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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.
Author Summary
Clonorchis sinensis is a significant pathogen that causes clonorchiasis, which is endemic to East Asian countries. This fluke provokes acute inflammation and chronic hyperplasic changes in the biliary tracts. C. sinensis promotes cholangiocarcinoma, and has been classified as a Group 1 biological carcinogen, alongside Opisthorchis viverrini, by the World Health Organization. Recently, transcriptomes for adult liver flukes have been reported with the molecular functionalities predicted on the bases of their transcriptomic data sets. We generated the developmental C. sinensis transcriptome for three different developmental stages, revealing that most functional genes were differentially expressed in each developmental stage; only a small proportion of the expressed genes were shared between the three stages. The developmental transcriptome describes the gene expression landscapes of C. sinensis adults, metacercariae, and eggs, and provides insight into how this fluke adapts to the distinctly different environments provided by its various hosts. We anticipate that the transcriptome will contribute significantly to the identification of intervention points along the developmental stages and allow the exploitation of novel potential targets for diagnostic, drug, and vaccine development purposes.
Citation: Yoo WG, Kim D-W, Ju J-W, Cho PY, Kim TI, Cho S-H, et al. (2011) Developmental Transcriptomic Features of the Carcinogenic Liver Fluke, Clonorchis sinensis. PLoS Negl Trop Dis 5(6): e1208. doi:10.1371/journal.pntd.0001208
Editor: Banchob Sripa, Khon Kaen University, Thailand
Received: February 26, 2011; Accepted: April 17, 2011; Published: June 28, 2011
Copyright: © 2011 Yoo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by Korea National Institutes of Health grants 2008-E54003-00 and 2009-E54004-00 to Sung-Jong Hong and 2006-E54005-00 and 2007-E54004-00 to Hong-Seog Park. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Clonorchis sinensis causes clonorchiasis, which is endemic to Korea, China, Taiwan and Vietnam; approximately 15 million people are estimated to be infected [1]–[3]. C. sinensis is a significant pathogen both from an epidemiological and clinical perspective, as people who develop clonorchiasis are debilitated, thereby negatively impacting socio-economic activities. In C. sinensis endemic areas, inhabitants become infected by eating raw or inappropriately cooked fresh water fish caught from water bodies near their villages [4]. Fresh water fish are the hosts of C. sinensis metacercariae, which is the infective stage to humans.
Once C. sinensis eggs reach a fresh water body, they develop into miracidiae. When ingested by freshwater snails, the miracidia escapes from the egg and transforms into sporocysts, and then into rediae within several weeks. The cercariae emerge into fresh water and swim in search of freshwater fish, the second intermediate host. The cercaria penetrates the skin of a freshwater fish and its body becomes encysted by a cyst wall, followed by transformation into a metacercaria. Almost all freshwater fish can serve as the second intermediate hosts, with the highest infection rate and metacercarial burden found in the topmouth gudgeon, Pseudorasbora parva. When ingested by humans and other mammals, the metacercariae are retained for a while in the stomach and then passed down to the duodenum. The metacercariae excyst there and the hatched, juvenile C. sinensis migrate up into the bile duct. The juvenile flukes grow to adults that produce eggs in the biliary passages of the mammalian host.
C. sinensis flukes in the biliary tracts lacerate and apply pressure to the epithelia, and excrete waste products from their excretory bladder and regurgitate residual digests from their intestinal ceca. In addition, ovigerous C. sinensis adults excrete uterine fluid with high protein content when they ovulate. These various excretory and secretory products act as chemical irritants that provoke inflammation, epithelial hyperplasia, and periductal fibrosis in the biliary tracts. In human clonorchiasis patients, frequent symptoms are epigastric discomfort and dull pain, mild fever, loss of appetite, diarrhea, and jaundice [5]. Moreover, clonorchiasis has epidemiologically been reported to be associated with cholangiocarcinoma [6]–[8]. Furthermore, experimental studies have shown that C. sinensis infection induces the differentiation of liver oval cells into a bile duct cell lineage and promotes the development of cholangiocarcinoma in golden hamsters [9], [10]. Recently, C. sinensis was officially classified along with Opisthorchis viverrini as a Group 1 biological carcinogen by the World Health Organization [11]. Among the excretory-secretory products produced by liver flukes, granulin was identified as a mitogenic agent capable of stimulating cell proliferation and epithelial hyperplasia [12]. By binding to Toll-like receptors, the excretory-secretory products of adult flukes activate the NF-kB pathway resulting in increased expression of the pro-inflammatory cytokine, IL-6 [13], which in turn leads to the production of reactive oxygen radicals. The endogenous reactive radicals damage DNA and could initiate carcinogenesis [14].
Expressed sequence tags (ESTs) generated from cDNA libraries cover a large proportion of functional mRNAs and can be assembled into overlapping contigs coding for almost complete open reading frames [15]. Schistosoma mansoni and S. japonicum were the first flukes for which transcriptome data was published [16], [17]; these studies stimulated the generation of ESTs and functional cataloging of these ESTs from the human-infecting liver flukes, C. sinensis, O. viverrini, and Fasciola hepatica [18]–[22]. The recent widespread availability of next-generation sequencing technology has also stimulated high-throughput analyses of the transcriptomes of liver flukes with a focus on the pathobiological characteristics of these adult liver fluke transcriptomes [23], [24]. Given that C. sinensis adults are considered carcinogenic agents, they are predicted to express genes encoding proteins that are also known to be involved in cancer development [25].
To further elucidate the pathogenesis and carcinogenesis provoked by C. sinensis infection, comprehensive molecular and genetic information covering the different developmental stages of this parasite is required. In this study, we generated and sequenced transcriptome-scale ESTs from three developmental stages of C. sinensis and investigated the biological properties, growth, host adaptations, and pathogenic features of these different developmental stages.
Materials and Methods
Ethics statement
Rabbits were handled in an accredited Korea Food and Drug Administration animal facility in accordance with the AAALAC International Animal Care policies (Accredited Unit, Korea FDA; Unit Number 000996). Approval for animal experiments was obtained from Korea FDA animal facility (NIH-06-15, NIH-07-16 and NIH-08-19).
Parasite resources, culture and RNA extraction
C. sinensis metacercariae were collected from naturally infected P. parva caught in Jinju, Korea, and Shenyang, China. The fish were ground and digested artificially in gastric juice for 1 hr at 37°C. Particulate material was filtered out using a sieve with 0.15 mm mesh and washed several times with 0.85% saline. C. sinensis metacercariae were identified and collected under a dissecting microscope. Male New Zealand White rabbits, 1.5–3.0 kg (Samtaco Inc., Korea) were infected with 500 metacercariae each, and adult flukes were recovered from the bile ducts of these experimental rabbits 2 months after the infection. Bile juice collected from C. sinensis-infected rabbits was centrifuged at 2,000 g for 10 min and C. sinensis eggs were collected from the sediment. To extract total RNA, adult flukes, metacercariae, and eggs of C. sinensis were put into liquid nitrogen in a pre-chilled mortar on dry ice and pulverized using a Mixer Mill MM301 (Retsch GmbH, Haan, Germany). Total RNA was extracted from the ground tissues using TRI reagent (MRC, Inc., Cincinnati, OH, USA). Poly(A+) mRNA was selected from the total RNA using the Absolutely mRNA Purification Kit (Stratagene, La Jolla, CA, USA) according to the manufacturer's instruction. The amounts of total RNA and mRNA were determined by measuring the absorbance at 260 nm and the degree of protein contamination was assessed by calculating the ratio of the absorbance at 260 nm to that at 280 nm. RNA integrity was assessed by examining ribosomal RNA bands on 1% RNA agarose gels stained with ethidium bromide.
