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ABSTRACT Entamoeba histolytica is an enteric protozoan parasite that causes human amebic colitis and extraintestinal abscesses. As a prerequisite for parasite colonization and invasion, adherence of E. histolytica is predominantly mediated by galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-inhibitable lectins. The intermediate subunit (Igl) of Gal-/GalNAc-inhibitable lectin is a cysteine-rich protein containing multiple CXXC motifs and is considered a key factor affecting trophozoite’s pathogenicity. However, details of the function of Igl during parasite adherence remain unclear. Here, using segmentally expressed Igl proteins and a CHO cell model transfected with Igl fragments, we identified a carbohydrate-recognition domain (CRD)-like region between amino acids 989 and 1,088. Through single- and double-point mutations in the Igl segments, two core CXXC motifs responsible for carbohydrate recognition in the CRD-like region, which are highly conserved among several lectins, were confirmed. In addition to adhesion, the roles of CRD-like region and its core CXXC motifs in various pathogenic effects were further explored. To our knowledge, this is the first report showing an adhesion-related region in E. histolytica Igl. The identification and characterization of this CRD-like region provides further insights into molecular mechanisms underlying E. histolytica pathogenicity and also aids in the determination of a potential drug target in this parasite.IMPORTANCEEntamoeba histolytica adhesion mainly depends on galactose (Gal)-/N-acetyl-d-galactosamine (GalNAc)-inhibitable lectins, subsequently triggering a series of amebic reactions. Among the three subunits of Gal-/GalNAc-inhibitable lectin, heavy subunit and intermediate subunit (Igl) have exhibited lectin activity, but that of Igl remains poorly understood. In this study, we confirmed a carbohydrate-recognition domain (CRD)-like limiting region in E. histolytica Igl and further identified its two core CXXC motifs responsible for carbohydrate recognition. Moreover, the role of Igl's CRD-like region and its CXXC motifs in hemolysis and pathogenic effects was explored. This is the first study to determine an adhesion-related region in E. histolytica Igl protein, providing a new reference direction for subsequent research studies. Since the potential homogeneity of galectin-2 in several mammals and Igl CRD-like region, it could be meaningful to relate the corresponding pathogeneses and phenotypes of these two proteins. Except for adhesion, studies on the involvement of Igl CRD-like region in different parasite–host interactions are also promising.

Abstract Merozoite surface protein 3 of Plasmodium vivax (PvMSP3) contains a repertoire of protein members with unique sequence organization. While the biological functions of these proteins await elucidation, PvMSP3 has been suggested to be potential vaccine targets. To date, studies on natural immune responses to this protein family have been confined to two members, PvMSP3α and PvMSP3β. This study analyzed natural IgG antibody responses to PvMSP3γ recombinant proteins derived from two variants: one containing insert blocks (CT1230nF) and the other without insert domain (NR25nF). The former variant was also expressed as two subfragment proteins: one encompassing variable domain I and insert block A (CT1230N) and the other spanning from insert block B to conserved block III (CT1230C). Serum samples were obtained from 246 symptomatic vivax malaria patients in Tak (n = 50) and Ubon Ratchathani (n = 196) Provinces. In total, 176 (71.5%) patients could mount antibodies to at least one recombinant PvMSP3γ antigen. IgG antibodies directed against antigens CT1230nF, CT1230N, CT1230C and NR25nF occurred in 96.6%, 61.4%, 71.6% and 68.2% of samples, respectively, suggesting the widespread occurrence of B-cell epitopes across PvMSP3γ. The rates of seropositivity seemed to correlate with the number of previous malaria episodes. Isotype analysis of anti-PvMSP3γ antibodies has shown predominant cytophilic subclass responses, accounting for 75.4–81.7% for IgG1 and 63.6–77.5% for IgG3. Comparing with previous studies in the same cohort, the numbers of serum samples reactive to antigens derived from P. vivax merozoite surface protein 9 (PvMSP9) and thrombospondin-related anonymous protein (PvTRAP) were higher than those to PvMSP3γ, being 92.7% and 87.0% versus 71.5%, respectively. Three (1.22%) serum samples were nonresponsive to all these malarial proteins. Nevertheless, the relevance of naturally acquired antibodies to PvMSP3γ in host protection requires further studies.

The photodecomposition behavior of cationic porphyrin ZnTMAP4+ (zinc tetrakis-(N,N,N-trimethylanilinium-4-yl) porphyrin) in water and complexed with clay nanosheets was investigated by light irradiation to the Soret band of ZnTMAP4+. The decomposition of ZnTMAP4+ was observed by UV–visible absorption spectroscopy. While the decomposition quantum yield (ϕdec) was 3.4 × 10−4 in water, that was 9.4 × 10−7 on the exfoliated clay nanosheets. It was revealed that the photostability of ZnTMAP4+ was stabilized by the complex formation with clay. When ZnTMAP4+ was intercalated between the stacked clay nanosheets, ϕdec was further decreased to 4.9 × 10−7. The photostability increased by 361 times and 693 times for the exfoliated and stacked state, respectively. These results indicate that the flat clay surface has the potential to control intra- and intermolecular photochemical reactions.

