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Purpose Progranulin (PGRN) is a secreted glycoprotein growth factor with roles in wound healing, inflammation, angiogenesis and malignancy. An orthologue of the gene encoding human PGRN was identified in the carcinogenic liver fluke Opisthorchis viverrini. Methods Sequence structure, general characteristics and possible function of O. viverrini PGRN was analyzed using bioinformatics. Expression profiles were investigated with quantitative RT-PCR, western blot and immunolocalization. A specific peptide of Ov-PGRN was used to investigate a role for this molecule in pathogenesis. Results The structure of the gene coding for O. viverrini PGRN was 36,463 bp in length, and comprised of 13 exons, 12 introns, and a promoter sequence. The Ov-pgrn mRNA is 2,768 bp in length and encodes an 846 amino acids with a predicted molecular mass of 91.61 kDa. Ov-PGRN exhibited one half and seven complete granulin domains. Phylogenetic analysis revealed that Ov-PGRN formed its closest relationship with PGRN of liver flukes in the Opisthorchiidae. Transcripts of Ov-pgrn were detected in several developmental stages, with highest expression in the metacercaria, indicating that Ov-PGRN may participate as a growth factor in the early development of O. viverrini. Western blot analysis revealed the presence of detected Ov-PGRN in both soluble somatic or excretory/secretory products, and immunolocalization indicated high levels of expression in the tegument and parenchyma of the adult fluke. Co-culture of a human cholangiocyte cell line and a peptide fragment of Ov-PGRN stimulated proliferation of cholangiocytes and upregulation of expression of the cytokines IL6 and IL8. Conclusion Ov-PGRN is expressed throughout the life cycle of liver fluke, and likely plays a key role in development and growth.

Background The human liver fluke Opisthorchis viverrini is a food-borne trematode that causes hepatobiliary disease in humans throughout Southeast Asia. People become infected by consuming raw or undercooked fish containing metacercariae. Development of a vaccine to prevent or minimize pathology would decrease the risk of severe morbidity, including the development of bile duct cancer. Methods We produced an oral vaccine based on recombinant Bacillus subtilis spores expressing the large extracellular loop (LEL) of O. viverrini tetraspanin-2 (Ov-TSP-2), a protein that is abundant on the surface of O. viverrini secreted extracellular vesicles (EVs). Recombinant spores expressing Ov-TSP-2-LEL were orally administered to hamsters prior to challenge infection with O. viverrini metacercariae. Results Vaccinated hamsters generated serum IgG as well as bile IgG and IgA responses to Ov-TSP-2-LEL, and serum IgG from vaccinated hamsters blocked the uptake of fluke EVs by a human bile duct epithelial cell line. Vaccinated hamsters had 56% reductions in both adult flukes and fecal eggs compared to the control group. Conclusions These findings indicate that oral vaccination of hamsters with recombinant B. subtilis spores expressing Ov-TSP-2-LEL is efficacious at reducing infection intensity and could form the basis of a vaccine for control of carcinogenic liver fluke infection in humans.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor for cholangiocarcinoma (CCA) in the Mekong Basin countries of Thailand, Lao PDR, Vietnam, Myanmar and Cambodia. Using a novel model of CCA, involving infection with gene-edited liver flukes in the hamster during concurrent exposure to dietary nitrosamine, we explored the role of the fluke granulin-like growth factor Ov-GRN-1 in malignancy. We derived RNA-guided gene knockout flukes (ΔOv-grn-1) using CRISPR/Cas9/gRNA materials delivered by electroporation. Genome sequencing confirmed programmed Cas9-catalyzed mutations of the targeted genes, which was accompanied by rapid depletion of transcripts and the proteins they encode. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes. However, less hepatobiliary tract disease manifested during chronic infection with ΔOv-grn-1 worms in comparison to hamsters infected with control gene-edited and mock-edited parasites. Specifically, immuno- and colorimetric-histochemical analysis of livers revealed markedly less periductal fibrosis surrounding the flukes and less fibrosis globally within the hepatobiliary tract during infection with ΔOv-grn-1 genotype worms, minimal biliary epithelial cell proliferation, and significantly fewer mutations of TP53 in biliary epithelial cells. Moreover, fewer hamsters developed high-grade CCA compared to controls. The clinically relevant, pathophysiological phenotype of the hepatobiliary tract confirmed a role for this secreted growth factor in malignancy and morbidity during opisthorchiasis.

About half of the world’s population and 80% of the world’s biodiversity can be found in the tropics. Many diseases are specific to the tropics, with at least 41 diseases caused by endemic bacteria, viruses, parasites, and fungi. Such diseases are of increasing concern, as the geographic range of tropical diseases is expanding due to climate change, urbanization, change in agricultural practices, deforestation, and loss of biodiversity. While traditional medicines have been used for centuries in the treatment of tropical diseases, the active natural compounds within these medicines remain largely unknown. In this review, we describe infectious diseases specific to the tropics, including their causative pathogens, modes of transmission, recent major outbreaks, and geographic locations. We further review current treatments for these tropical diseases, carefully consider the biodiscovery potential of the tropical biome, and discuss a range of technologies being used for drug development from natural resources. We provide a list of natural products with antimicrobial activity, detailing the source organisms and their effectiveness as treatment. We discuss how technological advancements, such as next-generation sequencing, are driving high-throughput natural product screening pipelines to identify compounds with therapeutic properties. This review demonstrates the impact natural products from the vast tropical biome have in the treatment of tropical infectious diseases and how high-throughput technical capacity will accelerate this discovery process.

Opisthorchis viverrini causes severe pathology in the bile ducts of infected human hosts, and chronic infection can culminate in bile duct cancer. The prevention of infection by vaccination would decrease opisthorchiasis-induced morbidity and mortality. The tetraspanin protein, Ov-TSP-2, is located on the membrane of secreted extracellular vesicles (EVs), and is a candidate antigen for inclusion in a subunit vaccine. To address the role of anti-Ov-TSP-2 antibodies in protection, we assessed the protective capacity of anti-Ov-TSP-2 monoclonal antibodies (mAbs) against opisthorchiasis. Two anti-TSP-2 IgM mAbs, 1D6 and 3F5, and an isotype control were passively transferred to hamsters, followed by parasite challenge one day later. Hamsters that received 3F5 had 74.5% fewer adult flukes and 67.4% fewer eggs per gram of feces compared to hamsters that received the control IgM. Both 1D6 and 3F5 (but not the control IgM) blocked the uptake of fluke EVs by human bile duct epithelial cells in vitro. This is the first report of passive immunization against human liver fluke infection, and the findings portend the feasibility of antibody-directed therapies for liver fluke infection, bolstering the selection of TSPs as components of a subunit vaccine for opisthorchiasis and fluke infections generally.

