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Clonorchiasis caused by Clonorchis sinensis is endemic in East Asia; approximately 15 million people have been infected thus far. To diagnose the infection, serodiagnostic tests with excellent functionality should be performed. First, 607 expressed sequence tags encoding polypeptides with a secretory signal were expressed into recombinant proteins using an in vitro translation system. By protein array-based screening using C. sinensis-infected sera, 18 antigen candidate proteins were selected and assayed for cross-reactivity against Opisthorchis viverrini-infected sera. Of the six antigenic proteins selected, four were synthesized on large scale in vitro and evaluated for antigenicity against the flukes-infected human sera using ELISA. CsAg17 antigen showed the highest sensitivity (77.1%) and specificity (71.2%). The sensitivity and specificity of the bacterially produced CsAg17-28GST fusion antigen was similar to those of CsAg17 antigen. CsAg17 antigen can be used to develop point-of-care serodiagnostic tests for clonorchiasis., Author summary Human clonorchiasis is a parasitic disease caused by the Chinese liver fluke, Clonorchis sinensis. Humans are infected through eating raw freshwater fishes carrying C. sinensis metacercariae, the encysted larvae. They excyst in the duodenum, move into the liver via bile duct and grow to adult worms. Excretory-secretory products of the worm damage the liver causing various inflammatory pathological changes and may lead to bile duct cancer. Although there exists an anthelmintic choice praziquantel to kill the fluke, emphasis is placed on early diagnosis and prevention before the infection becomes disease. Microscopic stool examination is the standard diagnostic method but is cumbersome and time consuming. Blood serum antibodies from clonorchiasis patients could provide a simple and fast diagnosis. However, antibody detecting diagnostics developed so far have a low specificity and sensitivity. In the present study we selected 607 antigenic candidate proteins from the genomic database and synthesized them through an integrated high-throughput proteogenomic tools. We identified several antigenic proteins and evaluated their diagnostic potential for clonorchiasis. One of them, CsAg17, showed a high sensitivity and specificity. This antigen deserves development of point-of-care serodiagnostics for C. sinensis infections.

Malaria remains one of the leading health burdens in the developing world, especially in several sub-Saharan Africa countries; and Uganda has some of the highest recorded measures of malaria transmission intensity in the world. It is evident that the prevalence of malaria infection, the incidence of disease, and mortality from severe malaria remain very high in Uganda. Although the recent stable political and economic situation in the last few decades in Uganda supported for a fairly good appreciation of malaria control, the declines in infection, morbidity, and mortality are not sufficient to interrupt transmission and this country is among the top 4 countries with cases of malaria, especially among children under 5 years of age. In fact, Uganda, which is endemic in over 95% of the country, is a representative of challenges facing malaria control in Africa. In this study, we evaluated an active case detection program in 6 randomly selected villages, Uganda. This program covered a potential target population of 5,017 individuals. Our team screened 12,257 samples of malaria by active case detection, every 4 months, from February 2015 to January 2017 in the 6 villages (a total of 6 times). This study assessed the perceptions and practices on malaria control in Kiyuni Parish of Kyankwanzi district, Uganda. Our study presents that the incidence of malaria is sustained high despite efforts to scale-up and improve the use of LLINs and access to ACDs, based on the average incidence confirmed by RDTs.

The paleoenvironmental history of the artificial Reservoir Gonggeomji in Sangju City, Korea, was reconstructed using fossil diatom analysis of four sediment cores: GG01, 02, 03, and 04. Accelerator mass spectrometer (AMS) radiocarbon dating results suggest that Reservoir Gonggeomji was maintained from ca. 1350 to 150 cal year BP, and the diatom record in core sediment samples revealed well-defined hydrological changes and paleoenvironmental conditions during this period. The fossil diatom assemblages were predominantly benthic freshwater diatoms. The highest diatom concentrations in all cores were in diatom zone II, in which species richness and diversity were also very high. Cymbella, Eunotia, Gomphonema, Gyrosigma, Navicula, and Pinnularia were the dominant genera in all core sediments. The long-term trend in diatom species abundance and species diversity showed a stronger relationship to temperature anomalies in the northern hemisphere during the past 2000 years with changes in precipitation. These diatom changes could have been controlled by natural climate change, despite anthropogenic activities linked to construction of the dyke in the artificial reservoir.

Waterborne parasitic protozoa, particularly Giardia lamblia and Cryptosporidium spp., are common causes of diarrhea and gastroenteritis worldwide. The most frequently identified source of infestation is water, and exposure involves either drinking water or recreation in swimming pools or natural bodies of water. In practice, studies on Cryptosporidium oocysts and Giardia cysts in surface water are challenging owing to the low concentrations of these microorganisms because of dilution. In this study, a 3-year monitoring of Cryptosporidium parvum, Giardia lamblia, and Naegleria fowleri was conducted from August 2014 to June 2016 at 5 surface water sites including 2 lakes, 1 river, and 2 water intake plants. A total of 50 water samples of 40 L were examined. Cryptosporidium oocysts were detected in 22% of samples and Giardia cysts in 32%. Water at the 5 sampling sites was all contaminated with Cryptosporidium oocysts (0–36/L), Giardia cysts (0–39/L), or both. The geometric mean concentrations of Cryptosporidium and Giardia were 1.14 oocysts/L and 4.62 cysts/L, respectively. Thus, effective monitoring plans must take into account the spatial and temporal parameters of contamination because they affect the prevalence and distribution of these protozoan cysts in local water resources.