Abstract In aging and aged countries, one of the main issues is to establish sufficient nursing homes for seniors. Japan, as one of the most rapidly aged countries, has been reforming nursing homes for the elderly since the 1990s and established a long‐term care insurance system. The purpose of this scoping review is to systematically map the evolution of researches for nursing homes in Japan since the 1990s, particularly with the post‐occupancy evaluation of the introduction of private rooms and small units. Forty‐three articles concerning privatization of rooms, shared spaces, and small unitization in nursing homes were extracted and analyzed. They were published between 1994 and 2021 in Journal of Architecture and Planning, AIJ Journal of Technology, JAABE, and JAR. The results revealed an evaluation of the privatized rooms mainly from the perspectives of residents' lives and care staffing, the effects and issues of the shared spaces introduced at the same time as private rooms, and the evaluation and issues on subsequent unit‐type floor plans. This study offers significant implications to Asian countries that will have aging and aged populations in the near future by summarizing Japan's experience in dealing with realistic and practical problems over the past 20 years.

Entamoeba histolytica is a parasitic protozoan with roles in pathogenicity of intestinal amoebiasis. E. histolytica trophozoites lack functional mitochondria and their energy production depends mostly on glycolysis. D-Glucose has a pivotal role in this process and trophozoites store this sugar as glycogen in glycogen granules. Rare sugars, which are defined as sugars present in nature in limited amounts, are of interest as natural low-calorie sweeteners for improving physical conditions of humans. One such rare sugar, D-allose, can be absorbed by a sodium-dependent glucose cotransporter as a substitute for D-glucose, and some rare sugars are known to inhibit growth of cancer cells, Caenorhabditis elegans and Tritrichomonas foetus. Based on these observations, we examined the effects of rare sugars on growth of E. histolytica trophozoites, together with those of D-galactose and D-fructose. The results indicate that treatment with D-allose or D-psicose (D-allulose) alone inhibits proliferation of E. histolytica trophozoites, but that these sugars enhance proliferation of trophozoites in the presence of D-glucose or D-galactose. The trophozoites could take up D-glucose and D-galactose, but not D-fructose, D-allose or D-psicose. Cell sizes of the trophozoites also differed depending on the culture medium.

Abstract Merozoite surface protein 9 (MSP9) constitutes a ligand complex involved in erythrocyte invasion by malarial merozoites and is a promising vaccine target. Plasmodium vivax MSP9 (PvMSP9) is immunogenic upon natural malaria exposure. To address whether sequence diversity in PvMSP9 among field isolates could affect natural antibody responses, the recombinant proteins representing two variants each for the N- and the C-terminal domains of PvMSP-9 were used as antigens to assess antibody reactivity among 246 P. vivax-infected patients’ sera from Tak and Ubon Ratchathani Provinces in Thailand. Results revealed that the seropositivity rates of IgG antibodies to the N-terminal antigens were higher than those to the C-terminal antigens (87.80% vs. 67.48%). Most seropositive sera were reactive to both variants, suggesting the presence of common epitopes. Variant-specific antibodies to the N- and the C-terminal antigens were detected in 15.85% and 16.70% of serum samples, respectively. These seropositivity rates were not significant difference between provinces. The seropositivity rates, levels and avidity of anti-PvMSP9 antibodies exhibited positive trends towards increasing malaria episodes. The IgG isotype responses to the N- and the C-terminal antigens were mainly IgG1 and IgG3. The profile of IgG responses may have implications for development of PvMSP9-based vaccine.

ABSTRACT Entamoeba histolytica is an intestinal protozoan parasite with remarkable ability to kill and phagocytose host cells, causing amoebic colitis and extraintestinal abscesses. The intermediate subunit (Igl) of galactose (Gal)- and N-acetyl-d-galactosamine (GalNAc)-specific lectins is considered an important surface antigen involved in the pathogenesis of E. histolytica. Here, we applied mass spectrometry-based quantitative proteomics technology to analyze the protein expression profile changes occurring in host Caco2 cells incubated with E. histolytica trophozoites or stimulated by purified native Igl protein. The expression levels of 1,490 and 489 proteins were significantly altered in the E. histolytica-treated and Igl-treated groups, respectively, among 6,875 proteins totally identified. Intriguingly, central carbon metabolism of host cells was suppressed in both E. histolytica-treated and Igl-treated groups, with evidence of decreased expression levels of several key enzymes, including pyruvate kinase muscle type 2, presenting a Warburg-like effect in host cells. Besides, Igl had potential physical interactions with central carbon metabolism enzymes and the proteolytic degradation family members proteasome subunit alpha and beta, which may be responsible for the degradation of key enzymes in carbon metabolism. These results provided a novel perspective on the pathogenic mechanism of E. histolytica and compelling evidence supporting the important role of Igl in the virulence of E. histolytica. IMPORTANCE Metabolic reprogramming is considered a hallmark of some infectious diseases. However, in amoebiasis, a neglected tropical disease caused by protozoan parasite E. histolytica, metabolic changes in host cells have yet to be proven. In this study, advanced data-independent acquisition mass spectrometry-based quantitative proteomics was applied to investigate the overall host cellular metabolic changes as high-throughput proteomics could measure molecular changes in a cell or tissue with high efficiency. Enrichment analysis of differentially expressed proteins showed biological processes and cellular pathways related to amoeba infection and Igl cytotoxicity. Specifically, central carbon metabolism of host cells was dramatically suppressed in both E. histolytica-treated and Igl-treated groups, indicating the occurrence of a Warburg-like effect induced by trophozoites or Igl from E. histolytica. Distinct differences in ubiquitin-mediated proteolysis, rapamycin (mTOR) signaling pathway, autophagy, endocytosis, and tight junctions provided novel perspectives on the pathogenic mechanism of E. histolytica.

