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Molecular Cloning and Characterization of Taurocyamine Kinase from Clonorchis sinensis: A Candidate Chemotherapeutic Target  [PDF]
Jing-ying Xiao equal contributor,Ji-Yun Lee equal contributor,Shinji Tokuhiro,Mitsuru Nagataki,Blanca R. Jarilla,Haruka Nomura,Tae Im Kim,Sung-Jong Hong ,Takeshi Agatsuma
PLOS Neglected Tropical Diseases , 2013, DOI: 10.1371/journal.pntd.0002548
Abstract: Background Adult Clonorchis sinensis lives in the bile duct and causes endemic clonorchiasis in East Asian countries. Phosphagen kinases (PK) constitute a highly conserved family of enzymes, which play a role in ATP buffering in cells, and are potential targets for chemotherapeutic agents, since variants of PK are found only in invertebrate animals, including helminthic parasites. This work is conducted to characterize a PK from C. sinensis and to address further investigation for future drug development. Methology/Principal findings A cDNA clone encoding a putative polypeptide of 717 amino acids was retrieved from a C. sinensis transcriptome. This polypeptide was homologous to taurocyamine kinase (TK) of the invertebrate animals and consisted of two contiguous domains. C. sinensis TK (CsTK) gene was reported and found consist of 13 exons intercalated with 12 introns. This suggested an evolutionary pathway originating from an arginine kinase gene group, and distinguished annelid TK from the general CK phylogenetic group. CsTK was found not to have a homologous counterpart in sequences analysis of its mammalian hosts from public databases. Individual domains of CsTK, as well as the whole two-domain enzyme, showed enzymatic activity and specificity toward taurocyamine substrate. Of the CsTK residues, R58, I60 and Y84 of domain 1, and H60, I63 and Y87 of domain 2 were found to participate in binding taurocyamine. CsTK expression was distributed in locomotive and reproductive organs of adult C. sinensis. Developmentally, CsTK was stably expressed in both the adult and metacercariae stages. Recombinant CsTK protein was found to have low sensitivity and specificity toward C. sinensis and platyhelminth-infected human sera on ELISA. Conclusion CsTK is a promising anti-C. sinensis drug target since the enzyme is found only in the C. sinensis and has a substrate specificity for taurocyamine, which is different from its mammalian counterpart, creatine.
Cholecystolithiasis Is Associated with Clonorchis sinensis Infection  [PDF]
Tie Qiao, Rui-hong Ma, Xiao-bing Luo, Zhen-liang Luo, Pei-ming Zheng
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0042471
Abstract: Background The objective of this study was to analyze gallbladder stones for direct evidence of a relationship between Clonorchis sinensis infection and gallbladder stones formation. Methodology We investigated one hundred eighty-three gallbladder stones for the presence of Clonorchis sinensis eggs using microscopy, and analyzed their composition using Fourier transform infrared spectroscopy. We confirmed the presence of Clonorchis sinensis eggs in the gallbladder stones using real-time fluorescent PCR and scanning electron microscopy. Principal Findings Clonorchis sinensis eggs were detected in 122 of 183 gallbladder stones based on morphologic characteristics and results from real-time fluorescent PCR. The proportion of pigment stones, cholesterol stones and mixed gallstones in the egg-positive stones was 79.5% (97/122), 3.3% (4/122) and 17.2% (21/122), respectively, while 29.5% (18/61), 31.1% (19/61) and 39.3% (24/61) in the egg-negative stones. The proportion of pigment stone in the Clonorchis sinensis egg-positive stones was higher than in egg-negative stones (P<0.0001). In the 30 egg-positive stones examined by scanning electron microscopy, dozens or even hundreds of Clonorchis sinensis eggs were visible (×400) showing a distinct morphology. Many eggs were wrapped with surrounding particles, and in some, muskmelon wrinkles was seen on the surface of the eggs. Also visible were pieces of texture shed from some of the eggs. Some eggs were depressed or without operculum while most eggs were adhered to or wrapped with amorphous particles or mucoid matter (×3000). Conclusion Clonorchis sinensis eggs were detected in the gallbladder stones which suggests an association between Clonorchis sinensis infection and gallbladder stones formation, especially pigment stones.