The vivax malaria epidemic has persisted in South Korea since its reemergence in 1993. Although there has been a significant decrease in the number of malaria cases in recent years, vivax malaria is still a major public health concern. To gain in-depth insight into the genetic makeup of Korean Plasmodium vivax, we analyzed polymorphic patterns of two major antigens, merozoite surface protein-1 (MSP-1) and MSP-3α, in 255 Korean P. vivax isolates collected over an extended period from 1998 to 2013. Combinational genetic analysis of polymorphic patterns of MSP-1 and MSP-3α in the isolates suggests that the P. vivax population in South Korea has been diversifying rapidly, with the appearance of parasites with new genotypes, despite the recent reduction of disease incidence. These results highlight the importance of molecular epidemiological investigations to supervise the genetic variation of the parasite in South Korea.

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 μM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 μM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

In recent years, rapid diagnostic tests (RDTs) have been widely used for malaria detection, primarily because of their simple operation, fast results, and straightforward interpretation. The Asan EasyTest™ Malaria Pf/Pan Ag is one of the most commonly used malaria RDTs in several countries, including Korea and India. In this study, we tested the diagnostic performance of this RDT in Uganda to evaluate its usefulness for field diagnosis of malaria in this country. Microscopic and PCR analyses, and the Asan EasyTest™ Malaria Pf/Pan Ag rapid diagnostic test, were performed on blood samples from 185 individuals with suspected malaria in several villages in Uganda. Compared to the microscopic analysis, the sensitivity of the RDT to detect malaria infection was 95.8% and 83.3% for Plasmodium falciparum and non-P. falciparum, respectively. Although the diagnostic sensitivity of the RDT decreased when parasitemia was ≤500 parasites/µl, it showed 96.8% sensitivity (98.4% for P. falciparum and 93.8% for non-P. falciparum) in blood samples with parasitemia ≥100 parasites/µl. The specificity of the RDT was 97.3% for P. falciparum and 97.3% for non-P. falciparum. These results collectively suggest that the accuracy of the Asan EasyTest™ Malaria Pf/Pan Ag makes it an effective point-of-care diagnostic tool for malaria in Uganda.

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient's blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.

For floristic study on diatoms from the source of Han and Nakdong Rivers, we collected samples in February, April, and July 2016 from Geomryongso and Neodeolsam Ponds, South Korea. Geomryongso and Neodeolsam Ponds are sources of Han and Nakdong Rivers, respectively. Thirty-eight infrageneric taxa of freshwater diatoms were identified in the collected samples from study areas. The diatom species were classified into 25 genera, 17 families, 10 orders, 5 subclasses, and 2 classes based on the taxonomic system proposed by Round et al . (1990). Nine diatom taxa— Achnanthes sinaensis , Achnanthidium atomus , Edtheriotia guizhoiana , Fragilaria recapitellata , Gyrosigma procerum , Navicula nipponica , Pinnularia pseudotabellaria , Pinnularia rabenhorstii , and Platessa barhlsii —were newly recorded, whose taxonomic features have been described; they are indicated by asterisk. We presented the taxonomic information, including the systematics, and dimension in the source of two major rivers in Korea. This study might contribute to the understanding of diversity and flora of diatoms in various freshwater environments in Korea.

The voltage-gated Ca(2+) channel β-subunit is a member of the membrane-associated guanylate kinase family and modulates kinetic properties of the Ca(2+) channels, such as their voltage-dependent activation and inactivation rates. Two cDNA clones were identified to encode each β-subunit isotype of the voltage-gated Ca(2+) channel of Clonorchis sinensis, CsCavβ1 and CsCavβ2, which consist of 606 and 887 amino acids, respectively. CsCavβ1 was found to be similar to the β-subunit containing two conserved serine residues that constitute the consensus protein kinase C phosphorylation site in the β-interaction domain (BID). CsCavβ2 had cysteine and alanine residues instead of the two serine residues conserved in BID and was homologous to variant β-subunit of Schistosoma mansoni and Schistosoma japonicum. CsCavβ1 and CsCavβ2 were almost equally expressed in the adults and metacercariae, but were more expressed in adult C. sinensis than in metacercariae. Collectively, our findings suggest that substitution of the two serine residues in BID of CsCavβ2 may render C. sinensis sensitive to praziquantel.

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity.

Background Accurate diagnosis of Plasmodium infection is crucial for prompt malaria treatment and surveillance. Microscopic examination has been widely applied as the gold standard for malaria diagnosis in most part of malaria endemic areas, but its diagnostic value has been questioned, particularly in submicroscopic malaria. In this study, the diagnostic performance of microscopic examination and nested polymerase chain reaction (PCR) was evaluated to establish optimal malaria diagnosis method in Myanmar. Methods A total of 1125 blood samples collected from residents in the villages and towns located in Naung Cho, Pyin Oo Lwin, Tha Beik Kyin townships and Mandalay of Upper Myanmar were screened by microscopic examination and species-specific nested PCR method. Results Among the 1125 blood samples, 261 samples were confirmed to be infected with malaria by microscopic examination. Evaluation of the 1125 samples by species-specific nested PCR analysis revealed that the agreement between microscopic examination and nested PCR was 87.3% (261/299). Nested PCR successfully detected 38 Plasmodium falciparum or Plasmodium vivax infections, which were missed in microscopic examination. Microscopic examinations also either misdiagnosed the infected Plasmodium species, or did not detect mixed infections with different Plasmodium species in 31 cases. Conclusions The nested PCR method is more reliable than conventional microscopic examination for the diagnosis of malaria infections, and this is particularly true in cases of mixed infections and submicroscopic infections. Given the observed higher sensitivity and specificity of nested PCR, the molecular method holds enormous promise in malaria diagnosis and species differentiation, and can be applied as an effective monitoring tool for malaria surveillance, control and elimination in Myanmar.

