In silico analysis of endogenous siRNAs associated transposable elements and NATs in Schistosoma japonicum reveals their putative roles during reproductive development.

Schistosomiasis is a neglected tropical disease caused by trematode of the genus Schistosoma. Successful reproductive development is critical for the production of eggs, which are responsible for host pathology and disease dissemination. Endogenous small non-coding RNAs play important roles in many biological processes such as protection against foreign pathogens, cell differentiation, and chromosomal stability by regulating target gene expression at the transcriptional and post-transcriptional levels. In this study, we performed in silico analysis of endogenous small non-coding RNAs in different stages, and sex of S. japonicum focusing on endogenous small interfering RNAs (endo-siRNAs) generated from transposable elements (TEs) and natural antisense transcripts (NATs). Both total and unique siRNA populations show 18-30 nt in length, but the predominant size was 20 nt and the leading first base was adenosine. Sense TE-derived endo-siRNAs reads were higher than antisense reads at different relative positions of TEs, whereas no such difference was observed for NAT-derived endo-siRNAs. TE- and NAT-derived endo-siRNAs were more enriched in the male compared to female worms, with the higher relative expression in early phase of pairing. Putative targets of endo-siRNAs indicated more of them in males (106 and 66) than in females (6 and 23) for TE- and NAT-derived endo-siRNAs, respectively. Our preliminary study revealed vital role of endo-siRNAs during the reproductive development of S. japonicum and provide clues for putative novel targets to suppress worm reproduction and direction for effective anti-schistosomal drug development.

Molecular characterization, expression profile, and preliminary evaluation of diagnostic potential of CD63 in Schistosoma japonicum.

Schistosomes are the causative agents of human schistosomiasis, which is endemic in tropical and subtropical zones. CD63 is a member of the tetraspanin protein family widely expressed among eukaryotes. Previously, we identified a CD63 homolog from extracellular vesicles isolated from Schistosoma japonicum. In this study, we characterized this CD63 homolog using a molecular approach and evaluated the potential of its recombinant protein for the diagnosis of schistosomiasis. A sequence alignment indicated that S. japonicum CD63 (SjCD63) has sequence identities of 76 and 28% with S. mansoni and human CD63, respectively. A phylogenetic analysis displayed that S. japonicum CD63 is related to S. mansoni and Opisthorchis viverrini CD63. The cDNA of SjCD63 was 740 bp long with an expected protein size of 23.58 kDa. A RT-qPCR analysis revealed significantly higher expression of SjCD63 mRNA in adult worms on days 21, 28, and 35 than in 7-day schistosomula, cercariae, and eggs. In addition, recombinant SjCD63 protein detected by ELISA revealed significantly higher optical density values compared to that of the negative control in both S. japonicum-infected mouse and rabbit sera, providing preliminary evidence for its diagnostic potential. Overall, these results provide insight into the molecular properties of SjCD63, its expression profiles, and its preliminary diagnostic potential.

Molecular characterization and expression profile of nanos in Schistosoma japonicum and its influence on the expression several mammalian stem cell factors.

Pluripotent stem cells, called neoblasts, are well known for the regenerative capability and developmental plasticity in flatworms. Impressive advancement has been made in free-living flatworms, while in case of its parasitic counterpart, neoblast-like stem cells have attracted recent attention for its self-renewal and differentiation capacity. Nanos is a key conserved post-transcriptional regulator critical for the formation, development, and/or maintenance of the pluripotent germ line stem cell systems in many metazoans including schistosomes. In the present study, we report the molecular cloning and expression of nanos orthologous genes nanos in Schistosoma japonicum (Sjnanos). The cDNA of Sjnanos is 826 bp long, containing an open reading frame (ORF) for 223 amino acid long protein. qRT-PCR analysis shown that Sjnanos was differently expressed in several stages of schistosomes with relatively high level in schistosomula. Additionally, Sjnanos was expressed highly in adult females compared to adult males. Transfection of recombinant plasmid for expressing Sjnanos resulted in significant proliferation and increased expression of several stem cell factors in mammalian cells. Overall, our preliminary study provides the molecular basis to further functionally characterize Sjnanos in S. japonicum.

