Malarial hemozoin: From target to tool

Background

Malaria is an extremely devastating disease that continues to affect millions of people each year. A distinctive attribute of malaria infected red blood cells is the presence of malarial pigment or the so-called hemozoin. Hemozoin is a biocrystal synthesized by Plasmodium and other blood-feeding parasites to avoid the toxicity of free heme derived from the digestion of hemoglobin during invasion of the erythrocytes.

Scope of review

Hemozoin is involved in several aspects of the pathology of the disease as well as in important processes such as the immunogenicity elicited. It is known that the once best antimalarial drug, chloroquine, exerted its effect through interference with the process of hemozoin formation. In the present review we explore what is known about hemozoin, from hemoglobin digestion, to its final structural analysis, to its physicochemical properties, its role in the disease and notions of the possible mechanisms that could kill the parasite by disrupting the synthesis or integrity of this remarkable crystal.

Major conclusions

The importance and peculiarities of this biocrystal have given researchers a cause to consider it as a target for new antimalarials and to use it through unconventional approaches for diagnostics and therapeutics against the disease.

General significance

Hemozoin plays an essential role in the biology of malarial disease. Innovative ideas could use all the existing data on the unique chemical and biophysical properties of this macromolecule to come up with new ways of combating malaria.     

Oral delivery of curcumin bound to chitosan nanoparticles cured Plasmodium yoelii infected mice

Curcumin has been shown to have anti malarial activity, but poor bioavailability and chemical instability has hindered its development as a drug. We have bound curcumin to chitosan nanoparticles to improve its bioavailability and chemical stability. We found that curcumin bound to chitosan nanoparticles did not degrade that rapidly in comparison to free curcumin when such particles were incubated in mouse plasma in vitro at room temperature. The uptake of bound curcumin from chitosan nanoparticles by mouse RBC was much better than from free curcumin. Oral delivery of curcumin bound chitosan nanoparticles to normal mice showed that they can cross the mucosal barrier intact and confocal microscopy detected the nanoparticles in the blood. Curcumin loaded chitosan nanoparticles when delivered orally improved the bioavailability of curcumin in the plasma and RBC. While mice infected with a lethal strain of Plasmodium yoelii (N-67) died between 8 and 9 days post infection, feeding of chitosan nanoparticles alone made them to survive for five more days. Feeding 1mg of native curcumin to infected mice per day for seven days resulted in survival of one third of mice but under the same condition when 1mg of curcumin bound to chitosan nanoparticles was fed all the mice survived. Like chloroquine, curcumin inhibited parasite lysate induced heme polymerization in vitro in a dose dependent manner and curcumin had a lower IC(50) value than chloroquine. We believe that binding of curcumin to chitosan nanoparticles increases its chemical stability and enhances its bioavailability when fed to mice. In vitro data suggest that it can inhibit hemozoin synthesis which is lethal for the parasite.     

Leishmania mexicana: inhibition and stimulation of phagosome-lysosome fusion in infected macrophages

The polyanionic compound poly-d-glutamic acid was found to inhibit significantly the fusion of secondary lysosomes to phagosomes containing Leishmania mexicana mexicana amastigotes for at least 96 hr. This process was viewed both by dark-field vital fluorescence microscopy and transmission electron microscopy. In poly-d-glutamic acid-treated macrophages parasites multiplied at a significantly greater rate than in untreated macrophages. Conversely, the secondary amine chloroquine caused a marked reduction in parasite growth. When L. m. mexicana promastigotes were substituted for amastigotes these results were strikingly more pronounced.     

Morphological Characterization and Fusion Properties of Triglyceride-rich Lipoproteins Obtained from Cells Transduced with Hepatitis C Virus Glycoproteins

The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of β-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP.     

Chloroquine resistant malaria: Association with enhanced plasmodial protease activity

Chloroquine resistant Plasmodium berghei has several unusual features including (i) lack of malaria “pigment”, (ii) more efficient host catabolism of heme from infected erythrocytes, and (iii) relatively inefficient uptake of external chloroquine by infected red cells. The malaria pigment produced by chloroquine sensitive P. berghei is probably incompletely catabolized hemoglobin, the heme group of which is unavailable for subsequent catabolism by the host's reticuloendothelial system. This pigment has been suggested by others as the site of high affinity chloroquine binding. We hypothesized that all three characteristics of chloroquine resistant infections might be explained by enhanced proteolytic digestion of host cell hemoglobin. In confirmation, we report that chloroquine resistant P. berghei has 700–800% greater protease activity than the chloroquine sensitive form. This greatly elevated protease activity may explain the aforementioned characteristics of chloroquine resistant P. berghei and may help elucidate the basis of chloroquine resistance in human P. falciparum.     

