecules had bound on the surface of the stainless steel firmly. The contact angle of the modified surfaces was significantly changed. Modified steel samples exhibited improved antibiofilm properties in comparison to untreated and dopamine-only counterpart, with the peptide 1 modification displaying the best antibiofilm effect. The modified surfaces showed antibacterial capacity. The antibiofilm capacity of the modified surfaces was also surface topography sensitive. The steel sample surfaces polished with 600# sandpaper exhibited stronger antibiofilm capacity than those polished with other types of sandpapers after peptide modification. These findings present valuable information for future antifouling material research.

Lemierre’s syndrome is a rare complication following an acute oropharyngeal infection. The aetiological agent is typically anaerobic bacteria of the genusFusobacterium. The syndrome is characterised by a primary oropharyngeal infection followed by metastatic spread and suppurative thrombophlebitis of the internal jugular vein. If left untreated, Lemierre’s syndrome carries a mortality rate of over 90%. Whilst relatively common in the preantibiotic era, the number of cases of Lemierre’s syndrome subsequently declined with the introduction of antibiotics. With the increase of antibiotic resistance and a greater reluctance to prescribe antibiotics for minor conditions such as tonsillitis, there are now concerns developing about the reemergence of the condition. This increasing prevalence in the face of an unfamiliarity of clinicians with the classical features of this “forgotten disease” may result in the misdiagnosis or delay in diagnosis of this potentially fatal illness. This case report illustrates the delay in diagnosis of probable Lemierre’s syndrome in a 17-year-old female, its diagnosis, and successful treatment which included the use of anticoagulation therapy. Whilst there was a positive outcome, the case highlights the need for a suspicion of this rare condition when presented with distinctive signs and symptoms.

Despite recent successful control efforts, malaria remains a leading global health burden. Alarmingly, resistance to current antimalarials is increasing and the development of new drug families is needed to maintain malaria control. Current antimalarials target the intraerythrocytic developmental stage of the Plasmodium falciparum life cycle. However, the invasive extracellular parasite form, the merozoite, is also an attractive target for drug development. We have previously demonstrated that heparin-like molecules, including those with low molecular weights and low anticoagulant activities, are potent and specific inhibitors of merozoite invasion and blood-stage replication. Here we tested a large panel of heparin-like molecules and sulfated polysaccharides together with various modified chemical forms for their inhibitory activity against P. falciparum merozoite invasion. We identified chemical modifications that improve inhibitory activity and identified several additional sulfated polysaccharides with strong inhibitory activity. These studies have important implications for the further development of heparin-like molecules as antimalarial drugs and for understanding merozoite invasion.

a b s t r a c t Plasmodium falciparum transcripts contain long untranslated regions (UTR), with some of the longest in any eukaryote that uses monocistronic transcription. Owing to the extreme AT nucleotide bias within the intergenic regions that encode these UTR, attempts to characterise how they are apportioned over genes and to describe their contribution to the absolute and temporal control of gene expression have been limited. Here we describe a study using a typical house-keeping gene that encodes phosphoglutamase 2 (PFD0660w), whose expression is subject to developmentally linked control during intraerythrocytic development. We show that deletion of a significant proportion (80%) of the predicted 5� UTR has no apparent effect on the developmentally linked expression of a luciferase reporter cassette. Further, serial deletions reveal that whilst the absolute level of transcription is unaffected when up to 50% of the predicted 5 � UTR is removed, the subsequent efficiency of translation is affected. These data provide key insights into the interplay of transcriptional and post-transcriptional mechanisms in the control of gene expression in this important human pathogen.