Identification and characterization of microRNAs in Clonorchis sinensis of human health significance
Min-Jun Xu, Quan Liu, Alasdair J Nisbet, Xian-Quan Cai, Chao Yan, Rui-Qing Lin, Zi-Guo Yuan, Hui-Qun Song, Xian-Hui He, Xing-Quan Zhu
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-521
Abstract: Here we report the use of this approach to identify and clone 6 new and 62,512 conserved C. sinensis miRNAs which belonged to 284 families. There was strong bias on families, family members and sequence nucleotides in C. sinensis. Uracil was the dominant nucleotide, particularly at positions 1, 14 and 22, which were located approximately at the beginning, middle and end of conserved miRNAs. There was no significant "seed region" at the first and ninth positions which were commonly found in human, animals and plants. Categorization of conserved miRNAs indicated that miRNAs of C. sinensis were still innovated and concentrated along three branches of the phylogenetic tree leading to bilaterians, insects and coelomates. There were two miRNA strategies in C. sinensis for its parasitic life: keeping a large category of miRNA families of different animals and keeping stringent conserved seed regions with high active innovation in other places of miRNAs mainly in the middle and the end, which were perfect for the parasite to perform its complex life style and for host changes.The present study represented the first large scale characterization of C. sinensis miRNAs, which have implications for understanding the complex biology of this zoonotic parasite, as well as miRNA studies of other related species such as Opisthorchis viverrini and Opisthorchis felineus of human and animal health significance.Fish-borne clonorchiasis, caused by the oriental liver fluke Clonorchis sinensis, is endemic in many Asian countries and over 35 million people globally are infected with C. sinensis, including an estimated 15 million in China [1]. The parasite has major socioeconomic impacts in other parts of Asia as well. In Korea, infection in humans is one of the most prevalent [2]. While in Vietnam, the prevalence reaches 79% in the Haiphong and Hanoi area [3]. This infection is also becoming increasingly common in non-endemic regions and in developed countries due to growing international ma
The Carcinogenic Liver Fluke, Clonorchis sinensis: New Assembly, Reannotation and Analysis of the Genome and Characterization of Tissue Transcriptomes  [PDF]
Yan Huang, Wenjun Chen, Xiaoyun Wang, Hailiang Liu, Yangyi Chen, Lei Guo, Fang Luo, Jiufeng Sun, Qiang Mao, Pei Liang, Zhizhi Xie, Chenhui Zhou, Yanli Tian, Xiaoli Lv, Lisi Huang, Juanjuan Zhou, Yue Hu, Ran Li, Fan Zhang, Huali Lei, Wenfang Li, Xuchu Hu, Chi Liang, Jin Xu, Xuerong Li, Xinbing Yu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0054732
Abstract: Clonorchis sinensis (C. sinensis), an important food-borne parasite that inhabits the intrahepatic bile duct and causes clonorchiasis, is of interest to both the public health field and the scientific research community. To learn more about the migration, parasitism and pathogenesis of C. sinensis at the molecular level, the present study developed an upgraded genomic assembly and annotation by sequencing paired-end and mate-paired libraries. We also performed transcriptome sequence analyses on multiple C. sinensis tissues (sucker, muscle, ovary and testis). Genes encoding molecules involved in responses to stimuli and muscle-related development were abundantly expressed in the oral sucker. Compared with other species, genes encoding molecules that facilitate the recognition and transport of cholesterol were observed in high copy numbers in the genome and were highly expressed in the oral sucker. Genes encoding transporters for fatty acids, glucose, amino acids and oxygen were also highly expressed, along with other molecules involved in metabolizing these substrates. All genes involved in energy metabolism pathways, including the β-oxidation of fatty acids, the citrate cycle, oxidative phosphorylation, and fumarate reduction, were expressed in the adults. Finally, we also provide valuable insights into the mechanism underlying the process of pathogenesis by characterizing the secretome of C. sinensis. The characterization and elaborate analysis of the upgraded genome and the tissue transcriptomes not only form a detailed and fundamental C. sinensis resource but also provide novel insights into the physiology and pathogenesis of C. sinensis. We anticipate that this work will aid the development of innovative strategies for the prevention and control of clonorchiasis.
Molecular Cloning and Gene Expression Analysis of the Leptin Receptor in the Chinese Mitten Crab Eriocheir sinensis  [PDF]
Hui Jiang,Fei Ren,Jiangling Sun,Lin He,Weiwei Li,Yannan Xie,Qun Wang
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0011175
Abstract: Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals.