Plasmodium knowlesi: Morphology and course of infection in rhesus monkeys treated with clindamycin and its N-demethyl-4′-pentyl analog

The clinical and morphologic effects of clindamycin and N-demethyl-4′-pentyl clindamycin were evaluated using Plasmodium knowlesi in rhesus monkeys. Both compounds cured blood-induced infections when administered daily for five consecutive days. When the rapidity of action of these antibiotics was compared with chloroquine it was evident that although they were able to control fulminating infections in all treated monkeys their effect was about 2 days slower than chloroquine in decreasing parasitemias and 3 to 4 days slower in clearing parasites from the blood. Morphologic changes within the parasite associated with drug action were studied in time sequences by light and electron microscopy. Changes were observed in the parasite ribosomes 24 hr after drug administration. Affected ribosomes showed electron-lucent zones measuring ~50 Å in the center. During the following 24 hr these changes became more prominent with foci in which disintegrated ribosomes were replaced by fine fibrillar material and the cisternae of the endoplasmic reticulum became dilated. By 72 hr this dilation was more apparent and resulted in abundant coalescence of vacuoles in the cytoplasm. Mitochondria became dilated with fibrils in the matrix, although the degree of swelling was not a conspicuous and constant feature. The nucleus presented a fine fibrillar appearance and fewer granules were seen than in normal parasites. The latter two observed changes appear to be secondary to changes in the ribosomes and probably are not directly related to the action of the antibiotics. These studies indicate that clindamycin and its analog affect primarily the ribosomes and their mode of action is different from that of the commonly used antimalarials.     

Effect of far-red and green irradiation on the nyctinastic closure of albizzia leaflets

The nyctinastic closing of Albizzia julibrissin pinnules is delayed by exposure to far-red radiation at 710 and 730 nanometers, with the former more effective than the latter. Far-red radiation at 750 and 770 nanometers has no effect on the process. Red light at 660 nanometers, which by itself has no effect, delayed closure when given before or simultaneously with far-red radiation at 750 or 770 nanometers. Low doses of green light, on the other hand, prevented all far-red radiations from delaying closure when given together with one of them. Effectiveness peaks at 550 nanometers. Green light by itself has no effect on the closing process.

From these and previous results, it is concluded that phytochrome is one of two photoreceptors in the process, that the other photoreceptor is an unknown pigment, and that the unknown photoreceptor requires some prior effect of the far-red-absorbing form of phytochrome before its action. Predictions are made of some of the properties of the unidentified pigment.

     

Plasmodium berghei: Uptake and distribution of chloroquine in infected mouse erythrocytes

The capacity of mouse erythrocytes infected with Plasmodium berghei to accumulate chloroquine is developed with maturation of the parasites. This is shown by direct comparison of the early and mature stages, which are separated by density difference. After drug accumulation, infected cells were fractionated by saponin lysis or nitrogen decompression to study the drug distribution. Effectiveness of isolating intact parasites and host components was checked by SDS-polyacrylamide gel electrophoresis and by low leakage of parasite-specific lactate dehydrogenase used as a marker enzyme. At low external drug concentration (~10^?7M), chloroquine is principally accumulated in the parasites. However, at higher drug concentrations (~10^?5and ~10^?3M), the proportion of the drug found in the host cytosol fraction is increased. A small but significant proportion of the drug (<20%) is associated with the host cell membrane. The pellet fraction of the freed parasites, further fractionated by freeze-thaw lysis, contains a major proportion of the drug at low external concentrations. However, the pellet fraction obtained from prolonged sonication of the parasites, which contains the bulk of hemozoin pigment, carries only a small proportion of the drug. This indicates that parasite membrane components may bind most of the drug. As external chloroquine concentration is increased, the proportion of drug in the parasite supernatant increases, some or most of which is probably bound by soluble hemecontaining compounds. However, the presence of chloroquine in the parasite does not affect the partition of heme in particulate and soluble forms.     