Construction of cDNA libraries
Using the poly(A+) mRNAs generated as described above, cDNA libraries of the three developmental stages of C. sinensis (adult, metacercaria, and egg) were constructed using the directional λ ZAP cDNA synthesis/Gigapack Ш Gold cloning kit (Stratagene, La Jolla, CA, USA). First stand cDNAs were synthesized from mRNAs primed at the poly-A tail using reverse transcriptase and an oligo-dT linker-primer containing an XhoI restriction enzyme site. Following second strand synthesis, an EcoR I linker was ligated to the 5′-termini followed by digestion with the restriction enzyme XhoI. These synthesized and assembled double strand cDNAs were size-fractionated using Sepharose® CL-2B gel filtration column chromatography. cDNA fractions longer than 500 bp were ligated into the ZAP Express vector pBK-CMV and the ligation products were packaged in vitro into cDNA libraries using the ZAP Express cDNA Gigapack Ш Gold cloning Kit (Stratagene). cDNAs were directionally cloned into the pBK-CMV vector, which allows both prokaryotic and eukaryotic expression of large sequences and in vivo excision into a phagemid vector. The adult, metacercaria, and egg cDNA libraries were plated onto LB-kanamycin plates, 23.5 cm×23.5 cm, coated with X-gal/IPTG for blue/white selection. White colonies were randomly picked and inoculated into each well of a 384-well plate (Corning Co., Cortland, NY, USA) containing 40 µl Terrific Broth/kanamycin, followed by incubation for 16 hr at 37°C. For storage, the culture media in the 384-well plates were mixed with an equal volume of glycerol solution (65% glycerin, 0.1 M MgSO4, 0.025 M Tris-HCl, pH 8.0) and stored at −80°C. To assess cDNA quality, additional cDNA libraries were constructed in the pBluescript SK(+) vector for the adult and in the pAD-GAL42.1 vector for the metacercaria.
cDNA sequencing
A total of 60,768 colonies were picked: 30,144 from the adult cDNA library, 20,256 from the metacercaria cDNA library, and 10,368 from the egg cDNA library. Single plasmid colonies were transferred into 540 µl of Terrific Broth medium supplemented with 50 µg/ml kanamycin in a 96-deep well plate and incubated at 37°C overnight with gentle rotation (550 rpm). Plasmids containing C. sinensis cDNA were extracted using an alkaline lysis method [26], [27]. The sequences of the cloned C. sinensis cDNAs were determined using the BigDye Terminator Cycle Sequencing Kit, ver. 3.1 (Applied Biosystems, Foster City, CA, USA). Sequencing reactions were performed in a 3-µl volume containing 250 ng plasmid DNA, 0.5 pmole universal primer, 0.87 µl of 5X Sequencing buffer, and 1.38 µl of distilled water. The cycling profile consisted of 35 cycles of denaturation at 96°C for 10 seconds, annealing at 50°C for 5 seconds, and extension at 60°C for 4 min. Sequencing products were purified via ethanol precipitation and read on an ABI 3730XL DNA Analyzer (Applied Biosystems, Foster City, CA). The T3 forward primer and T7 reverse primer were used as sequencing primers.
DNA sequence trimming and assembly
Nucleotide sequences of the 60,768 clones were read once from the 5′-end. The vector and adapter sequences were trimmed off all reads, as well as nucleotide stretches with a Phred score of 20 or less and poly A/T stretches [28], [29]. Reads shorter than 100 bp were then filtered out of the analyses. A total of 52,745 reads that survived these quality control filters were assembled into clusters using the TGICL and CAP3 programs with the following parameters: an offset of 40 bp overlap, 95% minimum identity, and a maximum mismatched overhang of 30 bp [30], [31]. Nucleotide sequences of the reads reported in this paper are registered in the DDBJ/EMBL/GenBank databases under the accession numbers FS126466-FS179210.
Bioinformatic analyses of ESTs
To annotate the assembled EST clusters, the nucleotide sequences of the clusters were translated into putative polypeptide sequences and these sequences were blasted against the NCBI non-redundant nucleotide and protein databases using the parameters of more than 30 matched amino acids, an identity greater than 25%, and an E-value less than 1e−4 using BLASTX. The domain structures of the translated polypeptides were predicted using InterProScan (data version v14.0) and an E-value of less than 1e−4 [32] and their potential function was assessed by gene ontology (GO) analysis. Moreover, we manually curated the gene descriptions in the databases by selecting known genes with a significant E-value to prevent incorrect assignment of annotated genes. To further enhance the reliability of the data and provide more accurate gene predictions for C. sinensis, we chose the species closest to C. sinensis for which data were available.
Signal peptides in the EST clusters were searched and categorized based on their function. The open reading frame of a conceptual polypeptide was first translated from the cluster sequence using OrfPredictor [33] (https://fungalgenome.concordia.ca/tools/OrfPredictor.html). The signal peptide sequences of the conceptual polypeptides were predicted using the SignalP 3.0 server [34] (http://www.cbs.dtu.dk/services/SignalP) and the subcellular localizations of proteins were analyzed using PSORTb [35] (http://psort.nibb.ac.jp). Secondary structure features of the peptides such as alpha helices and intervening loops were predicted using TMHMM version 2.0 [36] (http://www.cbs.dtu.dk/services/TMHMM/). Putative B-cell epitopes in C. sinensis EST-encoding proteins were predicted using the ABCpred database [37] (http://www.imtech.res.in/raghava/abcpred/ABC_submission.html).
To generate developmental gene expression profiles of C. sinensis, the number of ESTs in each contig was counted according to developmental stage and analyzed using Fisher's exact test using a significance level of P<0.01 at IDEG6 [38] (http://telethon.bio.unipd.it/bioinfo/IDEG6_form/). Putative SNPs in the EST sequences were determined using the AutoSNP program [39].
To gain insight into the evolutionary history of C. sinensis and to investigate parasitism-related genes, the global similarity of C. sinensis whole ESTs at the amino acid sequence level were compared to those of other parasites and free-living platyhelminthes using the SimiTri program (cut-off score: 50) [40]. Whole EST sequences of comparator organisms were retrieved from the NCBI protein database. The relative similarities of the gene sequences of C. sinensis to those of other species were analyzed using TBLASTX and the SimiTri viewer. Bulk sequences of the comparator species were downloaded from the GenBank EST databases. Sequences with BLAST scores (bit score values) higher than 50 were collected from each large dataset. Nucleotide versus nucleotide comparisons of the dataset of interest to the different databases were performed using the TBLASTX algorithm. The primary data consisted of the similarity values of each sequence from the chosen database. The primary data was transformed into input for the SimiTri viewer. A gene is indicated as a square tile, and these tiles are colored by similarity scores to other datasets. Genes that were similar to genes in only one other database are not shown. Genes that showed similarity to genes in only two databases are shown as lines joining the two databases.
More detailed information and raw data are accessible at http://grc.kribb.re.kr/pipeline2/.
ID of genes
The ID numbers for genes and proteins mentioned in the text refer to tables. The others are as follow: K+-channel, CL3811; Ca+2-channels, CL6309, CSM01492; Na+-Channel, CSA10278; Cl− channel, CL2019; Sodium transporters, CL5256, CL25, CL6319, CL420, CSA00901, CSA10105, CSA10217, CSA19634; Na+/K+-ATPases, CL2552, CSA23629; Glucose transporters, CL272, CL25; Amino acid transporters, CL1075, CL1111; Zinc transporter, CL1482; Phosphate transporters, CL5278, CSA12737; Fatty acid transporter, CL384. Apoptosis, CL632, CL635, CL621; Cell proliferation, CL1924, CL134, CL6028, CL894; cancer development, CL644, CL557, CL3820, CL1644, CL1801, CL5519, CL5976, CL1379, CL2881, CL2147, CL684, CL894, CL25, CL1289, CL1087, CL545, CL1679, CL421, CL111; Neuroreceptors, CL1876, CSM05821, CSM11397; Neurotransmitter-related proteins, CL4260, CSA10291, CL1504, CL5536, CSM14836, CL5972.
Results and Discussion
The C. sinensis transcriptome
To generate transcriptomes and evaluate developmental gene expression in C. sinensis, 60,768 clones were selected randomly from cDNA libraries of the adult, metacercaria, and egg developmental stages. After stringent quality filtering through base calling, vector sequence trimming, repeat masking, and contaminant screening, 52,745 high quality reads remained (success index of 86.8%) consisting of 27,070 reads from the adult stage, 15,872 reads from the metacercaria stage, and 9,803 reads from the egg stage (Table 1). The high quality reads were assembled and clustered into 12,830 C. sinensis assembled EST sequences (CsAEs) comprising 7,184 contigs and 5,646 singletons [31]. The length of the CsAEs ranged from 100–3,328 bp with an average length of 724 bp. Over 50% of the CsAEs were between 500–799 bp. More than 70% of the CsAEs consisted of less than 30 EST members, while the largest single CsAE had 643 EST members.
Developmental gene expression
A total of 52,745 reads collected from the adult, metacercaria, and egg stages were assembled into 7,184 contigs. Of these contigs, 1,887 (26.3%) were shared by two developmental stages; 648 contigs between the adult and egg stages, 974 between the adult and metacercaria stages, and 261 between the metacercaria and egg stages. A small portion of the transcriptome (564 contigs; 7.9%) occurred in all three developmental stages, suggesting that some of these are housekeeping genes expressed constitutively across the life stages of C. sinensis. A large number of the contigs (4,733; 65.9%) occurred in one of the three developmental stages (Figure S1). This finding suggests that genes associated with growth can be identified from the C. sinensis developmental transcriptome. The expression levels of these genes, as determined by the number of ESTs contained within a cluster, were compared to investigate stage-specific patterns of transcription based on an arbitrary cut-off as well as the statistical significance of the expression differences. In C. sinensis, 119 contigs in the adult stage, 48 in the metacercaria stage, and 134 in the egg stage were significantly differentially expressed. The majority of CsAEs obtained from each developmental stage were non-annotated or hypothetical transcripts. The unknown CsAEs were broadly distributed at each stage, indicating that C. sinensis may have a unique developmental mechanism compared to other parasites.