Molecular cloning and functional analysis of Chinese sturgeon (Acipenser sinensis) growth hormone receptor
ZhiYong Liao,XiaoLi Chen,MingJiang Wu
Science China Life Sciences , 2009, DOI: 10.1007/s11427-009-0131-3
Abstract: A full length cDNA encoding the growth hormone receptor (GHR) of Chinese sturgeon was cloned in order to investigate the mechanism of growth hormone in regulating the growth of Chinese sturgeon. The open reading frame of the cloned Chinese sturgeon growth hormone receptor (csGHR) cDNA encodes a trans-membrane protein of 611 amino acids containing all the characteristic motifs of GHR. By sequence alignment, substitutions of amino acid residues highly conserved in other species were identified. Using the CHO cell culture system, the function of csGHR and the biological significance of the amino acid substitution in csGHR were examined. The promoter of serine protease inhibitor 2.1 (Spi2.1) was trans-activated upon stimulation of seabream GH (sbGH) in the csGHR-expressing CHO cells. Furthermore, CHO cells stably expressing csGHR were stimulated to proliferate by sbGH. In agreement with our previous report, Chinese sturgeon growth hormone-binding protein (csGHBP) was detected in the culture medium of CHO cells stably expressing csGHR. Mutation of Asp residue in the ligand binding motif in csGHR to Glu significantly enhanced csGHR’s biological function, whereas mutation of Asp residue to Ala decreased its biological function. The results demonstrated that the cloned csGHR was of full biological function and the csGHBP could be generated through proteolysis of csGHR. These findings might provide new insights into thoroughly understanding the regulatory mechanism of Chinese sturgeon growth.
Molecular cloning and functional analysis of Chinese sturgeon (Acipenser sinensis) growth hormone receptor

LIAO ZhiYong,CHEN XiaoLi&,WU MingJiang,

中国科学C辑(英文版) , 2009,
Abstract: A full length cDNA encoding the growth hormone receptor (GHR) of Chinese sturgeon was cloned in order to investigate the mechanism of growth hormone in regulating the growth of Chinese sturgeon. The open reading frame of the cloned Chinese sturgeon growth hormone receptor (csGHR) cDNA encodes a trans-membrane protein of 611 amino acids containing all the characteristic motifs of GHR. By sequence alignment, substitutions of amino acid residues highly conserved in other species were identified. Using the CHO cell culture system, the function of csGHR and the biological significance of the amino acid substitution in csGHR were examined. The promoter of serine protease inhibitor 2.1 (Spi2.1) was trans-activated upon stimulation of seabream GH (sbGH) in the csGHR-expressing CHO cells. Furthermore, CHO cells stably expressing csGHR were stimulated to proliferate by sbGH. In agreement with our previous report, Chinese sturgeon growth hormone-binding protein (csGHBP) was detected in the culture medium of CHO cells stably expressing csGHR. Mutation of Asp residue in the ligand binding motif in csGHR to Glu significantly enhanced csGHR’s biological function, whereas mutation of Asp residue to Ala decreased its biological function. The results demonstrated that the cloned csGHR was of full biological function and the csGHBP could be generated through proteolysis of csGHR. These findings might provide new insights into thoroughly understanding the regulatory mechanism of Chinese sturgeon growth.
Unlocking the Transcriptomes of Two Carcinogenic Parasites, Clonorchis sinensis and Opisthorchis viverrini  [PDF]
Neil D. Young ,Bronwyn E. Campbell,Ross S. Hall,Aaron R. Jex,Cinzia Cantacessi,Thewarach Laha,Woon-Mok Sohn,Banchob Sripa,Alex Loukas,Paul J. Brindley,Robin B. Gasser
PLOS Neglected Tropical Diseases , 2010, DOI: 10.1371/journal.pntd.0000719
Abstract: The two parasitic trematodes, Clonorchis sinensis and Opisthorchis viverrini, have a major impact on the health of tens of millions of humans throughout Asia. The greatest impact is through the malignant cancer ( = cholangiocarcinoma) that these parasites induce in chronically infected people. Therefore, both C. sinensis and O. viverrini have been classified by the World Health Organization (WHO) as Group 1 carcinogens. Despite their impact, little is known about these parasites and their interplay with the host at the molecular level. Recent advances in genomics and bioinformatics provide unique opportunities to gain improved insights into the biology of parasites as well as their relationships with their hosts at the molecular level. The present study elucidates the transcriptomes of C. sinensis and O. viverrini using a platform based on next-generation (high throughput) sequencing and advanced in silico analyses. From 500,000 sequences, >50,000 sequences were assembled for each species and categorized as biologically relevant based on homology searches, gene ontology and/or pathway mapping. The results of the present study could assist in defining molecules that are essential for the development, reproduction and survival of liver flukes and/or that are linked to the development of cholangiocarcinoma. This study also lays a foundation for future genomic and proteomic research of C. sinensis and O. viverrini and the cancers that they are known to induce, as well as novel intervention strategies.
