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1.  Reversible paralysis of Schistosoma mansoni by forchlorfenuron, a phenylurea cytokinin that affects septins 
Septins are guanosine-5′-triphosphate-binding proteins involved in wide-ranging cellular processes including cytokinesis, vesicle trafficking, membrane remodeling and scaffolds, and with diverse binding partners. Precise roles for these structural proteins in most processes often remain elusive. Identification of small molecules that inhibit septins could aid in elucidating the functions of septins and has become increasingly important, including the description of roles for septins in pathogenic phenomena such as tumorigenesis. The plant growth regulator forchlorfenuron (FCF), a synthetic cytokinin known to inhibit septin dynamics, likely represents an informative probe for septin function. This report deals with septins of the human blood fluke Schistosoma mansoni and their interactions with FCF. Recombinant forms of three schistosome septins, SmSEPT5, SmSEPT7.2 and SmSEPT10, interacted with FCF, leading to rapid polymerization of filaments. Culturing developmental stages (miracidia, cercariae, adult males) of schistosomes in FCF at 50 – 500 μM rapidly led to paralysis, which was reversible upon removal of the cytokinin. The reversible paralysis was concentration-, time- and developmental stage-dependent. Effects of FCF on the cultured schistosomes were monitored by video and/or by an xCELLigence-based assay of motility, which quantified the effect of FCF on fluke motility. The findings implicated a mechanism targeting a molecular system controlling movement in these developmental stages: a direct effect on muscle contraction due to septin stabilization might be responsible for the reversible paralysis, since enrichment of septins has been described within the muscles of schistosomes. This study revealed the reversible effect of FCF on both schistosome motility and its striking impact in hastening polymerization of septins. These novel findings suggested routes to elucidate roles for septins in this pathogen, and exploitation of derivatives of FCF for anti-schistosomal drugs.
doi:10.1016/j.ijpara.2014.03.010
PMCID: PMC4071124  PMID: 24768753
Schistosome; Forchlorfenuron; Septin; Cytokinin; Miracidium; Cercariae; xCELLigence; Phenylurea
2.  Intake of Erythrocytes Required for Reproductive Development of Female Schistosoma japonicum 
PLoS ONE  2015;10(5):e0126822.
The reproductive development and maturation of female schistosomes are crucial since their released eggs are responsible for the host immunopathology and transmission of schistosomiasis. However, little is known about the nutrients required by female Schistosoma japonicum during its sexual maturation. We evaluated the promoting effect of several nutrients (calf serum, red blood cells (RBCs), ATP and hypoxanthine) on the reproductive development of pre-adult females at 18 days post infection (dpi) from mixed infections and at 50 dpi from unisexual infections of laboratory mice in basic medium RPMI-1640. We found RBCs, rather than other nutrients, promoted the female sexual maturation and egg production with significant morphological changes. In 27% of females (18 dpi) from mixed infections that paired with males in vitro on day 14, vitelline glands could be positively stained by Fast Blue B; and in 35% of females (50 dpi) from unisexual infections on day 21, mature vitelline cells were observed. Infertile eggs were detected among both groups. To analyze which component of mouse RBCs possesses the stimulating effect, RBCs were fractionated and included in media. However, the RBC fractions failed to stimulate development of the female reproductive organs. In addition, bovine hemoglobin hydrolysate, digested by neutral protease, was found to exhibit the promoting activity instead of untreated bovine hemoglobin. The other protein hydrolysate, lactalbumin hydrolysate, exhibited a similar effect with bovine hemoglobin hydrolysate. Using quantitative RT-PCR, we found the expression levels of four reproduction-related genes were significantly stimulated by RBCs. These data indicate that RBCs provide essential nutrients for the sexual maturation of female S. japonicum and that the protein component of RBCs appeared to constitute the key nutrient. These findings would improve laboratory culture of pre-adult schistosomes to adult worms in medium with well-defined components, which is important to investigate the function of genes related to female sexual maturation.
doi:10.1371/journal.pone.0126822
PMCID: PMC4433235  PMID: 25978643
3.  A microRNA profile associated with Opisthorchis viverrini-induced cholangiocarcinoma in tissue and plasma 
BMC Cancer  2015;15:309.
Background
Intrahepatic cholangiocarcinoma (ICC) is a highly aggressive tumor of the bile duct, and a significant public health problem in East Asia, where it is associated with infection by the parasite Opisthorchis viverrini. ICC is often detected at an advanced stage and with a poor prognosis, making a biomarker for early detection a priority.
Methods
We have comprehensively profiled miRNA expression levels in ICC tumor tissue using small RNA-Seq and validated these profiles using quantitative PCR on matched plasma samples.
Results
Distinct miRNA profiles were associated with increasing histological differentiation of ICC tumor tissue. We also observed that histologically normal tissue adjacent to ICC tumor displayed miRNA expression profiles more similar to tumor than liver tissue from healthy donors. In plasma samples, an eight-miRNA signature associated with ICC, regardless of the degree of histological differentiation of its matched tissue, forming the basis of a circulating miRNA-based biomarker for ICC.
