| Jin-Hee Han | 14 Articles |
Clonorchis sinensis is a liver fluke that causes clonorchiasis, a significant public health concern in East Asia, closely associated with hepatobiliary diseases. Dopamine is an essential neurotransmitter involved in neuromuscular signaling, and its uptake by trematodes may contribute to parasite physiology and survival. This study aimed to characterize the dopamine transporter CsDAT in C. sinensis by synthesizing cDNA from adult worms and expressing it in Xenopus laevis oocytes; subsequently, uptake assays were conducted using radiolabeled dopamine. Functional assays confirmed that CsDAT mediates dopamine uptake in a sodium-dependent manner. The uptake was saturable and exhibited Michaelis-Menten kinetics with a Michaelis constant of 454.5 nM and a maximum uptake rate of 1,422.5 fmol/oocyte/h. CsDAT efficiently transported dopamine with high affinity, indicating its physiological relevance in the parasite. A 3-dimensional model of CsDAT was constructed to examine its structural features. The predicted structure contained a conserved substrate-binding pocket similar to that of other known neurotransmitter transporters. Molecular docking simulations showed that dopamine stably fits within the binding pocket. The key amino acid residues formed hydrogen bonds and hydrophobic interactions with dopamine. Interestingly, dopamine and several inhibitors demonstrated higher binding affinity to CsDAT than the human dopamine transporter. This study provides the first functional and structural insights into CsDAT. The higher inhibitor-binding affinity of CsDAT compared to human dopamine transporter suggests its potential for use in therapeutic exploration. Targeting CsDAT may facilitate the development of new therapeutic agents against clonorchiasis with minimal off-target effects on the human nervous system.
Baseline genetic characterization of malaria vector populations provides critical data for evidence-based surveillance in persistent transmission foci. This pilot study generated preliminary genetic baseline data for Anopheles populations in the Menoreh Hills border region between Central Java and Yogyakarta provinces, Indonesia, addressing a key geographic gap in regional vector research. Adult female mosquitoes were collected from 3 houses with reported malaria cases in Ngadirejo Village using standardized entomological methods, including human landing, animal landing, and resting collections. Specimens were morphologically identified and molecularly characterized via ITS2 gene sequencing. Phylogenetic analyses were assessed using maximum likelihood methods, and genetic diversity indices were calculated to examine population structure. A total of 62 specimens representing 3 species were collected exclusively through animal landing collections: Anopheles vagus (48 specimens, 77.4%), Anopheles maculatus (9 specimens, 14.5%), and Anopheles kochi (5 specimens, 8.1%). An. kochi exhibited high haplotype diversity (Hd=0.709) with low nucleotide diversity (π=0.004), while An. maculatus showed lower haplotype diversity (Hd=0.480) and higher nucleotide diversity (π=0.026). Phylogenetic analysis revealed Purworejo specimens clustered with regional populations: An. kochi grouped within Clade I with Indonesian isolates; An. maculatus distributed across multiple clades; An. vagus formed a cohesive unit with other Indonesian populations. The exclusive success of animal landing collections in the Menoreh Hills highlands provides key methodological insights. This study offers essential baseline reference data, validates cost-effective genetic surveillance approaches, and supports future large-scale population connectivity studies across the Menoreh Hills malaria transmission complex.
As many countries implement different programs aimed at eliminating malaria, attention should be given to asymptomatic carriers that may interrupt the progress. This was a community-based cross-sectional study conducted in Tanzania from December 2022 to July 2023 within 4 villages from each of the 3 regions, Geita and Kigoma, which are high malaria transmission, and Arusha, which is low transmission. Malaria was diagnosed in asymptomatic individuals aged 1 year and older using the malaria rapid diagnostic test and light microscope. A total of 2,365 of 3,489 (67.9%) participants were enrolled from high-transmission villages. The overall prevalence was 25.5% and 15.8% by malaria rapid diagnostic test and light microscope, respectively. Using the respective tools, the prevalence was significantly higher at 35.6% (confidence interval (CI)=23.6–49.9) and 23.1% (CI=16.2–35.1) in the high-transmission regions (Geita and Kigoma) compared with 2.9% (CI=1.1–3.5) and 1.1% (CI=0.7–1.8) in the low-transmission region (Arusha). Children younger than 15 years and males accounted for the greatest proportion of infections. In the study area, the prevalence of asymptomatic cases was higher than that of reported symptomatic cases in health facilities. We hypothesize that these parasite reservoirs may contribute to the persistence of malaria in the country. Therefore, to achieve comprehensive malaria control in the country, the surveillance and screening of asymptomatic malaria cases are vital.