Quinoline antimalarials decrease the rate of beta-hematin formation

The strength of inhibition of beta-hematin (synthetic hemozoin or malaria pigment) formation by the quinoline antimalarial drugs chloroquine, amodiaquine, quinidine and quinine has been investigated as a function of incubation time. In the assay used, beta-hematin formation was brought about using 4.5M acetate, pH 4.5 at 60 degrees C. Unreacted hematin was detected by formation of a spectroscopically distinct low spin pyridine complex. Although, these drugs inhibit beta-hematin formation when relatively short incubation times are used, it was found that beta-hematin eventually forms with longer incubation periods (<8h for chloroquine and >8h for quinine). This conclusion was supported by both infrared and X-ray powder diffraction observations. It was further found that the IC(50) for inhibition of beta-hematin formation increases markedly with increasing incubation times in the case of the 4-aminoquinolines chloroquine and amodiaquine. By contrast, in the presence of the quinoline methanols quinine and quinidine the IC(50) values increase much more slowly. This results in a partial reversal of the order of inhibition strengths at longer incubation times. Scanning electron microscopy indicates that beta-hematin crystals formed in the presence of chloroquine are more uniform in both size and shape than those formed in the absence of the drug, with the external morphology of these crystallites being markedly altered. The findings suggest that these drugs act by decreasing the rate of hemozoin formation, rather than irreversibly blocking its formation. This model can also explain the observation of a sigmoidal dependence of beta-hematin inhibition on drug concentration.     

Etude ultrastructurale comparée du processus de dégradation de l'hémoglobine par P. berghei (Vincke et Lips, 1948) en fonction de l'état de maturité de la cellule hǒte1

By serial sectioning and 3D reconstruction we have been able to demonstrate that the type of system for hemoglobin digestion in two strains of Plasmodium berghei, N and RC, is dependent on the maturity of the host cell. In parasites growing in erythrocytes, both systems for the endocytosis of hemoglobin—micropinocytosis and the cytostomal system (i.e. a cytostome budding a cytostomal tube that releases food vacuoles)—are fully functional and produce a great quantity of residual pigment. Parasites growing in reticulocytes have a disrupted cytostomal system; no tube is formed and only food vacuoles are visible in their cytoplasm. Residual pigment is smaller in size and in quantity. The reduced quantity of pigment in reticulocytes is explained by our observation of the exocytosis of pigment. We propose a hypothesis that relates the process of degradation of hemoglobin to the maturity of the host cell and a possible mechanism of protection against chloroquine, a drug known for its affinity for malarial pigment.     

Epifluorescence and atomic force microscopy: Two innovative applications for studying phage-host interactions in Lactobacillus helveticus

Bacteriophages attacking lactic acid bacteria (LAB) still represent a crucial problem in industrial dairy fermentations. The consequences of a phage infection against LAB can lead to fermentation delay, alteration of the product quality and, in most severe cases, the product loss. Phage particles enumeration and phage-host interactions are normally evaluated by conventional plaque count assays, but, in many cases, these methods can be unsuccessful. Bacteriophages of Lactobacillus helveticus, a LAB species widely used as dairy starter or probiotic cultures, are often unable to form lysis plaques, thus impairing their enumeration by plate assay. In this study, we used epifluorescence microscopy to enumerate L. helveticus phage particles from phage-infected cultures and Atomic Force Microscopy (AFM) to visualize both phages and bacteria during the different stages of the lytic cycle. Preliminary, we tested the sensitivity of phage counting by epifluorescence microscopy. To this end, phage particles of ΦAQ113, a lytic phage of L. helveticus isolated from a whey starter culture, were stained by SYBR Green I and enumerated by epifluorescence microscopy. Values obtained by the microscopic method were 10 times higher than plate counts, with a lowest sensitivity limit of ≥ 6 log phage/ml. The interaction of phage ΦAQ113 with its host cell L. helveticus Lh1405 was imaged by AFM after 0, 2 and 5 h from phage-host adsorption. The lytic cycle was followed by epifluorescence microscopy counting and the concomitant cell wall changes were visualized by AFM imaging. Our results showed that these two methods can be combined for a reliable phage enumeration and for studying phage and host morphology during infection processes, thus giving a complete overview of phage-host interactions in L. helveticus strains involved in dairy productions.     

W-reactivation and W-mutagenesis of gamma-irradiated phage lambda.