Functional annotation
Our manual curation of the search reports generated by BLASTX and InterPro increased the annotation rate and accuracy of the functional notations of the CsAEs. Of the 12,830 CsAEs, 7,132 (55.6%) were found to have significant sequence similarities to sequences in the NCBI NR database and/or in InterPro [41], while the remaining 5,698 (44.4%) CsAEs had no homolog (Figure S2). One-half of the proteins translated from the CsAEs were annotated by BLASTX and found to be similar to proteins found in Schistosoma japonicum. Among them, the sequences of hsp90, RNA binding protein, and actin were highly conserved between C. sinensis and S. japonicum. Another 44% of the CsAEs were unique gene transcripts with no homolog in the databases searched.
Gene ontology
To predict the functions of the C. sinensis genes, we independently classified a total of 12,830 CsAEs into functional categories by analyzing automated gene ontology assignments [42]. The most accurate method to identify new members of known gene function among gene transcripts is to retrieve a sequence-based homology of the translated transcripts using domains extracted from a multiple alignment of gene members with known functions [43]. To functionally categorize CsAEs using domain homology searches, we translated the 12,830 CsAEs in six reading frames and recruited them into InterProScan [32], which aligned 6,106 CsAEs to InterPro entries (E-value≤1e−4). Among these, 2,889 CsAEs were assigned to 23,178 GO accession numbers. The 23,178 accession numbers further generated 2,349 distinguished GO mappings in two major ontologies, molecular functions and biological processes. We assigned 2,679 CsAEs (20.9%) to 17 main molecular functional subcategories and 2,195 CsAEs (17.1%) to 23 main biological process subcategories (Figure 1). The most abundant groups represented under the molecular function category were assigned to the GO categories of nucleotide binding (9.1%), nucleic acid binding (6.2%), ion binding (5.7%), transferase activity (6.0%), and hydrolase activity (7.4%). The most abundant groups represented under the biological process category corresponded to the GO categories of cellular component organization and biogenesis (2.3%), transport (2.9%), localization (3.0%), biosynthetic processes (2.4%), metabolic processes of nucleobases, nucleosides, nucleotides and nucleic acids (2.7%), and protein metabolism (5.2%). The apparent discrepancies between these values may be due to the fact that one InterProScan number can be assigned to more than one GO accession number, and one GO accession number can be mapped to multiple parental categories and CsAEs [33].
Distribution of molecular functional categories (a) and biological process categories (b) according to homology to genes in Uniprot with a gene ontology classification.
Putative single nucleotide polymorphisms (SNPs)
SNPs are the most common and abundant type of genetic variation between individuals and are used in population and evolutionary biology studies [44]. In addition, SNPs can be used as markers for physical and genetic mapping. SNP discovery based on large-scale EST datasets has proven an efficient technique for identifying large numbers of SNPs. Among the 7,184 CsAE contigs, SNPs were discovered in 2,896 contigs (40%). A large majority (77%) of the SNPs were detected from contigs consisting of 2–4 ESTs, while the remainder of the SNPs were discovered in contigs consisting of more than 5 ESTs (Table 2). A total of 9,077 SNPs were detected from the 7,184 contigs, which is an average of 0.37 SNPs per 100 bp. The putative SNP frequency varied from 0.22 to 0.50 SNPs per 100 base pairs among contigs of different sizes. The genetic diversity of C. sinensis SNPs was similar to that reported for S. japonicum: an average of 0.35 SNPs per 100 bp [21]. In eukaryotic organisms, SNPs occur every 500 to 1,000 bp, at frequencies higher than those found in non-eukaryotic organisms [45]. Almost all of the predicted CsAEs with >0.19 SNPs/100 bp were no-match genes, and only several were homologous to S. mansoni genes of unknown function. In trematodes, SNPs in the first codon position can result in an amino acid substitution, which may lead to structural changes in the respective proteins and affect the formation of functional domains, antigenic epitopes, or drug binding sites [44].
Abundantly expressed gene transcripts in the different developmental stages
The 30 genes most abundantly expressed were investigated further (Table S1). Genes with significant p-values were analyzed by one-to-one comparison with the number of reads per the same gene in each stage. Eighteen genes in the adult stage and 12 genes each in the egg and metacercaria stages were annotated. Highly abundant genes should be conserved based on the reasoning that they are major constituents of the transcriptome of the organism and are therefore likely to be functional, but the highly abundant genes found in C. sinensis had few homologs, indicating that this organism is developmentally quite different to other organisms for which gene abundance information is available. Genes expressed abundantly across all three stages coded for ubiquitin family proteins, elongation factor 1-alpha, fructose bisphosphate aldolase, which are all proteins involved in basal and energy metabolisms. Genes highly expressed in the adult compared to the metacercaria and egg encoded structural and reproduction-associated proteins (beta-tubulin, ferritin), detoxification proteins (glutathione S-transferase), transportation proteins (clonorporin 1, sodium/glucose co-transporter), energy production proteins (GAPDH, mitochondrial malate dehydrogenase), and enzymes (cysteine protease, PHGPx isoform 1). In particular, cysteine proteases have previously been shown to be the most abundantly expressed proteins in C. sinensis adults [19], [20], [24]. In the egg stage, the gene encoding acyl-CoA synthetase long chain family member 5 (ACSL5) was highly expressed among the known genes. ACSL5 is a member of the highly conserved ACSL family. In protozoan parasites, ACSL5 is thought to catalyze the conversion of long-chain fatty acids to CoA derivatives to enable parasite growth [46]. The majority of highly expressed CsAEs was unknown or hypothetical genes and therefore deserved further study.
Evolutionary and functionality conservation
To investigate the evolutionary and functional conservation of the transcriptome of C. sinensis, we estimated gene numbers and the degree of conservation among C. sinensis genes and the genomes of diverse eukaryotic organisms. We performed pair-wise sequence comparisons of all C. sinensis transcripts using BLASTX with an E-value cut-off from 1e−10 to 1e−200. The C. sinensis transcriptome shared 22.9% genes with Homo sapiens, 23.0% with Mus musculus, 20.5% with Drosophila melanogaster, 17.8% with Caenorhabditis elegans, and 26.6% Schistosoma japonicum at an E-value≤10−20. The CsAEs showed a moderate degree of sequence homology to the genes of the comparator organisms and a higher degree of sequence homology to S. japonicum genes (Table 3). Genes highly conserved between the CsAEs and S. japonicum may be important for parasite survival. These genes are discussed in more detail in the next section. A small fraction of genes (37 genes; 0.3%) were highly conserved (E-value≤10−200) across all the animals compared in this study (Table 4). These genes encode proteins such as actin, tubulin, translation elongation factor 1, valosin-containing protein, glycogen phosphorylase, and heat shock protein 70.
Clonorchis sinensis and parasitism
SimiTri graphically displays the relative similarity of one organism to others using bulk datasets from the respective organisms. The degree of similarity among helminth sequences was determined using two-dimensional plots [40]. We used SimiTri to analyze the global relative similarity of CsAEs to other parasitic or free-living trematodes, cestode and nematode (Figure 2). C. sinensis (12,830 CsAEs) was more similar to the two parasitic flukes, S. japonicum and O. viverrini, than to the free-living helminths Schmidtea mediterranea (73,650 ESTs) and C. elegans (474,350 ESTs). When compared to both Opisthorchis viverrini (4,194 ESTs) and S. japonicum (99,069 ESTs), the closest neighbor of C. sinensis was O. viverrini, consistent with the general taxonomic classification of these trematodes and the recent molecular phylogeny of Digenean trematodes based on morphological characters and the sequence of the nuclear ribosomal small subunit (18S) [47].
. Each C. sinensis contigs and singlets were searched against the whole transcriptome using TBLASTX score (a cut-off of ≥50). Similarity comparison of parasitic organisms with a free-living flatworm (A) or with free-living nematode (B). Square tiles indicate genes, with the squares colored by their highest TBLASTX score to each of the databases: red ≥300; yellow ≥200; green ≥150, blue ≥100 and purple <100.
Based on the SimiTri analyses results, 23 CsAEs were considered to contain parasitism-related genes of C. sinensis (Table S2). The genes encoded by these 23 CsAEs showed higher sequence similarity to the parasitic platyhelminths, O. viverrini, S. japonicum, and Taenia solium than to free-living ones, S. mediterranea and C. elegans (Figures 2A and B). Of these, 12 CsAEs had an unknown function, whereas 11 CsAEs had cell communication, ion transport, metabolic processes, nucleotide and protein binding, or oxidoreduction functional annotations.