Conclusions
The association of unique miRNA profiles with different ICC subtypes suggests the involvement of specific miRNAs during ICC tumor progression. In plasma, an eight-miRNA signature associated with ICC could form the foundation of an accessible (plasma-based) miRNA-based biomarker for the early detection of ICC.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1270-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1270-5
PMCID: PMC4417245  PMID: 25903557
MicroRNA; Cholangiocarcinoma; Intrahepatic cholangiocarcinoma; Opisthorchis viverrini; RNA-seq
4.  Gene function in schistosomes: recent advances toward a cure 
Frontiers in Genetics  2015;6:144.
doi:10.3389/fgene.2015.00144
PMCID: PMC4397921  PMID: 25926850
schistosomiasis; economical impact; poverty; treatment; gene function
6.  How might flukes and tapeworms maintain genome integrity without a canonical piRNA pathway? 
Trends in parasitology  2014;30(3):123-129.
Surveillance by RNA interference is central to controlling the mobilization of transposable elements (TEs). In stem cells, Piwi argonaute (Ago) proteins and associated proteins repress mobilization of TEs to maintain genome integrity. This defense mechanism targeting TEs is termed the Piwi-interacting RNA (Piwi-piRNA) pathway. In this Opinion, we draw attention to the situation that the genomes of cestodes and trematodes have lost the piwi and vasa genes that are hallmark characters of the germline multipotency program. This absence of Piwi-like Agos and Vasa helicases prompts the question: how does the germline of these flatworms withstand mobilization of TEs? Here we present an interpretation of mechanisms likely to defend the germline integrity of parasitic flatworms.
doi:10.1016/j.pt.2014.01.001
PMCID: PMC3941195  PMID: 24485046
Platyhelminthes; Cestoda; Trematoda; piwi; vasa; argonaute; transposable elements; germline; piRNA pathway
7.  Retrotransposon OV-RTE-1 from the carcinogenic liver fluke Opisthorchis viverrini; potential target for DNA-based diagnosis 
Infections by the fish-borne liver flukes Opisthorchis viverrini and Clonorchis sinensis can lead to bile duct cancer. These neglected tropical disease pathogens occur in East Asia, with O. viverrini primarily in Thailand and Laos and C. sinensis in Cambodia, Vietnam, and China. Genomic information about these pathogens holds the potential to improve disease treatment and control. Transcriptome analysis indicates that mobile genetic elements are active in O. viverrini, including a novel non-Long Terminal Repeat (LTR) retrotransposon. A consensus sequence of this element, termed OV-RTE-1, was assembled from expressed sequence tags and PCR amplified genomic DNA. OV-RTE-1 was 3,330 bp in length, encoded 1,101 amino acid residues and exhibited hallmark structures and sequences of non-LTR retrotransposons including a single open reading frame encoding apurinic-apyrimidinic endonuclease (EN) and reverse transcriptase (RT). Phylogenetic analyses confirmed that OV-RTE-1 was member of the RTE clade of non-LTR retrotransposons. OV-RTE-1 is the first non-LTR retrotransposon characterized from the trematode family Opisthorchiidae. Sequences of OV-RTE-1 were targeted to develop a diagnostic tool for detection of infection by O. viverrini. PCR specific primers for detection of O. viverrini DNA showed 100% specificity and sensitivity for detection of as little as five femtograms of O. viverrini DNA whereas the PCR based approach showed 62% sensitivity and 100% specificity with clinical stool samples. The OV-RTE-1 specific PCR could be developed as a molecular diagnostic for Opisthorchis infection targeting parasite eggs in stool samples, especially in regions of mixed infection of O. viverrini and/or C. sinensis and minute intestinal flukes.
doi:10.1016/j.meegid.2013.12.015
PMCID: PMC3955739  PMID: 24394447
Opisthorchis viverrini; liver fluke; retrotransposon; diagnosis; feces
8.  Schistosome and liver fluke derived catechol-estrogens and helminth associated cancers 
Frontiers in Genetics  2014;5:444.
Infection with helminth parasites remains a persistent public health problem in developing countries. Three of these pathogens, the liver flukes Clonorchis sinensis, Opisthorchis viverrini and the blood fluke Schistosoma haematobium, are of particular concern due to their classification as Group 1 carcinogens: infection with these worms is carcinogenic. Using liquid chromatography-mass spectrometry (LC-MS/MS) approaches, we identified steroid hormone like (e.g., oxysterol-like, catechol estrogen quinone-like, etc.) metabolites and related DNA-adducts, apparently of parasite origin, in developmental stages including eggs of S. haematobium, in urine of people with urogenital schistosomiasis, and in the adult stage of O. viverrini. Since these kinds of sterol derivatives are metabolized to active quinones that can modify DNA, which in other contexts can lead to breast and other cancers, helminth parasite associated sterols might induce tumor-like phenotypes in the target cells susceptible to helminth parasite associated cancers, i.e., urothelial cells of the bladder in the case of urogenital schistosomiasis and the bile duct epithelia or cholangiocytes, in the case of O. viverrini and C. sinensis. Indeed we postulate that helminth induced cancers originate from parasite estrogen-host epithelial/urothelial cell chromosomal DNA adducts, and here we review recent findings that support this conjecture.