UV irradiation of Escherichia coli wild-type cells manifested the phenomena of W-reactivation (WR) and W-mutagenesis (WM) of phage lambda irradiated by 60Co gamma-rays in broth. WR of gamma-irradiated phage was half as efficient as that of UV-irradiated phage, although the frequency of c mutations in conditions of WR was about the same in both phages. The xthA and recBrecC sbcB mutants were practically identical with wild-type cells in respect of WR and WM of UV- and gamma-irradiated phage. As in UV-irradiated phage, WR and WM of gamma-irradiated phage were absolutely dependent on the recA+ and lexA+ genes of the host cell. WR and WM required much smaller doses of UV radiation for induction in polA1 and uvrB mutants. The lig-ts mutant, temperature sensitive in polynucleotide ligase, was deficient in WR and WM of UV- and gamma-irradiated phage at the semi-permissive temperature of 37 degrees. The uvrE502 mutant and the allelic recL152 strain were absolutely deficient in WR and WM of gamma-irradiated phage. In UV-irradiated phage WR was reduced, but not eliminated, in the uvrE mutant, and WM was entirely suppressed. This is another example of uncoupling of WR and WM which shows that several repair systems are active in WR but only some of them are mutagenic.     

Axenic Culture of a Candidate Division TM7 Bacterium from the Human Oral Cavity and Biofilm Interactions with Other Oral Bacteria

The diversity of bacterial species in the human oral cavity is well recognized, but a high proportion of them are presently uncultivable. Candidate division TM7 bacteria are almost always detected in metagenomic studies but have not yet been cultivated. In this paper, we identified candidate division TM7 bacterial phylotypes in mature plaque samples from around orthodontic bonds in subjects undergoing orthodontic treatment. Successive rounds of enrichment in laboratory media led to the isolation of a pure culture of one of these candidate division TM7 phylotypes. The bacteria formed filaments of 20 to 200 μm in length within agar plate colonies and in monospecies biofilms on salivary pellicle and exhibited some unusual morphological characteristics by transmission electron microscopy, including a trilaminated cell surface layer and dense cytoplasmic deposits. Proteomic analyses of cell wall protein extracts identified abundant polypeptides predicted from the TM7 partial genomic sequence. Pleiomorphic phenotypes were observed when the candidate division TM7 bacterium was grown in dual-species biofilms with representatives of six different oral bacterial genera. The TM7 bacterium formed long filaments in dual-species biofilm communities with Actinomyces oris or Fusobacterium nucleatum. However, the TM7 isolate grew as short rods or cocci in dual-species biofilms with Porphyromonas gingivalis, Prevotella intermedia, Parvimonas micra, or Streptococcus gordonii, forming notably robust biofilms with the latter two species. The ability to cultivate TM7 axenically should majorly advance understanding of the physiology, genetics, and virulence properties of this novel candidate division oral bacterium.     

Comparative ultrastructural study of the process of hemoglobin degradation by P. berghei (Vincke and Lips, 1948) as a function of the state of maturity of the host cell

By serial sectioning and 3D reconstruction we have been able to demonstrate that the type of system for hemoglobin digestion in two strains of Plasmodium berghei, N and RC, is dependent on the maturity of the host cell. In parasites growing in erythrocytes, both systems for the endocytosis of hemoglobin-micropinocytosis and the cytostomal system (i.e. a cytostome budding a cytostomal tube that releases food vacuoles)-are fully functional and produce a great quantity of residual pigment. Parasites growing in reticulocytes have a disrupted cytostomal system; no tube is formed and only food vacuoles are visible in their cytoplasm. Residual pigment is smaller in size and in quantity. The reduced quantity of pigment in reticulocytes is explained by our observation of the exocytosis of pigment. We propose a hypothesis that relates the process of degradation of hemoglobin to the maturity of the host cell and a possible mechanism of protection against chloroquine, a drug known for its affinity for malarial pigment.     

Angelica gigas Nakai and Soluplus-Based Solid Formulations Prepared by Hot-Melting Extrusion: Oral Absorption Enhancing and Memory Ameliorating Effects

Oral solid formulations based on Angelica gigas Nakai (AGN) and Soluplus were prepared by the hot-melting extrusion (HME) method. AGN was pulverized into coarse and ultrafine particles, and their particle size and morphology were investigated. Ultrafine AGN particles were used in the HME process with high shear to produce AGN-based formulations. In simulated gastrointestinal fluids (pH 1.2 and pH 6.8) and water, significantly higher amounts of the major active components of AGN, decursin (D) and decursinol angelate (DA), were extracted from the HME-processed AGN/Soluplus (F8) group than the AGN EtOH extract (ext) group (p < 0.05). Based on an in vivo pharmacokinetic study in rats, the relative oral bioavailability of decursinol (DOH), a hepatic metabolite of D and DA, in F8-administered mice was 8.75-fold higher than in AGN EtOH ext-treated group. In scopolamine-induced memory-impaired mice, F8 exhibited a more potent cognitive enhancing effect than AGN EtOH ext in both a Morris water maze test and a passive avoidance test. These findings suggest that HME-processed AGN/Soluplus formulation (F8) could be a promising therapeutic candidate for memory impairment.     