Membrane proteins, channels and transporters
In the adult C. sinensis cDNA library, EST encoding K+-channel was abundant but that of Ca+2-channel was rare (Table 5). No Na+ -channel EST was found in any of the three developmental stages. From an evolutionary point of view, K+-channels are ancient, occurring in all three domains of life, and are abundant in invertebrate animals. K+-channels are ubiquitous and maintain cell homeostasis in organisms. Ca+2-channels emerged later in evolutionary time. In more basal animals, Ca+2-channels are activated in response to action potentials of nerve systems and provoke slow actions and movements. Na+-channels are more elaborate and are often multimeric, and are generally rare in invertebrates compared to higher vertebrate animals [48]. The frequency distribution of these cation channels is consistent with phylogenetic placement of C. sinensis in the tree of life. Specific structural motifs in the interacting domains of the beta subunit of Ca+2 channels render flukes PZQ susceptible. These specific structural motifs were found in the beta-subunits of C. sinensis adults, which explain why they are vulnerable to PZQ.
A large number of glucose transporters were found in both the adult and egg stages (Table 5). Glucose/sodium co-transporters and Na+/K+-transporting ATPases were abundant only in adult C. sinensis, not in the metacercaria. This fluke species consumes large amounts of glucose to generate energy and metabolic intermediates for physiologic regulation. In adult C. sinensis, glucose appears to be imported actively from the environment through glucose/sodium co-transporters using ATP as an energy source. Glucose molecules may move passively through the glucose transporters between cells in fluke tissues. Anion channel proteins including chloride channels were 4-fold more frequent than cationic channels, which could compensate for the large amount of Na+ ions co-imported with exogenous glucose. These anionic channels may be suitable targets for vaccine or drug development.
CsAEs coding for bile acid beta-glucosidase and a sodium-bile acid cotransporter, a component of the bile acid transportation pathway, were present in the C. sinensis EST pool (Table 5). Bile acid beta-glucosidase converts bile acid to a soluble conjugated form to facilitate its secretion. The sodium-bile acid cotransporter, which imports bile salts with Na+-dependency, was abundantly expressed in the metacercaria stage. This type of transporter is responsible for the influx of conjugated bile acids into hepatocytes, ileal enterocytes, and cholangiocytes in mammals [49], [50]. The presence of these transporters in C. sinensis suggests that C. sinensis thrives in bile juice by utilizing bile acid and its derivatives for normal physiologic pathways. C. sinensis is expected to have a bile acid exporting system comprising bile salt export pumps, organic solute transporters, and/or multidrug resistance protein 2 to maintain cellular homeostasis [49], [50].
Neurotransmitters & receptors.
CsAEs encoding proteins involved in neurotransmission such as serotonin receptors, tryptophan hydroxylase, aromatic amino acid decarboxylase, glutamate receptors, glutaminase, GABA receptor-associated proteins, acetylcholine esterase, and DOPA-decarboxylase were found as rare species (Table S3). The presence of these neurotransmission-related proteins implies that C. sinensis has a web of serotoninergic, glutaminergic, and cholinergic neurons, which is consistent with the previous observation that the nervous systems of trematodes are highly conserved [51].
Proteases and protease inhibitors
Proteases of parasitic origin are known to be important virulence factors based on genomic and proteomic analysis of several major global helminth species [52], [53], [54]. Secretory proteases of parasites are ubiquitous enzymes that have been implicated in several diverse physiological and adaptive mechanisms, such as tissue penetration, larval migration, immunoevasion, digestion, and excystation [55]. Because these proteases are indispensable for parasite viability and growth, they have been suggested as potential targets for vaccines or chemotherapeutic agents [56], [57]. We classified the CsAE proteases into four functional groups based on the catalytic type, namely serine, threonine, aspartate, and metallo- or cysteine proteases (Figure 3A). Cysteine proteases showed the highest expression (68.8%) levels among the four types of proteases during all three developmental stages. Cysteine proteases of C. sinensis are developmentally controlled and essential for survival because they are involved in processes such as nutrient uptake, tissue invasion, and evasion from host immune attacks [58]. Of the C. sinensis cysteine proteases, the cathepsin F-like isoenzyme (CsCF-6) was expressed across all developmental stages, and we observed that transcript levels of this protein increased according to the developmental stage of the parasite [59]. Metalloproteases were detected in all three developmental stages of C. sinensis (Figure 3A). Metalloproteases are crucial proteases for invasion and immune evasion of flukes in addition to the general roles they play in catabolic reactions and protein processing [57].
Relative reads refer to the number of calculated reads in proportion to the read size for each developmental stage (adult: metacercaria : egg = 2.76∶1.62∶1). (A) Proteases, (B) protease inhibitors, (C) antioxidant enzymes, (D) stress response proteins. Dyp, dye-decolorizing peroxidase; GST, glutathione-s-transferase; SOD, superoxide dismutase; GPX, glutathione peroxidase; GRX, glutaredoxin; PRX, Peroxiredoxin; TRXR, thioredoxin reductase; TRX, thioredoxin; HSP, heat hock protein.
Parasites utilize protease inhibitors to survive in their hosts; protease inhibitors can prevent damage by mature proteases prior to their secretion from the parasite and protect them against the digestive proteases of the hosts [60]. Diverse proteins function as protease inhibitors, but they have a common biochemical mechanism and are characterized by rapid evolution of their sequences [61]. In our study, cysteine protease inhibitor expression was highest in the adult stage among all three stages (Figure 3B). Cysteine protease inhibitors (cystatins) regulate cysteine proteases and modulate host immune responses [60]. Cystatins of C. elegans have been shown to inhibit cathepsin B while filarial cystatins have been shown to inhibit the proliferation of murine and human T-cells. Because C. sinensis cysteine proteases are expressed most abundantly in the intestinal epithelium for uptake of nutrients [59], cystatins could be expressed in the intestinal epithelium to fine-tune the intracellular activities of cysteine proteases. Expression of serine protease inhibitors remained stable in all stages with slightly greater expression levels observed in the egg stage (Figure 3B). This finding is consistent with a previous study that demonstrated that serine protease inhibitors were present mainly in the eggs of C. sinensis [62]. Transcripts encoding metalloprotease inhibitors were found only in the metacercaria library and inhibitors of aspartic and threonine proteases were not identified in any of the developmental stages.
Antioxidant enzymes
Several antioxidant enzymes constituting the oxidoreduction system were encoded by the CsAEs, with the expression levels of the various enzymes varying according to developmental stage (Figure 3C). Antioxidant enzymes catalyze reactions that neutralize endogenous and exogenous reactive oxygen species (ROS) that are produced either by aerobic cellular metabolism or by host immune responses [63]. Regulation of the expression levels of these enzymes during each development stage is therefore important to cope with host-produced ROS [64]. In the C. sinensis transcriptome, glutathione-S-transferases (GSTs) were the most highly expressed antioxidant enzymes, especially in the adult and egg stages. In Fasciola hepatica, GSTs are expressed at much lower levels in juvenile worms than in adult worms living in the bile duct, implying that adult worms require more protection against host immune responses [63]. In C. sinensis, two GST isoenzymes have been identified: a 26 kDa GST and a 28 kDa GST [65]. Glutathione peroxidase (GPx) and thioredoxins (TRX) were expressed differentially at high levels in the adult stage (Figure 3C). C. sinensis GPx has been reported to be specifically localized in the vitellocytes of vitelline glands and in the premature eggs [66]. GPx defends against ROS and repairs ROS-induced damage in trematodes that do not have catalases [53]. TRX was highly expressed (Figure 3). In Schistosoma mansoni, TRX is secreted from eggs and plays a crucial role in protecting eggs from host-induced ROS production [63]. All antioxidant enzymes were expressed at low levels in the metacercaria stage (Figure 3), which can be explained by the fact that this is a dormant state with a depressed metabolism that is protected by a cyst wall from exogenous oxidative stresses.