Cysticercus fasciolaris infection induced oxidative stress and apoptosis in rat liver: a strategy for host-parasite cross talk.

Parasitic helminths have developed various strategies to induce or inhibit apoptosis in the cells of their host, thereby modulating the host's immune response and aiding dissemination to the host. Cysticercus fasciolaris, the larval form of Taenia taeniaeformis, parasitized different intermediate hosts like rats, rabbits, etc. and is cosmopolitan in distribution. In the present study, we have investigated host-parasite interactions and the resulting effect of C. fasciolaris in the liver of rat. Histology of the infected livers showed dilation and damages of hepatic cells near the parasite. Infected liver cells showed an increase in DNA fragmentation and chromatin condensation compared to the normal liver. Acridine orange and ethidium bromide dual staining revealed the presence of apoptotic cells in the infected liver. The decline in the mitochondrial membrane potential in the infected liver suggested that the observed apoptosis is mitochondria mediated. Occurrence of an elevated level of active executioner caspases 3/7 in the infected rat liver further confirms the occurrence of apoptosis. Different antioxidant enzymes were also evaluated and revealed a notable decline in the level of glutathione and glutathione-S-transferase activity leading to the augmented generation of reactive oxygen species. Results of the present study revealed that C. fasciolaris infection leads to apoptosis in the liver of rats which may be a surviving strategy for the parasitic larvae.

Resveratrol- and α-viniferin-induced alterations of acetylcholinesterase and nitric oxide synthase in Raillietina echinobothrida.

Phytostilbenes, like resveratrol and α-viniferin, which occur mainly in the plants and belong to the families Cyperaceae, Vitaceae, and Gnetaceae are extensively popular for their medicinal and nutritional properties. In Northeast India, the Jaintia tribes consume these phytochemicals through aqueous extract of the medicinal plant Carex baccans to control helminthiasis. The present study aimed to investigate the inhibitory effect of the phytochemicals on neurotransmitters and its related enzymes in helminth parasite Raillietina echinobothrida. Viability of the parasites exposed to the phytostilbenes and extent of inhibition of cholinergic and nitrergic enzymes were evaluated in comparison to reference anthelmintic drug praziquantel and two known enzyme inhibitors, namely Nω-nitro-L-arginine and pyridostigmine. On exposure to resveratrol, α-viniferin, and reference drug praziquantel, the parasites ceased movement at 9.37, 11.38, and 0.24 h followed by death at 23.65, 34.13, and 1.87 h, respectively. Exposed parasites also showed a significant decrease in the activity of acetylcholinesterase (46.101, 65.935, and 63.645%) and nitric oxide synthase (61.241, 55.046, and 29.618%) in comparison to the controls. In addition, a decreased trend in nitric oxide (NO) level was also detected in the tissue of different phytochemical-exposed parasites compared to control. The present study suggests that anthelmintic potential of both the phytochemicals is mediated through inhibition of two vital enzymes which play diverse role in intracellular communications through neuromuscular system.

In vivo anthelmintic activity of Carex baccans and its active principle resveratrol against Hymenolepis diminuta.

Anthelmintic resistance against most of the commercial drugs is a great threat to humans as well as the veterinary live stocks. Hence, new treatment strategies to control helminth infections are essential at this hour. Carex baccans Nees has been traditionally used by Jaintia tribes in Northeast India to get rid of intestinal worm infections. Therefore, the present study was conducted to evaluate in vivo cestocidal activity of root tuber extract of C. baccans and its active component resveratrol against the zoonotic cestode Hymenolepis diminuta in the experimental model rat. The cestocidal activity was determined by monitoring the eggs per gram (EPG) counts in faeces of different treated groups. The result showed that the highest dose of the plant extract (50 mg/kg) and resveratrol (4.564 mg/kg body weight) has significant anthelmintic efficacy against H. diminuta. Crude extract of the plant as well as resveratrol reduced EPG count (56.012 and 46.049 %) and also resulted in decreased worm burden by 44.287 and 31.034 %, respectively. The efficacy of the crude extract and resveratrol can be compared to the reference drug praziquantel. The results exhibits considerable cestocidal potential of root tuber crude extract of C. baccans and resveratrol and justify its folklore use.

