Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini.

Mucin plays a crucial role in safeguarding mucosal tissues by obstructing the translocation of microorganisms. Mucosal tissue-dwelling parasites must devise a strategy to surmount this mucin barrier in order to establish colonization. In a recent discovery, it was observed that the liver fluke Opisthorchis viverrini secretes two mucinases, namely Ov-M60-like-1 and Ov-M60-like-2. Ov-M60-like-1 was previously characterized. Here, we study the Ov-M60-like-2 by utilizing the wheat germ expression system to produce recombinant proteins and conducted a functional analysis of its enzymatic activity on bovine submaxillary mucin (BSM). Subsequently, we delved deeper into understanding the role of this enzyme in host-parasite interactions by evaluating its mucinase activity on mucins from the bile duct of O. viverrini-infected hamsters. Through successful production of recombinant proteins using the wheat germ expression system, we observed that this enzyme displayed mucinase activity over a wide pH range (pH 2 to pH 10) against BSM. Our investigations revealed it ability to digest mucin from the bile duct. These findings suggest that Ov-M60-like-2 possess a mucinase activity, together with Ov-M60-like-1, enabling the liver fluke to successful colonization of the host's bile duct.

Bayesian spatio-temporal modelling of environmental, climatic, and socio-economic influences on malaria in Central Vietnam.

BACKGROUND: Despite the successful efforts in controlling malaria in Vietnam, the disease remains a significant health concern, particularly in Central Vietnam. This study aimed to assess correlations between environmental, climatic, and socio-economic factors in the district with malaria cases. METHODS: The study was conducted in 15 provinces in Central Vietnam from January 2018 to December 2022. Monthly malaria cases were obtained from the Institute of Malariology, Parasitology, and Entomology Quy Nhon, Vietnam. Environmental, climatic, and socio-economic data were retrieved using a Google Earth Engine script. A multivariable Zero-inflated Poisson regression was undertaken using a Bayesian framework with spatial and spatiotemporal random effects with a conditional autoregressive prior structure. The posterior random effects were estimated using Bayesian Markov Chain Monte Carlo simulation with Gibbs sampling. RESULTS: There was a total of 5,985 Plasmodium falciparum and 2,623 Plasmodium vivax cases during the study period. Plasmodium falciparum risk increased by five times (95% credible interval [CrI] 4.37, 6.74) for each 1-unit increase of normalized difference vegetation index (NDVI) without lag and by 8% (95% CrI 7%, 9%) for every 1ºC increase in maximum temperature (TMAX) at a 6-month lag. While a decrease in risk of 1% (95% CrI 0%, 1%) for a 1 mm increase in precipitation with a 6-month lag was observed. A 1-unit increase in NDVI at a 1-month lag was associated with a four-fold increase (95% CrI 2.95, 4.90) in risk of P. vivax. In addition, the risk increased by 6% (95% CrI 5%, 7%) and 3% (95% CrI 1%, 5%) for each 1ºC increase in land surface temperature during daytime with a 6-month lag and TMAX at a 4-month lag, respectively. Spatial analysis showed a higher mean malaria risk of both species in the Central Highlands and southeast parts of Central Vietnam and a lower risk in the northern and north-western areas. CONCLUSION: Identification of environmental, climatic, and socio-economic risk factors and spatial malaria clusters are crucial for designing adaptive strategies to maximize the impact of limited public health resources toward eliminating malaria in Vietnam.

Characteristics of SARS-CoV-2 and Opisthorchis viverrini coinfections: insights into immune responses and clinical outcomes.