Faced with the new stage of water oxidation by molecular catalysts (MCs) in artificial photosynthesis to overcome the bottle neck issue, the “Photon-flux density problem of sunlight,” a two-electron oxidation process forming H2O2 in place of the conventional four-electron oxidation evolving O2 has attracted much attention. The molecular characteristics of tin(IV)-tetrapyridylporphyrin (SnTPyP), as one of the most promising MCs for the two-electron water oxidation, has been studied in detail. The protolytic equilibria among nine species of SnTPyP, with eight pKa values on the axial ligands’ water molecules and peripheral pyridyl nitrogen atoms in both the ground and excited states, have been clarified through the measurements of UV-vis, fluorescence, 1H NMR, and dynamic fluorescence decay behaviour. The oxidation potentials in the Pourbaix diagram and spin densities by DFT calculation of the one-electron oxidized form of each nine species have predicted that the fully deprotonated species ([SnTPyP(O−)2]2−) and the singly deprotonated one ([SnTPyP(OH)(O−)]−) serve as the most favourable MCs for visible light-induced two-electron water oxidation when they are adsorbed on TiO2 for H2 formation or SnO2 for Z-scheme CO2 reduction in the molecular catalyst sensitized system of artificial photosynthesis.

Macrophages promote early host responses to infection by releasing pro-inflammatory cytokines, and they are crucial to combat amoebiasis, a disease affecting millions of people worldwide. Macrophages elicit pro-inflammatory responses following direct cell/cell interaction of Entamoeba histolytica, inducing NLRP3 inflammasome activation with high-output IL-1β/IL-18 secretion. Here, we found that trophozoites could upregulate peroxiredoxins (Prx) expression and abundantly secrete Prxs when encountering host cells. The C-terminal of Prx was identified as the key functional domain in promoting NLRP3 inflammasome activation, and a recombinant C-terminal domain could act directly on macrophage. The Prxs derived from E. histolytica triggered toll-like receptor 4-dependent activation of NLRP3 inflammasome in a cell/cell contact-independent manner. Through genetic, immunoblotting or pharmacological inhibition methods, NLRP3 inflammasome activation was induced through caspase-1-dependent canonical pathway. Our data suggest that E. histolytica Prxs had stable and durable cell/cell contact-independent effects on macrophages following abundantly secretion during invasion, and the C-terminal of Prx was responsible for activating NLRP3 inflammasome in macrophages. This new alternative pathway may represent a potential novel therapeutic approach for amoebiasis, a global threat to millions.

Intestinal parasitic infections, including those caused by Entamoeba species, are a persistent problem in rural areas of Thailand. The aims of this study were to identify pathogenic Entamoeba species and to analyze their genotypic diversity. Stool samples were collected from 1,233 students of three schools located in the Thai-Myanmar border region of Tak Province, Thailand. The prevalence of Entamoeba infection was measured by polymerase chain reaction (PCR) using species-specific primers. Thirty-one (2.5%) positive cases were detected for E. histolytica, 55 (4.5%) for E. dispar, and 271 (22.0%) for E. coli. Positive samples for E. histolytica and E. dispar were exclusively obtained from a few school classes, whereas E. coli was detected in all grades. No infections caused by E. moshkovskii, E. nuttalli, E. chattoni, and E. polecki were detected in the students studied. The D-A locus of tRNA-linked short tandem repeats was analyzed in samples of E. histolytica (n = 13) and E. dispar (n = 47) to investigate their diversity and potential modes of transmission. Five genotypes of E. histolytica and 13 genotypes of E. dispar were identified. Sequences of the D-A were divergent, but several unique genotypes were significantly prevalent in limited classes, indicating that intra-classroom transmission has occurred. As it was unlikely that infection would have been limited within school classes if the mode of transmission of E. histolytica and E. dispar had been through the intake of contaminated drinking water or food, these results suggest a direct or indirect person-to-person transmission mode within school classes. Positive rates for three Entamoeba species were 2-fold higher in students who had siblings in the schools than in those without siblings, suggesting that transmission occurred even at home due to heavy contacts among siblings.