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with the liver fluke is a major risk factor for cholangiocarcinoma (CCA). The liver fluke Opisthorchis viverrini secretes a growth factor termed liver fluke granulin, a homologue of the human progranulin, which contributes significantly to biliary tract fibrosis and morbidity. Here, extracellular vesicle (EV)-mediated transfer of mRNAs from human cholangiocytes to naïve recipient cells was investigated following exposure to liver fluke granulin. To minimize the influence of endogenous progranulin, its cognate gene was inactivated using CRISPR/Cas9-based gene knock-out. Several progranulin-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of specific transcript and progranulin, both in gene-edited cells and within EVs released by these cells. Profiles of extracellular vesicle RNAs (evRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific evRNAs including MAPK/AKT pathway members were induced by exposure to liver fluke granulin. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 cells exposed to liver fluke granulin. Of these, CCA-associated evRNAs modified the CCA microenvironment in naïve cells co-cultured with EVs from ΔhuPGRN-H69 cells exposed to liver fluke granulin, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells in comparison with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition. Keywords: Opisthorchis viverrini, Liver fluke granulin, Extracellular vesicle, Cellular crosstalk

For the first time the impedance-based xCELLigence real-time cell analysis system was used to measure the myotoxicity of sea snake venom. With a focus on the spine-bellied sea snake (Hydrophis curtus), the venom of four sea snake species and three terrestrial snake species were compared for myotoxicity against a human skeletal muscle cell line (HSkMC). Hydrophis curtus venom was also tested on a human cardiac muscle cell line (HCM). Surprisingly, all four sea snake venoms tested on HSkMC produced an initial 100-280% rise in xCELLigence cell index that peaked within the first two hours before falling. The cell index rise of H. curtus venom was correlated with the WST-1 cell proliferation assay, which demonstrated an increase in mitochondrial metabolism. The myotoxicity of H. curtus was 4.7-8.2 fold less potent than the other sea snakes tested, the Australian beaked sea snake (Hydrophis zweifeli), the elegant sea snake (Hydrophis elegans) and the olive sea snake (Aipysurus laevis). If our cell-based results translate to H. curtus envenomations, this implies that H. curtus would be less myotoxic than the other three. Yet the myotoxicity of H. curtus venom to cardiac muscle cells was nine times weaker than for skeletal muscle cells, providing evidence that the venom has a selective effect on skeletal muscle cells. This evidence, combined with the slow-acting nature of the venom, supports a digestive role for sea snake myotoxins.

Historically, sustained control of Aedes aegypti, the vector of dengue, chikungunya, yellow fever, and Zika viruses, has been largely ineffective. Subsequently, two novel 'rear and release' control strategies utilizing mosquitoes infected with Wolbachia are currently being developed and deployed widely. In the incompatible insect technique, male Aedes mosquitoes, infected with Wolbachia, suppress populations through unproductive mating. In the transinfection strategy, both male and female Wolbachia-infected Ae. aegypti mosquitoes rapidly infect the wild population with Wolbachia, blocking virus transmission. It is critical to monitor the long-term stability of Wolbachia in host populations, and also the ability of this bacterium to continually inhibit virus transmission. Ongoing release and monitoring programs must be future-proofed should political support weaken when these vectors are successfully controlled.

Organisms embedded within food webs must balance arms races with their predators and prey. For venom users, venom may mediate each arms race, but the dynamical evolutionary changes in venom production in response to the two arms races are still poorly understood. Here, we use a simple model to evaluate the evolutionary response of a venomous consumer to the presence of an apex generalist predator and evolution of the consumer’s prey. We find that introduction of the apex predator can weaken the arms race between the two lower trophic levels. In addition, when consumer prey capture and predator defense venoms functionally overlap, a reduced evolutionary response in the prey population can drive investment in venom used for prey capture going beyond what is optimal for subduing prey. These dynamics suggest that interactions with multiple trophic levels can substantially alter the venom complexity in predatory venomous animals and may explain the paradox of the overkill hypothesis.

Granulins are a family of protein growth factors that are involved in cell proliferation. An orthologue of granulin from the human parasitic liver fluke Opisthorchis viverrini, known as Ov-GRN-1, induces angiogenesis and accelerates wound repair. Recombinant Ov-GRN-1 production is complex and poses an obstacle for clinical development. To identify the bioactive region(s) of Ov-GRN-1, four truncated N-terminal analogues were synthesized and characterized structurally using NMR spectroscopy. Peptides that contained only two native disulfide bonds lack the characteristic granulin β-hairpin structure. Remarkably, the introduction of a non-native disulfide bond was critical for formation of β-hairpin structure. Despite this structural difference, both two and three disulfide-bonded peptides drove proliferation of a human cholangiocyte cell line and demonstrated potent wound healing in mice. Peptides derived from Ov-GRN-1 are leads for novel wound healing therapeutics, as they are likely less immunogenic than the full-length protein and more convenient to produce.

Crosstalk between malignant and neighboring cells contributes to tumor growth. In East Asia, infection with fish-borne liver flukes is a major risk factor for cholangiocarcinoma (CCA). The parasite secretes a growth factor termed liver fluke granulin (Ov-GRN-1), a homologue of the human progranulin (huPGRN), which contributes significantly to biliary tract fibrosis and morbidity during infection. Here, exosome-mediated transfer of mRNAs from the human cholangiocyte cell line (H69) was investigated following exposure to Ov-GRN-1, to naïve recipient H69 cells. To minimize the influence of endogenous huPGRN, the gene encoding huPGRN was inactivated using CRISPR/Cas9-based gene knock-out. Several huPGRN-depleted cell lines, termed ΔhuPGRN-H69, were established. These lines exhibited >80% reductions in levels of huPGRN transcripts and protein, both in gene-edited cells and within exosomes released by these cells. Profiles of exosomal RNAs (exRNA) from ΔhuPGRN-H69 for CCA-associated characteristics revealed a paucity of transcripts for estrogen- and Wnt-signaling pathways, peptidase inhibitors and tyrosine phosphatase related to cellular processes including oncogenic transformation. Several CCA-specific mRNAs including MAPK/AKT pathway members were induced by exposure to Ov-GRN-1. By comparison, estrogen, Wnt/PI3K and TGF signaling and other CCA pathway mRNAs were upregulated in wild type H69 exposed to Ov-GRN-1. Of these, CCA-associated exRNAs modified the CCA microenvironment in naïve recipient cells co-cultured with exosomes from ΔhuPGRN-H69 exposed to Ov-GRN-1, and induced translation of MAPK phosphorylation related-protein in naïve recipient cells comparing with control recipient cells. Exosome-mediated crosstalk in response to liver fluke granulin promoted a CCA-specific program through MAPK pathway which, in turn, established a CCA-conducive disposition.