The effects of co-infections with SARS-CoV-2 and parasitic diseases have been little investigated in terms of immune response, disease dynamics, and clinical outcomes. This study aimed to explore the impact of co-infection with Opisthorchis viverrini and SARS-CoV-2 on the immune response concerning clinical symptoms and the severity of pulmonary abnormalities. A cross-sectional study was conducted, including healthy participants as controls, participants with opisthorchiasis, SARS-CoV-2 infection, and a co-infection group with both diseases. Characteristics of SARS-CoV-2 infection were assessed based on clinical parameters and severity of pulmonary abnormalities, whereas opisthorchiasis burden was evaluated by eggs-per-gram (EPG) counts. Immune responses were assessed by measuring levels of interferon-γ (IFN-γ), SARS-CoV-2 anti-spike receptor binding domain (RBD) IgG, and neutralizing antibody against SARS-CoV-2. In the co-infected group, clinical parameters and hospitalization rates were lower than in the SARS-CoV-2 group. Pulmonary abnormalities, such as bronchial fibrosis, were commonly observed in the SARS-CoV-2 group, leading to hospitalization in some cases. Participants with opisthorchiasis had higher IFN-γ levels than healthy individuals. IFN-γ levels were significantly lower in the co-infection group compared with the SARS-CoV-2 group (P = 0.002). There was a significant (P = 0.044) positive correlation between RBD-specific IgG and percent neutralization levels in the SARS-CoV-2 group. Levels of both were somewhat lower (not statistically significant) in the co-infection group. A negative correlation was observed between opisthorchiasis burden (EPG counts) and IFN-γ and RBD-specific IgG levels in the co-infected group. Following vaccination, the increase in IgG levels against the RBD protein was significantly lower in the co-infected group than in the SARS-CoV-2 group. These results suggest that O. viverrini infection suppresses immune responses and may lead to a reduction in severity in cases of SARS-CoV-2 co-infection.

T helper cell responses to Opisthorchis viverrini infection associate with host susceptibility.

Opisthorchis viverrini infection is endemic in the lower Mekong subregion. The liver is an organ that worms are drawn to and cause damage. However, the immune-related susceptibility in the liver is poorly understood. In this study, we investigated T helper (Th) cell responses in the liver of BALB/c mice and golden Syrian hamsters during 2-28 days post-infection (DPI). We found that Th cell responses were distinct between mice and hamsters in terms of dynamics and polarization. Mice exhibited the early induction of Th1, Th2, Th17, and regulatory T (Treg) cells responses after the presence of O. viverrini worms at 2 DPI. In hamsters, the late induction of Th1/Th17, downregulation of Th2/Treg responses and early elevation of suppressive cytokine interleukin (IL)-10 were found together with swift reduction of Th cell numbers. Interestingly, expressions of IL-4 (Th2 functional cytokine) and Foxp3 (Treg lineage) were completely different between mice and hamsters which elevated in mice but suppressed in hamsters. These results suggest that early induction and well-regulation are related to host resistance. In contrast, late induction of Th cell response might allow immature worms to develop in the host. Our findings provide a greater understanding in Th cell response-related susceptibility in O. viverrini infection which would be targeting immunity for the development of immune-based intervention such as vaccine.

Opisthorchis viverrini excretory-secretory products suppress GLUT8 of cholangiocytes.

Opisthorchis viverrini infection and the subsequent bile duct cancer it induces remains a significant public health problem in Southeast Asia. Opisthorchiasis has been reported to cause reduced plasma glucose levels among infected patients. The underlying mechanism for this phenomenon is unclear. In the present study, evidence is presented to support the hypothesis that O. viverrini exploits host cholangiocyte glucose transporters (GLUTs) in a similar manner to that of rodent intestinal nematodes, to feed on unabsorbed glucose in the bile for survival. GLUT levels in a cholangiocyte H69 cell line co-cultured with excretory-secretory products of O. viverrini were examined using qPCR and immunoblotting. GLUT 8 mRNA and expressed proteins were found to be downregulated in H69 cells in the presence of O. viverrini. This suggests that O. viverrini alters glucose metabolism in cells within its vicinity by limiting transporter expression resulting in increased bile glucose that it can utilize and potentially explains the previously reported anti-insulin effect of opisthorchiasis.

Distinct antibody response in susceptible and non-susceptible hosts of the carcinogenic liver fluke Opisthorchis viverrini infection.

Opisthorchis viverrini is a carcinogenic parasite that can cause bile duct cancer called cholangiocarcinoma. A study of the immune response of this parasite in susceptible and non-susceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamsters and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between 1 and 2 weeks post-infection, whereas it was positive between 2 and 4 weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice reacts strongly with the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted with a single protein band. Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host reacts quicker and stronger than the susceptible host.

Persistent advanced periductal fibrosis is associated with cagA-positive Helicobacter pylori infection in post-praziquantel treatment of opisthorchiasis.