Evidence of apoptosis in Raillietina echinobothrida induced by methanolic extracts of three traditional medicinal plants of Northeast India.

The therapeutic benefits of medicinal plants in terms of anthelmintic properties are known since time immemorial in India, particularly among natives of the Northeast India. However, only sporadic and scarce reports on scientific validation of these plants are available. The present study was conducted on the cestode Raillietina echinobothrida, to establish whether the anthelmintic activity of Potentilla fulgens, Alpinia nigra and Millettia pachycarpa was mediated by apoptosis or not. Light microscopic observation following MTT assay revealed the highest percentage of inhibition of viability among the worms by methanol extract of M. pachycarpa (89.33%), followed by A. nigra (65%) and P. fulgens (37%). Ultrastructural observations revealed swelling of mitochondria, disruption of mitochondrial membrane, vacuolization of mitochondria, appearance of apoptotic bodies in the cytoplasm, disintegration of nuclear membrane and nucleolus were very common throughout the tegument. DAPI stained specimens showed typical morphology of apoptosis, like nuclear condensation and fragmentation in the extracts treated parasites. A decrease in mitochondrial membrane potential was also recorded in the treated groups. Confirmatory TUNEL assay and DNA fragmentation assay of the extracts treated parasites also confirmed the apoptotic nature of cell death and is concluded to be responsible for paralysis and death of the parasite.

Ultrastructural and biochemical alterations in rats exposed to crude extract of Carex baccans and Potentilla fulgens.

The use of plants as a source of medicine is an important component of the health care system in rural India. Carex baccans (Cyperaceae) and Potentilla fulgens (Rosaceae) have been known since ancient times in northeast India for their antitumor, antidiabetic, and antihelmintic properties. The present study was designed to determine the subacute toxicity profile of the root tuber extract of C. baccans and root-peel extract of P. fulgens in Wistar rats. The subacute oral toxicity was conducted using sublethal doses of 40, 50, 100, 150, 200, and 400 mgkg-1 body weights. Surface topographical and ultrastructural observations of liver and intestinal microvilli showed remarkable deformation and disruption, accompanied by quantitative changes in the liver enzymes, i.e., aspartate aminotransferase and alanine aminotransferase in comparison to those of the control group. Apoptotic cell death was observed in the liver cells of rats exposed to both of the plant extracts. A significant increase in splenic lymphocyte count was also observed in rats exposed to the highest concentration of both extracts. The results showed that consumption of the plant extracts at higher doses may cause toxicological effect if treatment continues for a long time.

Transcriptional profiles of genes potentially involved in extracellular vesicle biogenesis in Schistosoma japonicum.

Schistosomiasis is a severe chronic disease caused by parasitic worms of the genus Schistosoma. Recent studies indicate that schistosomes can secrete extracellular vesicles (EVs), which play important regulatory roles in many biological processes. However, the mechanisms underlying EV biogenesis in schistosomes are poorly understood. In this study, we performed bioinformatic analyses and identified several genes putatively involved in EV biogenesis in Schistosoma japonicum, which were then confirmed by PCR. Quantitative transcriptional profiles of the selected genes indicated that they were differentially expressed in male and female worms as well as in the different developmental stages of S. japonicum. Thus, the highest expression of VAMP3 was detected in cercariae, whereas that of ARF6 was detected in eggs. RAB11A and the Syntenin-encoding gene SDCBP were highly expressed in 14-day schistosomula and VPS4A and RAB27A were highly expressed in 35-day-old adult schistosomes. The expression of RAB11A, CHMP4C, VPS4A, and SDCBP was higher in male worms, whereas that of ARF6, VAMP3, and RAB27A was higher in female worms. Our results are expected to provide important clues for understanding the role of EV biogenesis in S. japonicum development.