Seroepidemiological changes of Toxoplasma gondii infection among the residents of the islands of Gangwha-gun, Incheon for 2 years were surveyed and evaluated by ELISA using a crude extract antigen. In 2010, sera of 919 adult residents in Gyodong-myeon and 313 adults in Samsan-myeon were collected and checked for IgG antibody titers, which showed 14.5% (133 sera) and 19.8% (62 sera) positive rates, respectively. In 2011, sera of 955 adults in Gyodong-myeon and 341 adults in Samsan-myeon were examined, which showed an increase of positive rates to 23.8% (227 sera) and 31.7% (108 sera), respectively. Totally, the seroprevalence of the first year was 15.8% and it increased rapidly to 25.8% in the second year. The positive rates of both sexes increased simultaneously with the significant ratio of males to females by 1.7-2.2 fold (P

Antigenic proteins from Clonorchis sinensis have been previously purified and evaluated for their antigenicity to enable the serodiagnosis of clonorchiasis. Though they were of high specificity, molecularly defined proteins were reported to be less sensitive as single antigens than crude antigen. To resolve this issue, 11 clones were selected by immunoscreening an adult C. sinensis cDNA library using infected human sera. Mixed antigens were prepared using recombinant proteins of positive clones and investigated for antigenicity by immunoblotting against C. sinensis- and helminth-infected patient sera. A mixed antigen of recombinant 28 and 26 kDa glutathion S-transferases (Cs28GST and Cs26GST) produced 76% sensitivity and 95% specificity. Furthermore, a triple mix of recombinant Cs26GST and Cs28GST with vitelline precursor protein pushed up the sensitivity to 87% and maintained specificity at 95%. It is proposed that multiple antigen mixes should be further studied to develop rapid serodiagnostic test kits for the serodiagnosis of human clonorchiasis.

The onset, severity, and ultimate outcome of malaria infection are influenced by parasite-expressed virulence factors and individual host responses to these determinants. In both humans and mice, liver injury is involved after parasite entry, which persists until the erythrocyte stage after infection with the fatal strain Plasmodium falciparum (Pf). Hepatocyte growth factor (HGF) has strong anti-apoptotic effects in various kinds of cells, and also has diverse metabolic functions. In this work, Pf-subtilisin-like protease 2 (Pf-Sub2) 5'untranslated region (UTR) was analyzed and its transcriptional activity was estimated by luciferase expression. Fourteen TATA boxes were observed but only one Oct-1 and c-Myb were done. In addition, host HGF interaction with Pf-Sub2 was evaluated by co-transfection of HGF- and Pf-Sub2-cloned vector. Interestingly, -1,422/+12 UTR exhibited the strongest luciferase activity but -329 to +12 UTR did not exhibit luciferase activity. Moreover, as compared with the control of unexpressed HGF, the HGF protein suppressed luciferase expression driven by the 5'untranslated region of the Pf-Sub2 promoter. Taken together, it is suggested that HGF controls and interacts with the promoter region of the Pf-Sub2 gene.

The original version of this article unfortunately contained a mistake. The authors wish to revise the “Acknowledgements” section of their article.

Pf-calpain, a cysteine protease of Plasmodium falciparum, is believed to be one of the central mediators for essential parasitic activity. However, the roles of calpain on parasitic activity have not been determined in P. falciparum. In the present study, the localization of Pf-calpain was investigated using polyclonal antibodies (anti-Pf-calpain antibody A and B) against peptides that distinguished it from human calpain-7 and rat calpain-10 protein. Recombinant Pf-calpain (rPf-calpain) was identified as a 46 kDa protein using an anti-Pf-calpain antibody A, which can recognize the Pf-calpain binding site. Confocal microscopy revealed calpain within cytoplasmic localized parasites in the erythrocytic cycle. The findings suggested that the expression of Pf-calpain would be proportional to all different parasites in the erythrocytic cycle. On the other hand, anti-human calpain-7 antibody detected Pf-calpain in schizonts, and the immunofluorescence was stronger than with anti-rat calpain-10 antibody. However, the antibodies reacted with calpains in human red blood cells. These results show that anti-Pf-calpain antibody A and B specifically recognize only Pf-calpain. Taken together, the results suggest that Pf-calpain is expressed in all erythrocytic stages. In particular, the expression of Pf-calpain is increased much more when the late ring matures into the early trophozoite. Moreover, anti-Pf-calpain antibody A and B against synthetic peptides of the catalytic domain of Pf-calpain are useful to specifically detect Pf-calpain in all erythrocytic stages, while human and rat calpain antibody are not useful.