Infection with the food-borne liver fluke Opisthorchis viverrini is the principal risk factor (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans, 2012) for cholangiocarcinoma (CCA) in the Lower Mekong River Basin countries including Thailand, Lao PDR, Vietnam and Cambodia. We exploited this link to explore the role of the secreted growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions by undertaking programmed CRISPR/Cas9 knockout of the Ov-GRN-1 gene from the liver fluke genome. Deep sequencing of amplicon libraries from genomic DNA of gene-edited parasites revealed Cas9-catalyzed mutations within Ov-GRN-1. Gene editing resulted in rapid depletion of Ov-GRN-1 transcripts and the encoded Ov-GRN-1 protein. Gene-edited parasites colonized the biliary tract of hamsters and developed into adult flukes, but the infection resulted in reduced pathology as evidenced by attenuated biliary hyperplasia and fibrosis. Not only does this report pioneer programmed gene-editing in parasitic flatworms, but also the striking, clinically-relevant pathophysiological phenotype confirms the role for Ov-GRN-1 in virulence morbidity during opisthorchiasis., eLife digest In the rural regions alongside the Mekong River in South East Asia, traditional cuisines often use uncooked or under cooked fish, many of which carry a worm known as Opisthorchis viverrini. Once inside the body, this parasite settles in the human liver, causing a tropical disease known as liver fluke infection. Out of the 10 million people affected by O. viverrini, thousands will also develop a type of liver cancer that is triggered by the presence of the worm. In particular, the parasite secretes a protein known as granulin that may encourage certain liver cells to multiply, potentially raising the risk for cancer. A gene editing technique called CRISPR/Cas9 allows scientist to precisely target and then deactivate the genetic information a cell needs to produce a given protein. While the tool has been used in other species before, it was unknown if it could be applied to O. viverrini. Here, Arunsan et al. harnessed CRISPR/Cas9 to deactivate the gene that codes for granulin and create parasites that can only produce very little of the protein. Hamsters infected with the gene-edited worms had fewer symptoms of liver fluke infection compared to those carrying normal O. viverrini. The animals with parasites that cannot produce granulin also had fewer changes to the liver that are associated with cancer. These findings confirm that granulin has a role in promoting liver fluke infection and liver cancer. Alongside this work, Ittiprasert et al. used CRISPR/Cas9 to inactivate a gene in a species of worm that causes a human disease called schistosomiasis. Together, these findings demonstrate for the first time that the gene editing method can be adapted for use in parasitic worms, which are a major public health problem in tropical climates. This tool should help scientists understand how the parasites invade and damage our bodies, and provide new ideas for treatment and disease control.

Infection with helminth parasites causes morbidity and mortality in billions of people and livestock worldwide. Where anthelmintic drugs are available, drug resistance is a major problem in livestock parasites, and a looming threat to public health. Monitoring the efficacy of these medicines and screening for new drugs has been hindered by the lack of objective, high-throughput approaches. Several cell monitoring technologies have been adapted for parasitic worms, including video-, fluorescence-, metabolism enzyme- and impedance-based tools that minimize the screening bottleneck. Using the xCELLigence impedance-based system we previously developed a motility-viability assay that is applicable for a range of helminth parasites. Here we have improved substantially the assay by using diverse frequency settings, and have named it the xCELLigence worm real-time motility assay (xWORM). By utilizing strictly standardized mean difference analysis we compared the xWORM output measured with 10, 25 and 50 kHz frequencies to quantify the motility of schistosome adults (human blood flukes) and hatching of schistosome eggs. Furthermore, we have described a novel application of xWORM to monitor movement of schistosome cercariae, the developmental stage that is infectious to humans. For all three stages, 25 kHz was either optimal or near-optimal for monitoring and quantifying schistosome motility. These improvements in methodology sensitivity should enhance the capacity to screen small compound libraries for new drugs both for schistosomes and other helminth pathogens at large., Graphical abstract, Highlights • 25 kHz on the xCELLigence system dramatically improves the schistosome xWORM assay. • xWORM assay can efficiently determine viability of Schistome adults or eggs. • First time cercariae have been incorporated into an automated viability assay. • Other helminth monitoring may benefit from alternate xCELLigence frequency options.

Scorpion venoms are a rich source of bioactive molecules, but characterisation of toxin peptides affecting cytosolic Ca2+, central to cell signalling and cell death, is limited. We undertook a functional screening of the venom of the Australian scorpion Hormurus waigiensis to determine the breadth of Ca2+ mobilisation. A human embryonic kidney (HEK293) cell line stably expressing the genetically encoded Ca2+ reporter GCaMP5G and the rabbit type 1 ryanodine&nbsp, receptor (RyR1) was developed as a biosensor. Size-exclusion Fast Protein Liquid Chromatography separated the venom into 53 fractions, constituting 12 chromatographic peaks. Liquid chromatography mass spectroscopy identified 182 distinct molecules with 3 to 63 components per peak. The molecular weights varied from 258 Da&mdash, 13.6 kDa, with 53% under 1 kDa. The majority of the venom chromatographic peaks (tested as six venom pools) were found to reversibly modulate cell monolayer bioimpedance, detected using the xCELLigence platform (ACEA Biosciences). Confocal Ca2+ imaging showed 9/14 peak samples, with molecules spanning the molecular size range, increased cytosolic Ca2+ mobilization. H. waigiensis venom Ca2+ activity was correlated with changes in bio-impedance, reflecting multi-modal toxin actions on cell physiology across the venom proteome.

Plant-derived compounds that modulate the immune responses are emerging as frontline treatment agents for cancer, infectious diseases and autoimmunity. Herein we have isolated 40 phytochemicals from five Bhutanese Sowa Rigpa medicinal plants&mdash, Aconitum laciniatum, Ajania nubegina, Corydalis crispa, Corydalis dubia and Pleurospermum amabile&mdash, and tested 14 purified compounds for their immunomodulatory properties using a murine dendritic cell (DC) line, and cytotoxicity against a human cholangiocyte cell line using xCELLigence real time cell monitoring. These compounds were: pseudaconitine, 14-veratryolpseudaconitine, 14-O-acetylneoline, linalool oxide acetate, (E)-spiroether, luteolin, luteolin-7-O-&beta, d-glucopyranoside, protopine, ochrobirine, scoulerine, capnoidine, isomyristicin, bergapten, and isoimperatorin. Of the 14 compounds tested here, scoulerine had adjuvant-like properties and strongly upregulated MHC-I gene and protein expression whereas bergapten displayed immunosuppressive properties and strongly down-regulated gene and protein expression of MHC-I and other co-stimulatory molecules. Both scoulerine and bergapten showed low cytotoxicity against normal healthy cells that were consistent with their immunoregulatory properties. These findings highlight the breadth of immunomodulatory properties of defined compounds from Bhutanese medicinal plants and show that some of these compounds exert their mechanisms of action by modulating DC activity.

