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Jun Sun - One of the best experts on this subject based on the ideXlab platform.
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Schistosoma Hemozoin and its possible roles
International journal for parasitology, 2016Co-Authors: Shu-hua Xiao, Jun SunAbstract:More than 95years ago Schistosoma pigment had been deemed as a degradation product of haemoglobin. Until the 1950s, scientists initiated to pay attention to understand the hematophagous habit of schistosomes, and to study the degradation of haemoglobin as well as the formation of Hemozoin inside the gut of the worms. For a long time, the formation of Hemozoin in both Plasmodium and in Schistosoma was considered to be the major route of heme detoxification, and Hemozoin served a role in waste disposal. At the beginning of this century, the chemical structure of Schistosoma pigment was confirmed to be identical to that of malarial pigment (Hemozoin) and its synthetic analogue, β-hematin. Since then, studies on Schistosoma Hemozoin have been investigated by some workers and the results showed that Schistosoma Hemozoin may play important roles in pathogenicity, immune modulation, iron supply for egg formation, and interaction with some anti-schistosomal drugs. In this review, we briefly review and discuss the hematophagous habit of schistosomes, degradation of haemoglobin, formation of Hemozoin in the worm gut, and possible roles of Hemozoin.
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Organism-like formation of Schistosoma Hemozoin and its function suggest a mechanism for anti-malarial action of artemisinin
Scientific reports, 2016Co-Authors: Jun Sun, Su-wen WangAbstract:The current theories of antimalarial mechanism of artemisinin are inadequate to fully explain the observed effects. In our study, “organism-like” formation of Schistosoma Hemozoin granules by attaching to and utilizing erythrocytes to form new ones was observed. This indicates that heme iron is transferred from erythrocytes to Hemozoin granules during their formation. However, as a disposal product of heme detoxification, these granules are not completely expelled from the Schistosoma gut, but decomposed again between microvilli in the posterior portion of the gut to transfer iron to eggs. Based on the function of iron transport supported by our observation of the unique process of Schistosoma Hemozoin formation, here we propose a new viewpoint of antimalarial mechanism of artemisinin, which emphasizes the final outcome, i.e., interference of iron utilization in parasites by artemisinin, instead of focusing on the mode of interaction between artemisinin and heme or Hemozoin. This suggests that artemisinin and its endoperoxides derivatives likely hit the Achilles’ heel of Hemozoin-producing and iron-dependent organisms.
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Can Hemozoin alone cause host anaemia
Parasitology research, 2016Co-Authors: Jun Sun, Su-wen Wang, Chang-long Jin, Xiao-li Zeng, Xing-yu Piao, Ling Bai, Dan-li TangAbstract:Both schistosomes and malaria parasites produce Hemozoin and cause host anaemia. However, the relationship between anaemia and Hemozoin is unclear. Although some studies have proposed that Hemozoin is related to anaemia in malaria patients, whether Hemozoin alone can cause anaemia in patients infected by malaria parasites or schistosomes is uncertain. To investigate the effect of Hemozoin on hosts, β-haematin was injected intravenously to normal mice. Then, liver and spleen tissues were observed. Mouse blood was examined. Red blood cells (RBCs), white blood cells (WBCs) and haemoglobin were analysed. Macrophage changes in the spleens and marrow cells were compared using immunofluorescence and H&E or Giemsa stain, respectively. We found that after 15 injections of β-haematin, a large amount of β-haematin was observed to deposit in the livers and spleens. Splenomegaly and bone marrow mild hyperplasia were detected. The average number of RBCs, average number of WBCs and average concentration of haemoglobin decreased significantly from 9.36 × 1012 cells/L to 8.7 × 1012 cells/L, 3.8 × 109 cells/L to 1.7 × 109 cells/L and 142.8 g/L to 131.8 g/L, respectively. In specific, the number of macrophages in the spleens greatly increased after β-haematin infection. The results showed that injections of β-haematin alone can cause anaemia possibly through hypersplenism.