doi:10.3389/fgene.2014.00444
PMCID: PMC4274992  PMID: 25566326
urogenital schistosomiasis; opisthorchiasis; catechol-estrogens; oxysterols; DNA-adducts; neglected tropical disease-associated-cancer; squamous cell carcinoma of the bladder; cholangiocarcinoma
9.  P53 and Cancer-Associated Sialylated Glycans Are Surrogate Markers of Cancerization of the Bladder Associated with Schistosoma haematobium Infection 
Background
Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers.
Methodology/Principal Findings
Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed in the healthy urothelium.
Conclusion/Significance
This preliminary study suggests that p53 and sialylated glycans are surrogate biomarkers of bladder cancerization associated with S. haematobium, highlighting a missing link between infection and cancer development. Eggs of S. haematobium express sLea and sLex antigens in mimicry of human leukocytes glycosylation, which may play a role in the colonization and disease dissemination. These observations may help the early identification of infected patients at a higher risk of developing bladder cancer and guide the future development of non-invasive diagnostic tests.
Author Summary
Epidemiological studies associate infection with S. haematobium, an endemic parasitic flatworm in Africa and the Middle East, with the development of bladder cancer. Nevertheless, little molecular evidence exists supporting this association. This work draws attention to the common molecular pathways underlying these two events, highlighting a potentially unreported link between infection and cancer development. It has been demonstrated that a panel of biomarkers commonly associated with aggressive forms of bladder cancer is also present in non-malignant tissues infected with the parasite. This may offer a means of early identification of people with this parasitic infection who are at risk of developing of bladder cancer, and may guide the establishment of non-invasive diagnostic tests. Furthermore, we observed that parasite eggs mimic the molecular nature of human cells, providing a possible mechanism of immune escape and persistent infection. Such knowledge is considered pivotal to develop novel therapeutic strategies.
doi:10.1371/journal.pntd.0003329
PMCID: PMC4263606  PMID: 25502795
10.  Carcinogenic liver fluke Opistorchis viverrini oxysterols detected by LC-MS/MS survey of soluble fraction parasite extract 
Parasitology international  2013;62(6):535-542.
Liquid chromatography in tandem mass spectrometry (LC-MS/MS) has emerged as an informative tool to investigate oxysterols (oxidized derivatives of cholesterol) in helminth parasite associated cancers. Here, we used LC-MS/MS to investigate in soluble extracts of the adult developmental stage of O. viverrini from experimentally infected hamsters. Using comparisons with known bile acids and the metabolites of estrogens, the LC-MS data indicated the existence of novel oxysterol derivatives in O. viverrini. Most of these derivatives were ramified at C-17, in similar fashion to bile acids and their conjugated salts. Several were compatible with the presence of an estrogen core, and/or hydroxylation of the steroid aromatic ring A, hydroxylation of both C-2 and C-3 of the steroid ring and further oxidation into an estradiol-2,3-quinone.
doi:10.1016/j.parint.2013.08.001
PMCID: PMC3797210  PMID: 23973383
bile acids; flukes; Opisthorchis viverrini; oxysterols
11.  Approaches to genotyping individual miracidia of Schistosoma japonicum 
Parasitology research  2013;112(12):10.1007/s00436-013-3587-9.
Molecular genetic tools are needed to address questions as to the source and dynamics of transmission of the human blood fluke Schistosoma japonicum in regions where human infections have re-emerged, and to characterize infrapopulations in individual hosts. The life-stage that interests us as a target for collecting genotypic data is the miracidium, a very small larval stage that consequently yields very little DNA for analysis. Here, we report the successful development of a multiplex format permitting genotyping of 17 microsatellite loci in four sequential multiplex reactions using a single miracidium held on a Whatman Classic FTA indicating card. This approach was successful after short storage periods, but after long storage (>4 years) considerable difficulty was encountered in multiplex genotyping, necessitating the use of whole genome amplification (WGA) methods. WGA applied to cards stored for long periods of time resulted in sufficient DNA for accurate and repeatable genotyping. Trials and tests of these methods, as well as application to some field-collected samples, are reported, along with discussion of the potential insights to be gained from such techniques. These include recognition of sibships among miracidia from a single host, and inference of the minimum number of worm pairs that might be present in a host.
doi:10.1007/s00436-013-3587-9
PMCID: PMC3834234  PMID: 24013341
China; miracidia; FTA; multiplexing; microsatellites; Schistosoma japonicum
12.  Helminth.net: expansions to Nematode.net and an introduction to Trematode.net 
Nucleic Acids Research  2014;43(Database issue):D698-D706.