Granulins are a family of growth factors involved in cell proliferation. The liver-fluke granulin, Ov-GRN-1, isolated from a carcinogenic liver fluke Opisthorchis viverrini, can significantly accelerate wound repair in vivo and in vitro. However, it is difficult to express Ov-GRN-1 in recombinant form at high yield, impeding its utility as a drug lead. Previously we reported that a truncated analogue (Ov-GRN12–35_3s) promotes healing of cutaneous wounds in mice. NMR analysis of this analogue indicates the presence of multiple conformations, most likely as a result of proline cis/trans isomerization. To further investigate whether the proline residues are involved in adopting the multiple confirmations, we have synthesized analogues involving mutation of the proline residues. We have shown that the proline residues have a significant influence on the structure, activity, and folding of Ov-GRN12–35_3s. These results provide insight into improving the oxidative folding yield and bioactivity of Ov-GRN12–35_3s...

Infections with several flatworm parasites represent group 1 biological carcinogens, i.e. definite causes of cancer. Infection with the food-borne liver fluke Opisthorchis viverrini causes cholangiocarcinoma (CCA). Whereas the causative agent for most cancers, including CCA in the West, remains obscure, the principal risk factor for CCA in Thailand is opisthorchiasis. We exploited this established link to explore the role of the secreted parasite growth factor termed liver fluke granulin (Ov-GRN-1) in pre-malignant lesions of the biliary tract. We targeted the Ov-grn-1 gene for programmed knockout and investigated gene-edited parasites in vitro and in experimentally infected hamsters. Both adult and juvenile stages of the liver fluke were transfected with a plasmid encoding a guide RNA sequence specific for exon 1 of Ov-grn-1 and the Cas9 nuclease. Deep sequencing of amplicon libraries from genomic DNA from gene-edited parasites exhibited programmed, Cas9-catalyzed mutations within the Ov-grn-1 locus, and tandem analyses by RT-PCR and western blot revealed rapid depletion of Ov-grn-1 transcripts and protein. Newly excysted juvenile flukes that had undergone editing of Ov-grn-1 colonized the biliary tract, grew and developed over a period of 60 days, were active and motile, and induced a clinically relevant pathophysiological tissue phenotype of attenuated biliary hyperplasia and fibrosis in comparison to infection with wild type flukes. This is the first report of gene knock-out using CRISPR/Cas9 in a parasitic flatworm, demonstrating the activity and utility of the process for functional genomics in these pathogens. The striking clinical phenotype highlights the role in virulence that liver fluke growth factors play in biliary tract morbidity during chronic opisthorchiasis.

The Australian jellyfish Chironex fleckeri, belongs to a family of cubozoan jellyfish known for their potent venoms. CfTX-1 and -2 are two highly abundant toxins in the venom, but there is no structural data available for these proteins. Structural information on toxins is integral to the understanding of the mechanism of these toxins and the development of an effective treatment. Two regions of CfTX-1 have been predicted to have helical structures that are involved with the mechanism of action. Here we have synthesized peptides corresponding to these regions and analyzed their structures using NMR spectroscopy. The peptide corresponding to the predicted N-terminal amphiphilic helix appears unstructured in aqueous solution. This lack of structure concurs with structural disorder predicted for this region of the protein using the Protein DisOrder prediction System PrDOS. Conversely, a peptide corresponding to a predicted transmembrane region is very hydrophobic, insoluble in aqueous solution and predicted to be structured by PrDOS. In the presence of SDS-micelles both peptides have well-defined helical structures showing that a membrane mimicking environment stabilizes the structures of both peptides and supports the prediction of the transmembrane region in CfTX-1. This is the first study to experimentally analyze the structure of regions of a C. fleckeri protein.

Schistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and loss-of-function approaches can be expected to precipitate the discovery of novel interventions, and drug selection of transgenic schistosomes would facilitate the establishment of stable lines of engineered parasites. Sensitivity of developmental stages of schistosomes to the aminonucleoside antibiotic puromycin was investigated. For the schistosomulum and sporocyst stages, viability was quantified by fluorescence microscopy following dual staining with fluorescein diacetate and propidium iodine. By 6 days in culture, the 50% lethal concentration (LC 50 ) for schistosomula was 19 μg/ml whereas the sporocysts were 45-fold more resilient. Puromycin potently inhibited the development of in vitro -laid eggs (LC 50 , 68 ng/ml) but was less effective against liver eggs (LC 50 , 387 μg/ml). Toxicity for adult stages was evaluated using the xCELLigence-based, real-time motility assay (xWORM), which revealed LC 50 s after 48 h of 4.9 and 17.3 μg/ml for male and female schistosomes, respectively. Also, schistosomula transduced with pseudotyped retrovirus encoding the puromycin resistance marker were partially rescued when cultured in the presence of the antibiotic. Together, these findings will facilitate selection on puromycin of transgenic schistosomes and the enrichment of cultures of transgenic eggs and sporocysts to facilitate the establishment of schistosome transgenic lines. Streamlining schistosome transgenesis with drug selection will open new avenues to understand parasite biology and hopefully lead to new interventions for this neglected tropical disease.