The particular virulence of the human malaria parasite Plasmodium falciparum derives from export of parasite-encoded proteins to the surface of the mature erythrocytes in which it resides. The mechanisms and machinery for the export of proteins to the erythrocyte membrane are largely unknown. In other eukaryotic cells, cholesterol-rich membrane microdomains or “rafts” have been shown to play an important role in the export of proteins to the cell surface. Our data suggest that depletion of cholesterol from the erythrocyte membrane with methyl-β-cyclodextrin significantly inhibits the delivery of the major virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). The trafficking defect appears to lie at the level of transfer of PfEMP1 from parasite-derived membranous structures within the infected erythrocyte cytoplasm, known as the Maurer's clefts, to the erythrocyte membrane. Thus our data suggest that delivery of this key cytoadherence-mediating protein to the host erythrocyte membrane involves insertion of PfEMP1 at cholesterol-rich microdomains. GTP-dependent vesicle budding and fusion events are also involved in many trafficking processes. To determine whether GTP-dependent events are involved in PfEMP1 trafficking, we have incorporated non-membrane-permeating GTP analogs inside resealed erythrocytes. Although these nonhydrolyzable GTP analogs reduced erythrocyte invasion efficiency and partially retarded growth of the intracellular parasite, they appeared to have little direct effect on PfEMP1 trafficking.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts., Author Summary Malaria leads to the loss of over 440,000 lives annually; accelerating research to discover new candidate drugs is a priority. Medicines for Malaria Venture (MMV) has distilled over 25,000 compounds that kill malaria parasites in vitro into a group of 400 representative compounds, called the "Malaria Box". These Malaria Box sets were distributed free-of-charge to research laboratories in 30 different countries that work on a wide variety of pathogens. Fifty-five groups compiled >290 assay results for this paper describing the many activities of the Malaria Box compounds. The collective results suggest a potential mechanism of action for over 130 compounds against malaria and illuminate the most promising compounds for further malaria drug development research. Excitingly some of these compounds also showed outstanding activity against other disease agents including fungi, bacteria, other single-cellular parasites, worms, and even human cancer cells. The results have ignited over 30 drug development programs for a variety of diseases. This open access effort was so successful that MMV has begun to distribute another set of compounds with initial activity against a wider range of infectious agents that are of public health concern, called the Pathogen Box, available now to scientific labs all over the world (www.PathogenBox.org).

Gene expression during the intraerythrocytic development cycle of the human malarial parasite Plasmodium falciparum is subject to tight temporal control, resulting in a cascade of gene expression to meet the physiological demands of growth, replication, and reinvasion. The roles of the different molecular mechanisms that drive this temporal program of gene expression are poorly understood. Here we report the use of the bxb1 integrase system to reconstitute all aspects of the absolute and temporal control of the prototypical housekeeping gene encoding the proliferating cell nuclear antigen (Pfpcna) around an integrated luciferase reporter cassette. A quantitative analysis of the effect of the serial deletion of 5′ and 3′ genetic elements and sublethal doses of histone deacetylase inhibitors demonstrates that while the absolute control of gene expression could be perturbed, no effect on the temporal control of gene expression was observed. These data provide support for a novel model for the temporal control of potentially hundreds of genes during the intraerythrocytic development of this important human pathogen.

Ten years ago this journal published a review with an almost identical title detailing how the then recent introduction of transfection technology had advanced our understanding of the molecular control of transcriptional processes in Plasmodium falciparum, particularly in terms of promoter structure and function. In the succeeding years, sequencing of several Plasmodium spp. genomes and application of high throughput global postgenomic technologies have proven as significant, if not more, as has the ability to genetically manipulate these parasites in dissecting the molecular control of gene expression. Here we aim to review our current understanding of the control of gene expression in P. falciparum, including evidence available from other Plasmodium spp. and apicomplexan parasites. Specifically, however, we will address the current polarised debate regarding the level at which control is mediated, and attempt to identify some of the challenges this field faces in the next 10 years.

Microarray-based comparative genomic hybridization (CGH) provides a powerful tool for whole genome analyses and the rapid detection of genomic variation that underlies virulence and disease. In the field of Plasmodium research, many of the parasite genomes that one might wish to study in a high throughput manner are not laboratory clones, but clinical isolates. One of the key limitations to the use of clinical samples in CGH, however, is the miniscule amounts of genomic DNA available. Here we describe the successful application of multiple displacement amplification (MDA), a non-PCR-based amplification method that exhibits clear advantages over all other currently available methods. Using MDA, CGH was performed on a panel of NF54 and IT/FCR3 clones, identifying previously published deletions on chromosomes 2 and 9 as well as polymorphism in genes associated with disease pathology.

The trafficking of proteins from the malaria parasite into the erythrocyte host has an important role in both the adaptation by the parasite of its immediate environment and the pathophysiology of disease. The molecular basis of these trafficking processes, particularly export from the parasitophorous vacuole (PV), is poorly understood and a matter of some controversy. In this article, we highlight two recent, independent reports that have provided new insights into protein translocation across the PV membrane, characterizing a novel signalling motif as a key signature in cataloguing the parasite secretome.

Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug–protein interactions involving this mutant residue.