Helminth.net (http://www.helminth.net) is the new moniker for a collection of databases: Nematode.net and Trematode.net. Within this collection we provide services and resources for parasitic roundworms (nematodes) and flatworms (trematodes), collectively known as helminths. For over a decade we have provided resources for studying nematodes via our veteran site Nematode.net (http://nematode.net). In this article, (i) we provide an update on the expansions of Nematode.net that hosts omics data from 84 species and provides advanced search tools to the broad scientific community so that data can be mined in a useful and user-friendly manner and (ii) we introduce Trematode.net, a site dedicated to the dissemination of data from flukes, flatworm parasites of the class Trematoda, phylum Platyhelminthes. Trematode.net is an independent component of Helminth.net and currently hosts data from 16 species, with information ranging from genomic, functional genomic data, enzymatic pathway utilization to microbiome changes associated with helminth infections. The databases’ interface, with a sophisticated query engine as a backbone, is intended to allow users to search for multi-factorial combinations of species’ omics properties. This report describes updates to Nematode.net since its last description in NAR, 2012, and also introduces and presents its new sibling site, Trematode.net.
doi:10.1093/nar/gku1128
PMCID: PMC4383941  PMID: 25392426
13.  Mass spectrometry techniques in the survey of steroid metabolites as potential disease biomarkers: a review 
Mass spectrometric approaches have been fundamental to the identification of metabolites associated with steroid hormones, yet this topic has not been reviewed in depth in recent years. To this end, and given the increasing relevance of liquid chromatrography-mass spectrometry (LC-MS) studies on steroid hormones and their metabolites, the present review addresses this subject. This review provides a timely summary of the use of various mass spectrometry-based analytical techniques during the evaluation of steroidal biomarkers in a range of human disease settings. The sensitivity and specificity of these technologies are clearly providing valuable new insights into breast cancer and cardiovascular disease.
We aim to contribute to an enhanced understanding of steroid metabolism and how it can be profiled by LC-MS techniques.
doi:10.1016/j.metabol.2013.04.003
PMCID: PMC3755027  PMID: 23664145
Mass spectrometry; LC-MS; estradiol metabolites; cholesterol metabolites; cancer biomarkers
14.  RNA interference of Schistosoma mansoni cathepsin D, the apical enzyme of the hemoglobin proteolysis cascade 
The aspartic protease cathepsin D (Clan AA, Family A1) is expressed in the schistosome gut where it plays an apical role in the digestion of hemoglobin released from ingested erythrocytes. In this report, RNA interference approaches were employed to investigate the effects of knockdown of schistosome cathepsin D. Cultured schistosomules of Schistosoma mansoni were exposed by square wave electroporation to double stranded RNA (dsRNA) specific for cDNA encoding S. mansoni cathepsin D. RNAi mediated reductions in transcript levels led to phenotypic changes including significant growth retardation in vitro and suppression of aspartic protease enzyme activity. In addition, black-pigmented heme, the end point by-product of normal hemoglobin proteolysis that accumulates in the schistosome gut, was not apparent within the guts of the treated schistosomules. Rather, their guts appeared to be red in color, rather than black, apparently indicating the presence of intact rather than digested host hemoglobin. These phenotypic effects were apparent when either of two forms of dsRNA, a long form spanning the entire target transcript or a short form specific for the 3’-region were employed. Off-target effects were not apparent in transcript levels of the gut-localized cysteine protease cathepsin B1. Finally, cathepsin D may be an essential enzyme in the mammal-parasitic stages of schistosomes because schistosomules treated ex vivo with dsRNA did not survive to maturity after transfer into Balb/c mice. These and earlier findings suggest that, given its essential function in parasite nutrition, schistosome cathepsin D could be developed as a target for novel anti-schistosomal interventions.
doi:10.1016/j.molbiopara.2007.10.009
PMCID: PMC4130333  PMID: 18067980
schistosomes; cathepsin D; RNAi; schistosomules; hemoglobin proteolysis; RT-PCR
15.  Circumventing qPCR inhibition to amplify miRNAs in plasma 
Biomarker Research  2014;2:13.
Background
Circulating microRNAs (c-miRNAs) have be identified in saliva, urine and blood, which has led to increasing interest in their development as biomarkers for diverse diseases including cancers. One of the key advantages of c-miRNAs over other biomarkers is the ability to be amplified and quantified by quantitative PCR (qPCR). However, at phlebotomy when whole blood is dispensed into heparinized tubes, residual levels of the anti-coagulant lithium heparin may remain in the plasma and hence with RNA isolated from the plasma. This can confound the detection of c-miRNAs by qPCR because it inhibits reverse transcriptase (RT). Here we present a procedure, modified from earlier techniques, to detect c-miRNAs in plasma that improves sensitivity and streamlines performance.