ABSTRACT Entamoeba histolytica is an intestinal protozoan that causes human amoebic colitis and extraintestinal abscesses. Virulence variation is observed in the pathogenicity of E. histolytica trophozoites, but the detailed mechanism remains unclear. Here, a single trophozoite was cultured alone, and the progeny of the trophozoites of each generation were subjected to single-cell RNA sequencing (scRNA-seq) to study the transcriptional profiles of trophozoites. The scRNA-seq analysis indicated the importance of sulfur metabolism and the proteasome pathway in pathogenicity, whereas the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic analysis did not identify the bulk trophozoites. The trophozoite improved the synthesis of cysteine under cysteine-deficient conditions but downregulated the expression of the intermediate subunit of the lectin of E. histolytica trophozoites and retained the expression of the heavy subunit of lectin, resulting in decreased amoebic phagocytosis and cytotoxicity. The variation in the transmembrane kinase gene family might be critical in regulating the proteasome pathway. Thus, the scRNA-seq technique provided an improved understanding of the biological characteristics and the mechanism of virulence variation of amoebic trophozoites. IMPORTANCE Studies on the trophozoite of Entamoeba histolytica suggested this organism could accumulate polyploid cells in its proliferative phase and differentiate its cell cycle from that of other eukaryotes. Therefore, a single-cell sequencing technique was used to study the switching of the RNA transcription profiles of single amoebic trophozoites. We separated individual trophozoites from axenic cultured trophozoites, CHO cell-incubated trophozoites, and in vivo trophozoites. We found important changes in the sulfur and cysteine metabolism in pathogenicity. The trophozoites strategically regulated the expression of the cysteine-rich protein-encoding genes under cysteine-deficient conditions, thereby decreasing amoebic phagocytosis and cytotoxicity. The single-cell sequencing technique shows evident advantages in comparison with the isobaric tags for relative and absolute quantitation (iTRAQ) proteomic technology (bulk trophozoite level) and reveals the regulation strategy of trophozoites in the absence of exogenous cysteine. This regulation strategy may be the mechanism of virulence variation of amoebic trophozoites.

Abstract Background Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. Methods The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. Results The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. Conclusions This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.

Abstract Entamoeba histolytica is an anaerobic parasitic protist and possesses mitosomes, one of the most highly divergent mitochondrion-related organelles (MROs). Although unique metabolism and protein/metabolite transport machinery have been demonstrated in Entamoeba mitosomes, the mechanism of mitosomal fusion and fission remains to be elucidated. In this study, we demonstrate that two dynamin-related proteins (DRPs) are cooperatively involved in the fission of Entamoeba mitosomes. Expression of a dominant negative form of EhDrpA and EhDrpB, and alternatively, repression of gene expression of EhDrpA and EhDrpB genes, caused elongation of mitosomes, reflecting inhibition of mitosomal fission. Moreover, EhDrpA and EhDrpB formed an unprecedented hetero-oligomeric complex with an approximate 1:2 to 1:3 ratio, suggesting that the observed elongation of mitosomes is likely caused by the disruption and instability of the complex caused by an imbalance in the two DRPs. Altogether, this is the first report of a hetero-oligomeric DRP complex which participates in the fission of mitochondria and MROs.

The enteric protozoa Entamoeba histolytica is the causative agent of amebiasis, which is one of the most common parasitic diseases in developed and developing countries. Entamoeba nuttalli is the genetically closest species to E. histolytica in current phylogenetic analyses of Entamoeba species, and is prevalent in wild macaques. Therefore, E. nuttalli may be a key organism in which to investigate molecules required for infection of human or non-human primates. To explore the molecular signatures of host-parasite interactions, we conducted de novo assembly of the E. nuttalli genome, utilizing self-correction of PacBio long reads and polishing corrected reads using Illumina short reads, followed by comparative genomic analysis with two other mammalian and a reptilian Entamoeba species. The final draft assembly of E. nuttalli included 395 contigs with a total length of approximately 23 Mb, and 9,647 predicted genes, of which 6,940 were conserved with E. histolytica. In addition, we found an E. histolytica-specific repeat known as ERE2 in the E. nuttalli genome. GO-term enrichment analysis of mammalian host-related molecules indicated diversification of transmembrane proteins, including AIG1 family and BspA-like proteins that may be involved in the host-parasite interaction. Furthermore, we identified an E. nuttalli-specific protein that contained 42 repeats of an octapeptide ([G,E]KPTDTPS). This protein was shown to be localized on the cell surface using immunofluorescence. Since many repeat-containing proteins in parasites play important roles in interactions with host cells, this unique octapeptide repeat-containing protein may be involved in colonization of E. nuttalli in the intestine of macaques. Overall, our draft assembly provides a valuable resource for studying Entamoeba evolution and host-parasite selection.

Autophagy, an evolutionarily conserved mechanism to remove redundant or dangerous cellular components, plays an important role in innate immunity and defense against pathogens, which, in turn, can regulate autophagy to establish infection within a host. However, for Entamoeba histolytica, an intestinal protozoan parasite causing human amoebic colitis, the interaction with the host cell autophagy mechanism has not been investigated. In this study, we found that E. histolytica peroxiredoxin (Prx), an antioxidant enzyme critical for parasite survival during the invasion of host tissues, could activate autophagy in macrophages. The formation of autophagosomes in macrophages treated with recombinant Prx of E. histolytica for 24 h was revealed by immunofluorescence and immunoblotting in RAW264.7 cells and in mice. Prx was cytotoxic for RAW264.7 macrophages after 48-h treatment, which was partly attributed to autophagy-dependent cell death. RNA interference experiments revealed that Prx induced autophagy mostly through the toll-like receptor 4 (TLR4)–TIR domain-containing adaptor-inducing interferon (TRIF) pathway. The C-terminal part of Prx comprising 100 amino acids was the key functional domain to activate autophagy. These results indicated that Prx of E. histolytica could induce autophagy and cytotoxic effects in macrophages, revealing a new pathogenic mechanism activated by E. histolytica in host cells.