Cysteine proteases of helminth parasites play essential roles in parasite physiology as well as in a variety of important pathobiological processes. In this study, we identified a multigene family of cathepsin F cysteine proteases in Clonorchis sinensis (CsCFs). We identified a total of 12 CsCF genes through cDNA cloning using degenerate PCR primers followed by RACE. Sequence and phylogenetic analysis of the genes suggested they belonged to the cathepsin F-like enzyme family and further clustered into three different subfamilies. Enzymatic and proteomic analysis of C. sinensis excretory and secretory products (ESP) revealed that multiple isoforms of CsCF were the major proteins present in the ESP and the proteolytic activity of the ESP is mainly attributable to the enzymes. Comparative analysis of representative enzymes for each subfamily, CsCF-4, CsCF-6, and CsCF-11, showed that they share similar biochemical properties typical for cathepsin F-like enzymes, but significant differences were also identified. The enzymes were expressed throughout various developmental stages of the parasite and the transcripts increased gradually in accordance with the maturation of the parasite. Immunolocalization analysis of CsCFs showed that they were mainly localized in the intestine and intestinal contents of the parasite. These results collectively suggested that CsCFs, which are apparently synthesized in the epithelial cells lining the parasite intestine and secreted into the intestinal lumen of the parasite, might have a cooperative role for nutrient uptake in the parasite. Furthermore, they were eventually secreted into outside of the parasite and may perform additional functions for host-parasite interactions.

Bile stimulates many intestinal parasites, and newly excysted juvenile Clonorchis sinensis (CsNEJ) responds chemotactically to bile and matures in the bile duct. In this study, using annealing control primer-based polymerase chain reaction (PCR), 16 differentially expressed genes (DEGs) were found to be upregulated in C. sinensis metacercariae incubated in bile. Using contigs retrieved from a C. sinensis-expressed sequence tag pool, DEG sequences were extended further by DNA-walking. Of these, five DEGs were annotated to functional genes and confirmed to have been upregulated by more than twofold by quantitative real-time PCR. The gene products of these DEGs were cytochrome c oxidase subunit 2, phosphoenolpyruvate carboxykinase 2, and mitochondrial phosphate carrier protein, which are involved in energy generation, and HLA-B-associated transcript 3 and zinc finger protein, which are regulatory proteins associated with apoptosis and/or proliferation signaling pathways. Based on these results, it is suggested that bile stimulates the expressions of genes that produce the energy required by CsNEJs to migrate to the bile duct and to modulate the regulatory signals of cell proliferation associated with adult development.

Clonorchis sinensis thrives on bile juice. The effects of bile and bile acids on newly excysted juvenile C. sinensis (CsNEJ) were studied in terms of survival. Survival of CsNEJs maintained in 1x Locke's solution, Dulbecco's modified Eagle's medium, NCTC 109, Eagle's, RPMI 1640, and 0.1% glucose was high, but dropped rapidly in 2x Locke's, 0.85% NaCl, and phosphate-buffered saline. Most amino acids in the media favored CsNEJ survival; however, aspartic and glutamic acids and adenine reduced survival. Survival was also significantly lower in media containing more than 0.1% bile. CsNEJs preconditioned in low bile media survived longer in higher bile media. All bile acids and conjugated bile salts were found to favor CsNEJ survival, except for lithocholic acid (LCA) which was toxic. NCTC 109 medium was found to be optimal for the in vitro maintenance of CsNEJs and 1x Locke's solution to be suitable for analyzing the biological effects of bioactive compounds and molecules. Based on these results, we propose that bile acids enhance activity of CsNEJs, but LCA deteriorate CsNEJs.

Gamma-rays are a form of ionizing radiation and produce serious cellular damage to nuclei and organelles. Gamma irradiation induces the expressions of genes involved in DNA repair. Clonorchis sinensis resides in and provokes pathophysiologic changes in the bile ducts of mammals. The C. sinensis metacercariae are unsusceptible or resistant to gamma irradiation with LD50 of 16.5 Gy. Using the annealing control primer-based polymerase chain reaction (PCR) method, 19 genes were found to be up-regulated in C. sinensis metacercariae exposed to gamma rays. Contigs of up-regulated genes (URGs) were retrieved in a C. sinensis expressed sequence tag pool and extended by DNA-walking. Of the 13 URGs annotated putatively as functional genes, five URGs were associated with energy metabolism, six with protein processing, and the other two with DNA repair protein RAD23 and inhibitor of apoptosis protein. Four URGs were confirmed up-regulated by gamma irradiation by quantitative real-time PCR. One unknown gene, which was up-regulated to the greatest extent, might contribute to early recovery from gamma-irradiation-induced damage. The up-regulations of genes encoding DNA repair, protein processing, and energy metabolism proteins suggests that increases in gene products orchestrate DNA lesion repair and recover cellular functions in gamma-irradiated C. sinensis metacercariae.

Clonorchis sinensis develop through miracidium, sporocyst, redia, cercaria, and metacercaria stages before becoming egg-laying adult flukes. The authors undertook this analysis of gene expression profiles during developmental stages to increase our understanding of the biology of C. sinensis and of host-parasite relationships. From a C. sinensis metacercariae complementary deoxyribonucleic acid library, 419 expressed sequence tags (ESTs) of average length of 668 bp were collected and assembled into 322 genes containing 70 clusters and 252 singletons. The genes were annotated using BLAST searches and categorized into ten major functional categories. Genes expressed abundantly were those of proteases and metabolic, transcription, and translation housekeeping proteins. Genes expressed higher in C. sinensis metacercariae than in adults coded structural and cytoskeletal proteins, transcription and translation machinery proteins, and energy metabolism-related proteins. This EST information supports the notion that C. sinensis metacercariae in fish hosts have a physiology and metabolism that is quite different from that of its adult form in mammals.