The invertebrate cell line, Bge, from embryos of the snail Biomphalaria glabrata, remains to date the only established cell line from any species of the Phylum Mollusca. Since its establishment in 1976 by Eder Hansen, few studies have focused on profiling its cytometrics, growth characteristics or sensitivity to xenobiotics. Bge cells are reputed to be challenging to propagate and maintain. Therefore, even though this cell line is a noteworthy resource, it has not been studied widely. With growing interest in functional genomics, including genetic transfection, to elucidate molecular aspects of the snail intermediate hosts responsible for transmission of schistosomiasis, and aiming to enhance the convenience of maintenance of this molluscan cell line, we deployed the xCELLigene real time approach to study Bge cells. Doubling times for three isolates of Bge, termed CB, SL and UK, were longer than for mammalian cell lines - longer than 40 h in complete Bge medium supplemented with 7% fetal bovine serum at 25°C, ranging from ∼42 h to ∼157 h when 40,000 cells were seeded. To assess the potential of the cells for genetic transformation, antibiotic selection was explored. Bge cells were sensitive to the aminonucleoside antibiotic puromycin (from Streptomyces alboniger) from 5 μg/ml to 200 ng/ml, displaying a half maximal inhibitory concentration (IC50) of ∼1.91 μg/ml. Sensitivity to puromycin, and a relatively quick kill time (

The liver fluke Opisthorchis viverrini is a food-borne, zoonotic pathogen endemic to Thailand and adjacent countries in Southeast Asia. The adult developmental stage of the O. viverrini parasite excretes and secretes numerous proteins within the biliary tract including the gall bladder. Lesions caused by the feeding activities of the liver fluke represent wounds that undergo protracted cycles of healing and re-injury during chronic infection, which can last for decades. Components of the excretory/secretory (ES) complement released by the worms capably drive proliferation of bile duct epithelial cells and are implicated in establishing the oncogenic milieu that leads to bile duct cancer, cholangiocarcinoma. An ES protein, the secreted granulin-like growth factor termed Ov-GRN-1, accelerates wound resolution in mice and in vitro. To investigate angiogenesis (blood vessel development) that may contribute to wound healing promoted by liver fluke granulin and, by implication, to carcinogenesis during chronic opisthorchiasis, we employed an in vitro tubule formation assay (TFA) where human umbilical vein endothelial cells were grown on gelled basement matrix. Ten and 40 nM Ov-GRN-1 significantly stimulated angiogenesis as monitored by cellular proliferation and by TFA in real time. This demonstration of potent angiogenic property of Ov-GRN-1 bolsters earlier reports on the therapeutic potential for chronic non-healing wounds of diabetics, tobacco users, and the elderly and, in addition, showcases another of the hallmark of cancer characteristic of this carcinogenic liver fluke.

Background Schistosomiasis affects over 200 million people and there are concerns whether the current chemotherapeutic control strategy (periodic mass drug administration with praziquantel (PZQ)—the only licenced anti-schistosome compound) is sustainable, necessitating the development of new drugs. Methodology/Principal findings We investigated the anti-schistosome efficacy of polypyridylruthenium(II) complexes and showed they were active against all intra-mammalian stages of S. mansoni. Two compounds, Rubb12-tri and Rubb7-tnl, which were among the most potent in their ability to kill schistosomula and adult worms and inhibit egg hatching in vitro, were assessed for their efficacy in a mouse model of schistosomiasis using 5 consecutive daily i.v. doses of 2 mg/kg (Rubb12-tri) and 10 mg/kg (Rubb7-tnl). Mice treated with Rubb12-tri showed an average 42% reduction (P = 0.009), over two independent trials, in adult worm burden. Liver egg burdens were not significantly decreased in either drug-treated group but ova from both of these groups showed significant decreases in hatching ability (Rubb12-tri—68%, Rubb7-tnl—56%) and were significantly morphologically altered (Rubb12-tri—62% abnormal, Rubb7-tnl—35% abnormal). We hypothesize that the drugs exerted their activity, at least partially, through inhibition of both neuronal and tegumental acetylcholinesterases (AChEs), as worms treated in vitro showed significant decreases in activity of these enzymes. Further, treated parasites exhibited a significantly decreased ability to uptake glucose, significantly depleted glycogen stores and withered tubercules (a site of glycogen storage), implying drug-mediated interference in this nutrient acquisition pathway. Conclusions/Significance Our data provide compelling evidence that ruthenium complexes are effective against all intra-mammalian stages of schistosomes, including schistosomula (refractory to PZQ) and eggs (agents of disease transmissibility). Further, the results of this study suggest that schistosome AChE is a target of ruthenium drugs, a finding that can inform modification of current compounds to identify analogues which are even more effective and selective against schistosomes., Author summary Schistosomiasis is a neglected tropical disease which affects over 200 million people and there is only one licensed drug, praziquantel, currently available for treatment. In a search for new drugs to control schistosomiasis, we tested the anti-schistosome efficacy of a series of ruthenium compounds and found that a number of them were able to inhibit parasite eggs from hatching and kill adult worms and praziquantel-refractory juvenile worms in vitro. We demonstrated that the compounds inhibit schistosome acetylcholinesterase (the enzyme that breaks down the neurotransmitter acetylcholine), which could potentially result in paralysis of the parasite, likely due to uncontrolled neuromuscular function caused by acetylcholine excess. Moreover, we showed that drug-treated worms had a significantly reduced ability to uptake exogenous glucose and markedly depleted glycogen stores, presumably through inhibition of the acetylcholinesterase-mediated glucose scavenging pathway. Lastly, we found that two of the drugs—Rubb12-tri and Rubb7-tnl—when used to treat schistosome-infected mice, were able to reduce worm burdens and significantly affect the viability of parasite eggs in vivo, which would have a marked impact on disease transmission. We believe that these complexes are desirable drug lead scaffolds which could be used to develop effective and selective compounds to control and treat schistosomiasis and, potentially, other parasitic diseases.

Objectives: To investigate the effects of temperature and hot water immersion time on neutralising venom lethality of the Australian estuarine stonefish (Synanceia horrida). Design: Depths of the spines were measured while venom was extracted from S. horrida individuals. The venom was then exposed to temperatures of 4°C, 37.0°C, 40.1°C, 42.3°C, 45.0°C, 47.7°C, 55.2°C, and 60.0°C for either five or 20 minutes incubation periods. Venom samples were added to cultured human cardiomyocytes and cell viability curves were produced using the ACEA's xCELLigence real-time cell monitoring system. Main outcome measures: Determination of venom lethality on cardiomyocytes at a range of temperatures. Results: The average depth of the spine required to go into a victims' flesh before the venom gland compressed and expelled venom was 18 mm. Cardiomyocytes exposed to heat-treated venom for five minutes required higher temperatures to neutralise 99% of the venom, namely 44.6°C in comparison to 42.1°C with an incubation time of 20 minutes. Conclusion: This study supports the use of hot water immersion therapy in the treatment of S. horrida stings. It is suggested that due to the depth of the puncture wound longer incubation times should be sought to allow heat to penetrate the deeper portions of the dermis and effectively begin venom deactivation.