Entamoeba histolytica is the causative agent of amoebiasis. This disease results in 40,000 to 100,000 deaths annually. The pathogenic molecules involved in the invasion of trophozoites had been constantly being clarified. This study explored the role of elongation factor 1 alpha (EF1a) in E. histolytica pathogenicity. Biolayer interferometry binding and pull-down assays suggest that EF1a and intermediate subunit of lectin (Igl) binding are specific. Submembranous distribution of EF1a closely aligns with the localization of Igl, which appear in abundance on membranes of trophozoites. Messenger RNA (mRNA) expression of EF1a is positively correlated with trends in Igl levels after co-incubation with Chinese hamster ovary (CHO) cells in vitro, suggesting a regulatory linkage between these proteins. Erythrophagocytosis assays also imply a role for EF1a in phagocytosis. Finally, EF1a and actin are collocated in trophozoites. These results indicated elongation factor 1a is associated with E. histolytica phagocytosis, and the relationships between EF1a, Igl, and actin are worth further study to better understand the pathogenic process.

Entamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica.Trophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1β) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro.The results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

[This corrects the article DOI: 10.1371/journal.pone.0110463.].

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica has roles in pathogenicity and induction of protective immunity in rodent models of amoebiasis. Recently, the intermediate subunit of the lectin, Igl1, of E. histolytica has been shown to have hemolytic activity. However, the corresponding lectin is also expressed in a non-virulent species, Entamoeba dispar, and another subunit, Igl2, is expressed in the protozoa. Therefore, in this study, we compared the activities of Igl1 and Igl2 subunits from E. histolytica and E. dispar using various regions of recombinant Igl proteins expressed in Escherichia coli. The recombinant E. dispar Igl proteins had comparable hemolytic activities with those of E. histolytica Igl proteins. Furthermore, Igl1 gene-silenced E. histolytica trophozoites showed less hemolytic activity compared with vector-transfected trophozoites, indicating that the expression level of Igl1 protein influences the activity. These results suggest that the lower hemolytic activity in E. dispar compared with E. histolytica reflects the lower expression level of Igl1 in the E. dispar parasite.

Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.

Thrombospondin-related adhesive protein (TRAP) of malaria parasites is essential for sporozoite motility and invasions into mosquito's salivary gland and vertebrate's hepatocyte; thereby, it is a promising target for pre-erythrocytic vaccine. TRAP of Plasmodium vivax (PvTRAP) exhibits sequence heterogeneity among isolates, an issue relevant to vaccine development. To gain insights into variation in the complete PvTRAP sequences of parasites in Thailand, 114 vivax malaria patients were recruited in 2006-2007 from 4 major endemic provinces bordering Myanmar (Tak in the northwest, n = 30 and Prachuap Khirikhan in the southwest, n = 25), Cambodia (Chanthaburi in the east, n = 29) and Malaysia (Yala and Narathiwat in the south, n = 30). In total, 26 amino acid substitutions were detected and 9 of which were novel, resulting in 44 distinct haplotypes. Haplotype and nucleotide diversities were lowest in southern P. vivax population while higher levels of diversities were observed in other populations. Evidences of positive selection on PvTRAP were demonstrated in domains II and IV and purifying selection in domains I, II and VI. Genetic differentiation was significant between each population except that between populations bordering Myanmar where transmigration was common. Regression analysis of pairwise linearized Fst and geographic distance suggests that P. vivax populations in Thailand have been isolated by distance. Sequence diversity of PvTRAP seems to be temporally stable over one decade in Tak province based on comparison of isolates collected in 1996 (n = 36) and 2006-2007. Besides natural selection, evidences of intragenic recombination have been supported in this study that could maintain and further generate diversity in this locus. It remains to be investigated whether amino acid substitutions in PvTRAP could influence host immune responses although several predicted variant T cell epitopes drastically altered the epitope scores. Knowledge on geographic diversity in PvTRAP constitutes an important basis for vaccine design provided that vaccination largely confers variant-specific immunity.

BACKGROUND: A strategy to combat infectious diseases, including neglected tropical diseases (NTDs), will depend on the development of reliable epidemiological surveillance methods. To establish a simple and practical seroprevalence detection system, we developed a microsphere-based multiplex immunoassay system and evaluated utility using samples obtained in Kenya. METHODS: We developed a microsphere-based immuno-assay system to simultaneously measure the individual levels of plasma antibody (IgG) against 8 antigens derived from 6 pathogens: Entamoeba histolytica (C-IgL), Leishmania donovani (KRP42), Toxoplasma gondii (SAG1), Wuchereria bancrofti (SXP1), HIV (gag, gp120 and gp41), and Vibrio cholerae (cholera toxin). The assay system was validated using appropriate control samples. The assay system was applied for 3411 blood samples collected from the general population randomly selected from two health and demographic surveillance system (HDSS) cohorts in the coastal and western regions of Kenya. The immunoassay values distribution for each antigen was mathematically defined by a finite mixture model, and cut-off values were optimized. FINDINGS: Sensitivities and specificities for each antigen ranged between 71 and 100%. Seroprevalences for each pathogen from the Kwale and Mbita HDSS sites (respectively) were as follows: HIV, 3.0% and 20.1%; L. donovani, 12.6% and 17.3%; E. histolytica, 12.8% and 16.6%; and T. gondii, 30.9% and 28.2%. Seroprevalences of W. bancrofti and V. cholerae showed relatively high figures, especially among children. The results might be affected by immunological cross reactions between W. bancrofti-SXP1 and other parasitic infections; and cholera toxin and the enterotoxigenic E. coli (ETEC), respectively. INTERPRETATION: A microsphere-based multi-serological assay system can provide an opportunity to comprehensively grasp epidemiological features for NTDs. By adding pathogens and antigens of interest, optimized made-to-order high-quality programs can be established to utilize limited resources to effectively control NTDs in Africa.