BACKGROUND: Liver fluke infection caused by Opisthorchis viverrini is associated with several hepatobiliary diseases including advanced periductal fibrosis (APF) and cholangiocarcinoma. Recently, we demonstrated a persistent APF in over one-third of opisthorchiasis patients after worm removal by praziquantel (PZQ) treatment. However, the underlying mechanism(s) of this phenomena is unclear. Given a co-infection with Helicobacter pylori (H. pylori) especially cagA-positive strain enhances APF, we hypothesized that H. pylori with CagA virulent factor contributes to persistent APF. MATERIALS AND METHODS: Seventy-five opisthorchiasis patients who underwent ultrasonography and treatment with PZQ were recruited in the 2-year follow-up study. Helicobacter and its cagA in the feces were examined by conventional and qPCR. Correlations between prevalence or bacterial loads of Helicobacter spp., H. pylori, and cagA-positive H. pylori before and after PZQ treatment were analyzed among resolved, slowly resolved, relapsed, and persistent APF groups. RESULTS: Overall, prevalence of Helicobacter spp., H. pylori, and cagA-positive H. pylori declined after PZQ treatment. However, only the prevalence and bacterial loads of cagA-positive H. pylori detected at 2-year post-treatment were significantly lower than those before treatment (p < .05). In addition, both prevalence and bacterial loads of cagA-positive H. pylori were significantly lower in the resolved APF group after PZQ treatment, while there were no significant changes in the slowly resolved, relapsed, and persistent APF groups. Among the APF subgroups, cagA-positive H. pylori prevalence in both relapsed and persistent APF groups were significantly higher than the resolved APF group. CONCLUSION: The results support our hypothesis that H. pylori, especially cagA-positive strain, contributes to the relapsed and persistent APF. A supplementary antibiotic treatment for H. pylori to reduce persistent APF and eventually CCA is warranted.

Does Opisthorchis viverrini circulate between humans and domestic cats in an endemic area in Thailand?

The liver fluke Opisthorchis viverrini is a foodborne trematode that, in chronic infection, is a leading cause of bile-duct cancer ­ cholangiocarcinoma. Cats and dogs are acknowledged as reservoir hosts of this parasite. However, this assumption is based on morphological similarity of flukes recovered from these hosts, without any molecular genetic evidence. The aim of this study was to obtain molecular data from O. viverrini eggs present in feces of humans and cats in the same locality in Thanya sub-district, Kalasin, Thailand. The mitochondrial cytochrome c oxidase subunit 1 (cox1) gene was used as the marker for a population-genetic study. A DNA fragment of the cox1 gene was amplified from stool samples and subjected to nucleotide sequencing. Phylogenetic and haplotype network analyses were performed. The cox1 sequences of O. viverrini eggs from humans and cats largely formed separate clades on the phylogenetic trees, with an Fst value of 0.64 (P < 0.05), indicating largely distinct populations in the 2 species. However, 5 samples from cats were placed in the human cluster and 1 sample from a human was placed in the cat cluster. This suggests that host specificity of 'human' and 'cat' clades is not absolute. These results indicate that there are 2 populations of O. viverrini, one circulates primarily in humans and the other in cats. However, cross-transmission can occur between these 2 hosts. Taken altogether, the population-genetic evidence from this study partially supports the assumption that the cat can act as a reservoir host of O. viverrini.

Adherence of Helicobacter pylori to Opisthorchis viverrini gut epithelium and the tegument mediated via L-fucose binding adhesin.

Recent reports implicate both the liver fluke Opisthorchis viverrini as a reservoir of Helicobacter pylori within the human gastrointestinal tract and H. pylori in the pathogenesis of opisthorchiasis-associated cholangiocarcinoma. We postulated that adherence of bacterial ligands to host receptors initiates colonization of the live fluke by H. pylori and here we aimed to assess the molecular interaction between O. viverrini and H. pylori by investigating host receptors for H. pylori in the fluke. Several known receptors of H. pylori including Lewis B, sialyl-Lewis X, Toll-like receptor 4 and L-fucose were detected immunohistochemically and histochemically by focusing analysis on the gut epithelium and tegument of the adult stage of the fluke. The frequency of detection of Lewis B, sialyl-Lewis X, TLR4 and L-fucose in 100 individual worms was 3, 3, 19 and 70%, respectively. Detection of H. pylori by a diagnostic ureA gene-based PCR assay revealed the presence of H. pylori in individual O. viverrini worms in 41 of 49 (79%) worms examined. In addition, numbers of bacteria decreased in a dose- and time-dependent fashion following exposure to fucosidase. These findings suggested that L-fucose represents a tractable receptor for H. pylori that can mediate bacterial colonization of the gut of O. viverrini.

Immunolocalization and functional analysis of Opisthorchis viverrini-M60-like-1 metallopeptidase in animal models.

