Biological activities and functional analysis of macrophage migration inhibitory factor in Oncomelania hupensis, the intermediate host of Schistosoma japonicum.

Schistosomiasis is a destructive parasitic zoonosis caused by agents of the genus Schistosoma, which afflicts more than 250 million people worldwide. The freshwater amphibious snail Oncomelania hupensis serves as the obligate intermediate host of Schistosoma japonicum. Macrophage migration inhibitory factor (MIF) has been demonstrated to be a pleiotropic immunoregulatory cytokine and a key signaling molecule involved in adaptive and innate immunity. In the present study, we obtained the full-length cDNA of OhMIF and analyzed the characteristics of the ORF and the peptide sequence in O. hupensis. Next we have successfully expressed and purified the recombinant OhMIF protein (rOhMIF) together with a site-directed mutant rOhMIFP2G, in which the N-terminal Proline (Pro2) was substituted by a Gly. Our results indicated that rOhMIF displayed the conserved D-dopachrome tautomerase activity which is dependent on Pro2, and this enzymatic activity can be significantly inhibited by the MIF antagonist ISO-1. Moreover, we also measured and compared the steady state kinetic values for D-dopachrome tautomerase activity of rOhMIF and rHsMIF, and the results showed that the reaction rate, catalytic efficiency and substrate affinity of rOhMIF are significantly lower than those of rHsMIF. Additionally, we also showed that rOhMIF had the oxidoreductase activity which can utilize DTT as reductant to reduce insulin. Furthermore, the results obtained from the in vitro injection assay demonstrated that rOhMIF and its mutant rOhMIFP2G can also induce the phosphorylation and activation of ERK1/2 pathway in O. hupensis circulating hemocytes, indicating that the tautomerase activity is not required for this biological function. These results are expected to produce a better understanding of the internal immune defense system in O. hupensis, and help to further explore the interaction between O. hupensis and its natural parasite S. japoniucm.

Heat shock protein 90 inhibitor mycoepoxydiene modulates kinase signaling in cervical cancer cells and inhibits in-vivo tumor growth.

Heat shock protein 90 (Hsp90) functions within multiple signaling pathways on the basis of its ability to serve as a chaperone for more than 100 client proteins. Thus, inhibition of Hsp90 alone can trigger numerous pathways. Mycoepoxydiene (MED) can inhibit Hsp90 function and induce apoptosis in cervical cancer cells. However, the antitumor efficacy of MED in vivo is still not clear. We examined the efficacy of MED in a mouse xenograft model to further elucidate HeLa cell fate and also assessed the mechanism of altered protein signaling in response to this compound in vitro. Our data showed that Hsp90 inhibition simultaneously triggers signaling that regulates both cell death and cell proliferation, and that HeLa cell death may be a result of the disequilibrium of these signals. MED induces cell death as a result of the destabilization of Akt and IKK, which may promote cell death through a reduction in the activation of Bad and nuclear factor-κB. However, MED also induces the MEK/ERK pathway, which is classically considered to promote cell survival. MEK/ERK activation leads to an increase in p21, a cyclin-dependent kinase inhibitor, and is independent of Raf, but is shown to be mediated by p53. MED also leads to a decrease in several additional G2/M regulatory proteins independent of the MEK/ERK pathway. These results indicate an interesting mechanism of cross-talk between the inhibition of Akt phosphorylation and the activation of the MEK pathway by MED and provide in-vivo evidence for the potential of inhibiting Hsp90 as a candidate anticancer treatment.

Size-dependent enhancement of gene expression by Plasmodium 5'UTR introns.

BACKGROUND: Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5' and 3' untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown. METHODS: In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5'UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5'UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5'UTR intron were generated using the CRISPR/Cas9 system. RESULTS: We showed that promoters with 5'UTR introns had higher activities in driving gene expression than those without 5'UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5'UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5'UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages. CONCLUSIONS: Plasmodium 5'UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5'UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation.

RURAL LEFT-BEHIND CHILDREN ARE SERIOUSLY AFFLICTED WITH INTESTINAL HELMINTH INFECTIONS IN SOUTHERN SICHUAH, CHINA.