Stress responsive genes
Heat shock proteins (HSPs) function as molecular chaperones and play an important role in the stress response to a variety of biological stresses such as heat shock, hypoxia, mechanical stimuli, lack of glucose, and UV exposure by assisting in the refolding of denatured proteins into active forms or targeting them for degradation [67]. We identified six HSPs from the CsAEs: HSP110, HSP90, HSP70, HSP60, HSP DnaJ, and HSP20. Most HSPs were expressed at higher levels in the metacercaria and egg stages than in adult worms. The higher molecular weight HSPs such as HSP110, HSP90, HSP70 and HSP60 were more highly expressed in the metacercaria stage, while the lower molecular weight HSPs, HSP DnaJ and HSP20, were expressed at higher levels in the egg stage. In C. sinensis adults, HSPs were expressed at low levels relative to the metacercaria and egg stages (Figure 3D). In the life cycle of C. sinensis, the metacercariae experience a large thermal change when they move from the environment (ambient temperature) to the stomach of the mammalian host (37°C). Furthermore, as the metacercariae pass down and excyst in the duodenum, they face the osmotic stress of intestinal secretions, and then when they migrate into the bile duct, they are exposed to bile juices. The higher molecular weight C. sinensis HSPs are likely to responsd to these thermally- and environmentally-induced stresses [68], [69]. The C. sinensis eggs that ovulate in the bile duct are carried down the intestine and passed out in the feces of the mammalian host into the environment, thereby experiencing cold shock. In the C. sinensis eggs, the low molecular weight HSPs may function to protect the eggs against cold thermal shock [70], while the high molecular weight HSPs might contribute to recovery from the cold shock [71].
Cell proliferation and cholangiocarcinoma-related genes
The C. sinensis transcriptome had several contigs encoding proteins associated with cell proliferation and apoptosis such as granulin, epidermal growth factor (EGF), tumor growth factor (TGF) interacting protein, and inhibitors and regulators of apoptosis. Among these proteins, granulin, encoded for by 67 reads, was most abundantly expressed with a 8.6-fold higher expression level in adults than metacercariae. Similarly, the EGF gene was also expressed at 2.5-fold higher levels in the adult stage than the metacercaria stage (Table 6). Transcriptomic datasets of C. sinensis and O. viverrini were previously analyzed for proteins common to carcinogenesis and a large number of the amino acid sequences of these trematodes were inferred to have homologs to genes involved in human cancer development [25]. The C. sinensis genes associated with apoptosis, cell proliferation, and cancer development encoded laminins, c-Jun N-terminal kinase, catenins, cyclin-dependent kinases, histone deacetylases, MFS transporters, serine/threonine kinases, and transcription factors (Table 6). C. sinensis infection provokes both acute and chronic pathological changes such as proliferation of the bile duct epithelium and periductal fibrosis that is disseminated over the biliary tree from proximal to remote biliary capillaries [5]. The mitogen-like proteins secreted or excreted from C. sinensis are likely to be provocative agents that cause biliary epithelial alterations, as has been documented for the granulin-like growth factor of O. viverrini [72], [73]. Proliferating cholangiocytes could be vulnerable to DNA damage from endogenous and exogenous carcinogens, bioreactive free radicals, and nitrosocompounds. The apoptosis inhibitor and regulatory proteins identified in the transcriptome of C. sinensis in this study (Table 6) may prevent the death of DNA-damaged cells and possibly facilitate their transformation into cancerous cells [73]. Epidemiologically, the incidence of cholangiocarcinoma is significantly higher in clonorchiasis endemic areas than in non-endemic areas [74]. Experimentally, infection with C. sinensis and the ingestion of dimethylnitrosamine by Syrian golden hamsters resulted in cholangiocarcinoma [9], [75].
Drug target candidates
Praziquantel is currently used to treat clonorchiasis. However, the efficacy of praziquantel against clonorchiasis has been reported to be poor in northern Vietnam [76]. Tribendimidine has recently emerged as a promising alternative to praziquantel for the treatment of human opisthorchiasis [77]. Transcriptomic datasets facilitate the search for new drug targets by mining them with bioinformatic tools that employ statistical and network analyses, parasite-specific physico-metabolic pathways and developmental regulation can be found. Membrane proteins including channels, transporters, and permeases, which play important roles in host-parasite interactions, were some of the first novel targets identified using transcriptome data [78], [79]. A total of 435 CsAEs that had more than two transmembrane domains were screened using the TMHMM algorithm [36], and the localization of the predicted proteins was determined using PSORTb based on homology to proteins of known localization [35]. CsAEs homologous to proteins of the free-living flatworm S. mediterranea, or of humans were selected using a cut-off E-value≤1e−10 using BLASTX. Five genes not present in vertebrates were identified as putative drug targets using the screening strategy described above (Table S4). Tetraspanins, four-transmembrane-domain proteins, are present on the outer tegument of trematodes and function as receptors for host molecules [80]. Tetraspanins are a recognized vaccine target for S. mansoni [78]. ADP ribosylation-like factor-6 (ARL6) interacting protein, which interacts with the ARL6 protein [81], is involved in hematopoietic maturation processes such as protein transport, membrane trafficking, and cell signaling [82]. Myelin proteolipid protein is a transmembrane protein that has been suggested to serve as a structural component of myelin, contributing to both the stability and compact lamellar structure of myelin [83].
Diagnostic antigen candidates
Serodiagnostic methods are used to screen for patients infected with C. sinensis and as supportive diagnosis tools in individual patients. Antigen proteins have been identified from the excretory-secretory products of C. sinensis and have been purified from crude extracts [84]. As antigenic preparations, crude extracts of C. sinensis show high sensitivity but low specificity toward the sera of clonorchiasis patients. In contrast, some of the recombinant antigenic proteins used for screening have high specificity but low sensitivity [84]. Potent serodiagnostic antigens for clonorchiasis are therefore still lacking. As the first step to find antigen candidates, the C. sinensis transcriptome data was filtered for secretory signal peptides using the SignalP 3.0 server and a neural network/hidden Markov model [34], while proteins secreted into the extracellular space were searched for using PSORTb [35]. After removing proteins with transmembrane domains using TMHMM, 411 CsAEs were designated 'secretory'. To determine which of these proteins is potentially antigenic [36], the secretory candidates were filtered using the ABCpred server [85] with default parameters, and 43 CsAEs with two or more B-cell epitopes and a score of 0.82 or higher were obtained. By removing proteins homologous to mammalian and nuclear proteins, a total of 19 CsAEs were identified as putative antigen candidates. Examples of these candidates include a male sterility protein, cathepsin L-like cysteine proteinase A, protein disulfide isomerase-related protein P5 precursor, cryptosporidial mucin, and TGF-beta receptor interacting protein 1 (Table 7). Recombinant antigenic proteins encoded by the selected CsAEs could be synthesized in vitro using a high-throughput cell-free system [86], and their antigenicity for the serodiagnosis of clonorchiasis could be determined using various methods such as a protein chip [87].
Supporting Information
Figure S1.
Contigs and singlets of the assembled C. sinensis EST pool according to developmental stage.
Figure S2.
The annotation of CsAEs using Interpro and BLASTX. Whole 12,830 CsAEs were searched for homologs in the NCBI NR database and the InterPro database, and the retrieved homologs were manually curated. A total of 7,132 CsAEs were annotated with homologs, but the remaining 5,698 (orange) found to have no homolog. Of the annotateds, 5,387 CsAEs (violet) matched homologs in both databases, 719 ones (green) in the InterPro, and 1,026 ones (blue) in the BLASTX.
Table S1.
The 30 most abundantly expressed genes in the adult, metacercaria, and egg stages of C. sinensis.
Table S2.
Putative parasitism-related genes of C. sinensis.
Table S3.
CsAEs of neuro-receptors and neurotransmitter producing enzymes according to C. sinensis developmental stage.
Table S4.
Putative drug targets of C. sinensis
We thank Dr. Shunyu Li (Chung-Ang University) and Dr. Sung-Hwa Chae (GnCBIO Co., LTD.) for help with the laboratory experiments.
Author Contributions
Conceived and designed the experiment: JWJ, TSK, HSP, SJH. Performed the experiments: WGY, PYC, TIK, SH Choi, SH Cho. Material provided: JWJ, PYC, SH Cho, SJH. Analyzed the data: WGY, DWK, TIK, HSP, SH Choi. Wrote the paper: WGY, DWK, JWJ, PYC, TSK, HSP, SJH.
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USDA Rural Development Programs Making a Big Impact in Western Iowa
On Friday, April 8th I had the opportunity to see first-hand the many ways USDA Rural Development programs are improving the lives of rural Americans when I visited a variety of USDA-funded projects in Anita, Logan and Woodbine in western Iowa.
In Anita, I met Duane Murphy, the local fire chief and all-around community champion. He told me how the grant we are giving his community to assist with the purchase of a new fire truck will help keep the local residents safe. He also mentioned how his department will now be better equipped to provide rescue services to travelers along Interstate 80 and visitors to Lake Anita State Park. Read more »

subset: commoncrawl


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Ralph Lauren Collection Women's Oren Boot
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Item # 229209
The Ralph Lauren Collection Oren is a stunning display of quality and craftsmanship. With its classic design and versatility, you'll covet this boot for years to come.