Host mucins have crucial physical roles in preventing the parasitic establishment and maturation, and also in expelling the invading parasites. However, some parasites utilize mucinase enzymes to facilitate the infection. Recently, we have identified a mucinase enzyme of the liver fluke Opisthorchis viverrini, Ov-M60-like-1, which exhibits metallopeptidase activity against bovine submaxillary mucin substrate. Here, we aimed to study the localization of this enzyme in O. viverrini and the bile duct of hamsters using immunohistochemistry and functional analysis by mucin digestion in hamsters and mice tissues. The results showed that Ov-M60-like-1 was detected strongly in the tegument, tegumental cells, vitelline glands and mature eggs with miracidium. Expression in the gut, ovary and testis of the parasite was moderate while parenchyma showed slight colour intensity. In addition, the mucinase was also detected in the host biliary epithelial cells and goblet cells surrounding the worm. The mucinase assay revealed that the Ov-M60-like-1 could digest neutral mucin in the parenchyma, testis and seminal receptacle, but not the mucin in the tegument, tegumental cells and vitelline glands of the worm. The enzyme can also digest mucin in the cholangiocytes and modified the mixture type in the bile duct goblet cells of the infected hamsters, a susceptible host. In contrast, the enzyme was unable to digest neutral, acid and mixture mucin in the bile duct of the mice, a non-susceptible host. These findings indicate that Ov-M60-like-1 may have functions in both housekeeping tasks and host­parasite interactions, especially in modification of host susceptibility.

Helicobacter pylori GroEL Seropositivity Is Associated with an Increased Risk of Opisthorchis viverrini-Associated Hepatobiliary Abnormalities and Cholangiocarcinoma.

Despite the synergistic effect of Opisthorchis viverrini and Helicobacter pylori co-infection on pathogenesis of severe hepatobiliary abnormalities (HBA) including advanced periductal fibrosis and replace with cholangiocarcinoma (CCA) have been established, the immune response to H. pylori in O. viverrini infected population has never been explored. Hence, this study aimed to investigate the antibody responses to 2 immunogenic H. pylori proteins in O. viverrini-infected patients with HBA and CCA. The risk analysis by multinomial logistic regression revealed that GroEL seropositivity was associated with higher risks of hepatobiliary abnormalities and CCA with adjusted odds ratios (95% confidence intervals) of 2.11 (95% CI=1.20-3.71, P=0.008) and 2.13 (95% CI=1.21-3.75, P=0.009), respectively. These findings indicate that GroEL seropositivity might be a biomarker for early detection of O. viverrini associated HBA and CCA.

High macrophage activities are associated with advanced periductal fibrosis in chronic Opisthorchis viverrini infection.

Liver fluke infection caused by Opisthorchis viverrini induces several hepatobiliary conditions including advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA), but >25% of the infected population develops APF and 1% develop CCA. The innate immune response is the first line of defence, and macrophages are critical regulators of fibrosis. We hypothesized that macrophages from infected individuals have different capacities to either promote or suppress periductal fibrosis. We compared phagocytic activities of macrophages of healthy individuals and O viverrini-infected individuals ± APF, and found that macrophages from infected individuals with APF ingested significantly higher numbers of beads compared with healthy controls and O viverrini-infected individuals without APF. To further investigate proteolytic activity, we monitored real-time phagosomal proteolysis of beads conjugated to DQ-BODIPY-BSA using live cell imaging. We show that macrophages from O viverrini-infected individuals with APF also have elevated phagosomal proteolysis activity, which is consistent with their increased phagocytic activity. Additionally, stimulated ROS production by blood monocytes was higher in individuals with APF compared with healthy controls and infected individuals without APF. These results suggest that during O viverrini infection, macrophages with high phagocytic and proteolytic activities together with elevated ROS production are the phenotypes that can promote tissue damage, which results in periductal fibrosis.

HIV-1 Integrates Widely throughout the Genome of the Human Blood Fluke Schistosoma mansoni.

Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.

A comparative proteomic analysis of bile for biomarkers of cholangiocarcinoma.