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Conditional inhibition of Hemozoin formation by chloroquine in vitro
Zhongguo ji sheng chong xue yu ji sheng chong bing za zhi = Chinese journal of parasitology & parasitic diseases, 2009Co-Authors: Jun Sun, Bo Chen, Yan-yu LongAbstract:To study the characteristics of inhibition on Hemozoin formation by chloroquine under in vitro condition. Under different concentrations (0.5-2 mol/L) of sodium acetate (NaAc) and at the pH range of 4.0-5.0, chloroquine was tested for inhibition of beta-hematin (hemozion) formation by using the HPIA (heme polymerization inhibitory activity) assay. The morphology of beta-hematin crystals was determined by light microscopy. Ultraviolet spectrophotometry was employed to measure beta-hematin content, and the size of beta-hematin crystal was analyzed by X-ray diffraction (XRD). Chloroquine exhibited varied effect on beta-hematin formation, depending on pH value and Na+ concentration. When the NaAc concentration increased from 0.5 mol/L (pH 4.2) to 2 mol/L (pH 4.8), the chloroquine inhibitory effect also increased. Results suggested that there exists a threshold pH, below which the beta-hematin formation escalates and chloroquine inhibition declines, and at or above which chloroquine exerts a stronger inhibitory effect on beta-hematin formation. With the increase of pH from 4.4 to 4.8, the crystallinity and the size of crystal changed from 6.93% and 357 angstrom to 6.32% and 264 angstrom, respectively. When pH reached to 5, no more beta-hematin formed. Chloroquine could reduce the crystallinity and crystal size of beta-hematin at same pH value. Morphology analysis on the samples was consistent with the above results. Chloroquine inhibits Hemozoin formation only when the pH value is at or above threshold pH.
Babu L. Tekwani - One of the best experts on this subject based on the ideXlab platform.
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Targeting the Hemozoin Synthesis Pathway for New Antimalarial Drug Discovery: Technologies for In Vitro β-Hematin Formation Assay
Combinatorial chemistry & high throughput screening, 2005Co-Authors: Babu L. Tekwani, Larry A. WalkerAbstract:Clinical manifestations of malaria primarily result from proliferation of the parasite within the hosts' erythrocytes. During this process, hemoglobin is utilized as the predominant source of nutrition. The malaria parasite digests hemoglobin within the digestive vacuole through a sequential metabolic process involving multiple proteases. Massive degradation of hemoglobin generates large amount of toxic heme. Malaria parasite, however, has evolved a distinct mechanism for detoxification of heme through its conversion into an insoluble crystalline pigment, known as Hemozoin. Hemozoin is identical to beta-hematin, which is constituted of cyclic heme dimers arranged in an ordered crystalline structure through intermolecular hydrogen bonding. The exact mechanism of biogenesis of Hemozoin in malaria is still obscure and is the subject of intense debate. Hemozoin synthesis is an indispensable process for the parasite and is the target for action of several known antimalarials. The pathway has therefore attracted significant interest for new antimalarial drug discovery research. Formation of beta-hematin may be achieved in vitro under specific chemical and physiochemical conditions through a biocrystallization process. Based on these methods several experimental approaches have been described for the assay of formation of beta-hematin in vitro and screening of compounds as inhibitors of Hemozoin synthesis. These assays are primarily based on differential solubility and spectral characteristics of monomeric heme and beta-hematin. Different factors viz., the malaria parasite lysate, lipids extracts, preformed beta-hematin, malarial histidine rich protein II and some unsaturated lipids have been employed for promoting beta-hematin formation in these assays. The assays based on spectrophotometric quantification of beta-hematin or incorporation of (14)C-heme yield reproducible results and have been applied to high throughput screening. Several novel antimalarial pharmacophores have been discovered through these assays.
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Spectrophotometric determination of de novo Hemozoin/β-hematin formation in an in vitro assay
Analytical biochemistry, 2004Co-Authors: Abhai K. Tripathi, Shabana I. Khan, Larry A. Walker, Babu L. TekwaniAbstract:Formation of Hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of Hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to Hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo Hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of Hemozoin formation as potential blood schizontocidal antimalarials, has been developed.