- Suede upper
- 12" shaft height, lace up
- 12" circumference
- Fully lined with shearling
- Flexible sole for maximum comfort
- Imported
By: AK123456789
Location: New York
January 01, 2012
Love them!
"I got these for Christmas and have barely taken them off since! The inside is real wool and the outside is real leather, they're so comfortable and warm for the winter time! They're the cutest boots I own, I recomend them to any girl looking for some cute calf-covering new shoes! :)"
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Mix No. 6 Wispy Wedge Sandal
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Looks like you've got more shoe shopping to do - have fun!
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Item # 278379
Stroll through summer in a superb platform sandal. The Mix No. 6 Wispy wedges are the perfect sandal to give your look a boost in height and style.
- Faux patent leather T-strap upper
- Ankle strap with adjustable buckle
- 1½" platform, 5¼" stacked wedge heel
- Synthetic sole
- Imported
- View more Women's Mix No.6 Shoes
By: Shoes4life24
Location: Maryland
July 27, 2013
"Loved the nude color and so cute!!!"
By: Blasiangyrl
Location: Sacramento
June 27, 2013
Perfect Nude wedge!!
"Wearing my shoes today and absolutely love them. The fit is perfect for me as I have a wide foot and a bunion on one of my feet lol I know probably TMI!!!! Anyhow I have no aches with my one bunion as I tend to have with my other heels. If your foot is narrow then I wouldnt suggest these beauties. These are a little higher than what I usually wear but are bearable as long as I dont plan on walking long. I say two thumbs up."
By: ILuvsshoes777
Location: Sault Ste. Marie ON
June 26, 2013
Great Shoes
"Love these shoes/ can be worn out with denim short or to the office! Very versatile and the wedge is comfortable."
By: Dartyy
Location: Austin
June 11, 2013
Cute Summer Wedges
"These shoes are perfect, they will match with anything! They are also extremely comfortable!"
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Steve Lansky
December 17 2010 09:33AM
You know how kids are. When they go out, they forget to call home and tell you they got there safely. On bitterly cold days, they forget their hats and mitts. They climb on the hood of your car when you are about to drive to Peterborough. And they forget their homework. That's exactly what the Leafs' kids did Thursday night in Calgary. They forgot their homework…for 57 seconds. But the kids had lots on their minds. The gang leader, Dion Phaneuf, was returning to Calgary for the first time. Oh, the excitement!
And the Leafs proved to their fans once again that patience is everything with this club. In those 57 seconds, Flames Alex Tanguay, Olli Jokinen and Rene Bourque pumped pucks past J-S Giguère…and the party was over. Dion's big night went splat. And those 57 negated some head-shaking saves by Jiggy. But that's what kids do. They can concentrate for 59 minutes and 3 seconds and then – poof! And that's the problem with winning the first game of a three-game trip.
Those two points are in the bag so, whether you consciously want to or not, you will let down in the second game. Saturday is the real test, anyway. Calgary and Edmonton will likely be also-rans this season. The Canucks are the genuine NHL article.
And, in Calgary, Phil Kessel (once again) proved he's just a kid by pulling the old disappearing act. Note to all print sports editors: Two goals in two games does not mean "Kessel's slump is over!" Call me when it's seven goals in eight games.
"Neon Dion," as his former Flames' veteran teammates called him (revealed by Iron Mike Keenan to TSN's Darren Dreger yesterday), had a strong start to his Calgary return, then disappeared too. Right up until he hauled down Jarome Iginla, providing Iggy with a third-period penalty shot…long after the game had already been decided. But some of the kids showed up.
Mikhail Grabovski seems to have decided he's Wayne Gretzky – shooting and scoring at will. And Grandpa Jiggy was outstanding until those stinky 57. Saturday in Vancouver is going to be a whole different ballgame. Everyone will have to bring their homework that night.
Against the Canucks, there can't be any lapses. No running around in their own end. No hanging the goaltender out to dry. No weak clearing attempts around the boards (hear that, Keith Aulie?...assuming you play). Often, this season, the Leafs play their best against the best. Makes sense. You fight hardest against the toughest bully in the yard.
And that's what the kids are going to have to do in B.C. It will be a game that determines if the growth pattern continues, or if the Maple Leafs fly home with their tails between their legs, begging their fans for even more patience.
December 17 2010, 09:39AM
One thing Dad and Mom (Burke & Wilson) need to remind the kids about: the amount of work you put into something is equal to what you get back. This game was a perfect example of that. Work harder. You're all A+ students, why would you EVER settle for anything less?
December 17 2010, 12:44PM
As an Oilers fan we have seen some similarities with the Leafs, flashes of brilliance salted with some bonehead moves. Not a game goes by that we are saying WOW, but... My question is; in your opinion Steve, who is having more WOW! moments and will that translate into success in the future?


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AK-47 with a strapby RownRoger
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GTA IV Pack 2016 (Low PC)
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I can't seem to find the download link. Please point me to it :))by Shufa
M3 Lee 4 for World of Tanks
25 November 2012
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Skip to comments.Chicago sex-ed will begin in kindergarten, promote gay lifestyle
Posted on 08/30/2013 12:48:43 PM PDT by SeekAndFind
Sex education including same sex relationships is on the curriculum for Chicago public school students this year, and a leading Illinois conservative group thinks its only the beginning of practice that could become a new national norm.
And even though its radically different from the current sex ed programs, that begin in fifth or sixth grades, its a norm President Obama has backed since his days in the Illinois legislature.
kindergartenclassAccording to CNS News, the new Chicago curriculum has been booted around in Springfield since at least 2003, when Obama was a state senator working with Planned Parenthood to push a sex education bill that not only pushed moved instruction from sixth-grade to kindergarten, it removed any mention of traditional marriage from the curriculum.
The bill didnt pass until Obama left the legislature, though, for bigger things like getting a brief Democrat supermajority in Congress to impose a takeover of the American health-care system. (If they can do that with the health system, you think they wont try it with sex ed?)
The new sex ed policy was adopted by the Chicago Board of Education in February to begin this school year.
In a spring interview with the John Birch Societys New American magazine, Illinois Family Institute cultural analylist Laurie Higgins said the curriculum is aimed at advancing acceptance of gay households.
Comprehensive sex-ed dogmatists believe its appropriate for kindergartners to learn about families that are headed by homosexuals, whereas many parents believe that no child in early elementary school should hear anything about homosexuality, Higgins said. Whats worse, comprehensive sex ed proponents will present these disordered family structures positively.
Higgins said the Chicago schools curriculum is part a National Sexuality Education Standards curriculum liberals are pushing around the country, developed by groups including the Gay, Lesbian, and Straight Education Network, the Sexuality Information and Education Council of the United States, the abortion giant Planned Parenthood, and the National Education Association.
Supporters, like Stephanie Whyte, chief health officer of Chicago Public Schools, say the curriculum is just a way of exposing children to different family structures. Whether that means theres two moms at home, everyones home life is different, and we introduce the fact that we all have a diverse background, Whyte said.
Higgins doesnt think thats a decision for parents not a public school system to make.
It is not the obligation of public schools to teach about every sexuality-related phenomenon that exists, Higgins told the New American. And it is neither the obligation nor the right of public schools to affirm phenomena, like homosexual relationships, that many believe are immoral.
Well, the schools have to prepare the children for the new liberal push to "modernize thinking" about pedophiles and their "sexual orientation."
Recruit new people who will unfailingly vote socialist.
Destroy the family as the source of moral values.
Just what Stalin ordered.
Gays recuiting children.
So, homosexuals got laws passed that prevent people from converting homosexuals from their mentally deviant sexual practices, yet, they demand to recruit children this way.
I am sick and tired of these perverts. Nothing but pedophiles.
Another step to insure the destruction of society and introduce the reign of the dictatorship of savagery.
"For whosoever bringeth scandal to these little ones, it were better that a great millstone be hung around his neck, and he be drowned in the depths of the sea."
"Gays recuiting children."
That's how they perpetuate their perversion.
In the meantime, the Lesbian Mayoral Candidate in NYC, Christine Quinn (who is "married"), has other Progressive ideas too:
Christine Quinn may ok morning-after contraceptive pill for 11-year-old middle school children
Anyone that leaves their child in public school is harming that child.
> Gays recuiting children.
They can now recruit in schools, behind closed tents in the Boy Scouts, and even on the battlefield. The new norm - sexual deviants.
I recently saw a "sex-ed" book for little kids where the author went through great pains to essentially eliminate identifiable gender. Just merely stating that some "people" have eggs and others have sperm cells. As if it is a totally random thing! All to make it seem like reproduction is just a matter of mixing stuff in a bowl and a baby pops out of nowhere!
Must really grate those people to know that two guys or women cant create a child.
Well NYC is like Sodom & Gomarrah so its not surprising its politicians want to sexualize children.