Preliminary evaluation of the diagnostic potential of Schistosoma japonicum extracellular vesicle proteins for Schistosomiasis japonica.

Schistosomiasis is a chronic parasitic disease caused by the genus Schistosoma and poses a great threat to human and animal health. Identification of effective biomarkers would facilitate evaluation of drug efficacy and recognition of infected hosts, which are crucial for effective schistosomiasis control. Extracellular vesicle (EV) proteins are considered ideal biomarkers for developing invasive diagnostic tools. In this study, we evaluated the potential of Schistosoma japonicum EV (SjEV) proteins as biomarkers for diagnosing schistosomiasis. Several SjEV proteins were subject to epitope prediction using DNASTAR software, and the diagnostic potential of selected peptides was evaluated using enzyme-linked immunosorbent assay (ELISA). The results indicated that the sera showed detectable antibody levels against the two antigens in mice, rabbits, and humans infected with S. japonicum. Further analysis of the combined epitope protein demonstrated a modest sensitivity for detection of Schistosomiasis japonica. Our preliminary study suggests that S. japonicum EV proteins could serve as potential biomarkers for developing diagnostic tools for schistosomiasis.

RNA sequencing analysis of altered expression of long noncoding RNAs associated with Schistosoma japonicum infection in the murine liver and spleen.

BACKGROUND: Schistosomiasis is a chronic, debilitating infectious disease caused by members of the genus Schistosoma. Previous findings have suggested a relationship between infection with Schistosoma spp. and alterations in the liver and spleen of infected animals. Recent reports have shown the regulatory role of noncoding RNAs, such as long noncoding RNAs (lncRNAs), in different biological processes. However, little is known about the role of lncRNAs in the mouse liver and spleen during Schistosoma japonicum infection. METHODS: In this study, we identified and investigated lncRNAs using standard RNA sequencing (RNA-Seq). The biological functions of the altered expression of lncRNAs and their target genes were predicted using bioinformatics. Ten dysregulated lncRNAs were selected randomly and validated in reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) experiments. RESULTS: Our study identified 29,845 and 33,788 lncRNAs from the liver and spleen, respectively, of which 212 were novel lncRNAs. We observed that 759 and 789 of the lncRNAs were differentially expressed in the respective organs. The RT-qPCR results correlated well with the sequencing data. In the liver, 657 differentially expressed lncRNAs were predicted to target 2548 protein-coding genes, whereas in the spleen 660 differentially expressed lncRNAs were predicted to target 2673 protein-coding genes. Moreover, functional annotation showed that the target genes of the differentially expressed lncRNAs were associated with cellular processes, metabolic processes, and binding, and were significantly enriched in metabolic pathways, the cell cycle, ubiquitin-mediated proteolysis, and pathways in cancer. CONCLUSIONS: Our study showed that numerous lncRNAs were differentially expressed in S. japonicum-infected liver and spleen compared to control liver and spleen; this suggested that lncRNAs may be involved in pathogenesis in the liver and spleen during S. japonicum infection.

Host miR-148 regulates a macrophage-mediated immune response during Schistosoma japonicum infection.

Extracellular vesicles are critical regulators of host-parasite interactions. We previously demonstrated that Schistosoma japonicum EVs contain a remarkably high abundance of host miR-148a. Here, we characterised the abundance of miR-148a in circulation, in peripheral immune cells, and in plasma EVs of S. japonicum-infected mice. The results suggested the high abundance of miR-148a in macrophages to be likely linked to S. japonicum EVs. Additionally, miR-148a was found to target PTEN through the PI3K/AKT pathway to regulate cytokine production in macrophages. Consequently, our findings suggest that high abundance of miR-148a in macrophages may be associated with S. japonicum EVs, and regulate the host immune response during schistosome infection.

14-3-3 protein and ubiquitin C acting as SjIAP interaction partners facilitate tegumental integrity in Schistosoma japonicum.