International audience; Conotoxins are a large family of disulfide-rich peptides that contain unique cysteine frameworks that target a broad range of ion channels and receptors. We recently discovered the 33-residue conotoxin Φ-MiXXVIIA from Conus miles with a novel cysteine framework comprising three consecutive cysteine residues and four disulfide bonds. Regioselective chemical synthesis helped decipher the disulfide bond connectivity and the structure of Φ-MiXXVIIA was determined by NMR spectroscopy. The 3D structure displays a unique topology containing two β-hairpins that resemble the N-terminal domain of granulin. Similar to granulin, Φ-MiXXVIIA promotes cell proliferation (EC50 17.85 μm) while inhibiting apoptosis (EC50 2.2 μm). Additional framework XXVII sequences were discovered with homologous signal peptides that define the new conotoxin superfamily G2. The novel structure and biological activity of Φ-MiXXVIIA expands the repertoire of disulfide-rich conotoxins that recognize mammalian receptors.

Schistosomiasis and trichuriasis are two of the most common neglected tropical diseases (NTD) that affect almost a billion people worldwide. There is only a limited number of effective drugs to combat these NTD. Medicinal plants are a viable source of parasiticides. In this study, we have investigated six of the 19 phytochemicals isolated from two Bhutanese medicinal plants, Corydalis crispa and Pleurospermum amabile, for their anthelmintic properties. We used the xWORM technique and Scanning Electron Microscope-based imaging to determine the activity of the compounds. Of the six compounds tested, isomyristicin and bergapten showed significant anthelmintic activity against Schistosoma mansoni and Trichuris muris with bergapten being the most efficacious compound one against both parasites (S. mansoni IC50 = 8.6 μg/mL and T. muris IC50 = 10.6 μg/mL) and also against the schistosomulum stage of S. mansoni. These two compounds induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. The efficacy against multiple phylogenetically distinct parasites and different life stages, especially the schistosomulum where praziquantel is ineffective, makes isomyristicin and bergapten novel scaffolds for broad-spectrum anthelmintic drug development that could be used for the control of helminths infecting humans and animals.

Background Whipworms and blood flukes combined infect almost one billion people in developing countries. Only a handful of anthelmintic drugs are currently available to treat these infections effectively; there is therefore an urgent need for new generations of anthelmintic compounds. Medicinal plants have presented as a viable source of new parasiticides. Ajania nubigena, the Bhutanese daisy, has been used in Bhutanese traditional medicine for treating various diseases and our previous studies revealed that small molecules from this plant have antimalarial properties. Encouraged by these findings, we screened four major compounds isolated from A. nubigena for their anthelmintic properties. Methodology/Principal Findings Here we studied four major compounds derived from A. nubigena for their anthelmintic properties against the nematode whipworm Trichuris muris and the platyhelminth blood fluke Schistosoma mansoni using the xWORM assay technique. Of four compounds tested, two compounds—luteolin (3) and (3R,6R)-linalool oxide acetate (1)—showed dual anthelmintic activity against S. mansoni (IC50 range = 5.8–36.9 μg/mL) and T. muris (IC50 range = 9.7–20.4 μg/mL). Using scanning electron microscopy, we determined luteolin as the most efficacious compound against both parasites and additionally was found effective against the schistosomula, the infective stage of S. mansoni (IC50 = 13.3 μg/mL). Luteolin induced tegumental damage to S. mansoni and affected the cuticle, bacillary bands and bacillary glands of T. muris. Our in vivo assessment of luteolin (3) against T. muris infection at a single oral dosing of 100 mg/kg, despite being significantly (27.6%) better than the untreated control group, was markedly weaker than mebendazole (93.1%) in reducing the worm burden in mice. Conclusions/Significance Among the four compounds tested, luteolin demonstrated the best broad-spectrum activity against two different helminths—T. muris and S. mansoni—and was effective against juvenile schistosomes, the stage that is refractory to the current gold standard drug, praziquantel. Medicinal chemistry optimisation including cytotoxicity analysis, analogue development and structure-activity relationship studies are warranted and could lead to the identification of more potent chemical entities for the control of parasitic helminths of humans and animals., Author Summary Schistosomiasis and trichuriasis affects millions of people worldwide and are caused by blood flukes and whipworms, respectively. Only a handful of anthelmintic drugs exist to treat these infections and the pipeline for the next generation of anthelmintic drugs is sparse, precipitating the need for new drug development. In this context, medicinal plants present a viable source of novel anthelmintic compounds. This inspired us to study the selected naturally occurring compounds derived from a Bhutanese daisy medicinal plant, Ajania nubigena for their anthelmintic activities. Here, using the xWORM motility assay, we demonstrate that two compounds, luteolin (3) and (3R,6R)-linalool oxide acetate (1), display significant broad-spectrum anthelmintic activity against two of the most important genera of human helminth parasites, the nematode whipworm and the platyhelminth blood fluke. Luteolin exhibited the best activities with IC50 values of 5.8 μg/mL against schistosomes and 9.7 μg/mL against whipworms. Using scanning electron microscopy we showed that luteolin damages the tegument of blood flukes and induces abnormalities in the bacillary bands/glands and cuticles of whipworms. Intriguingly, our previous study showed that luteolin (3) was effective against multi-drug resistant Plasmodium falciparum malaria. Due to its broad-spectrum anti-parasitic activities, luteolin (3) is a desirable drug lead scaffold, which could be used for developing effective compounds to control and treat numerous tropical diseases.

The association between liver fluke infection caused by Opisthorchis viverrini and cholangiocarcinoma (CCA — hepatic cancer of the bile duct epithelium) has been well established. Multiple mechanisms play a role in the development of CCA, but the excretory/secretory products released by O. viverrini (OvES) represent the major interface between the parasite and its host, and their uptake by biliary epithelial cells has been suggested to be responsible for proliferation of cholangiocytes, the cells that line the biliary epithelium. Despite recent progress in the study of the molecular basis of O. viverrini–host interactions, little is known about the effects that OvES induces upon internalization by host cells. In the present study we incubated non-cancerous human cholangiocytes (H69) and human colon cancer (CaCo-2) cells with OvES and performed a time-course quantitative proteomic analysis on the cells to determine the early changes induced by the parasite. Different KEGG pathways were altered in H69 cells compared to Caco-2 cells: glycolysis/gluconeogenesis and protein processing in the endoplasmic reticulum. In addition, the Reactome pathway analysis showed a predominance of proteins involved in cellular pathways related to apoptosis and apoptotic execution phase in H69 cells after incubation with OvES. The present study provides the first proteomic analysis to address the molecular mechanisms by which OvES products interact with host cells, and Sheds light on the cellular processes involved in O. viverrini-induced CCA.