A survey of the prevalence of Trypanosoma cruzi infection was carried out in Oitis, a small community in the State of Piaui, Brazil. Two hundred and sixty five individuals were screened by microscopic examination, hémoculture, indirect immunofluorescence (IFA), enzyme-linked immunosorbent assay (ELISA), and competitive enzyme-linked immunosorbent assay (C-ELISA) using the monoclonal antibody TCF87 against to a 25kd T. cruzi antigen. Seropositivity was 14.3% by the IFA test, 14.7% by ELISA, and 13.2% by C-ELISA. The C-ELISA using the TCF87 monoclonal antibody seems to be applicable in serodiagnosis of Chagas' disease.A prevalência da infecção pelo Trypanosoma cruzi foi pesquisada em Oitis, uma pequena comunidade do Estado do Piauí. Duzentos e sessenta e cinco indivíduos foram investigados pelos seguintes métodos: pesquisa direta, hemocultura, imunofluorescência indireta (IFA), teste imunoabsorvente ligado a enzima (ELISA), e ELISA de competição (C-ELISA) com a utilização do anticorpo monoclonal TCF87 contra um antígeno do T. cruzi com 25kd. A IFA foi positiva em 14,3% dos indivíduos, ELISA em 14,7% e C-ELISA em 13,2%. Este último teste mostrou-se aplicável no diagnóstico sorológico da Doença de Chagas.

Cellular functions, such as differentiation and migration, are regulated by the extracellular microenvironment, including the extracellular matrix (ECM). Cells adhere to ECM through focal adhesions (FAs) and sense the surrounding microenvironments. Although FA proteins have been actively investigated, little is known about the lipids in the plasma membrane at FAs. In this study, we examine the lipid composition at FAs with imaging and biochemical approaches. Using the cholesterol-specific probe D4 with total internal reflection fluorescence microscopy and super-resolution microscopy, we show an enrichment of cholesterol at FAs simultaneously with FA assembly. Furthermore, we establish a method to isolate the lipid from FA-rich fractions, and biochemical quantification of the lipids reveals that there is a higher content of cholesterol and phosphatidylcholine with saturated fatty acid chains in the lipids of the FA-rich fraction than in either the plasma membrane fraction or the whole-cell membrane. These results demonstrate that plasma membrane at FAs has a locally distinct lipid composition compared to the bulk plasma membrane. [ABSTRACT FROM AUTHOR]

Myiasis refers to the infestation of living humans and vertebrate animals by dipterous larvae. Many organs can be infested by fly larvae, but cutaneous and wound myiases are the most frequently encountered clinical forms. Persistent ulcer or non-healing wound is one of the symptoms of squamous cell carcinoma which is the second most common skin cancer in the world. Here we report a case of an elderly man with a severe wound myiasis in a squamous cell carcinoma lesion of the scalp. The maggots were confirmed to be Lucilia sericata which are widespread flies in Japan. Human myiasis is rarely reported in Japan, but patients with necrotic, hemorrhaging, or pus-filled wounds are susceptible to infestation. It is necessary for doctors and nurses to ensure that their patients change their dressings daily and keep their wounds clean. [ABSTRACT FROM AUTHOR]

The deactivation process of excited 1-(dimethylamino)-9-fluorenone (1-DMAF) was investigated by means of steady-state and time-resolved fluorescence spectroscopy. Fluorescence decay profiles for 1-DMAF, which has a relatively short lifetime are much affected by the fluidity of the surrounding solvent.

Ticks have a cosmopolitan distribution and, as such, are also found in Japan. Ticks are typically ectoparasites of wild animals, however, humans can also be bitten when visiting environments inhabited by ticks. Herein, we describe two cases with atypical tick bites. Case 1 was an elderly Japanese male patient who presented with a fully engorged tick measuring 20 × 17 × 8 mm; it is rare for ticks to attain a length of 20 mm. Case 2 was an elderly Japanese female with severe dementia who presented with multiple tick bites, which is rare, after going missing for 6 days before being found in a densely wooded area. Ticks are responsible for the transmission of many infectious agents, such as bacteria, viruses and parasites. The National Institute of Infectious Diseases and the Ministry of Health, Labour and Welfare regularly inform citizens of the risks posed by tick bites. However, the tick bites could not be prevented in our patients. Further edification about tick bites, tick-borne diseases, and their prevention are considered necessary in Japan. [ABSTRACT FROM AUTHOR]

Cryptosporidiosis has been reported to be associated with HIV/acquired immune deficiency syndrome, which greatly reduces the quality of life and shortens the life expectancy of HIV-infected patients. In order to properly treat the infected patients, accurate and automatic diagnostic tools need to be developed. In this study, a novel microfluidic immunochip system was presented for the surveillance and the rapid detection of Cryptosporidium infection in 190 HIV-infected patients from Guangxi, China, using the P23 antigen of Cryptosporidium. The procedure of detection can be completed within 10 min with 2 μl sample consumption. The system also was evaluated using the standard ELISA method. Among 190 HIV-infected individuals, the rate of P23 positivity was 13.7%. Seropositivity in HIVinfected individuals was higher in female patients. The seropositivity to P23 was higher in HIV-infected individuals with high viral load, although the difference was statistically insignificant. Significantly higher Cryptosporidium seropositivity was observed in HIV-infected individuals with a CD4+ T-cell count of <200cells/μl than in those with ≥200 cells/μl. Our results also demonstrate that a lower CD4+ T-cell count may reflect an increased accumulated risk for cryptosporidiosis. The detection system was further validated using the standard ELISA method and good correlation between the two methods was found (r = 0.80). Under the same sensitivity, this new microfluidic chip device had a specificity of 98.2%. This developed system may provide a powerful platform for the fast screening of Cryptospordium infection in HIV-infected patients. [ABSTRACT FROM AUTHOR]