Austin isn't Texas anymore, it's a suburb of San Fransico.
Government gets away with telling children things in school that if a person said it to a child in a park they'd be arrested.
liberal progressive rot, eating at the foundation of a nation
THIS is what homo marriage was about.
On another note, parents will do nothing.
SEE HERE: http://hotair.com/archives/2013/08/29/slate-only-bad-people-wont-sacrifice-their-children-on-altar-of-public-education-or-something/
Slate: Only bad people wont sacrifice their children on altar of public education, or something
google fistgate
they've been pushing to corrupt kids starting as young as 5 for years
Does this mean they don't really believe their "born that way" nonsense......
I love Red states. Why cant we just separate?
More proof that Satan rules the liberal mind. Anything to poke a stick in they eye of Christianity. So much hate from the group that decries hate.
They will pervert children and use the result to demand a lower age of consent. Libertarians are going to be on their side AGAIN
See Post #10. They are already proposing morning after pills for children as young as 11 in our public schools.
What until their graduation in high school is dependent upon showing "competency" on the gay lifestyle and on being tolerant of homosexuality.
That is what they are moving toward.
Take you children OUT of government indoctrination centers (schools) or YOU ARE AN ABUSER.
I hope decent Chicagoans will remove their precious kids from the propaganda mill. Sure, it's nice to have somebody bus and teach the children while both parents bring in incomes, but we just didn't think that in twenty years we'd like the results.
Private Christhian school or homeschool!
Pedophiles on the prowl
So, why no mass outrage? Does the entire city accept this now?
So, homosexuals got laws passed that prevent people from converting homosexuals from their mentally deviant sexual practices, yet, they demand to recruit children this way.
I am sick and tired of these perverts. Nothing but pedophiles.
And the ones who aren't G'hey who support it are also Pedophile enablers.
And home to these two nuts, LOL
Might be necessary to teach sex ed to Chicago kindergarten - kids may not graduate to first grade before age 12 & puberty.
This was expected.
NYC, CA, MA already have pro-sex sex-ed courses for kids that age!
These are the days you are tempted to pray to God.."Nuk the whole d— thing from orbit, its the only way to be sure"
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Just imagine what the kids who go through this will be like when they're teenagers!
More "mature" than and eager than Miley Cyrus??
That is the only answer. Two separate countries.
Disgusting. I pray for God's judgment and soon.
To hell with lawyers,
1st lets go after the educators
"They will pervert children and use the result to demand a lower age of consent. Libertarians are going to be on their side AGAIN"
Geron, take your dislike of libertarians, and stick it where the sun don't shine.
That is no more correct than saying that all women are pacifists, or that all Republicans support Juan McCain.
There are delusional people of every political leaning - and I have to say that I think you're one of them . ;-)
I saw something in which a homo activist referred to heterosexuality as the "binary model". So describing humanity as consisting of men and women is now called the "binary model" just one of many other possiblities and it seems that they are inventing new possibilities every day.
Disclaimer: Opinions posted on Free Republic are those of the individual posters and do not necessarily represent the opinion of Free Republic or its management. All materials posted herein are protected by copyright law and the exemption for fair use of copyrighted works.


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Posts Tagged for La Caja China
Birthday Bash: La Caja China Pig Roast
Posted by Jason on August 2, 2011
Birthdays only come once a year, and milestone birthdays only happen once in a lifetime. It just so happens that I recently passed one of those milestone birthdays and ticked over into the big 30′s. Some people like to spend these occasions relaxing on a beach, or sulking about father time catching up with them, but I saw this as a prime opportunity to expand my barbecue horizons into a category of cooking that every great pitmaster must dabble in. So I treated myself to a pre-birthday present by purchasing a La Caja China roasting box and set out on my first pig roast!


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Cardiff Blues overturned a half-time deficit to eek out victory 22-15 over Connacht in Saturday's PRO12 clash in Galway.
Cardiff Blues overturned a half-time deficit to eek out victory 22-15 over Connacht in Saturday's PRO12 clash in Galway.
Not since February 2011 have the Welsh capital club enjoyed such a purple patch in the league, raising hopes of better times ahead under the coaching team of Paul John and Dale McIntosh.
For the first hour of the match though Cardiff were behind and at 15-3 down looked set to return home from Ireland empty-handed. But two tries in the space of three minutes – from a penalty try and replacement Lewis Jones – saw a turnaround in their fortunes.
Led out by Matthew Rees for the first time since the hooker successfully underwent treatment for testicular cancer, the Blues showed good early touches in attack but were unable to find a way through during a scoreless first quarter.
Instead it was Connacht who took the lead on 21 minutes through the boot of fly-half Miah Nikora. And worse was to come for the visitors when wing Matt Healy touched down Kieran Marmion's kick ahead for a try which Nikora converted. Gareth Davies' penalty on the stroke of half-time finally got the Blues on the scoreboard as they went into the break 10-3 down.
With the second half only five minutes old Connacht stretched their lead to 12 points after centre Robbie Henshaw crossed for their second try of the night. Blues' dominance at scrum time eventually saw them get a foothold in the game with the award of a penalty try, and three minutes later Lewis Jones dotted down for a try with Davies adding both conversions to edge them ahead at 17-15.
Aly Muldowney saw yellow on 69 minutes after referee Neil Paterson penalised Connacht for another scrum offence and it didn't take long for the Blues to take advantage, Dan Fish crossing wide out with what proved to be the final score of the game. The Blues move up to 7th as a result of the win in Galway.
The scorers:
For Connacht:
Tries: Healy, Henshaw, Fish
Con: Nikora
Pen: Nikora
Yellow cards: Muldowney, McKeon
For Cardiff:
Tries: Penalty Try, Jones
Cons: Davies
Pen: Davies
The teams:
Connacht: 15 Darragh Leader, 14 Danie Poolman, 13 Robbie Henshaw, 12 Eoin Griffin, 11 Matt Healy, 10 Miah Nikora, 9 Kieran Marmion, 8 Eoin McKeon, 7 Willie Faloon, 6 John Muldoon (capt), 5 Mick Kearney, 4 Aly Muldowney, 3 Rodney Ah You, 2 Jason Harris-Wright, 1 Ronan Loughney.
Replacements: 16 Dave Heffernan, 17 Denis Buckley, 18 Finlay Bealham, 19 Michael Swift, 20 Andrew Browne, 21 Frank Murphy, 22 Dan Parks, 23 Gavin Duffy.
Cardiff Blues: 15 Dan Fish, 14 Alex Cuthbert, 13 Cory Allen, 12 Dafydd Hewitt, 11 Harry Robinson, 10 Gareth Davies, 9 Lloyd Williams, 8 Ellis Jenkins, 7 Josh Navidi, 6 Macauley Cook, 5 Filo Paulo, 4 Bradley Davies, 3 Taufa'ao Filise, 2 Matthew Rees (c), 1 Gethin Jenkins.
Replacements: 16 Kristian Dacey, 17 Thomas Davies, 18 Scott Andrews, 19 Chris Dicomidis, 20 Luke Hamilton, 21 Lewis Jones, 22 Simon Humberstone, 23 Isaia Tuifua.
Referee: Neil Paterson (Scotland)
Assistant referees: Dudley Phillips (Ireland), Olly Hodges (Ireland)


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Bills Beat Blog
By Joe Buscaglia
Brought to you by:
Bills release DE Mark Anderson
by Joe Buscaglia
posted Jul 23 2013 5:39PM
Less than a week to go before the start of training camp for the Buffalo Bills, the team has released one of their big signings from the 2012 off-season. Taking only one sentence to explain in the press release, the Bills announced they have released defensive end Mark Anderson after just one year of his four-year contract.
When the agreement first happened, Anderson inked the contract which would have paid him $19.5 million over the four seasons if he saw the end of the deal. Only $7.9 million, however, was qualified as guaranteed money.
Now with Anderson's release, he made a total of $8,104,850 from Buffalo in his 16 months as a member of the roster. The Bills parting ways with Anderson now means they'll be on the hook for a cap hit of $4.5 million at some point. They could choose to take it all on the 2013 salary cap, or they could instead elect to prorate it.
In real cash however, Anderson will not see a dime of the remaining $11.4 million (his remaining three base salaries and final two years of workout bonuses) that he would have had he been with the Bills all four seasons.
In one injury-plagued season, the 30-year old played in just five games. Anderson amassed only 12 tackles and one sack in 2012.
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- Las Vegas Wedding Specials
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Viva Las Vegas Weddings
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I just wanted to THANK YOU for our amazing wedding!! I was a little scared something would go wrong with it being 11-11-11, but you all proved me wrong!!