The var multicopy gene family encodes Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) variant antigens, which, through their ability to adhere to a variety of host receptors, are thought to be important virulence factors. The predominant expression of a single cytoadherent PfEMP1 type on an infected red blood cell, and the switching between different PfEMP1 types to evade host protective antibody responses, are processes thought to be controlled at the transcriptional level. Contradictory data have been published on the timing of var gene transcription. Reverse transcription-polymerase chain reaction (RT-PCR) data suggested that transcription of the predominant var gene occurs in the later (pigmented trophozoite) stages, whereas Northern blot data indicated such transcripts only in early (ring) stages. We investigated this discrepancy by Northern blot, with probes covering a diverse var gene repertoire. We confirm that almost all var transcript types were detected only in ring stages. However, one type, the well-conserved varCSA transcript, was present constitutively in different laboratory parasites and does not appear to undergo antigenic variation. Although varCSA has been shown to encode a chondroitin sulphate A (CSA)-binding PfEMP1, we find that the presence of full-length varCSA transcripts does not correlate with the CSA-binding phenotype.

Genomic DNA is organised at its simplest level within phased arrays of nucleosomes, a structure key to the correct transcriptional regulation of the encoded genes. Here we studied chromatin formation on DNA transfected into Plasmodium falciparum either as an episomal plasmid or following integration by homologous recombination. We show that stably maintained and replicated plasmid assembles phased arrays of nucleosomes and that a reporter gene is transcribed in an appropriate temporal manner. These data provide a key observation for the future investigation of promoter structure and function with transfected DNA in Plasmodium spp.

The 49th Annual Spring Meeting of the British Society of Parasitology was held at Nottingham University, UK, on 11–14 April 2011. Although a meeting of a national parasitological society, the meeting attracted approximately 375 delegates from 30 countries worldwide. Of the various themes at this meeting, two were focused on the biology, etiology and development of therapeutics for the parasitic diseases; malaria and schistosomiasis. Here we report on three aspects of this meeting that are focused on the development of novel therapeutics to address the significant health burden imposed by these tropical diseases.

Background Homopolymeric tracts, particularly poly dA.dT, are enriched within the intergenic sequences of eukaryotic genomes where they appear to act as intrinsic regulators of nucleosome positioning. A previous study of the incomplete genome of the human malarial parasite Plasmodium falciparum reports a higher than expected enrichment of poly dA.dT tracts, far above that anticipated even in this highly AT rich genome. Here we report an analysis of the relative frequency, length and spatial arrangement of homopolymer tracts for the complete P. falciparum genome, extending this analysis to twelve additional genomes of Apicomplexan parasites important to human and animal health. In addition, using nucleosome-positioning data available for P. falciparum, we explore the correlation of poly dA.dT tracts with nucleosome-positioning data over key expression landmarks within intergenic regions. Results We describe three apparent lineage-specific patterns of homopolymeric tract organization within the intergenic regions of these Apicomplexan parasites. Moreover, a striking pattern of enrichment of overly long poly dA.dT tracts in the intergenic regions of Plasmodium spp. uniquely extends into protein coding sequences. There is a conserved spatial arrangement of poly dA.dT immediately flanking open reading frames and over predicted core promoter sites. These key landmarks are all relatively depleted in nucleosomes in P. falciparum, as would be expected for poly dA.dT acting as nucleosome exclusion sequences. Conclusions Previous comparative studies of homopolymer tract organization emphasize evolutionary diversity; this is the first report of such an analysis within a single phylum. Our data provide insights into the evolution of homopolymeric tracts and the selective pressures at play in their maintenance and expansion. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-848) contains supplementary material, which is available to authorized users.

Antigenic variation is a key survival strategy employed by a wide range of infectious organisms, allowing them to colonize and persist in a vertebrate host in the face of an evolving immune response. Variant surface antigens (VSA) of the parasite responsible for the most severe form of human malaria, Plasmodium falciparum, also contribute significantly to the pathology of disease. Characterization of var gene coding sequences is difficult due to their extreme diversity, but there are certain key conserved features in var gene structure. The conservation in upstream sequences, in terms of both sequence and organization, may indicate some evolutionary pressure that restricts recombination between limited subsets of var genes. The adhesion of different Duffy-binding-like (DBL) and cysteine-rich interdomain region (CIDR) domain types to host cell-surface molecules has been investigated in heterologous expression studies, establishing domains responsible for particular adhesive phenotypes and pinpointing regions critical for binding. Clonal switching of var gene expression during chronic infection reflects the sum of several molecular processes, including temporal regulation of var gene expression during intraerythrocytic development, mutually exclusive expression of only one var variant per infected erythrocytes (IE), and the ability to switch var expression in progeny parasites. Chronic infection with malaria is characterized by periodic peaks of parasitemia. A consensus model for variant expression during chronic infections can now be proposed. Following merozoite release from the liver, an initial P. falciparum erythrocyte membrane protein 1 (PfEMP1) variant dominates the first cycle of intraerythrocytic infection.