Findings
Treatment of total RNA isolated from human blood plasma with Bacteroides heparinase I during reverse transcription at 37°C for one hour improved sensitivity and performance of the qPCR. This is in comparison to no treatment or treatment of the RNA prior to RT, which is the current suggested method and exposes plasma to Flavobacterium heparinum heparinase I for up to 2 hours before RT. This modest alteration improved qPCR performance and resulted in lowered threshold cycles (Ct) for detection of the target sequence, candidate c-miRNA biomarkers, and controls. It also reduced the expense and number of processing steps, shortening the duration of the assay and minimizing exposure of RNA to elevated temperatures.
Conclusion
Incorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers.
doi:10.1186/2050-7771-2-13
PMCID: PMC4114091  PMID: 25075309
miRNA; Interference; Plasma; Reverse transcriptase; qPCR; Biomarker; Anti-coagulant; Heparin; Heparinase; Eliminase; Bacteroides heparinase I
16.  Ultrasonography assessment of hepatobiliary abnormalities in 3,359 subjects with Opisthorchis viverrini infection in endemic areas of Thailand 
Parasitology international  2011;61(1):208-211.
A cross sectional study on hepatobiliary abnormalities in opisthorchiasis was performed in 8,936 males and females aged from 20 to 60 years from 90 villages of Khon Kaen province, Northeast Thailand. All were stool-examined for Opisthorchis viverrini infection by standard quantitative formalin/ethyl acetate concentration technique. Of these, 3,359 participants with stool egg positive were underwent ultrasonography of the upper abdomen. The hepatobiliary abnormalities detected by ultrasound are described here. This study found a significantly higher frequency of advanced periductal fibrosis in persons with chronic opisthorchiasis (23.6%), particularly in males. Risks of the fibrosis included intensity of infection, and age younger than 30 years. Height of left lobe of the liver, cross-section of the gallbladder dimensions post fatty meal, sludge, and, interestingly, intrahepatic duct stones were significantly associated with the advanced periductal fibrosis. Eleven suspected cholangiocarcinoma (CCA) cases were observed. This study emphasizes the current status of high O. viverrini infection rate and the existence of hepatobiliary abnormalities including suspected CCA in opisthorchiasis endemic areas of Thailand.
doi:10.1016/j.parint.2011.07.009
PMCID: PMC4096033  PMID: 21771664
ultrasonography; opisthorchiasis; hepatobiliary abnormalities; cholangiocarcinoma; Opisthorchis viverrini
17.  Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium 
BMC Genomics  2014;15(1):428.
Background
Cysticercosis remains a major neglected tropical disease of humanity in many regions, especially in sub-Saharan Africa, Central America and elsewhere. Owing to the emerging drug resistance and the inability of current drugs to prevent re-infection, identification of novel vaccines and chemotherapeutic agents against Taenia solium and related helminth pathogens is a public health priority. The T. solium genome and the predicted proteome were reported recently, providing a wealth of information from which new interventional targets might be identified. In order to characterize and classify the entire repertoire of protease-encoding genes of T. solium, which act fundamental biological roles in all life processes, we analyzed the predicted proteins of this cestode through a combination of bioinformatics tools. Functional annotation was performed to yield insights into the signaling processes relevant to the complex developmental cycle of this tapeworm and to highlight a suite of the proteases as potential intervention targets.
Results
Within the genome of this helminth parasite, we identified 200 open reading frames encoding proteases from five clans, which correspond to 1.68% of the 11,902 protein-encoding genes predicted to be present in its genome. These proteases include calpains, cytosolic, mitochondrial signal peptidases, ubiquitylation related proteins, and others. Many not only show significant similarity to proteases in the Conserved Domain Database but have conserved active sites and catalytic domains. KEGG Automatic Annotation Server (KAAS) analysis indicated that ~60% of these proteases share strong sequence identities with proteins of the KEGG database, which are involved in human disease, metabolic pathways, genetic information processes, cellular processes, environmental information processes and organismal systems. Also, we identified signal peptides and transmembrane helices through comparative analysis with classes of important regulatory proteases. Phylogenetic analysis using Bayes approach provided support for inferring functional divergence among regulatory cysteine and serine proteases.
Conclusion
Numerous putative proteases were identified for the first time in T. solium, and important regulatory proteases have been predicted. This comprehensive analysis not only complements the growing knowledge base of proteolytic enzymes, but also provides a platform from which to expand knowledge of cestode proteases and to explore their biochemistry and potential as intervention targets.
Electronic supplementary material
The online version of this article (doi: 10.1186/1471-2164-15-428) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-428
PMCID: PMC4070553  PMID: 24899069
Proteases; Taenia solium; Drug target; Vaccine candidate antigen; Genome-wide analysis; Cysticercosis; Platyhelminth
18.  Urinary Estrogen Metabolites and Self-Reported Infertility in Women Infected with Schistosoma haematobium 
PLoS ONE  2014;9(5):e96774.
Background
Schistosomiasis is a neglected tropical disease, endemic in 76 countries, that afflicts more than 240 million people. The impact of schistosomiasis on infertility may be underestimated according to recent literature. Extracts of Schistosoma haematobium include estrogen-like metabolites termed catechol-estrogens that down regulate estrogen receptors alpha and beta in estrogen responsive cells. In addition, schistosome derived catechol-estrogens induce genotoxicity that result in estrogen-DNA adducts. These catechol estrogens and the catechol-estrogen-DNA adducts can be isolated from sera of people infected with S. haematobium. The aim of this study was to study infertility in females infected with S. haematobium and its association with the presence of schistosome-derived catechol-estrogens.