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spectrophotometric determination of de novo Hemozoin β hematin formation in an in vitro assay
Analytical Biochemistry, 2004Co-Authors: Abhai K. Tripathi, Shabana I. Khan, Larry A. Walker, Babu L. TekwaniAbstract:Formation of Hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of Hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to Hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo Hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of Hemozoin formation as potential blood schizontocidal antimalarials, has been developed.
Torsten Frosch - One of the best experts on this subject based on the ideXlab platform.
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Morphology-sensitive Raman modes of the malaria pigment Hemozoin.
The Analyst, 2009Co-Authors: Torsten Frosch, Sasa Koncarevic, Katja Becker, Jürgen PoppAbstract:Resonance Raman spectroscopy was applied for investigating the malaria pigment Hemozoin, which is an important target structure of antimalarialdrugs. Morphology-sensitive low wavenumber modes of Hemozoin were selectively enhanced with help of excitation wavelengths at λ = 633 nm and λ = 647 nm. The assignment of the most prominent bands in the Raman spectra at 343 cm−1 and 368 cm−1 was assisted by DFT calculations of the Hemozoin dimer. The mode at 343 cm−1 in the Raman spectrum of Hemozoin is strongly enhanced with λexc. = 647 nm and is represented by a combined, symmetric doming mode of the two hematin units in the Hemozoin dimer. The enhancement of this vibration is stronger in the resonance Raman spectrum of Hemozoin compared with less crystalline β-hematin. The selective resonance enhancement of the morphology-sensitive Raman modes of Hemozoin is caused by absorption bands in the UV-VIS-NIR spectrum. This absorption spectrum of the crystalline malaria pigment Hemozoin shows a strong band at 655 nm. Another broad absorption band at 870 nm is the reason for the strong relative resonance enhancement of the mode at 1372 cm−1 in the Raman spectrum of crystalline Hemozoin with λexc. = 830 nm. In conclusion, resonance Raman micro-spectroscopy with λexc. = 647 nm was shown to have great potential as an analytical tool to probe the morphology of hematin samples.
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in situ localization and structural analysis of the malaria pigment Hemozoin
Journal of Physical Chemistry B, 2007Co-Authors: Torsten Frosch, Sasa Koncarevic, Katja Becker, Linda Zedler, Michael Schmitt, Karla Schenzel, Jiirgen PoppAbstract:Raman microspectroscopy was applied for an in situ localization of the malaria pigment Hemozoin in Plasmodium falciparum-infected erythrocytes. The Raman spectra (λexc = 633 nm) of Hemozoin show very intense signals with a very good signal-to-noise ratio. These in situ Raman signals of Hemozoin were compared to Raman spectra of extracted Hemozoin, of the synthetic analogue β-hematin, and of hematin and hemin. β-Hematin was synthesized according to the acid-catalyzed dehydration of hematin and the anhydrous dehydrohalogenation of hemin which lead to good crystals with lengths of about 5−30 μm. The Raman spectra (λexc = 1064 nm) of Hemozoin and β-hematin show almost identical behaviors, while some low wavenumber modes might be used to distinguish between the morphology of differently synthesized β-hematin samples. The intensity pattern of the resonance Raman spectra (λexc = 568 nm) of Hemozoin and β-hematin differ significantly from those of hematin and hemin. The most striking difference is an additional b...
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In situ localization and structural analysis of the malaria pigment Hemozoin.