Objectives: A future treatment for uncomplicated malaria will contain at least one component that exerts a rapid rate of kill. We describe here the validation and application of a simple, robust and rapid bioluminescence-based assay for the determination of the initial rate of kill in intra-erythrocytic asexual stages of Plasmodium falciparum.\ud \ud Methods: A modification to the concentration–response bioluminescence [here termed bioluminescence relative rate of kill (BRRoK)] assay, utilizing exposure to fold-IC50 concentrations (0.33× to 9×), was used to monitor the immediate cytocidal effect of 372 open-source compounds for antimalarial drug discovery available through the Medicines for Malaria Venture Malaria Box.\ud \ud Results: Antimalarial drugs that exert a rapid cytocidal effect produce a concentration-dependent loss of bioluminescence signal that correlates with available in vitro and in vivo estimates of parasite clearance time and parasite reduction ratio. Following the measurement of IC50 for the Malaria Box compounds in Dd2luc, the BRRoK assay was used to identify and rank 372 compounds for their initial cytocidal activity. Fifty-three compounds in the Malaria Box show an initial relative rate of kill greater than that of chloroquine, with 17 of these having an initial relative rate of kill greater than that of dihydroartemisinin.\ud \ud Conclusions: The BRRoK assay provides a rapid assay format for the estimation of a key pharmacodynamic property of antimalarial drug action. The simplicity and robustness of the assay suggests it would be readily scalable for high-throughput screening and a critical decision-making tool for antimalarial drug development.

A gene (hap) transcribed during the intra-erythrocytic life cycle stages of the human malaria parasite Plasmodium falciparum was cloned and sequenced. It was found to encode a protein belonging to the aspartic proteinase family but which carried replacements of catalytically crucial residues in the hallmark sequences contributing to the active site of this type of proteinase. Consideration is given as to whether this protein is the first known parasite equivalent of the pregnancy-associated glycoproteins that have been documented in ungulate mammals. Alternatively, it may be operative as a new type of proteinase with a distinct catalytic mechanism. In this event, since no counterpart is known to exist in humans, it affords an attractive potential target against which to develop new anti-malarial drugs.

Here we describe the functional characterization of a Plasmodium falciparum promoter region, identifying a discrete five base pair sequence element that is responsible for efficient promoter activity. This sequence element binds nuclear factors in a sequence-specific manner. It shares no homology with any known eukaryotic transcription factor binding site, supporting the notion that the protozoan parasite P. falciparum has evolved a transcriptional machinery distinct from that of its human and mosquito hosts. This report represents the first description of a minimal and necessary cis-acting sequence element for efficient promoter activity in P. falciparum.

Tosh, K., Cheesman, S., Horrocks, P., and Kilbey, B. 1999. Plasmodium falciparum: Stage-related expression of topoisomerase I. Experimental Parasitology 91, 126–132. The expression and activity of topoisomerase I (PfTopoI) has been examined during the intraerythrocytic stages of the Plasmodium falciparum life cycle. The promoter is inactive during the early ring stage and becomes active only during the later trophozoite and schizont stages. The Pf TOP1 transcript starts to accumulate in the trophozoite stage parasite, decreasing again in the schizont stage. Using both stage-specific Western analysis and immunofluorescent assays we show that PfTopoI is present at low levels in rings and accumulates to approximately equal levels in the trophozoite and schizont stages. Experiments to determine the activity of PfTopoI, using a topoisomerase I relaxation assay, show that there is a low level of PfTopoI activity in both ring and trophozoite stages, but activity increases dramatically in the schizont stage. The PfTopoI activity can be inhibited by treatment with specific antiserum and by the type I topoisomerase-specific inhibitor camptothecin.

Transfection has facilitated a functional analysis of transcriptional processes in the human malarial parasite Plasmodium falciparum, providing the first fascinating glimpses into the mechanisms regulating parasite development and pathogenicity. Here we review our rapidly evolving knowledge of what constitutes a promoter, what factors regulate promoter activity and how this activity affects the manifestation of the disease.

The development of a transfection system for the protozoan parasite Plasmodium falciparum hailed a breakthrough in understanding one of the world's most prevalent human pathogens. One hundred years after the discovery by Sir Ronald Ross that the etiological agent of malaria is transmitted to humans by the bite of infected Anopheline mosquitoes, we now have the tools at hand with which to investigate the biology and pathogenicity of P. falciparum , at the molecular level, in an attempt to develop sustainable means to control this important infectious disease. Here we review the recent progress facilitated by transfection in malariology and discuss the future opportunities this technique offers.