WD40-repeat proteins have four to eight repeating units flanked by Gly-His (GH) and Trp-Asp (WD) at both termini and folds into a beta-propeller. A polypeptide deduced from a Clonorchis sinensis cDNA clone analyzed to have seven WD40-repeats and predicted to form a beta-propeller (CsWD1). The CsWD1 protein was expressed stage-specifically in the metacercariae and localized in the tegumental syncytium. The CsWD1 protein is suggested to serve as a platform for interacting partner proteins in the tegumental syncytium of C. sinensis metacercariae.

Codon usage in Clonorchis sinensis was analyzed using 12,515 codons from 38 coding sequences. Total GC content was 49.83%, and GC1, GC2 and GC3 contents were 56.32%, 43.15% and 50.00%, respectively. The effective number of codons converged at 51-53 codons. When plotted against total GC content or GC3, codon usage was distributed in relation to GC3 biases. Relative synonymous codon usage for each codon revealed a single major trend, which was highly correlated with GC content at the third position when codons began with A or U at the first two positions. In codons beginning with G or C base at the first two positions, the G or C base rarely occurred at the third position. These results suggest that codon usage is shaped by a bias towards G or C at the third base, and that this is affected by the first and second bases.

Background Plasmodium vivax apical membrane antigen-1 (PvAMA-1) is a leading candidate antigen for blood stage malaria vaccine. However, antigenic variation is a major obstacle in the development of an effective vaccine based on this antigen. In this study, the genetic structure and the effect of natural selection of PvAMA-1 among Korean P. vivax isolates were analysed. Methods Blood samples were collected from 66 Korean patients with vivax malaria. The entire PvAMA-1 gene was amplified by polymerase chain reaction and cloned into a TA cloning vector. The PvAMA-1 sequence of each isolate was sequenced and the polymorphic characteristics and effect of natural selection were analysed using the DNASTAR, MEGA4, and DnaSP programs. Results Thirty haplotypes of PvAMA-1, which were further classified into seven different clusters, were identified in the 66 Korean P. vivax isolates. Domain II was highly conserved among the sequences, but substantial nucleotide diversity was observed in domains I and III. The difference between the rates of non-synonymous and synonymous mutations suggested that the gene has evolved under natural selection. No strong evidence indicating balancing or positive selection on PvAMA-1 was identified. Recombination may also play a role in the resulting genetic diversity of PvAMA-1. Conclusions This study is the first comprehensive analysis of nucleotide diversity across the entire PvAMA-1 gene using a single population sample from Korea. Korean PvAMA-1 had limited genetic diversity compared to PvAMA-1 in global isolates. The overall pattern of genetic polymorphism of Korean PvAMA-1 differed from other global isolates and novel amino acid changes were also identified in Korean PvAMA-1. Evidences for natural selection and recombination event were observed, which is likely to play an important role in generating genetic diversity across the PvAMA-1. These results provide useful information for the understanding the population structure of P. vivax circulating in Korea and have important implications for the design of a vaccine incorporating PvAMA-1.

Expressed sequence tag (EST) pools represent partial profiles of the gene expressions of organisms. In an effort to construct a Clonorchis sinensis EST pool, 2,387 ESTs were collected from an adult C. sinensis cDNA library and assembled into 1,573 clusters. Of these clusters, 1,225 ESTs (51%) were singletons and 348 clusters consisted of more than two ESTs. There were 848 clusters (54%) that shared significant identity with previously reported proteins, and of these, 401 clusters were categorized into 11 major functional protein classes. Three cDNA clones of fructose-1,6-bisphosphate (FBP) aldolase were selected from the C. sinensis EST pool and analyzed for phylogenic clustering. FBP clones encoded a complete polypeptide, which shared significant identity to those of vertebrate and invertebrate animals and clustered with those of trematodes. We believe that the EST pool described can be confidently used as a platform in multigene researches on C. sinensis gene expression.

In trematodes, vitelline precursor proteins are required for eggshell formation. A cDNA clone of Clonorchis sinensis (CsVpB1) was selected from an EST pool, encoding a polypeptide of 245 amino acids. The CsVpB1 polypeptide demonstrated homology with vitelline precursor proteins from trematodes with high sequential identities. In a phylogenic tree, CsVpB1 clustered with trematode VpB proteins. The CsVpB1 polypeptide was found to be rich in tyrosine residues, including putative pre-dihydroxyphenyl alanine (DOPA) residues, involved in cross-linking of the precursor proteins. Mouse immune sera were raised against a recombinant CsVpB1 protein. In adult C. sinensis, CsVpB1 protein was exclusively localized in vitelline follicles. Based on these results, the CsVpB1 cDNA is believed to encode a VpB of C. sinensis.

One cDNA clone was purified from an adult Clonorchis sinensis cDNA library, and its deduced polypeptide sequence was found to be homologous with myosin regulatory light chain (MRLC) of invertebrates and vertebrates. Two amino-acid residues, Thr and Ser, were conserved at the phosphorylation sites that regulate the function of MRLCs. Recombinant C. sinensis MRLC (rCsMRLC) protein was produced and purified from Escherichia coli, and mouse anti-CsMRLC immune sera recognized a protein of molecular weight 24 kDa from a soluble protein preparation of C. sinensis. The CsMRLC protein was immunohistochemically localized to the muscle fibers of the subtegumental muscle layer and to the muscles of oral and ventral suckers. However, the rCsMRLC protein proved to be less useful antigen for the serodiagnosis of human clonorchiasis.