Methodology/Principal Findings
A cross-sectional study was undertaken of female residents of a region in Bengo province, Angola, endemic for schistosomiasis haematobia. Ninety-three women and girls, aged from two (parents interviewed) to 94 years were interviewed on present and previous urinary, urogenital and gynecological symptoms and complaints. Urine was collected from the participants for egg-based parasitological assessment of schistosome infection, and for liquid chromatography diode array detection electron spray ionization mass spectrometry (LC/UV-DAD/ESI-MSn) to investigate estrogen metabolites in the urine. Novel estrogen-like metabolites, potentially of schistosome origin, were detected in the urine of participants who were positive for eggs of S. haematobium, but not detected in urines negative for S. haematobium eggs. The catechol-estrogens/ DNA adducts were significantly associated with schistosomiasis (OR 3.35; 95% CI 2.32–4.84; P≤0.001). In addition, presence of these metabolites was positively associated with infertility (OR 4.33; 95% CI 1.13–16.70; P≤0.05).
Conclusions/Significance
Estrogen metabolites occur widely in diverse metabolic pathways. In view of the statistically significant association between catechol-estrogens/ DNA adducts and self-reported infertility, we propose that an estrogen-DNA adduct mediated pathway in S. haematobium-induced ovarian hormonal deregulation could be involved. In addition, the catechol-estrogens/ DNA adducts described here represent potential biomarkers for schistosomiasis haematobia.
doi:10.1371/journal.pone.0096774
PMCID: PMC4029575  PMID: 24848950
19.  Suppression of aquaporin, a mediator of water channel control in the carcinogenic liver fluke, Opisthorchis viverrini 
Parasites & Vectors  2014;7:224.
Background
Opisthorchiasis and Opisthorchis viverrini-associated bile duct cancer represent major public health threats in Thailand and Laos. The tegument of this food borne fluke plays pivotal roles in parasite metabolism, homeostasis and osmoregulation. Excretory/secretory products also pass from the fluke to the biliary environment, products that likely underlie pathogenesis of liver fluke infection. Aquaporins (AQPs), belong to the major intrinsic protein superfamily of integral plasma membrane channel proteins that selectively transport water across cell membranes. AQPs play key roles as water and ion transport channels through the tegument of helminth parasites.
Methods
Here, two forms of AQP mRNAs from the adult developmental stage of O. viverrini, termed O. viverrini aquaporin-1 and -2 (Ov-aqp-1 and -2) were investigated. Roles of Ov-aqp-1 and -2 in the movement of water across the tegument of this carcinogenic liver fluke were investigated using RNA interference.
Results
Ov-AQP-1 and Ov-AQP-2 contain unique characteristic asparagine-proline-alanine (NPA) motifs of AQP transmembrane proteins. Phylogenetic analysis indicated that Ov-AQPs belong to an expanding group of aquaglyceroporin-like water channel proteins characterized from helminth and protozoan parasites, which is pivotal to the specialized requirements of water and solute control during parasitism. Elevated transcription of Ov-aqp-1 was evident in the egg, cercaria, metacercaria and adult stages of O. viverrini, whereas Ov-aqp-2 transcripts were detected at higher level in egg, metacercaria, cercaria and adult stage, respectively. RNA interference using electroporated dsRNA suppressed transcript levels of Ov-aqp-1 and Ov-aqp-2 in adult worms by 58-99% over periods of up to 16 days in vitro. Suppression of Ov-aqp-1 and Ov-aqp-2 in vitro disabled water transport in adult flukes.
Conclusion
The apparently pivotal roles of Ov-AQP in solute homeostasis at the fluke surface suggest that deeper investigation will be informative for the pathophysiology of O. viverrini, and may uncover intervention targets, particularly in view of the singularly notable predilection of this pathogen for residence within ducts of the biliary tree.
doi:10.1186/1756-3305-7-224
PMCID: PMC4033688  PMID: 24885060
O. viverrini; Cholangiocarcinoma; RNA interference; Aquaporin; Liver fluke; Tegument
20.  Bioclojure: a functional library for the manipulation of biological sequences 
Bioinformatics  2014;30(17):2537-2539.
Motivation: BioClojure is an open-source library for the manipulation of biological sequence data written in the language Clojure. BioClojure aims to provide a functional framework for the processing of biological sequence data that provides simple mechanisms for concurrency and lazy evaluation of large datasets.
Results: BioClojure provides parsers and accessors for a range of biological sequence formats, including UniProtXML, Genbank XML, FASTA and FASTQ. In addition, it provides wrappers for key analysis programs, including BLAST, SignalP, TMHMM and InterProScan, and parsers for analyzing their output. All interfaces leverage Clojure’s functional style and emphasize laziness and composability, so that BioClojure, and user-defined, functions can be chained into simple pipelines that are thread-safe and seamlessly integrate lazy evaluation.