Schistosomiasis, caused by trematodes of the genus Schistosoma, remains an important public health issue. Adult schistosomes can survive in the definitive host for several decades, although they are subject to the host immune response. Consequently, understanding the mechanism underlying worm survival in the definitive hosts could aid in developing novel strategies against schistosomiasis. We previously found that an inhibitor of apoptosis in Schistosoma japonicum (SjIAP) could negatively regulate apoptosis by inhibiting caspase activity, which plays a critical role in maintaining tegument integrity. The current study aimed to further analyze the mechanism related to SjIAP governing worm tegument integrity; therefore, we used a yeast two-hybrid screen and identified a series of putative interacting partners of SjIAP, including 14-3-3 (Sj14-3-3) and ubiquitin C (SjUBC). Quantitative real time PCR (qRT-PCR) analysis indicated that transcript profiles of Sj14-3-3 and SjUBC increased together with worm development in definitive hosts, which corresponds to those of SjIAP in S. japonicum. Immunohistochemical analysis showed Sj14-3-3 and SjUBC were located in the tegument of adult parasites while they were also ubiquitously distributed in the bodies of worms. Silencing of Sj14-3-3/SjUBC expression led to increased caspase activity and induced worm death. Inhibition of Sj14-3-3 or SjUBC resulted in significant morphological alterations in the schistosome tegument. Overall, our findings indicated that Sj14-3-3 and SjUBC interacting with SjIAP may belong to another strategy of S. japonicum to maintain the tegument integrity.

Preliminary evaluation of neoblast-like stem cell factor and transcript expression profiles in Schistosoma japonicum.

Neoblast-like stem cell factors and transcripts are essential for cell proliferation, self-renewal, and differentiation. Recent studies have demonstrated that nanos, sox, and vasa-like transcription factors are associated with neoblast-like stem cells in Schistosoma mansoni and play crucial roles in the regulation of worm development. However, these neoblast-like stem cell factors and transcripts and their expression profiles remain unknown in Schistosoma japonicum. In this study, we identified orthologs of 11 neoblast-like stem cell factors and transcripts in S. japonicum using bioinformatics and confirmed them by PCR. The expression profiles of neoblast-like stem cell factors and transcripts revealed that some of them were highly expressed in certain stages. Sex-based expression analysis revealed that nanos, polo-like kinase, PCNA, cyclin B, and H2A showed significantly higher expression in female worms, whereas ago and bruli showed higher expression in male worms. In addition, we noted that ago, bruli, and pp32 exhibited higher expression in the testes, while nanos, polo-like kinase, cyclin B, H2A, and H2B showed notable higher expression in both isolated ovaries and testes. Our preliminary results are expected to provide important information about the regulatory roles of these stem cell factors in parasite development and sexual maturation.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum.

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Praziquantel induced oxidative stress and apoptosis-like cell death in Raillietina echinobothrida.

Praziquantel (PZQ) is an anthelmintic drug used against trematode and cestode parasites of humans and veterinary animals. Since praziquantel was introduced as a broadspectrum anthelmintic, numerous studies described its successful use against helminth parasites, but its exact mechanism of action is feebly understood. Therefore, the present study was carried out to evaluate the possible role of PZQ induced oxidative stress in apoptosis-like cell death in the poultry tapeworm Raillietina echinobothrida. Parasite viability assay revealed a time-dependent reduction in the worm viability compared to the control. Transmission electron microscopy showed typical apoptotic features like condensed nucleus, damaged nuclear envelope and altered mitochondrial membrane in PZQ exposed parasites. Results revealed chromatin condensation and DNA fragmentation in PZQ exposed parasites. There was a notable decline in the level of glutathione and glutathione-s-transferase activity leading to the augmented generation of reactive oxygen species. This led to the alterations in the mitochondrial membrane potential with increased active caspase-3/7, confirms the involvement of mitochondria in the event. The present study suggests that PZQ exerts oxidative stress leading to apoptosis-like events in the parasites resulting their death.

α-Viniferin and resveratrol induced alteration in the activities of some energy metabolism related enzymes in the cestode parasite Raillietina echinobothrida.