Granulins (GRNs) are potent growth factors that are upregulated in many aggressive cancers from a wide range of organs. GRNs form tight, disulphide bonded, beta hairpin stacks, making them difficult to express in recombinant form. We recently described Ov-GRN-1, a GRN family member secreted by the carcinogenic liver fluke of humans, Opisthorchis viverrini, and showed that recombinant Ov-GRN-1 expressed and refolded from Escherichia coli caused proliferation of mammalian cell lines at nanomolar concentrations. We now report on an optimized method to express and purify monomeric Ov-GRN-1 in E. coli using a straightforward and scalable purification and refolding process. Purified monomeric protein caused proliferation at nanomolar concentrations of cancerous and non-cancerous cell lines derived from human bile duct tissue. The expression and purification method we describe herein will serve as a backbone upon which to develop expression and purification processes for recombinant GRNs from other organisms, accelerating research on this intriguing family of proteins.

Granulins are a family of protein growth factors that are involved in a range of biological functions, including wound repair, inflammation, and tumor growth. They are often expressed as part of large precursor proteins containing multiple granulin domains. Individual granulin domains are characterized by a conserved arrangement of 12 cysteine residues that form six disulfide bonds. Despite the conservation of the cysteine residues, there is significant sequence variation between granulins from different species. The initial structure determined for this family indicated the presence of a well‐defined structure with a laddered arrangement of the six disulfide bonds and a β‐hairpin stack. However, subsequent studies have shown the structure‐function relationships of granulins are quite complex. Recent studies have indicated some granulins might have potential as wound healing agents, and studies aimed at understanding the structure‐function relationships of this family are likely to enhance this potential in drug design. This review provides an overview of the structure‐based studies of granulins, including the folding of truncated peptides derived from granulins from different species.

Infection with the human liver fluke, Opisthorchis viverrini, is a serious public health problem in Thailand, Laos and nearby locations in Southeast Asia. Both experimental and epidemiological evidence strongly implicate liver fluke infection in the etiology of one of the liver cancer subtypes, cholangiocarcinoma (CCA). To identify parasite proteins critical for liver fluke survival and the etiology of CCA, OFFGEL electrophoresis and Multiple reaction monitoring were employed to characterize 300 parasite proteins from the O. viverrini excretory/secretory products (ES) and, utilizing selective labeling and sequential solubilization, from the host-exposed tegument. The ES included a complex mixture of proteins that have been associated with cancers, including proteases of different mechanistic classes and orthologues of mammalian growth factors and anti-apoptotic proteins. Also identified was a cysteine protease inhibitor which, in other helminth pathogens, induces nitric oxide production by macrophages, and, hence may contribute to malignant transformation of inflamed cells. Proteases in ES included cathepsins, calpain and a protein with homology to autoimmune prostatitis antigen 2. More than 160 tegumental proteins were identified using sequential solubilization of isolated teguments, and a subset of these was localized to the surface membrane of the tegument by labeling living flukes with biotin and confirming surface localization with fluorescence microscopy. These included annexins, which are potential immuno-modulators, and orthologues of the schistosomiasis vaccine antigens Sm29 and tetraspanin-2. Novel roles in pathogenesis were suggested for the tegument-host interface since more than ten biotinylated surface proteins had no homologues in the public databases. The O. viverrini proteins identified here provide an extensive catalogue of novel leads for research on the pathogenesis of opisthorchiasis and the development of novel interventions for this disease and CCA, as well as providing a scaffold for sequencing the genome of this fluke.

© The Author 2015. Background. Throughout Asia, there is an unprecedented link between cholangiocarcinoma and infection with the liver fluke Opisthorchis viverrini. Multiple processes, including chronic inflammation and secretion of parasite proteins into the biliary epithelium, drive infection toward cancer. Until now, the mechanism and effects of parasite protein entry into cholangiocytes was unknown. Methods. Various microscopy techniques were used to identify O. viverrini extracellular vesicles (EVs) and their internalization by human cholangiocytes. Using mass spectrometry we characterized the EV proteome and associated changes in cholangiocytes after EV uptake, and we detected EV proteins in bile of infected hamsters and humans. Cholangiocyte proliferation and interleukin 6 (IL-6) secretion was measured to assess the impact of EV internalization. Results. EVs were identified in fluke culture medium and bile specimens from infected hosts. EVs internalized by cholangiocytes drove cell proliferation and IL-6 secretion and induced changes in protein expression associated with endocytosis, wound repair, and cancer. Antibodies to an O. viverrini tetraspanin blocked EV uptake and IL-6 secretion by cholangiocytes. Conclusions. This is the first time that EVs from a multicellular pathogen have been identified in host tissues. Our findings imply a role for O. viverrini EVs in pathogenesis and highlight an approach to vaccine development for this infectious cancer.

© 2015 Australian Society for Parasitology Inc. Liver fluke infection caused by Opisthorchis viverrini remains a major public health problem in many parts of Asia including Thailand, Lao PDR, Vietnam and Cambodia, where there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium). Among other factors, uptake of O. viverrini excretory/secretory products (OvES) by biliary epithelial cells has been postulated to be responsible for chronic inflammation and proliferation of cholangiocytes, but the mechanisms by which cells internalise O. viverrini excretory/secretory products are still unknown. Herein we incubated normal human cholangiocytes (H69), human cholangiocarcinoma cells (KKU-100, KKU-M156) and human colon cancer (Caco-2) cells with O. viverrini excretory/secretory products and analysed the effects of different endocytic inhibitors to address the mechanism of cellular uptake of ES proteins. Opisthorchis viverrini excretory/secretory products was internalised preferentially by liver cell lines, and most efficiently/rapidly by H69 cells. There was no evidence for trafficking of ES proteins to cholangiocyte organelles, and most of the fluorescence was detected in the cytoplasm. Pretreatment with clathrin inhibitors significantly reduced the uptake of O. viverrini excretory/secretory products, particularly by H69 cells. Opisthorchis viverrini excretory/secretory products induced proliferation of liver cells (H69 and CCA lines) but not intestinal (Caco-2) cells, and proliferation was blocked using inhibitors of the classical endocytic pathways (clathrin and caveolae). Opisthorchis viverrini excretory/secretory products drove IL6 secretion by H69 cells but not Caco-2 cells, and cytokine secretion was significantly reduced by endocytosis inhibitors. This the first known study to address the endocytosis of helminth ES proteins by host epithelial cells and sheds light on the pathways by which this parasite causes one of the most devastating forms of cancer in south-eastern Asia.