Availability and implementation: BioClojure is distributed under the Lesser GPL, and the source code is freely available from GitHub (https://github.com/s312569/clj-biosequence).
Contact: jason.mulvenna@qimrberghofer.edu.au or jason.mulvenna@qimr.edu.au
doi:10.1093/bioinformatics/btu311
PMCID: PMC4147884  PMID: 24794932
21.  Methods and matrices: approaches to identifying miRNAs for Nasopharyngeal carcinoma 
Background
Nasopharyngeal carcinoma (NPC) is a solid tumor of the head and neck. Multimodal therapy is highly effective when NPC is detected early. However, due to the location of the tumor and the absence of clinical signs, early detection is difficult, making a biomarker for the early detection of NPC a priority. The dysregulation of small non-coding RNAs (miRNAs) during carcinogenesis is the focus of much current biomarker research. Herein, we examine several miRNA discovery methods using two sample matrices to identify circulating miRNAs (c-miRNAs) associated with NPC.
Methods
We tested two miRNA discovery workflows on two sample sources for miRNAs associated with NPC. In the first workflow, we assumed that NPC tumor tissue would be enriched for miRNAs, so we compared miRNA expression in FFPE from NPC cases and controls using microarray and RNA-Seq technologies. Candidate miRNAs from both technologies were verified by qPCR in FFPE and sera from an independent NPC sample set. In a second workflow, we directly interrogated NPC case and control sera by RNA-Seq for c-miRNAs associated with NPC, with candidate c-miRNAs verified by qPCR in the sera from the same independent NPC sample set.
Results
Both microarray and RNA-Seq narrowed the miRNA signature to 1-5% of the known mature human miRNAs. Moreover, these two methods produced similar results when applied to the same sample type (FFPE), with RNA-Seq additionally indicating “unknown” miRNAs associated with NPC. However, we found different miRNA profiles in NPC sera compared to FFPE using RNA-Seq, with the few overlapping miRNAs found to be significantly up-regulated in FFPE significantly down-regulated in sera (and vice versa). Despite the different miRNA profiles found in FFPE and sera, both profiles strongly associated with NPC, providing two potential sources for biomarker signatures for NPC.
Conclusions
We determined that the direct interrogation of sera by RNA-Seq was the most informative method for identifying a c-miRNA signature associated with NPC. We also showed that there are different miRNA expression profiles associated with NPC for tumor tissue and sera. These results reflect on the methods and meaning of miRNA biomarkers for NPC in tissue and peripheral blood.
doi:10.1186/1479-5876-12-3
PMCID: PMC3895762  PMID: 24393330
Nasopharyngeal carcinoma; Methods; Biomarker; MicroRNAs; qPCR; Next generation sequencing; RNA-Seq; Tumor
22.  Septins of Platyhelminths: Identification, Phylogeny, Expression and Localization among Developmental Stages of Schistosoma mansoni 
Septins are a family of eukaryotic GTP binding proteins conserved from yeasts to humans. Originally identified in mutants of budding yeast, septins participate in diverse cellular functions including cytokinesis, organization of actin networks, cell polarity, vesicle trafficking and many others. Septins assemble into heteroligomers to form filaments and rings. Here, four septins of Schistosoma mansoni are described, which appear to be conserved within the phylum Platyhelminthes. These orthologues were related to the SEPT5, SEPT10 and SEPT7 septins of humans, and hence we have termed the schistosome septins SmSEPT5, SmSEPT10, SmSEPT7.1 and SmSEPT7.2. Septin transcripts were detected throughout the developmental cycle of the schistosome and a similar expression profile was observed for septins in the stages examined, consistent with concerted production of these proteins to form heterocomplexes. Immunolocalization analyses undertaken with antibodies specific for SmSEPT5 and SmSEPT10 revealed a broad tissue distribution of septins in the schistosomulum and colocalization of septin and actin in the longitudinal and circular muscles of the sporocyst. Ciliated epidermal plates of the miracidium were rich in septins. Expression levels for these septins were elevated in germ cells in the miracidium and sporocyst. Intriguingly, septins colocalize with the protonephridial system of the cercaria, which extends laterally along the length of this larval stage. Together, the findings revealed that schistosomes expressed several septins which likely form filaments within the cells, as in other eukaryotes. Identification and localization demonstrating a broad distribution of septins across organs and tissues of schistosome contributes towards the understanding of septins in schistosomes and other flatworms.