Developmental Transcriptomic Features of the Carcinogenic Liver Fluke, Clonorchis sinensis  [PDF]
Won Gi Yoo equal contributor,Dae-Won Kim equal contributor,Jung-Won Ju equal contributor,Pyo Yun Cho,Tae Im Kim,Shin-Hyeong Cho,Sang-Haeng Choi,Hong-Seog Park ,Tong-Soo Kim ,Sung-Jong Hong
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001208
Abstract: Clonorchis sinensis is the causative agent of the life-threatening disease endemic to China, Korea, and Vietnam. It is estimated that about 15 million people are infected with this fluke. C. sinensis provokes inflammation, epithelial hyperplasia, and periductal fibrosis in bile ducts, and may cause cholangiocarcinoma in chronically infected individuals. Accumulation of a large amount of biological information about the adult stage of this liver fluke in recent years has advanced our understanding of the pathological interplay between this parasite and its hosts. However, no developmental gene expression profiles of C. sinensis have been published. In this study, we generated gene expression profiles of three developmental stages of C. sinensis by analyzing expressed sequence tags (ESTs). Complementary DNA libraries were constructed from the adult, metacercaria, and egg developmental stages of C. sinensis. A total of 52,745 ESTs were generated and assembled into 12,830 C. sinensis assembled EST sequences, and then these assemblies were further categorized into groups according to biological functions and developmental stages. Most of the genes that were differentially expressed in the different stages were consistent with the biological and physical features of the particular developmental stage; high energy metabolism, motility and reproduction genes were differentially expressed in adults, minimal metabolism and final host adaptation genes were differentially expressed in metacercariae, and embryonic genes were differentially expressed in eggs. The higher expression of glucose transporters, proteases, and antioxidant enzymes in the adults accounts for active uptake of nutrients and defense against host immune attacks. The types of ion channels present in C. sinensis are consistent with its parasitic nature and phylogenetic placement in the tree of life. We anticipate that the transcriptomic information on essential regulators of development, bile chemotaxis, and physico-metabolic pathways in C. sinensis that presented in this study will guide further studies to identify novel drug targets and diagnostic antigens.
Prevalence of Clonorchis sinensis infection in dogs and cats in subtropical southern China
Rui-Qing Lin, Jian-Dong Tang, Dong-Hui Zhou, Hui-Qun Song, Si-Yang Huang, Jia-Xu Chen, Mu-Xin Chen, Han Zhang, Xing-Quan Zhu, Xiao-Nong Zhou
Parasites & Vectors , 2011, DOI: 10.1186/1756-3305-4-180
Abstract: A total of 503 dogs and 194 cats from 13 administrative regions in Guangdong province were examined by post-mortem examination. The worms were examined, counted, and identified to species according to existing keys and descriptions. The average prevalences of C. sinensis infection in dogs and cats were 20.5% and 41.8%, respectively. The infection intensities in dogs were usually light, but in cats the infection intensities were more serious. The prevalences were higher in some of the cities located in the Pearl River Delta region which is the most important endemic area in Guangdong province, but the prevalences were relatively lower in seaside cities.The present investigation revealed a high prevalence of C. sinensis infection in its reservoir hosts dogs and cats in China's subtropical Guangdong province, which provides relevant "base-line" data for conducting control strategies and measures against clonorchiasis in this region.Clonorchiasis, caused by Clonorchis sinensis, is considered as one of the major parasitic zoonoses in some parts of Asia, which can cause a series of hepatic diseases such as periductal inflammation, fibrosis, pyogenic cholangitis, biliary calculi, cholecystitis, liver cirrhosis and pancreatitis, and C. sinensis is currently classified as carcinogenic to humans for cholangiocarcinoma [1-3]. It is estimated that about 35 million people are infected globally, and of whom approximately 15 million are in China [4,5], where human clonorchiasis is endemic in 27 provinces (including municipal cities and autonomous regions) [4-6].Snails serve as the first intermediate hosts for C. sinensis and the prevalence is high (up to 15%) in some areas of China, and approximately 140 species of freshwater fishes and four species of shrimp have been recognized as the second intermediate hosts for C. sinensis in China [6]. Many mammals, such as cats, dogs and pigs, serve as definitive hosts or reservoir hosts for C. sinensis [4,7,8]. "Ingesting raw fish" is cons
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