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Clonorchis sinensis, which causes clonorchiasis, is prevalent in East Asian countries and poses notable health risks, including bile duct complications. Although praziquantel is the primary treatment for the disease, the emerging resistance among trematodes highlights the need for alternative strategies. Understanding the nutrient uptake mechanisms in trematodes, including C. sinensis, is crucial for developing future effective treatments. This study aimed to characterize the function of C. sinensis glucose transporter 4 (CsGTP4) and determine its role in nutrient uptake employing synthesized cDNA of adult C. sinensis worms. The functional characterization of CsGTP4 involved injecting its cRNA into Xenopus laevis oocytes and analyzing the deoxy-D-glucose uptake levels. The results demonstrated that deoxy-D-glucose uptake depended on the deoxy-D-glucose incubation and CsGTP4 expression time, but not sodium-dependent. The concentration-dependent uptake followed the Michaelis–Menten equation, with a Km value of 2.7 mM and a Vmax value of 476 pmol/oocyte/h based on the Lineweaver–Burk analysis. No uptake of radiolabeled α-ketoglutarate, p-aminohippurate, taurocholate, arginine, or carnitine was observed. The uptake of deoxy-D-glucose by CsGTP4 was significantly inhibited by unlabeled glucose and galactose in a concentration-dependent manner. It was significantly inhibited under strongly acidic and basic conditions. These insights into the glucose uptake kinetics and pH dependency of CsGTP4 provide a deeper understanding of nutrient acquisition in trematodes. This study contributes to the development of novel antiparasitic agents, addressing a considerable socioeconomic challenge in affected regions.
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Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis–Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 μM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.
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Plasmodium vivax exhibits dormant liver-stage parasites, called hypnozoites, which can cause relapse of malaria. The only drug currently used for eliminating hypnozoites is primaquine. The antimalarial properties of primaquine are dependent on the production of oxidized metabolites by the cytochrome P450 isoenzyme 2D6 (CYP2D6). Reduced primaquine metabolism may be related to P. vivax relapses. We describe a case of 4 episodes of recurrence of vivax malaria in a patient with decreased CYP2D6 function. The patient was 52-year-old male with body weight of 52 kg. He received total gastrectomy and splenectomy 7 months before the first episode and was under chemotherapy for the gastric cancer. The first episode occurred in March 2019 and each episode had intervals of 34, 41, and 97 days, respectively. At the first and second episodes, primaquine was administered as 15 mg for 14 days. The primaquine dose was increased with 30 mg for 14 days at the third and fourth episodes. Seven gene sequences of P. vivax were analyzed and revealed totally identical for all the 4 samples. The CYP2D6 genotype was analyzed and intermediate metabolizer phenotype with decreased function was identified.
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The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2-6.7% for Plasmodium knowlesi and 0.3-4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson’s correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r2=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.
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Plasmodium vivax reemerged in 1993. It has been sustained for more than 25 years and become one of the important indigenous parasitic diseases in northern and western parts of the Republic of Korea near the demilitarized zone. In particular, relapse is a significant concern for the control of malaria, as short- and long-term incubation periods vary among those infected in Korea. In this study, the prevalence of asymptomatic carriers was examined among residents of high endemic areas of vivax malaria during nonseasonal transmission of mosquitoes. Blood samples from 3 endemic regions in northwestern Korea were evaluated by microscopic examination, rapid diagnostic testing, and nested PCR to identify asymptomatic patients carrying malaria parasites in the community. However, no positive malaria case among residents of endemic areas was detected. Additionally, serological analysis was carried out to measure antibodies against 3 antigenic recombinant proteins of P. vivax, merozoite surface protein 1-19, circumsporozoite surface protein-VK210, and liver-stage antigen (PvLSA-N), by the protein array method. Interestingly, seropositivity of sera between previous exposure and samples without exposure to malaria was significantly higher using the PvLSA-N antigen than the other antigens, suggesting that PvLSA-N can be used as a serological marker to analyze the degree of exposure for malaria transmission in endemic areas. This indicates a very low asymptomatic carrier prevalence during the nonmalaria season in the endemic areas of Korea.
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Plasmodium lactate dehydrogenase (pLDH) is a strong target antigen for the determination of infection with Plasmodium species specifically. However, a more effective antibody is needed because of the low sensitivity of the current antibody in many immunological diagnostic assays. In this study, recombinant Plasmodium vivax LDH (PvLDH) was experimentally constructed and expressed as a native antigen to develop an effective P. vivax-specific monoclonal antibody (mAb). Two mAbs (2CF5 and 1G10) were tested using ELISA and immunofluorescence assays (IFA), as both demonstrated reactivity against pLDH antigen. Of the 2 antibodies, 2CF5 was not able to detect P. falciparum, suggesting that it might possess P. vivax-specificity. The detection limit for a pair of 2 mAbs-linked sandwich ELISA was 31.3 ng/ml of the recombinant antigen. The P. vivax-specific performance of mAbs-linked ELISA was confirmed by in vitro-cultured P. falciparum and P. vivax-infected patient blood samples. In conclusion, the 2 new antibodies possessed the potential to detect P. vivax and will be useful in immunoassay.