Toxoplasma gondii, an apicomplexan protozoa, can infect a broad range of host animals, including humans. This infection passes by asymptomatically and opportunistically in most infected hosts, but can cause toxoplasmosis in some individuals with such symptoms as encephalitis, chorioretinitis, and lymphadenitis in acquired infections and abortion and neonatal mortality in congenital infections [1-3]. In the Republic of Korea, seroprevalence of toxoplasmosis has been reported sporadically among various groups of outpatients in hospitals. It has been recognized generally that the positive rates of Koreans are in the range from 1.9 to 7.7% [4-6], except for patient groups or residents in Jeju island of 12.9% [6] or more recently 13.2% [7], which is maintained higher than the other regions of Korea. However, all positive rates in Korea are still significantly lower than, approximately 1/10 of, those of other endemic countries of 30-70% [1]. Here, we present a survey which has screened the seroprevalence of toxoplasmosis among the residents in several villages of Cheorwon-gun, Gangwon-do, which are localized near the demilitarized zone (DMZ) facing the North Korea and resulted in a 17.0% of the positive rate, the highest among the surveys ever done in Korea. A total of 1,661 sera were collected from residents (866 males and 795 females) of villages scattered over the 6 administrative regions of Cheorwon-gun, including urban and rural environments, from November to December 2010 and the same 2 months of 2011, under the regulation of the IRB Committee of Chung-Ang University (No. 2010-06-03) held on June 18, 2010. The sera were checked for IgG antibody titers by ELISA using a crude extract RH strain antigen of T. gondii according to the method of [4], which resulted in a 17.0% positive rate (282 sera) (Table 1). The positive rate was significantly different (P

Peptides pore-forming in cell membrane have been identified from a wide range of animals. A putative pore-forming peptide deduced from a cDNA clone of Clonorchis sinensis (clonorin) was predicted to consist of four amphipathic α-helices. Clonorin contained six invariably conserved cysteine residues, identified to form three disulfide bonds. These predicted structural features are highly homologous with pore-forming peptides, the amoebapores. Recombinant clonorin showed hemolytic activity toward rabbit erythrocytes. The hemolytic activity of C. sinensis extract increased dose-dependently and was inhibited by anti-clonorin immune sera. The clonorin was expressed developmentally in juvenile and adult flukes and localized in the intestinal epithelium of adult flukes. It is proposed that, through lysing host cellular components, clonorin could enhance proteolytic digestion in the intestine of C. sinensis .

Summary Previous studies have shown that spatiotemporal regulation of extracellular matrix (ECM) by proteinases is implicated in the initial step of regeneration. In amphibian regeneration, the up-regulation of proteinases such as metalloproteinases (MMPs) and cathepsin D, and proteinase-related proteins such as proteinase tissue inhibitors and activators has been demonstrated. Since the earthworm could provide a unique and valuable model to investigate the mechanism of regeneration, we studied the developmental change in proteinase expression during earthworm tail regeneration. Zymographic analysis revealed that proteinase activities began to increase within 1 h after amputation and reached a maximum at 7 days post-amputation. This peak in activity was approximately 22-fold greater than the unamputated controls. Thereafter, the proteinase activities tended to decrease followed by another peak at 30 days before returning to control levels. At least four types of proteinase were distinguishable at 7 and 30...

Background Plasmodium vivax re-emerged in 1993. Although the number of infections has been steadily decreasing, it is likely to continue to affect public health until it is eradicated. The aim of this study is to measure anti-circumsporozoite protein (CSP) antibody and compare malaria prevalence. As to understand the prevalence, an epidemiology study has to be conducted in the Republic of Korea. Methods A total of 1,825 and 1,959 blood samples were collected in 2010 and 2011, respectively, from the inhabitants of Ganghwa and Cheorwon counties. The antibody titers of the inhabitants were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant protein purified from Escherichia coli transformed with a CSP gene-inserted pET-28a(+) expression vector. Microscopic examination was performed to identify malaria parasites. Results The annual parasite incidence (API) in Ganghwa decreased from 4.28 in 2010 to 2.23 in 2011, and that in Cheorwon decreased from 1.88 in 2010 to 1.15 in 2011. The antibody-positive CSP rate in these areas also decreased from 18.14% (331/1825) in 2010 to 15.36% (301/1959) in 2011. Pearson analysis showed a strong correlation between the API and the antibody-positive CSP rate in these areas (r = 1.000, P

Plasmodium vivax reemerged in the Republic of Korea (ROK) in 1993, and is likely to continue to affect public health. The purpose of this study was to measure levels of anti-P. vivax antibodies using indirect fluorescent antibody test (IFAT) in border areas of ROK, to determine the seroprevalence of malaria (2003-2005) and to plan effective control strategies. Blood samples of the inhabitants in Gimpo-si, Paju-si, and Yeoncheon-gun (Gyeonggi-do), and Cheorwon-gun (Gangwon-do) were collected and kept in Korea Centers for Disease Control and Prevention (KCDC). Out of a total of 1,774 serum samples tested, the overall seropositivity was 0.94% (n=17). The seropositivity was the highest in Paju-si (1.9%, 7/372), followed by Gimpo-si (1.4%, 6/425), Yeoncheon-gun (0.67%, 3/451), and Cheorwon-gun (0.19%, 1/526). The annual parasite incidence (API) in these areas gradually decreased from 2003 to 2005 (1.69, 1.09, and 0.80 in 2003, 2004, and 2005, respectively). The highest API was found in Yeoncheon-gun, followed by Cheorwon-gun, Paju-si, and Gimpo-si. The API ranking in these areas did not change over the 3 years. The seropositivity of Gimpo-si showed a strong linear relationship with the API of 2005 (r=0.9983, P=0.036). Seropositivity data obtained using IFAT may be useful for understanding malaria prevalence of relevant years, predicting future transmission of malaria, and for establishing and evaluating malaria control programs in affected areas.