The journal of physical chemistry. B, 2007Co-Authors: Torsten Frosch, Sasa Koncarevic, Katja Becker, Linda Zedler, Michael Schmitt, Karla Schenzel, Jiirgen PoppAbstract:Raman microspectroscopy was applied for an in situ localization of the malaria pigment Hemozoin in Plasmodium falciparum-infected erythrocytes. The Raman spectra (lambdaexc=633 nm) of Hemozoin show very intense signals with a very good signal-to-noise ratio. These in situ Raman signals of Hemozoin were compared to Raman spectra of extracted Hemozoin, of the synthetic analogue beta-hematin, and of hematin and hemin. beta-Hematin was synthesized according to the acid-catalyzed dehydration of hematin and the anhydrous dehydrohalogenation of hemin which lead to good crystals with lengths of about 5-30 microm. The Raman spectra (lambdaexc=1064 nm) of Hemozoin and beta-hematin show almost identical behaviors, while some low wavenumber modes might be used to distinguish between the morphology of differently synthesized beta-hematin samples. The intensity pattern of the resonance Raman spectra (lambdaexc=568 nm) of Hemozoin and beta-hematin differ significantly from those of hematin and hemin. The most striking difference is an additional band at 1655 cm(-1) which was only observed in the spectra of Hemozoin and beta-hematin and cannot be seen in the spectra of hematin and hemin. Raman spectra of the beta-hematin dimer were calculated ab initio (DFT) for the first time and used for an assignment of the experimentally derived Raman bands. The calculated atomic displacements provide valuable insight into the most important molecular vibrations of the Hemozoin dimer. With help from these DFT calculations, it was possible to assign the Raman band at 1655 cm(-1) to a mode located at the propionic acid side chain, which links the Hemozoin dimers to each other. The Raman band at 1568 cm(-1), which has been shown to be influenced by an attachment of the antimalarial drug chloroquine in an earlier study, could be assigned to a C=C stretching mode spread across one of the porphyrin rings and is therefore expected to be influenced by a pi-pi-stacking to the drug.
Jiirgen Popp - One of the best experts on this subject based on the ideXlab platform.
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in situ localization and structural analysis of the malaria pigment Hemozoin
Journal of Physical Chemistry B, 2007Co-Authors: Torsten Frosch, Sasa Koncarevic, Katja Becker, Linda Zedler, Michael Schmitt, Karla Schenzel, Jiirgen PoppAbstract:Raman microspectroscopy was applied for an in situ localization of the malaria pigment Hemozoin in Plasmodium falciparum-infected erythrocytes. The Raman spectra (λexc = 633 nm) of Hemozoin show very intense signals with a very good signal-to-noise ratio. These in situ Raman signals of Hemozoin were compared to Raman spectra of extracted Hemozoin, of the synthetic analogue β-hematin, and of hematin and hemin. β-Hematin was synthesized according to the acid-catalyzed dehydration of hematin and the anhydrous dehydrohalogenation of hemin which lead to good crystals with lengths of about 5−30 μm. The Raman spectra (λexc = 1064 nm) of Hemozoin and β-hematin show almost identical behaviors, while some low wavenumber modes might be used to distinguish between the morphology of differently synthesized β-hematin samples. The intensity pattern of the resonance Raman spectra (λexc = 568 nm) of Hemozoin and β-hematin differ significantly from those of hematin and hemin. The most striking difference is an additional b...
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In situ localization and structural analysis of the malaria pigment Hemozoin.
The journal of physical chemistry. B, 2007Co-Authors: Torsten Frosch, Sasa Koncarevic, Katja Becker, Linda Zedler, Michael Schmitt, Karla Schenzel, Jiirgen PoppAbstract:Raman microspectroscopy was applied for an in situ localization of the malaria pigment Hemozoin in Plasmodium falciparum-infected erythrocytes. The Raman spectra (lambdaexc=633 nm) of Hemozoin show very intense signals with a very good signal-to-noise ratio. These in situ Raman signals of Hemozoin were compared to Raman spectra of extracted Hemozoin, of the synthetic analogue beta-hematin, and of hematin and hemin. beta-Hematin was synthesized according to the acid-catalyzed dehydration of hematin and the anhydrous dehydrohalogenation of hemin which lead to good crystals with lengths of about 5-30 microm. The Raman spectra (lambdaexc=1064 nm) of Hemozoin and beta-hematin show almost identical behaviors, while some low wavenumber modes might be used to distinguish between the morphology of differently synthesized beta-hematin samples. The intensity pattern of the resonance Raman spectra (lambdaexc=568 nm) of Hemozoin and beta-hematin differ significantly from those of hematin and hemin. The most striking difference is an additional band at 1655 cm(-1) which was only observed in the spectra of Hemozoin and beta-hematin and cannot be seen in the spectra of hematin and hemin. Raman spectra of the beta-hematin dimer were calculated ab initio (DFT) for the first time and used for an assignment of the experimentally derived Raman bands. The calculated atomic displacements provide valuable insight into the most important molecular vibrations of the Hemozoin dimer. With help from these DFT calculations, it was possible to assign the Raman band at 1655 cm(-1) to a mode located at the propionic acid side chain, which links the Hemozoin dimers to each other. The Raman band at 1568 cm(-1), which has been shown to be influenced by an attachment of the antimalarial drug chloroquine in an earlier study, could be assigned to a C=C stretching mode spread across one of the porphyrin rings and is therefore expected to be influenced by a pi-pi-stacking to the drug.