α-Viniferin (AVF) and its monomer resveratrol (RESV) are natural phytostilbenes produced by several plants in response to injury or under the influence of pathogens such as bacteria or fungi. Our earlier studies have revealed that both the compounds exert anthelmintic activity through alterations of cestode tegument and its associated enzymes. The present study investigates the effects of these phytochemicals on some energy metabolism related enzymes in the fowl tapeworm, Raillietina echinobothrida. The phytostilbenes AVF, RESV and the reference drug praziquantel (PZQ) were tested against some selected enzymes i.e., phosphoenolpyruvate carboxykinase (PEPCK), lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) of R. echinobothrida. Exposure of the tapeworm to AVF, RESV and PZQ causes reduction in activity of PEPCK to the extent of 40.57/41.96, 24.58/23.75 and 41.11/13.47%, respectively, and LDH up to 48.95/16.25, 38.31/38.42 and 45.67/41.87%, respectively, at the time of paralysis. Whereas activity of MDH decreased by 34.22/37.7, 39.1/35.24 and 28.83/19.26%, respectively. Decrease in activities of LDH and MDH was also visible through histochemical observations. The results suggest that both the phytochemicals interfere with the energy transducing pathways by inhibiting the studied energy metabolism related enzymes of the parasite.

Apoptosis like cell death in Raillietina echinobothrida induced by resveratrol.

Northeast India is geographically nestled as one of the biodiversity hotspots, rich in traditionally used medicinal plants. Resveratrol, a naturally occurring phytoalexin found in berries, peanuts, grapes, red wine and also in numerous anthelmintic plants, has attracted wide interest because of its diverse pharmacological characteristics. Recently, anthelmintic potential of the compound is established. The present study was carried out to understand the possible mechanism of action of resveratrol on poultry tapeworm Raillietina echinobothrida. Resveratrol showed excellent cestocidal activity in a dose dependent manner as revealed through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. The progressive ultrastructural alterations followed by complete disruption of nuclear membrane, chromosomal condensation and in situ DNA fragmentation confirm the occurrence of apoptosis like cell death. Increased pro-apoptotic caspase activity and significant decreases in mitochondrial membrane potential in R. echinobothrida exposed to resveratrol confirm the involvement of mitochondria in the process of apoptosis.

Resveratrol induced structural and biochemical alterations in the tegument of Raillietina echinobothrida.

The root tuber of Carex species has been used as an anthelmintic medicine for intestinal helminthic infections in the Northeast region of India. The main compound present in the root tuber of the genus Carex is resveratrol. Therefore, the present study was conducted to evaluate the anthelmintic effects of resveratrol in Raillietina echinobothrida by using motility observation, electron microscopy, histochemical and biochemical analysis. Resveratrol causes complete inactivation and flaccid paralysis of the cestode, followed by death. The treated parasites also exhibited extensive distortion of the surface fine topography and decrease in the activities of major tegumental enzymes compared to that of control parasite. Ultrastructural alterations include changes in cell organelles present in the tegument and sub-tegumental cyton. Extensive alterations in the surface topography of the treated parasites resulted in a decrease in the activities of tegumental enzyme which suggest that, resveratrol may be useful as a therapeutic agent to treat cestode parasites.

Ultrastructural observations on Raillietina echinobothrida exposed to crude extract and active compound of Securinega virosa.

Securinega virosa has been used traditionally by the natives of Mizoram, India, to control intestinal worm infections. In the present study, the crude ethanol extract of the plant and its active component virosecurinine were tested in vitro on Raillietina echinobothrida to evaluate its potential anthelmintic efficacy and ultrastructural changes. The test parasites were exposed to different concentrations of the plant extract, active compound virosecurinine and reference drug praziquantel. Scanning and transmission electron microscopic observations on the paralyzed worms revealed wide scale destruction of the tegument with intense vacuolization of the syncytium and swellings of the basal lamina accompanied by deformities in the cell organelles. Extensive structural alteration of tegument indicates that the plant extract and its active component alter membrane permeability of the parasite leading to paralysis and subsequent death, as confirmed by in vitro tests.