Rural children are seriously afflicted with intestinal helminth infections in China. Of note, the term rural children includes rural left-behind children (LBC) and rural non-left-behind children (NLBC); the difference in the prevalence of intestinal helminths between the 2 groups remains unclear. In this study, Gulin and Xuyong counties in southern Sichuan were chosen for investigation in 2019. The Kato Katz thick smear method was used to detect the presence of intestinal helminth eggs in rural children. For children aged 3-6 yr, the adhesive tape perianal swab method was used to detect Enterobius vermicularis and tapeworm eggs. Statistical differences in infection rates between the 2 groups were determined by the chi-square test. In total, 1,608 rural children, 911 LBC and 697 NLBC, participated in the investigation. Six species of intestinal helminths were detected. A total of 358 (39.3%) and 130 (18.7%) intestinal helminth positives were found among LBC and NLBC, respectively; the former had a higher (P < 0.05) infection level. Moreover, an analysis of double worm infection rates among intestinal helminth positive LBC and NLBC showed a difference between the 2 groups that was also statistically significant. These surveys indicated that the risk of intestinal helminth infection was substantially higher and the severity of infection much worse in rural LBC in southern Sichuan. More attention should be paid to the parasitic infection of LBC.

Digenean parasites of Chinese marine fishes: a list of species, hosts and geographical distribution.

In the literature, 630 species of Digenea (Trematoda) have been reported from Chinese marine fishes. These belong to 209 genera and 35 families. The names of these species, along with their hosts, geographical distribution and records, are listed in this paper.

Combined transcriptomics and proteomics to identify differential proteins involved in the immune response to the parasite schistosoma japonicum in snail hosts pre-infected with exorchis sp.

Oncomelania hupensis is the obligate intermediate host of Schistosoma japonicum, and it also serves as the first intermediate host for Exorchis sp., which uses Parasilurus asoyus as its definitive host rather than humans. In previous studies, Tang et al. found that all S. japonicum larvae can be blocked and killed in O. hupensis pre-infected with Exorchis sp. eggs. However, the molecular and cellular mechanisms involved in this process remain unclear. Therefore, in the present study, a combined transcriptomic and proteomic analysis was performed to identify the differential proteins involved in the immune response to the parasite S. japonicum in the O. hupensis snail host pre-infected with Exorchis sp. trematodes. The results showed that a total of 46,162 unigenes were obtained with 23,535 (50.98%) unigenes annotated in relevant databases, and 3811 proteins from O. hupensis were identified. In addition, iTRAQ-based quantitative proteomic analysis demonstrated that among three groups (OhSj-1_vs_OhN-1, OhE-1_vs_OhN-1 and OhES-1_vs_OhN-1), there were 146 common differential proteins including 44 up-regulated proteins and 90 down-regulated proteins, and 195 differential proteins exclusive to only one experimental group, including 91 up-regulated proteins and 104 down-regulated proteins, which were defined as the Common group and the Only group, respectively. KEGG analysis showed that 15 and 11 differential proteins were annotated in "Infectious diseases" in the Common group and the Only group, respectively, indicating that these proteins may be involved in the snail host immune response to parasite infection. These data will be helpful for better understanding the host-parasite interaction, and could pave the way towards exploring the mechanisms involved in the biological control on S. japonicum in O. hupensis. They also provide valuable information about developing new anti-schistosomiasis strategies.

Cep85 Relays Plk1 Activity to Phosphorylated Nek2A for Its Timely Activation in Centrosome Disjunction.

Timely centrosome separation is critical for accurate chromosome separation. It is initiated by Nek2A at the onset of mitosis, but the mechanism for the strict requirement of phosphorylated Nek2A for its own activation remains unclear. In this study, we have found that Plk1 interacts with Cep85 and forms a ternary complex with Cep85-Nek2A. Nek2A binding, but not its kinase activity, is pre-required for Cep85 to be phosphorylated by Plk1. Nek2A-dependent Cep85 phosphorylation, in turn, leads to the dissociation of phosphorylated Cep85 exclusively from phospho-Nek2A, thereby increasing the freed phospho-Nek2A activity. Both kinases are also required for phosphorylating endogenous Cep85 in cells, and timely phosphorylation of Cep85 and Nek2A is crucial for initiating centrosome disjunction at G2/M. Overall, our study has uncovered a previously unrecognized role of Plk1 and Nek2A and identified Cep85 as a missing piece directly relaying Plk1 activity to Nek2A for its activation in centrosome disjunction.