Doug and Jen - 11-11-11 at 12:30 PM in the Main Chapel
Las Vegas
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Old Tradition, the groom shouldn't see the bride until she walks down the aisle! New Tradition, getting hitched in Fabulous Las Vegas! Our "First Look" packages marry the old with the new.
At Viva Las Vegas Weddings brides can ride in a separate limousine, primp with their wedding attendants in a luxury suite and enjoy individual wedding coordination.
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Las Vegas Weddings are Romantic, Elegant & Exciting
Is your dream wedding in Las Vegas romantic and tastefully traditional, or are you looking for a unique, fantasy- themed ceremony that expresses who you are as a couple? Maybe your idea of the perfect Vegas wedding is one that includes fun-filled vows with Elvis. No matter how you choose to tie the knot, Viva Las Vegas Wedding Chapel has the perfect wedding package for you!
Besides offering the most popular, creative, and affordable wedding ceremonies in Las Vegas, our wedding couples receive a free Wedding Website, including a free digital Weddng Invitation. Live Video Web Stream of your ceremony is also available. State-of-the-art technology throughout our chapels allows your family and friends back home to view your Las Vegas wedding in real time on the Internet. That means no one needs to miss out on your beautiful wedding! Visit our Wedding Website page for complete details.
Same Sex Marriage Is Now Legal In Nevada!
On October 7, the 9th Circuit Court of Appeals ruled in favor of the freedom to marry allowing same-sex couples to legally marry in Nevada. We are thrilled about the legalization of same-sex marriage in Nevada, come join us when it's time to celebrate your marriage.
Viva Las Vegas Wedding Chapels
One of our five distinctive Las Vegas wedding chapels will create the perfect backdrop for any of our popular wedding packages.
Our Main Chapel is the largest, most affordable wedding chapel on the Las Vegas Strip and is the setting for many of our traditional, themed and Elvis weddings. This exquisitely-appointed venue is warm and elegant, but also boasts technologically-advanced theatrical lighting and fog effects. In fact, the entire chapel can be dramatically transformed with imaginative sets to accompany your themed Las Vegas wedding.
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Our Doo Wop Diner Chapel transports you and your guests back to the 1950's. It's just right for Elvis weddings, and great for vow renewals in Vegas, too! For cozier weddings, we sometimes suggest our Little Chapel. It's ideal when it's just the two of you, or if you have a small number of guests and want an intimate ambiance.
Traditional Las Vegas Weddings
Our many and varied traditional wedding packages are all-inclusive, with options that make your ceremony affordable and uniquely your own. Some packages even include limo service to and from the chapel, which is an unforgettable way to cruise The Strip after your ceremony. Whatever you're looking for, you're sure to find a Viva Las Vegas wedding package that's just right for you. Whether your special wedding will be indoors or outdoors (we even offer packages at exotic locations), our friendly staff will work with you to make sure everything is just the way you've imagined your Las Vegas wedding to be.
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Another fantastic option is our Elvis Blue Hawaii Wedding. This package is highlighted by theatrical fog and a hula girl dancing to Elvis's rendition of the Hawaiian Wedding Song. You can also choose to step back in time and get married by Elvis in our Doo Wop Diner. Get out your Hawaiian shirts, poodle skirts and bobby socks, folks. We even offer diner-style reception packages with sliders and root beer floats!
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With over 30 different themes to choose from, Viva Las Vegas Weddings is the number one wedding chapel for memorable and wildly imaginative wedding packages. If you don't see one that's perfect for the two of you, just ask. We'll create a custom wedding for you, or we can simply combine different elements of existing packages. We want you to have a wedding that expresses your unique relationship. Our corny motto here in the office? "If you can dream it, we can theme it." And, all of our weddings can be performed as commitment ceremonies or vow renewals. Have a look by clicking on the themes that intrigue you most! Or, take a video tour for a taste of the exciting themed weddings Viva Las Vegas Weddings has to offer.
Viva Las Vegas Wedding Chapel's Awards and Accolades
Viva Las Vegas Weddings Won Best of Nevada 2014!
Each year the readers of Nevada Magazine vote for their favorite attractions in the state of Nevada. In order to vote, Nevada Magazine's readers are required to write in their selections for what they consider the Best of the Best in Nevada.
We are proud and honored to share with you that Viva Las Vegas Wedding Chapel was voted 2014's Best Wedding Chapel in Southern Nevada. We would like to thank all of the readers, wedding couples and fans who took the time to cast their votes for us. This year's selection is extra special because it is Viva Las Vegas Wedding Chapel's tenth Best of Nevada award. Thank you, thank you very much!!!
Viva Las Vegas Weddings Won Best of Las Vegas 2011!
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Does our chapel look familiar? Viva Las Vegas Wedding Chapel has been featured in CSI Las Vegas, Fox'sThe Best Dam Sports Show With Tom Arnold, Love Nest on CMT, The Daily Show with Jon Stewart, Extra, The Sharon Osborne Show, The Ultimate Revenge, Dinner and a Movie on TBS, Entertainment Tonight, NBC Nightly News, The Tonight Show with Jay Leno, The Christopher Lowell Show, The Billboard Awards, VH1, MTV, NBC News, A&E, The Learning Channel, CBS Morning Show, Animal Planet, Soap Talk, The View and The Today Show! Katie Couric of The Today Show was so intrigued by the variety and style of our ceremonies that she sent a film crew to feature us on her show.


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By COREY BLUEMELStaff writer
The Emery County Commissioners met in their regularly scheduled meeting in Green River. Consideration was given, and discussion was held concerning a resolution to establish the Emery County Trails Committee. The resolution states:
"Whereas, the Emery County Commission deems it timely and in the best interest of the citizens of Emery County to establish a committee to oversee the management of properly recognized routes within Emery County that are not managed by the Emery County Road Department; and,
Whereas, there are both motorized and non-motorized routes in the county that are officially designated by federal agencies while other existing routes may not be formally recognized; and,
Whereas, the establishment of a Trails Committee will aid in the development and recognition of such routes;
Now, therefore, be it resolved by the Emery County Commission as follows:
1. Establishes the Emery County Trails Committee.
2. The Committee shall consist of five members with the following composition:
A. Two members representing the Emery County Travel Bureau.
B. Two members representing the Public Lands Council.
C. One representative from the Emery County Commission.
3. The initial Committee shall consist of the following individuals as currently assigned:
A. LaMar Guymon and Mark H Williams from the Emery County Travel Bureau.
B. Randal Stilson and Mistie Christiansen from the Public Lands Council.
C. Gary Kofford representing the Emery County Commission.
4. The Committee shall, with the aid of the Public Lands Director, Economic Development Director and Travel Bureau Secretary as a professional staff, establish bylaws and procedures to govern the affairs of the Committee.
5. The purpose of the Committee shall be to develop and promote non-motorized trails and shall have oversight responsibility for recognized motorized trails.
6. The Committee shall work under the direction of the Emery County Travel Bureau and funds for any projects shall be part of the budgeted Bureau funds or from grants.
This Resolution shall become effective immediately upon adoption by the Emery County Commission."
Following discussion of the resolution, the resolution was adopted.
In other business conducted by the commissioners, a waiver of building permit fee for Green River Medical Center was approved. Revisions to the Emery County personnel/travel policy which have taken a year and a half to work out were approved. These revisions were made necessary by the Internal Revenue Service policy that states employer benefits, such as supplied cars, must be considered into the employee pay at the rate of $3 per day. At the next commission meeting, resolutions separating the personnel and travel policies will be discussed.
A cooperative agreement between Emery County and the Utah Department of Transportation was entered into for the Temple Mountain Road. The agreement for the road which leads into Goblin Valley State Park is for phase III of that project. This will complete the road from Goblin Valley to U-24 and it will be completed in Spring 2010.
A memorandum of understanding was signed between Emery County and the Bureau of Land Management, with Emery County as the cooperating agency, for the Gateway South and TransWest Express transmission line projects. Emery County was contacted by the Wyoming State office and was asked to be the cooperating agency. Rocky Mountain Power and PacifiCorp are establishing new corridors for power lines to get power from Wyoming to Nevada. A portion of these lines may come through Emery County and by agreeing to be a cooperating agency, it will give Emery County a seat at the table for all discussions concerning these projects.
The agreement between Emery County and the BLM for a right of way application was signed for the Emery County communication site. This agreement is a renewal of a previous agreement.
Emery County is hosting a senior citizen lamb fry at Bear Creek Campground on Sept. 11 beginning at 11 a.m. Seniors are welcome to attend this luncheon and entertainment.
A cooperative fire rate agreement was signed designating rates for county equipment use in the event of a fire. This agreement is between Emery County and the State Division of Forestry, Fire and State Lands.
The next Emery County Commission meeting will be Sept. 8 at 9 a.m. in the old courthouse building in the commission chambers.