Quantitative characterization of clumping in Scots pine crowns

Background and Aims

Proper characterization of the clumped structure of forests is needed for calculation of the absorbed radiation and photosynthetic production by a canopy. This study examined the dependency of crown-level clumping on tree size and growth conditions in Scots pine (Pinus sylvestris), and determined the ability of statistical canopy radiation models to quantify the degree of self-shading within crowns as a result of the clumping effect.

Methods

Twelve 3-D Scots pine trees were generated using an application of the LIGNUM model, and the crown-level clumping as quantified by the crown silhouette to total needle area ratio (STAR_crown) was calculated. The results were compared with those produced by the stochastic approach of modelling tree crowns as geometric shapes filled with a random medium.

Key Results

Crown clumping was independent of tree height, needle area and growth conditions. The results supported the capability of the stochastic approach in characterizing clumping in crowns given that the outer shell of the tree crown is well represented.

Conclusions

Variation in the whole-stand clumping index is induced by differences in the spatial pattern of trees as a function of, for example, stand age rather than by changes in the degree of self-shading within individual crowns as they grow bigger.     

A Simple Protocol for Platelet-mediated Clumping of Plasmodium falciparum-infected Erythrocytes in a Resource Poor Setting

P. falciparum causes the majority of severe malarial infections. The pathophysiological mechanisms underlying cerebral malaria (CM) are not fully understood and several hypotheses have been put forward, including mechanical obstruction of microvessels by P. falciparum-parasitized red blood cells (pRBC). Indeed, during the intra-erythrocytic stage of its life cycle, P. falciparum has the unique ability to modify the surface of the infected erythrocyte by exporting surface antigens with varying adhesive properties onto the RBC membrane. This allows the sequestration of pRBC in multiple tissues and organs by adhesion to endothelial cells lining the microvasculature of post-capillary venules ¹. By doing so, the mature forms of the parasite avoid splenic clearance of the deformed infected erythrocytes ² and restrict their environment to a more favorable low oxygen pressure ³. As a consequence of this sequestration, it is only immature asexual parasites and gametocytes that can be detected in peripheral blood.Cytoadherence and sequestration of mature pRBC to the numerous host receptors expressed on microvascular beds occurs in severe and uncomplicated disease. However, several lines of evidence suggest that only specific adhesive phenotypes are likely to be associated with severe pathological outcomes of malaria. One example of such specific host-parasite interactions has been demonstrated in vitro, where the ability of intercellular adhesion molecule-1 to support binding of pRBC with particular adhesive properties has been linked to development of cerebral malaria ^4,5. The placenta has also been recognized as a site of preferential pRBC accumulation in malaria-infected pregnant women, with chondrotin sulphate A expressed on syncytiotrophoblasts that line the placental intervillous space as the main receptor ⁶. Rosetting of pRBC to uninfected erythrocytes via the complement receptor 1 (CD35)^7,8 has also been associated with severe disease ⁹.One of the most recently described P. falciparum cytoadherence phenotypes is the ability of the pRBC to form platelet-mediated clumps in vitro. The formation of such pRBC clumps requires CD36, a glycoprotein expressed on the surface of platelets. Another human receptor, gC1qR/HABP1/p32, expressed on diverse cell types including endothelial cells and platelets, has also been shown to facilitate pRBC adhesion on platelets to form clumps ¹⁰. Whether clumping occurs in vivo remains unclear, but it may account for the significant accumulation of platelets described in brain microvasculature of Malawian children who died from CM ¹¹. In addition, the ability of clinical isolate cultures to clump in vitro was directly linked to the severity of disease in Malawian ¹² and Mozambican patients ¹³, (although not in Malian ¹⁴).With several aspects of the pRBC clumping phenotype poorly characterized, current studies on this subject have not followed a standardized procedure. This is an important issue because of the known high variability inherent in the assay ¹⁵. Here, we present a method for in vitro platelet-mediated clumping of P. falciparum with hopes that it will provide a platform for a consistent method for other groups and raise awareness of the limitations in investigating this phenotype in future studies. Being based in Malawi, we provide a protocol specifically designed for a limited resource setting, with the advantage that freshly collected clinical isolates can be examined for phenotype without need for cryopreservation.     