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Plasmodium vivax produces numerous caveola-vesicle complex (CVC) structures beneath the membrane of infected erythrocytes. Recently, a member helical interspersed subtelomeric (PHIST) superfamily protein, PcyPHIST/CVC-8195, was identified as CVCs-associated protein in Plasmodium cynomolgi and essential for survival of this parasite. Very little information has been documented to date about PHIST/CVC-8195 protein in P. vivax. In this study, the recombinant PvPHIST/CVC-8195 N and C termini were expressed, and immunoreactivity was assessed using confirmed vivax malaria patients sera by protein microarray. The subcellular localization of PvPHIST/CVC-8195 N and C termini in blood stage parasites was also determined. The antigenicity of recombinant PvPHIST/CVC-8195 N and C terminal proteins were analyzed by using serum samples from the Republic of Korea. The results showed that immunoreactivities to these proteins had 61% and 43% sensitivity and 96.9% and 93.8% specificity, respectively. The N terminal of PvPHIST/CVC-8195 which contains transmembrane domain and export motif (PEXEL; RxLxE/Q/D) produced CVCs location throughout the erythrocytic-stage parasites. However, no fluorescence was detected with antibodies against C terminal fragment of PvPHIST/CVC-8195. These results suggest that the PvPHIST/CVC-8195 is localized on the CVCs and may be immunogenic in natural infection of P. vivax.
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The discovery and understanding of antigenic proteins are essential for development of a vaccine against malaria. In Plasmodium falciparum, Pf92 have been characterized as a merozoite surface protein, and this protein is expressed at the late schizont stage, but no study of Pv92, the orthologue of Pf92 in P. vivax, has been reported. Thus, the protein structure of Pv92 was analyzed, and the gene sequence was aligned with that of other Plasmodium spp. using bioinformatics tools. The recombinant Pv92 protein was expressed and purified using bacterial expression system and used for immunization of mice to gain the polyclonal antibody and for evaluation of antigenicity by protein array. Also, the antibody against Pv92 was used for subcellular analysis by immunofluorescence assay. The Pv92 protein has a signal peptide and a sexual stage s48/45 domain, and the cysteine residues at the N-terminal of Pv92 were completely conserved. The N-terminal of Pv92 was successfully expressed as soluble form using a bacterial expression system. The antibody raised against Pv92 recognized the parasites and completely merged with PvMSP1-19, indicating that Pv92 was localized on the merozoite surface. Evaluation of the human humoral immune response to Pv92 indicated moderate antigenicity, with 65% sensitivity and 95% specificity by protein array. Taken together, the merozoite surface localization and antigenicity of Pv92 implicate that it might be involved in attachment and invasion of a merozoite to a new host cell or immune evasion during invasion process.
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In the era of (pre) elimination setting, the prevalence of malaria has been decreasing in most of the previously endemic areas. Therefore, effective cost- and time-saving validated pooling strategy is needed for detection of malaria in low transmission settings. In this study, optimal pooling numbers and lowest detection limit were assessed using known density samples prepared systematically, followed by genomic DNA extraction and nested PCR. Pooling strategy that composed of 10 samples in 1 pool, 20 ?l in 1 sample, was optimal, and the parasite density as low as 2 p/?l for both falciparum and vivax infection was enough for detection of malaria. This pooling method showed effectiveness for handling of a huge number of samples in low transmission settings (<9% positive rate). The results indicated that pooling of the blood samples before DNA extraction followed by usual nested PCR is useful and effective for detection of malaria in screening of hidden cases in low-transmission settings.
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Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.
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The study of Plasmodium falciparum intraerythrocytic developmental cycle relies on synchronized parasite cultures. This study aimed to quantitatively evaluate 4 common synchronization methods for the culture of P. falciparum 3D7, incorporating a novel nanomaterial-based tool to
objective ly assess late-stage parasite enrichment. The 4 methods including 5% D-sorbitol, isosmotic Percoll-Sorbitol (60% PS), discontinuous Percoll-Sorbitol gradients (70%–40% PS), and the Plasmion method. Synchronization efficiency was assessed through microscopic examination and flow cytometry. In addition to SYBR Green I as the stain for flow cytometry, we innovatively employed 20 nm polystyrene fluorescent microspheres (PFMs20), which bind to parasites following the production of new permeability pathways, to provide an objective validating the enrichment of mature stages. The binding rate was defined as the ratio of PFMs20 positivity to SYBR Green I positivity. Microscopic examination of Giemsa-stained blood smears served as the gold standard for assessing the stage-specific profiles achieved by each synchronization method. The 4 methods yielded distinct efficiency profiles: 5% D-sorbitol achieved 88.37% ring-stage purity; 60% PS demonstrated high late-stage enrichment with PFMs20 binding rates reaching 87.47%; 70%–40% PS uniquely enabled visible hemozoin-rich layer separation and showed 88.12% binding efficiency in its schizont-enriched fraction; while Plasmion yielded a binding rate of 73.37% in its late-stage fraction. Our results formulate a clear decision matrix for synchronization protocol selection. The sorbitol method is ideal for basic ring-stage studies, Percoll-based gradients support rigorous stage-specific investigations, and the Plasmion method is well-suited for resource-limited settings. This comparative analysis provides preliminary guidelines for synchronization method selection that require validation in diverse laboratory and field contexts.
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