Background Transmission-blocking vaccines (TBVs), which target the sexual stages of malaria parasites to interfere with and/or inhibit the parasite’s development within mosquitoes, have been regarded as promising targets for disrupting the malaria transmission cycle. In this study, genetic diversity of four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, among Plasmodium vivax Korean isolates was analysed. Methods A total of 86 P. vivax- infected blood samples collected from patients in Korea were used for analyses. Each of the full-length genes encoding four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, were amplified by PCR, cloned into T&A vector, and then sequenced. Polymorphic characteristics of the genes were analysed using the DNASTAR, MEGA4, and DnaSP programs. Results Polymorphism analyses of the 86 Korean P. vivax isolates revealed two distinct haplotypes in Pvs25 and Pvs48/45, and three different haplotypes in PvWARP. In contrast, Pvs28 showed only a single haplotype. Most of the nucleotide substitutions and amino acid changes identified in all four TBV candidate antigens were commonly found in P. vivax isolates from other geographic areas. The overall nucleotide diversities of the TBV candidates were much lower than those of blood stage antigens. Conclusions Limited sequence polymorphisms of TBV candidate antigens were identified in the Korean P. vivax population. These results provide baseline information for developing an effective TBV based on these antigens, and offer great promise for applications of a TBV against P. vivax infection in regions where the parasite is most prevalent.

Plasmodium vivax Duffy binding protein (PvDBP) is a micronemal type I membrane protein that plays an essential role in erythrocyte invasion of merozoites. PvDBP is a prime blood stage vaccine candidate antigen against P. vivax, but its polymorphic nature represents a major obstacle to the successful design of a protective vaccine against vivax malaria. In this study, we analyzed the genetic polymorphism and natural selection at the N-terminal cysteine-rich region of PvDBP (PvDBPII) among 70 P. vivax isolates collected from Korean patients during 2005-2010. Seventeen single nucleotide polymorphisms (SNP), which resulted in 14 non-synonymous and 3 synonymous mutations, were found in PvDBPII among the Korean P. vivax isolates. Sequence analyses revealed that 13 different PvDBPII haplotypes, which were clustered into 3 distinct clades, were identified in Korean P. vivax isolates. The difference between the rates of nonsynomyous and synonymous mutations suggested that the region has evolved under natural selection. High selective pressure preferentially acted on regions identified or predicted to be B- and T-cell epitopes and MHC binding regions of PvDBPII. Recombination may also contribute to genetic diversity of PvDBPII. Our results suggest that PvDBPII of Korean P. vivax isolates display a limited genetic polymorphism and are under selective pressure. These results have significant implications for understanding the nature of the P. vivax population circulating in Korea and provide useful information for development of malaria vaccines based on this antigen.

Rapid in-field diagnosis is very important to prevent the outbreak of various infectious and contagious diseases. Highly sensitive and quantitative detection of diseases can be performed using fluorescent immunochemical assay with specific antigen-antibody binding and a good quality fluorophore. This can lead to the development of a small, portable, quantitative biosensor to transmit diagnostic results to a control center in order to systematically prevent disease outbreaks. In this study, we developed a novel fluorophore, coumarin-derived dendrimer, with high emission intensity, strong signal brightness, and high photostability. It is easily coupled with biomolecules and emits strong and stable fluorescence at 590 nm with excitation at 455 nm. Application to fluorescent immunochromatographic test (FICT) showed that the novel coumarin-derived dendrimer bioconjugate could detect antigens at amount as low as 0.1 ng. The clinical results and the spectral characteristics of the novel coumarin-derived dendrimer open, for the first time, the possibility of developing a cost/energy efficient LED-based portable quantitative biosensor for point-of-care (POC) disease diagnosis, which can permit real time monitoring (U-healthcare system) by a disease control center.

The fluorescent compound (VI) can be used for the detection of Swine flu virus antigen with selectivity and specificity.

Anthelmintic resistance is a serious global problem because of the worldwide spread of resistant nematodes in animals and humans. This has triggered increasing investment in research for new anthelmintics. Over the past decade, Caenorhabditis elegans has become a popular model organism for parasitic nematode research, and many examples have been published to illustrate its use. In this study, we investigated the effect of KSI-4088 on the egg hatching, larval development, and migration of the nematode worm C. elegans compared with ivermectin and levamisole (well-known anthelmintic drugs). KSI-4088 demonstrated anthelmintic activity on all assays of C. elegans. The anthelmintic activity of KSI-4088 on egg hatching and larval development showed especially strong activity, but assays showed that ivermectin and levamisole had no effects on C. elegans. In addition, KSI-4088 was capable of producing a change in the timing of the development of the worms at the L1-L3 and L4 stage. Also, we demonstrate that C. elegans L3-4 are more sensitive than adults to KSI-4088 in assay of migration. Our results indicate that KSI-4088 is an active anthelmintic compound that should be further investigated with the aim of developing a potent drug against nematodes.