Identification and functional characterization of thioredoxin-related protein of 14 kDa in Oncomelania hupensis, the intermediate host of Schistosoma japonicum.

Oncomelania hupensis is the unique intermediate host of the blood fluke Schistosoma japonicum, which causes schistosomiasis. In snails, highly toxic reactive oxygen species (ROS) can be continually generated by hemocytes in response to foreign particles or pathogens, and may be involved in damaging and eliminating digenean larvae. Thioredoxin-related protein of 14 kDa (TRP14) is a member of the Trx superfamily, and plays an important role in the scavenging of ROS. This study was designed to identify and characterize TRP14 from O. hupensis (OhTRP14), and investigate the involvement of OhTRP14 in the scavenging of ROS in snail host immune response to the parasite S. japonicum. Here we expressed and purified the recombinant OhTRP14 and its mutant, and rOhTRP14 displayed oxidoreductase activity dependent on the CPDC motif. OhTRP14 protein was ubiquitously present in all the tested snail tissues, and especially immunolocalized in the cytoplasm of immune cell types (hemocytes). Both the expression of OhTRP14 and ROS level increased significantly in snails following challenge with S. japonicum. The dsRNA-mediated knockdown of OhTRP14 was successfully conducted by oral feeding, and ROS production was increased by OhTRP14 knockdown, implying that OhTRP14 was involved in the scavenging of ROS in O. hupensis circulating hemocytes. Therefore, we conclude that OhTRP14 may be involved in the scavenging of ROS in snail host immune response to the parasite S. japonicum. The results expand our understanding of the interaction between this parasite and host, and lay a foundation for the establishment of Oncomelania-schistosome infection models.

Identification and functional characterization of Oncomelania hupensis macrophage migration inhibitory factor involved in the snail host innate immune response to the parasite Schistosoma japonicum.

Schistosomiasis, caused by parasitic trematodes of the genus Schistosoma, remains a devastating public health problem, with over 200 million people infected and 779 million people at risk worldwide, especially in developing countries. The freshwater amphibious snail Oncomelania hupensis is the obligate intermediate host of Schistosoma japonicum. This unique and long-standing host-parasite interaction highlights the biomedical importance of the molecular and cellular mechanisms involved in the snail immune defense response against schistosome infection. In recent years, a number of immune-related effectors and conserved signalling pathways have been identified in molluscs, especially in Biomphalaria glabrata, which is an intermediate host for Schistosoma mansoni, but few have been reported in O. hupensis. Here we have successfully identified and functionally characterized a homologue of mammalian macrophage migration inhibitory factor (MIF) from O. hupensis (OhMIF). MIF, a pleiotropic regulator of innate immunity, is a constitutively expressed mediator in the host's antimicrobial defense system and stress response that promotes the pro-inflammatory functions of immune cells. In the present study, we detected the distribution of OhMIF in various snail tissues, especially in immune cell types (hemocytes) and found that OhMIF displays significantly increased expression in snails following challenge with S. japonicum. Knockdown of OhMIF was conducted successfully in O. hupensis and significantly reduced the percentage of phagocytic cell populations in circulating hemocytes. Furthermore, OhMIF is not only implicated in the activation and differentiation of hemocytes, but also essential to promote the migration and recruitment of hemocytes towards the infected sites. These results provide the first known functional evidence in exploring the molecular mechanisms involved in the O. hupensis innate immune defense response to the parasite S. japonicum and help to better understand the complex host-parasite interaction.

Integrating Morphology, Breeding Ground and Mitochondrial COI Gene Analysis for Species Identification of Bellamya lithophaga (Gastropoda: Viviparidae) in China.