István Kézsmárki - One of the best experts on this subject based on the ideXlab platform.
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Rapid and quantitative antimalarial drug efficacy testing via the magneto-optical detection of Hemozoin.
Scientific reports, 2020Co-Authors: Petra Molnár, Ágnes Orbán, Ádám Butykai, Lívia Marton, Beáta G. Vértessy, Richard Izrael, Réka Babai, Stephan Karl, István KézsmárkiAbstract:Emergence of resistant Plasmodium species makes drug efficacy testing a crucial part of malaria control. Here we describe a novel assay for sensitive, fast and simple drug screening via the magneto-optical detection of Hemozoin, a natural biomarker formed during the hemoglobin metabolism of Plasmodium species. By quantifying Hemozoin production over the intraerythrocytic cycle, we reveal that Hemozoin formation is already initiated by ~ 6-12 h old ring-stage parasites. We demonstrate that the new assay is capable of drug efficacy testing with incubation times as short as 6-10 h, using synchronized P. falciparum 3D7 cultures incubated with chloroquine, piperaquine and dihydroartemisinin. The determined 50% inhibitory concentrations agree well with values established by standard assays requiring significantly longer testing time. Accordingly, we conclude that magneto-optical Hemozoin detection provides a practical approach for the quick assessment of drug effect with short incubation times, which may also facilitate stage-specific assessment of drug inhibitory effects.
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Highly Sensitive and Rapid Characterization of the Development of Synchronized Blood Stage Malaria Parasites Via Magneto-Optical Hemozoin Quantification.
Biomolecules, 2019Co-Authors: Mária Pukáncsik, Petra Molnár, Ágnes Orbán, Ádám Butykai, Lívia Marton, István Kézsmárki, Beáta G. Vértessy, Mohd Kamil, Amanah Abraham, Ahmed S. I. AlyAbstract:The rotating-crystal magneto-optical diagnostic (RMOD) technique was developed as a sensitive and rapid platform for malaria diagnosis. Herein, we report a detailed in vivo assessment of the synchronized Plasmodium vinckei lentum strain blood-stage infections by the RMOD method and comparing the results to the unsynchronized Plasmodium yoelii 17X-NL (non-lethal) infections. Furthermore, we assess the Hemozoin production and clearance dynamics in chloroquine-treated compared to untreated self-resolving infections by RMOD. The findings of the study suggest that the RMOD signal is directly proportional to the Hemozoin content and closely follows the actual parasitemia level. The lack of long-term accumulation of Hemozoin in peripheral blood implies a dynamic equilibrium between the Hemozoin production rate of the parasites and the immune system's clearing mechanism. Using parasites with synchronous blood stage cycle, which resemble human malaria parasite infections with Plasmodium falciparum and Plasmodium vivax, we are demonstrating that the RMOD detects both Hemozoin production and clearance rates with high sensitivity and temporal resolution. Thus, RMOD technique offers a quantitative tool to follow the maturation of the malaria parasites even on sub-cycle timescales.