The accuracies of relative gene expressions as determined by quantitative real-time polymerase chain reaction are largely dependent on the variabilities of the reference genes used. Validation of the stabilities of reference genes under experimental conditions is an essential initial step for comparative studies on the expression levels of target genes in experimental groups. Using three total RNA samples extracted independently from Clonorchis sinensis metacercariae and adults, we determined the gene expression stabilities of eight reference gene candidates and the relative transcript levels of three target genes using the geNorm program. The reference genes found to be stably expressed in metacercariae and adults were phosphoglycerate kinase, beta-actin, and calcyphosine; reference genes found to be stably expressed under gamma-irradiated and non-irradiated conditions were succinate dehydrogenase, small nuclear ribonucleoprotein, and beta-actin; and those stably expressed regardless of bile treatment were small nuclear ribonucleoprotein, phosphoglycerate kinase, and succinate dehydrogenase. According to our data, the expression levels of target genes are dependent on normalization factors, such as the C (T) values of single reference genes and the geometric mean of the C (T) values of three reference genes. When comparing C. sinensis gene expressions, we propose to employ the geometric mean of the C (T) values of more than three reference genes validated in the same experimental setting.

The WD40-repeat proteins serve as a platform coordinating partner proteins and are involved in a range of regulatory cellular functions. A WD40-repeat protein (CsWD1) of Clonorchis sinensis previously cloned is expressed stage-specifically in the tegumental syncytium of C. sinensis metacercariae. In the present study, interact- ing proteins with the CsWD1 protein was purified by immunoprecipitation and 2 dimension gel electrophoresis from the C. sinensis metacercaria soluble extract, and tryptic peptides were analyzed by LC/ESI-MS. Putative partner pro- teins were annotated to be actin-2, glyceraldehyde-3-phosphate dehydrogenase, and hypothetical and unmanned proteins. The CsWD1 protein was predicted to contain 3 conserved actin-interacting residues on its functional sur- face. With these results, the CsWD1 protein is suggested to be an actin-interacting protein of C. sinensis.

Elongation factor-1 (EF-1) plays a primary role in protein synthesis, e.g., in the regulation of cell growth, aging, motility, embryogenesis, and signal transduction. The authors identified a clone CsIH23 by immunoscreening a Clonorchis sinensis cDNA library. The cDNA of CsIH23 was found to have a putative open reading frame containing 461 amino acids with a predicted molecular mass of 50.5 kDa. Its polypeptide sequence was highly homologous with EF-1alpha of parasites and vertebrate animals. CsIH23 polypeptide contained three GTP/GDP-binding sites, one ribosome-binding domain, one actin-binding domain, one tRNA-binding domain, and two glyceryl-phosphoryl-ethanolamine attachment sites. Based on these primary and secondary structural similarities, it was concluded that CsIH23 cDNA encodes C. sinensis EF-1alpha (CsEF-1alpha). In a molecular phylogenic tree, CsEF-1alpha clustered with the EF-1alpha of helminthic parasites. Subsequently, CsEF-1alpha recombinant protein was bacterially overexpressed and purified by Ni-NTA affinity column chromatography. Immunoblotting using CsEF-1alpha recombinant protein produced positive signals for all serum samples tested from clonorchiasis, opisthorchiasis viverinii, and paragonimiasis westermani patients and normal healthy controls. These findings suggest that recombinant CsEF-1alpha is of limited usefulness as serodiagnostic antigen for clonorchiasis.

WD40-repeat proteins have four to eight repeat units, which have Gly–His (GH) and Trp–Asp (WD) at both termini and fold into a beta-propeller. In particular, the WD40-repeat protein of Clonorchis sinensis (CsWD1) has seven WD-repeat units and is expressed stage-specifically in metacercariae. By yeast two-hybrid screening, putative interacting protein cDNAs were cloned from a C. sinensis metacercaria cDNA library and purified further by higher stringency screening and lacZ colony-lift assay. After assessing their nucleotide and polypeptide sequences, 21 putative partner protein cDNAs were selected and assembled into 14 clones. Using YRG2 strain yeast, 12 putative partner protein clones were confirmed to interact with CsWD1 protein. These 12 proteins were grouped into functional categories, i.e., signal proteins, transporters, proteases, and muscle proteins. These results suggest that CsWD1 protein is associated with intracellular protein translocation and cell cycle control in C. sinensis metacercaria.

Ferritin is an intracellular protein that is involved in iron metabolism. A cDNA clone of Clonorchis sinensis (CsFtn), 565 bp long, encoded a putative polypeptide of 166 amino acids. CsFtn cDNA revealed a putative loop–stem structure similar to iron-responsive element (IRE). CsFtn polypeptide appeared homologous to the ferritin of trematodes with high sequential identity. Phylogenetic tree analysis showed that CsFtn clustered with the ferritins of other flukes. Recombinant CsFtn protein was produced and purified from an Escherichia coli system, and immune mouse serum was raised against CsFtn. Recombinant CsFtn showed iron-uptake ability. In adult C. sinensis, CsFtn protein was found to localize in vitelline follicles and eggs. Based on these results, CsFtn cDNA is considered to encode a C. sinensis yolk ferritin.