Author Summary
Schistosoma mansoni is one of the causative agents of schistosomiasis, a neglected tropical disease affecting over 230 million people in the developing world. Research on new therapies for this parasitic disease has been facilitated by the recent publication of a curated draft sequence of the schistosome genome. Here, we describe proteins from the septin family found in the genome of S. mansoni. The septins are increasingly recognized as central components of the cytoskeleton of eukaryotic cells. They are linked to numerous cellular functions, although the precise role(s) of these proteins is not fully understood. Schistosome septins were seen in the miracidium and sporocyst larval stages, on superficial structures, within epidermal plates and in muscles. Notably, septins were prominently expressed in the germ cells of larval stages of the blood fluke. In addition, septins were ubiquitously immuno-localized throughout the organs and tissues of the schistosomulum stage of the parasite. This is the first report on septins in schistosomes; these proteins are broadly distributed among organs and tissues of the parasite where they likely perform diverse functions. Identification and localization demonstrating a broad distribution of septins across organs and tissues of schistosome contributes towards the understanding of septins in schistosomes and other flatworms.
doi:10.1371/journal.pntd.0002602
PMCID: PMC3868516  PMID: 24367716
23.  The current status of opisthorchiasis and clonorchiasis in the Mekong Basin 
Parasitology international  2011;61(1):10.1016/j.parint.2011.08.014.
This review highlights the current status and control of liver fluke infections in the Mekong Basin countries where Opisthorchis and Clonorchis are highly endemic. Updated data on prevalence and distribution have been summarized from presentations in the “96 Years of Opisthorchiasis. International Congress of Liver Flukes”. It is disturbing that despite treatment and control programs have been in place for decades, all countries of the Lower Mekong Basin are still highly endemic with O. viverrini and/or C. sinensis as well as alarmingly high levels of CCA incidence. A common pattern that is emerging in each country is the difference in transmission of O. viverrini between lowlands which have high prevalence versus highlands which have low prevalence. This seems to be associated with wetlands, flooding patterns and human movement and settlement. A more concerted effort from all community, educational, public health and government sectors is necessary to successfully combat this fatal liver disease of the poor.
doi:10.1016/j.parint.2011.08.014
PMCID: PMC3836690  PMID: 21893213
24.  Transduction of Schistosoma japonicum schistosomules with vesicular stomatitis virus glycoprotein pseudotyped murine leukemia retrovirus and expression of reporter human telomerase reverse transcriptase in the transgenic schistosomes 
Molecular and biochemical parasitology  2010;174(2):10.1016/j.molbiopara.2010.07.007.
Although draft genome sequences of two of the major human schistosomes, Schistosoma japonicum and S. mansoni are available, the structures and characteristics of most genes and the influence of exogenous genes on the metabolism of schistosomes remain uncharacterized. Furthermore, which functional genomics approaches will be tractable for schistosomes are not yet apparent. Here, the vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped pantropic retroviral vector pBABE-puro was modified to incorporate the human telomerase reverse transcriptase gene (hTERT) as a reporter, under the control of the retroviral long terminal repeat (LTR). Pseudotyped virions were employed to transduce S. japonicum to investigate the utility of retrovirus-mediated transgenesis of S. japonicum and the activity of human telomerase reverse transcriptase as a reporter transgene in schistosomes. Schistosomules perfused from experimentally infected rabbits were cultured for six days after exposure to the virions after which genomic DNAs from virus-exposed and control worms were extracted. Analysis of RNA from transduced parasites and immunohistochemistry of thin parasite sections revealed expression of hTERT in the transduced worms. Expression of hTERT was also confirmed by immunoblot analysis. These findings indicated that S. japonicum could be effectively transduced by VSVG pseudotyped retrovirus carrying the hTERT gene. Given the potential of hTERT to aid in derivation of immortalized cells, these findings suggest that this pantropic retroviral approach can be employed to transduce cells from specific tissues and organs of schistosomes to investigate the influence of transgene hTERT on growth and proliferation of schistosome cells.
doi:10.1016/j.molbiopara.2010.07.007
PMCID: PMC3836731  PMID: 20692298
Schistosoma japonicum; glycoprotein of vesicular stomatitis virus (VSVG); pantropic retrovirus; murine leukemia virus; transgene; telomerase
25.  The tumorigenic liver fluke Opisthorchis viverrini –multiple pathways to cancer 
Trends in parasitology  2012;28(10):395-407.
Liver fluke infection caused by Opisthorchis viverrini is a major public health problem in Thailand and adjacent countries. In addition to infection-associated morbidity, infection with O. viverrini and the related Clonorchis sinensis are unarguable risk factors for cholangiocarcinoma, bile duct cancer. Here we review the pathogenesis of opisthorchiasis and the association of O. viverrini infection and bile duct cancer, focusing on the molecular parallels between wound healing, chronic inflammation and cancer development. We review a schema for human disease progression from fluke infection, chronic opisthorchiasis, advanced periductal fibrosis, and cholangiocarcinogenesis, and present a rationale for biomarker discovery to facilitate early intervention. We conclude by addressing post-genomic advances with a view to developing new control strategies to combat this infectious cancer.
doi:10.1016/j.pt.2012.07.006
PMCID: PMC3682777  PMID: 22947297
Opisthorchis; liver fluke; cholangiocarcinoma; liver cancer; granulin; periductal fibrosis; excretory/secretory; wound healing

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