Two key reaction intermediates in the photochemical oxygenation of alkene sensitized by carbonyl-coordinated ruthenium(ii)–porphyrin complex, with water acting both as an electron and oxygen atom donor, are postulated. Under the low concentration of hydroxide ion (<2 × 10−3M) added to the reaction mixture of tetra(2,4,6-trimethyl)phenylporphyrinatoruthenium(ii) (RuIITMP(CO)), K4PtCl6as a sacrificial electron acceptor, and cyclohexene as a substrate in aqueous acetonitrile, the major reaction product was cyclohexaneoxide (“Epoxide”), while it drastically decreased along with an increase of 2-cyclohexenol (“Alcohol”) by increasing the amount of hydroxide ion (>2 × 10−3M). The tendency was more obvious in the case of tetrasodium tetra(4-sulfonate)phenylporphyrinatoruthenium(ii) (RuIITSPP(CO)) in aqueous solution. The “Alcohol” was exclusively formed in the higher concentration region of OH−, strongly suggesting the presence of acid–base equilibrium among two reaction intermediates. Theoretical DFT calculation indicates that the hydroxyl-coordinated one-electron oxidized Ru–porphyrin (Intermediate (I)), which is formed by the axial ligation of hydroxide ion to the cation radical of Ru–porphyrin generated through electron transfer from the excited triplet state of the sensitizer porphyrins, suffers deprotonation of its axial hydroxide group to lead to an oxo-type complex (Intermediate (II)) formation. The DFT calculation also indicates that the electron spin on the Intermediate (I) is shared by the axial oxygen atom and the central Ru metal, while it is mostly localized on the axial oxygen atom to behave as an oxygen radical in the case of the Intermediate (II). These are very strong indications towards understanding how OH−(water molecule) is oxidatively activated on the Ru center: the water molecule is serving as an electron donor ion in the redox cycles. Theoretical calculation predicts that Intermediate (I) allows the epoxidation of alkene and Intermediate (II) can proceed through hydrogen abstraction from the substrate and is rebound to form hydroxylated compound, “Alcohol.” [ABSTRACT FROM AUTHOR]

A novel photofunctional material composed of polyfluorinated cationic azobenzene and layered potassium titanoniobate was synthesized and its photochemical behavior was investigated. Although polyfluorinated cationic azobenzene could not be intercalated into the interlayer region of layered potassium titanoniobate directly, the intercalation compound was obtained by guest–guest-exchange with the hexylammonium-TiNbO5 intercalation compound. X-Ray diffraction, TGA, IR, TEM, UV-visible spectroscopy and elemental analysis results indicated that the polyfluorinated cationic azobenzene was intercalated into the interlayer spaces of the potassium titanoniobate. The AFM image for the intercalated compound with a basal spacing of 3.90 nm was consistent with the X-ray diffraction data. The spectral properties as well as X-ray diffraction results have revealed that the adsorbed polyfluorinated cationic azobenzene molecules form J-like aggregates with bi-layers in the interlayer space of the potassium titanoniobate. The intercalation compound exhibited an excellent reversible cis–trans photoisomerization by successive illumination with UV light at 365 nm and visible light at 458 nm. The basal spacing changed reversibly upon photoisomerization of the intercalated polyfluorinated cationic azobenzene. [ABSTRACT FROM AUTHOR]

Abstract  We recently produced human monoclonal antibody Fab fragments specific for the 19-kDa C-terminal merozoite surface protein 1 of Plasmodium falciparum in a bacterial expression system. The effect of single amino acid modifications in the third complementarity-determining regions of the heavy and light chains on affinity was examined in one of the Fab fragments, Pf25. Recombination polymerase chain reaction was used to modify Tyr92 or Ile97 in the light chain and Val101 or Trp107 in the heavy chain. No effective replacements for Tyr92 and Val101 were found, but possible substitutions of Ile97 with Gly, Leu, Glu, Ala and Ser, and of Trp107 with Arg and Ser were demonstrated. Of these modified Fab fragments, the affinities of Fabs with Ile97–Leu and Trp107–Ser mutations were slightly higher than that of the original Fab. The effects of these modifications on the antigen–antibody interaction are discussed. [ABSTRACT FROM AUTHOR]