Respiratory syncytial virus (RSV) is a ubiquitous human pathogen and the leading cause of lower respiratory tract infections in infants. Infection of cells and subsequent formation of syncytia occur through membrane fusion mediated by the RSV fusion protein (RSV-F). A novel in vitro assay of recombinant RSV-F function has been devised and used to characterize a number of escape mutants for three known inhibitors of RSV-F that have been isolated. Homology modeling of the RSV-F structure has been carried out on the basis of a chimera derived from the crystal structures of the RSV-F core and a fragment from the orthologous fusion protein from Newcastle disease virus (NDV). The structure correlates well with the appearance of RSV-F in electron micrographs, and the residues identified as contributing to specific binding sites for several monoclonal antibodies are arranged in appropriate solvent-accessible clusters. The positions of the characterized resistance mutants in the model structure identify two promising regions for the design of fusion inhibitors.

Surface and secreted proteins of schistosomes orchestrate the basic physiologic requirements of a parasitic existence. These proteins are often exposed to host tissues during penetration, migration, feeding, and immune evasion, and they are obvious targets for control strategies. Signal sequence trap (SST) represents a novel approach that selects for cDNAs encoding secreted and surface proteins with N-terminal signal peptides, so we constructed a randomly primed adult Schistosoma mansoni cDNA library fused to a signalless reporter gene encoding placental alkaline phosphatase. The library was used to transfect COS-7 cells, which were then assayed for the presence of reporter at the cell surface. Eighteen S. mansoni cDNA fragments were isolated and sequenced. Expression profiles of the novel clones were determined for different developmental stages; some transcripts were restricted to single-sex adult worms, while others were ubiquitously distributed. Most clones contained signal peptides or signal anchors as determined by the SignalP algorithm. Open reading frames (ORFs) were categorized as follows: (i) previously identified S. mansoni cDNAs encoding proteins of known function; (ii) cDNAs encoding proteins of known function in other organisms but novel for Schistosoma ; (iii) S. mansoni expressed sequence tags (ESTs) of unknown function; and (iv) completely novel ORFs without homologues (including ESTs) from any phylum. Clones of particular interest included tetraspanins similar to human cell surface antigens, a protein kinase, and ORFs transcribed in the antisense orientation to previously characterized S. mansoni cDNAs. This is the first report describing the use of SST as a tool for identifying secreted proteins from any pathogenic organism.

Animals embedded between trophic levels must simultaneously balance pressures to deter predators and acquire resources. Venomous animals may use venom toxins to mediate both pressures, and thus changes in this balance may alter the composition of venoms. Basic theory suggests that greater exposure to a predator should induce a larger proportion of defensive venom components relative to offensive venom components, while increases in arms races with prey will elicit the reverse. Alternatively, reducing the need for venom expenditure for food acquisition, for example because of an increase in scavenging, may reduce the production of offensive venom components. Here, we investigated changes in scorpion venom composition using a mesocosm experiment where we manipulated scorpions' exposure to a surrogate vertebrate predator and live and dead prey. After six weeks, scorpions exposed to surrogate predators exhibited significantly different venom chemistry compared with naive scorpions. This change included a relative increase in some compounds toxic to vertebrate cells and a relative decrease in some compounds effective against their invertebrate prey. Our findings provide, to our knowledge, the first evidence for adaptive plasticity in venom composition. These changes in venom composition may increase the stability of food webs involving venomous animals.

Food allergies are increasing worldwide, demonstrating a considerable public health concern. Shellfish allergy is one of the major food groups causing allergic sensitization among adults and children, affecting up to 2% of the general world population. Tropomyosin (TM) is the major allergen in shellfish and frequently used in the diagnosis of allergic sensitization and the detection of cross-contaminated food. To improve and establish better and more sensitive diagnostics for allergies and immunotherapeutics, large quantities of pure allergens are required. To establish a reproducible method for the generation of pure recombinant tropomyosin we utilized in this study different Escherichia coli strains (NM522, TOP10 and BL21(DE3)RIPL). In addition, isopropyl-β-d-thiogalactoside (IPTG) induction was compared with a novel auto-induction system to allow the generation of larger quantities of recombinant allergen. We demonstrated that the B-strain of E. coli is better for the expression of TM compared to the K-strain. Moreover, a higher yield could be achieved when using the auto-induction system, with up to 62 mg/l. High yield expressed recombinant TM from King prawn (KP) was compared to recombinant TM from Black tiger prawn (Pen m 1). We demonstrated that recombinant TM from KP and known isoallergen Pen m 1 have very similar molecular and immunological characteristics. Overall, we demonstrate that auto-induction can be used to express larger quantities of recombinant allergens for the development of diagnostic, to quantify allergens as well as immunotherapeutics employing isoallergens.

Septins are guanosine-5'-triphosphate-binding proteins involved in wide-ranging cellular processes including cytokinesis, vesicle trafficking, membrane remodelling 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, 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 forchlorfenuron. Recombinant forms of three schistosome septins, SmSEPT5, SmSEPT7.2 and SmSEPT10, interacted with forchlorfenuron, 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 forchlorfenuron on the cultured schistosomes were monitored by video and/or by an xCELLigence-based assay of motility, which quantified the effect of forchlorfenuron 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 forchlorfenuron 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 forchlorfenuron for anti-schistosomal drugs.

The vertebrate cardiotoxic components of the venom produced by the Australian box jellyfish, Chironex fleckeri, have not previously been isolated. We have uncovered for the first time, three distinct cytotoxic crude fractions from within the vertebrate cardiotoxic peak of C. fleckeri venom by monitoring viability of human muscle cells with an impedance based assay (ACEA xCELLigence system) measuring cell detachment as cytotoxicity which was correlated with a reduction in cell metabolism using a cell proliferation (MTS) assay. When the effects of the venom components on human cardiomyocytes and human skeletal muscle cells were compared, two fractions were found to specifically affect cardiomyocytes with distinct temporal profiles (labelled Crude Toxic Fractions (CTF), α and β). A third fraction (CTF-γ) was toxic to both muscle cell types and therefore not cardio specific. The vertebrate, cardio specific CTF-α and CTF-β, presented distinct activities; CTF-α caused rapid but short term cell detachment and reduction in cell metabolism with enhanced activity at lower concentrations than CTF-β. This activity was not permanent, with cell reattachment and subsequent increased metabolism of heart muscle cells observed when exposed to all but the highest concentrations of CTF-α tested. The cytotoxic effect of CTF-β took twice as long to act on the cells compared to CTF-α, however, the activity was permanent. Furthermore, we showed that the two fractions combined have a synergistic effect causing a much stronger and faster cell detachment (death) when combined than the sum of the individual effects of each toxin. These data presented here improves the current understanding of the toxic mechanisms of the Australian box jellyfish, C. fleckeri, and provides a basis for in vivo research of these newly isolated toxic fractions.