Antifungal treatment for invasive Candida infections: a mixed treatment comparison meta-analysis

Background

Malaria remains one of the most important tropical diseases of human with 1–2 million deaths annually especially caused by P. falciparum. During malarial life cycle, they exposed to many environmentally stresses including wide temperature fluctuation and pharmacological active molecules. These trigger malarial evolutionarily adaptive responses. The effect of febrile temperature on malarial growth, development and drug susceptibility by mimicking patient in treatment failure before and after drug uptake was examined.

Methods

Sensitivities of P. falciparum to antimalarial drug (chloroquine, mefloquine, quinine and artesunate) were investigated based on the incorporation of [³H] hypoxanthine into parasite nucleic acids or radioisotopic technique. The number of parasites was examined under microscope following Giemsa staining and the parasite development at the end of each phase was counted and comparison of parasite number was made. The proteome was separated, blotted and hybridized with anti-Hsp70s primary antibody. The hybridized proteins were separately digested with trypsin and identified by MALDI-TOF peptide mass fingerprint.

Results

The results show that febrile temperature is capable of markedly inhibiting the growth of field isolate P. falciparum but not to K1 and 3D7 standard strains. K1 and 3D7 grown under heat shock developed greater and the reinfection rate was increased up to 2-folds when compared to that of non-heat shock group. The IC₅₀ value of K1 toward chloroquine, mefloquine and quinine under heat shock was higher than that of K1 under non-heat shock which is opposite to that of 3D7. Heat shock caused death in field isolated parasite. It was also found that the febrile temperature coped with chloroquine uptake had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine shows extremely effect toward 3D7 and field isolate PF91 as shown by higher number of dead parasites compared to that of control group. After culture under high temperature with artesunate, the total parasite number of all strains including K1, 3D7 and PF91 was extremely decreased and the parasite was not found at the end. Additionally, the expression of pfHsp70s was found in all strains and conditions as shown in 120 kDa hybridized band. However, the proteome extracted from K1 grown under heat shock with chloroquine, anti-pfHsp70 interacted with additional three bands identified by MALDI-TOF as elongation factor-1α (83 kDa), pfHsp86 (60 kDa) and phosphoethanolamine N-methyltransferase (43 kDa).

Conclusion

In conclusion, febrile temperature was capable of markedly inhibiting the growth of field isolate P. falciparum while the development, reinfection rate and drug (chloroquine, mefloquine and quinine) resistant level of standard strain K1 was enhanced. However, the febrile temperature coped with chloroquine had no effect to the development, drug sensitivity and the parasite number of K1 strain. In the opposite way, heat shock and chloroquine showed extremely effect toward 3D7 and field isolate PF91 as shown by some died parasites. Heat shock protein 70 (pfHSP70) of strain K1 under heat shock with chloroquine might involved in many pathways in order to sustain the parasite.     

New pyrazolylpyrazoline derivatives as dual acting antimalarial-antileishamanial agents: synthesis,biological evaluation and molecular modelling simulations

Promising inhibitory activities of the parasite multiplication were obtained upon evaluation of in vivo antimalarial activities of new pyrazolylpyrazoline derivatives against Plasmodium berghei infected mice. Further evaluation of 5b and 6a against chloroquine-resistant strain (RKL9) of P. falciparum showed higher potency than chloroquine. In vitro antileishmanial activity testing against Leishmania aethiopica promastigote and amastigote forms indicated that 5b, 6a and 7b possessed promising activity compared to miltefosine and amphotericin B deoxycholate. Moreover, antileishmanial activity reversal of the active compounds via folic and folinic acids showed comparable results to the positive control trimethoprim, indicating an antifolate mechanism via targeting leishmanial DHFR and PTR1. The compounds were non-toxic at 125, 250 and 500 mg/kg. In addition, docking of the most active compound against putative malarial target Pf-DHFR-TS and leishmanial PTR1 rationalised the observed activities. Molecular dynamics simulations confirmed a stable and high potential binding of 7a against leishmanial PTR1.