Abstract  Three protein-coding loci in DNA of an Entamoeba histolytica strain (EHMfas1) isolated from cynomolgus monkey (Macaca fascicularis) were sequenced; these loci corresponded to the genes for chitinase, the serine-rich E. histolytica protein (SREHP), and the 16 S-like small subunit ribosomal RNA (16S-like SSUrRNA). The nucleotide and deduced amino-acid sequences of chitinase and SREHP were compared with sequences from human isolates. EHMfas1 had several specific mutations in units in the polymorphic regions of the chitinase and SREHP loci, with some repetition of these mutated units. The sequence of the 16S-like SSUrRNA gene (16S-like SSUrDNA) was compared with other Entamoeba species. In phylogenetic analysis, EHMfas1 was not categorized in the E. histolytica cluster but between E. histolytica and E. dispar. To our knowledge, this is the first molecular characterization of E. histolytica isolated from cynomolgus monkey, and our results indicate that EHMfas1 may be a subspecies of E. histolytica that infects cynomolgus monkey. [ABSTRACT FROM AUTHOR]

Three hundred and three stool samples of cynomolgus monkeys (Macaca fascicularis) imported from China and the Philippines were examined for Entamoeba histolytica/Entamoeba dispar infections. Microscopy detected E. histolytica/E. dispar cysts in 41 samples. Positive rates were higher in the monkeys from China (37.5%) than in the monkeys from the Philippines (3.7%). PCR analysis of 25 samples successfully cultured from the cysts demonstrated that 24 were E. dispar, one of the samples from China was E. histolytica. The one sample was also identified as E. histolytica by an antigen detection kit, although the monkey was asymptomatic and serology was negative. To our knowledge, this is the first report of E. histolytica isolation from cynomolgus monkeys based on the discrimination between E. histolytica and E. dispar. [ABSTRACT FROM AUTHOR]

A fluorescence immunochromatography (FIC) kit was developed recently using fluorescent silica nanoparticles coated with a recombinant C-terminal fragment of the surface lectin intermediate subunit (C-Igl) of Entamoeba histolytica to establish rapid serodiagnosis of amebiasis. We further evaluated the system using serum samples from 52 Thai patients with amebiasis. Of the patients, 50 (96%) tested positive using FIC. The samples were also tested using enzyme-linked immunosorbent assay (ELISA) with C-Igl as the antigen. Two samples were negative on ELISA but positive on FIC. The correlation coefficient between the fluorescence intensity using FIC and the optical density value using ELISA was 0.5390, indicating a moderate correlation between the two tests. Serum samples from 20 patients with malaria and 22 patients with Clostridioides difficile infection were also tested using FIC. The false-positive rates were 4/20 (20%) and 1/22 (4%) in patients with malaria and C. difficile infection, respectively. Combining the data from the present study with our previous study, the sensitivity and specificity of FIC were determined to be 98.5% and 95.2%, respectively. The results of the 50 samples were studied using a fluorescence scope and a fluorescence intensity reader, and the findings were compared. Disagreements were found in only two samples showing near-borderline fluorescence intensity, indicating that the use of scope was adequate for judging the results. These results demonstrate that FIC is a simple and rapid test for the serodiagnosis of amebiasis. [ABSTRACT FROM AUTHOR]

Entamoeba nuttalli infection is prevalent in captive and wild macaques. Recent studies have suggested that genotypes of E. nuttalli isolates are correlated with the geographical distribution of host macaques. Correlation of amoebic genotypes with genetic diversity of host macaques was analyzed in present study. Sixty fresh stool samples were obtained from wild Tibetan macaques living in Mount Huang (HS) of the An-hui Province in China. PCR analysis revealed that the most prevalent Entamoeba species was E. chattoni (E. polecki ST2) (86.7%) followed by E. nuttalli (58.3%) and E. coli (25%). Six E. nuttalli HS isolates were successfully cultured. The tRNA-linked short tandem repeat (STR) loci and serine-rich protein gene of E. nuttalli isolates from four different regions of China (Mount Long-hu, Gui-yang, Mount E-mei, and HS, the former three isolates were obtained in previous studies) were studied and high numbers of polymorphisms were detected. When genetic diversity of different populations of E. nuttalli isolates was compared with geographical distance, an r² value of 0.919 was assigned by a Mantel test based on the tRNA-STR loci. In host macaques, the mtDNA HVS-I gene was also highly polymorphic in each of the genomes. Multiple regression analysis using E. nuttalli tRNA-STR loci genetic, macaque mtDNA HVS-I gene, and geographic distances showed an r² value of 0.943, indicating that a higher relevance was demonstrated when geographic and host gene factors were considered. Analysis of genetic factor of host would benefit for better understanding of the evolution of E. nuttalli. [ABSTRACT FROM AUTHOR]

This study is the first to isolate an Entamoeba histolytica strain from Chinese amoebic patients and to conduct a detailed examination of its virulence. A fecal sample that contains cysts of E. histolytica was obtained from Guangxi province. The sample was cultured axenically and then cloned by limiting dilution, and named as XLAC. In vitro and in vivo tests were conducted to evaluate the virulence of the Entamoeba isolate. The E. histolytica strain XLAC was successfully cloned and cultured axenically. DNA regions that contain hexokinase, glucose-6-phosphate isomerase, phosphoglucomutase, and heavy subunit of lectin genes were amplified by PCR. The PCR products were then sequenced. Virulence analysis suggested that the XLAC strain was similar to the HM1:IMSS strain at the genetic level. In vitro and in vivo tests also implicated these strains to be similar. These findings may be attributed to the low expression levels of pathogenic genes obtained through realtime PCR. The XLAC strain restored its virulence after it was injected into hamster liver. This study may be a good model for studying virulence changes in E. histolytica. [ABSTRACT FROM AUTHOR]