BACKGROUND: Angiostrongylus cantonensis is a zoonotic public health concern that causes human severe eosinophilic meningitis in Southeast Asia and China. As a medically important intermediate host of A. cantonensis, Bellamya lithophaga (Gastropoda: Viviparidae) is often confused with other morphologically similar sibling species of genus Bellamya, such as B. aeruginosa and B. purificata in the past. Hence, the aim of the present study was to investigate evidences to discriminate these equivocal Bellamya species. METHODS: This study was carried out by getting Bellamya snail samples from Fujian Province in the South-East of China. The snail morphological features, breeding grounds and phylogenetic relationship according to mitochondrial cytochrome c oxidase subunit I (COI) gene marker were analyzed. RESULTS: Based on external morphology, radular shape and cusp formula, as well as major breeding environment, B. lithophaga could be distinguished from B. aeruginosa, B. purificata. The phylogenetic tree also unconfirmed that B. lithophaga belongs to a different genetic clade from other morphologically similar species. CONCLUSION: Our findings demonstrate the significant differences in B. lithophaga and other sibling species, which supports the traditional species delimitation in the genus Bellamya.

Multi-strain infections and 'relapse' of Leucocytozoon sabrazesi gametocytes in domestic chickens in southern China.

Leucocytozoon parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss to the poultry industry. Although the prevalence and distribution of two Leucocytozoon species (L. sabrazesi and L. caulleryi) have been reported in China previously, there are many questions related to the parasite infection that remain unanswered, including population diversity and transmission dynamics in domestic chickens. Here we surveyed chicken blood samples from seven sites in four provinces of China to identify Leucocytozoon infection, characterized parasite diversity within individual infected hosts and between sampling sites, and investigated the dynamics of gametocytemia in chickens over time. We found high infection rates in three of the seven sites. Clustering parasite sequences of the mitochondrial cytochrome oxidase III (coxIII) and cytochrome b (cytb) genes showed lack of grouping according to geographic origins and individual hosts carrying large numbers of L. sabrazesi strains. Monitoring gametocytemia in blood samples from infected chickens over time showed 'relapse' or persistence of low-level gametocytemia for 4-5 months, which could be explored as an in vivo model for testing drugs against liver stages of Apicomplexan parasites. This study provides important information on population diversity and transmission dynamics of L. sabrazesi and for disease control.

Efficient editing of malaria parasite genome using the CRISPR/Cas9 system.

Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. Importance: Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.

Praziquantel synergistically enhances paclitaxel efficacy to inhibit cancer cell growth.

The major challenges we are facing in cancer therapy with paclitaxel (PTX) are the drug resistance and severe side effects. Massive efforts have been made to overcome these clinical challenges by combining PTX with other drugs. In this study, we reported the first preclinical data that praziquantel (PZQ), an anti-parasite agent, could greatly enhance the anticancer efficacy of PTX in various cancer cell lines, including PTX-resistant cell lines. Based on the combination index value, we demonstrated that PZQ synergistically enhanced PTX-induced cell growth inhibition. The co-treatment of PZQ and PTX also induced significant mitotic arrest and activated the apoptotic cascade. Moreover, PZQ combined with PTX resulted in a more pronounced inhibition of tumor growth compared with either drug alone in a mouse xenograft model. We tried to investigate the possible mechanisms of this synergistic efficacy induced by PZQ and PTX, and we found that the co-treatment of the two drugs could markedly decrease expression of X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptotic protein. Our data further demonstrated that down-regulation of XIAP was required for the synergistic interaction between PZQ and PTX. Together, this study suggested that the combination of PZQ and PTX may represent a novel and effective anticancer strategy for optimizing PTX therapy.

Development of larval Schistosoma japonicum blocked in Oncomelania hupensis by pre-infection with larval Exorchis sp.

Schistosomiasis continues to be a significant public health threat in the world. In the area of parasitic diseases, it is widely considered second only to malaria as a global health problem, with an incalculable drain on the economic resources of countries where it is endemic. Schistosoma japonicum is widespread in eastern and southeastern Asia, where the amphibious snail, Oncomelania hupensis, is the intermediate host. In the present study, we found that infection of O. hupensis with the mature eggs of another trematode, Exorchis sp., inhibited development of S. japonicum mother sporocysts in O. hupensis. Exorchis sp. commonly infects the edible fish Parasilurus asotus in China, but it is harmless to humans. This discovery provides an opportunity for possible biological control of S. japonicum infection and transmission. Additionally, it has the potential to substantially reduce the impact of the global S. japonicum that is independent of antihelminthic use. The mechanisms used by Exorchis sp. to inhibit infection by S. japonicum in the snail require further investigation.