The stage-specific relationship between promoter activity, transcript production, protein expression and enzyme activity has been investigated for the gene encoding Plasmodium falciparum topoisomerase II (PfTopoII). Nuclear run-on experiments have shown that the P. falciparum topoisomerase II gene (PfTOP2) promoter is active at low levels in ring stage parasites, but reaches high levels of activity as the parasites progress into trophozoite/schizont asexual stages. Steady-state PfTOP2 transcripts are present at low levels in rings, accumulate in trophozoites, but are completely undetectable in schizonts. An antiserum raised against the species-divergent carboxy-terminus of PfTopoII, which neutralised the decatenation activity in parasite extracts, was used to probe Western blots of ring, trophozoite and schizont stage parasite extracts. Relatively low levels of PfTopoII were seen in rings compared with those in trophozoite and schizont preparations. Parasite extracts were also used to compare the patterns of protein accumulation and enzyme activity at these stages. Complete decatenation of kinetoplast substrate DNA (KDNA) was found in schizont stages, very low levels of activity were observed in rings and trophozoites showed intermediate levels. These finding show that, as parasites progress towards the stages where DNA replication occurs, there is a concomitant increase in both topoisomerase II production and activity.

Plasmodium falciparum var genes encode a diverse family of proteins, located on the surfaces of infected erythrocytes, which are implicated in the pathology of human malaria through antigenic variation and adhesion of infected erythrocytes to the microvasculature. We have constructed a complete representative telomere-to-telomere yeast artificial chromosome (YAC) contig map of the P. falciparum chromosome 8 for studies on the chromosomal organization, distribution, and expression of var genes. Three var gene loci were identified on chromosome 8, two of which map close to the telomeres at either end of the chromosome. Analysis of the previously described chromosome 2 contig map and random P. falciparum telomeric YAC clones revealed that most, if not all, 14 P. falciparum chromosomes contain var genes in a subtelomeric location. Mapping the chromosomal location of var genes expressed in a long-term culture of the P. falciparum isolate Dd2 revealed that four of the five different expressed var genes identified map within subtelomeric locations. Expression of var genes from a chromosomal domain known for frequent rearrangements has important implications for the mechanism of var gene switching and the generation of novel antigenic and adhesive phenotypes.

3-(4-Chlorophenyl)-4-substituted pyrazole derivatives were synthesised and tested for their in vitro antifungal activity. Some compounds showed very good antifungal activity against four pathogenic strains of fungi. The same compounds exhibited an interesting activity against the tested strain of Mycobacterium tuberculosis H37Rv. The results suggest that 1,3,4-oxadiazoles and 5-pyrazolinones bearing a core pyrazole scaffold may be promising antifungal and antitubercular agents.

Review of Carlton JM, Perkins SL, Deitsch, KW: Malaria Parasites: Comparative Genomics, Evolution and Molecular Biology. Caister Academic Press; 2013. 280 pages. ISBN 190823007X

Background Transgenic Plasmodium falciparum expressing luciferase offers an attractive bioluminescence-based assay platform for the investigation of the pharmacological properties of anti-malarial drugs. Here a side-by-side comparison of bioluminescence and fluorescence-based assays, utilizing a luciferase reporter cassette that confers a strong temporal pattern of luciferase expression during the S-phase of intraerythrocytic development, is reported. Methods Key assay parameters for a range of commercially available luminogenic substrates are determined and compared to those measured using a Malaria Sybr Green I fluorescence assay. In addition, the short-term temporal effects of anti-malarial compounds are evaluated using both bioluminescent and fluorescent assay platforms. Results The Z’, % coefficient of variation and 50% inhibition concentrations are essentially the same for bioluminescent and fluorescent assays in transgenic parasites generated in both chloroquine-sensitive and -resistant genetic backgrounds. Bioluminescent assays, irrespective of the luminogenic agent employed, do, however, offer significantly enhanced signal-to-noise ratios. Moreover, the bioluminescent assay is more dynamic in terms of determining temporal effects immediately following drug perturbation. Conclusion This study suggests that opportunities for bioluminescence-based assays lie not in the measurement of 50% inhibition concentrations, where the cheaper fluorescence assay performs excellently and is not restricted by the need to genetically modify the parasite clone under investigation. Instead, assays that use the dynamic response of the luciferase reporter for semi-automated screening of additional pharmacological properties, such as relative rate-of-kill and lethal dose estimation, are a more attractive development opportunity.