Cholangiocarcinoma is a primary malignant tumor of the bile duct epithelium. Cholangiocarcinoma is usually detected at an advanced stage when successful treatment is no longer possible. As the tumor originates from the bile duct epithelium, bile is an ideal source of tumor biomarkers for cholangiocarcinoma. In this study, we used a quantitative proteomics approach to identify potential tumor-associated proteins in the bile fluid of six cholangiocarcinoma patients. Three different gross-appearance tumor types were used in the analysis: mass-forming type ( n = 2), periductal infiltrating type ( n = 2), and intraductal growth type ( n = 2). Two bile samples from non-cancerous patients were used as controls. Isobaric labeling, coupled with Tandem mass spectrometry, was used to quantify protein levels in the bile of cholangiocarcinoma and control patients. In all, 63 proteins were significantly increased in cholangiocarcinoma bile compared to normal bile. Alpha-1-antitrypsin was one of the overexpressed proteins that increased in cholangiocarcinoma bile samples. Immunohistochemical analysis revealed that alpha-1-antitrypsin was detected in 177 (50%) of 354 cholangiocarcinoma tissues from our Tissue Bank. Immunoblotting of 54 cholangiocarcinoma bile samples showed that alpha-1-antitrypsin was positive in 38 (70%) samples. Fecal enzyme-linked immunosorbent assay showed that alpha-1-antitrypsin level was able to distinguish cholangiocarcinoma patients from normal individuals. In conclusion, alpha-1-antitrypsin is a potential marker for early diagnosis of cholangiocarcinoma.

Subsets of Inflammatory Cytokine Gene Polymorphisms are Associated with Risk of Carcinogenic Liver Fluke Opisthorchis viverrini-Associated Advanced Periductal Fibrosis and Cholangiocarcinoma.

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF-, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1ß, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF- and APF+) cases. Polymorphisms in genes encoding IL-1ß-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α -308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α -308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 -174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.

Germline transgenesis and insertional mutagenesis in Schistosoma mansoni mediated by murine leukemia virus.

Functional studies will facilitate characterization of role and essentiality of newly available genome sequences of the human schistosomes, Schistosoma mansoni, S. japonicum and S. haematobium. To develop transgenesis as a functional approach for these pathogens, we previously demonstrated that pseudotyped murine leukemia virus (MLV) can transduce schistosomes leading to chromosomal integration of reporter transgenes and short hairpin RNA cassettes. Here we investigated vertical transmission of transgenes through the developmental cycle of S. mansoni after introducing transgenes into eggs. Although MLV infection of schistosome eggs from mouse livers was efficient in terms of snail infectivity, >10-fold higher transgene copy numbers were detected in cercariae derived from in vitro laid eggs (IVLE). After infecting snails with miracidia from eggs transduced by MLV, sequencing of genomic DNA from cercariae released from the snails also revealed the presence of transgenes, demonstrating that transgenes had been transmitted through the asexual developmental cycle, and thereby confirming germline transgenesis. High-throughput sequencing of genomic DNA from schistosome populations exposed to MLV mapped widespread and random insertion of transgenes throughout the genome, along each of the autosomes and sex chromosomes, validating the utility of this approach for insertional mutagenesis. In addition, the germline-transmitted transgene encoding neomycin phosphotransferase rescued cultured schistosomules from toxicity of the antibiotic G418, and PCR analysis of eggs resulting from sexual reproduction of the transgenic worms in mice confirmed that retroviral transgenes were transmitted to the next (F1) generation. These findings provide the first description of wide-scale, random insertional mutagenesis of chromosomes and of germline transmission of a transgene in schistosomes. Transgenic lines of schistosomes expressing antibiotic resistance could advance functional genomics for these significant human pathogens. DATABASE ACCESSION: Sequence data from this study have been submitted to the European Nucleotide Archive (http://www.ebi.ac.uk/embl) under accession number ERP000379.

Pseudotyped murine leukemia virus for schistosome transgenesis: approaches, methods and perspectives.

Draft genome sequences for the human schistosomes, Schistosoma japonicum, S. mansoni and S. haematobium are now available. The schistosome genome contains ~11,000 protein encoding genes for which the functions of few are well understood. Nonetheless, the newly described gene products and novel non-coding RNAs represent potential intervention targets, and molecular tools are being developed to determine their importance. Over the past decade, noteworthy advances has been reported towards development of tools for gene manipulation of schistosomes, including gene expression perturbation by RNAi, and transient and stable transfection including transgenesis mediated by genome integration competent vectors. Retrovirus-mediated transgenesis is an established functional genomic approach for model species. It offers the means to establish gain- or loss-of-function phenotypes, supports vector-based RNA interference, and represents a powerful forward genetics tool for insertional mutagenesis. Murine leukemia virus (MLV) pseudotyped with vesicular stomatitis virus glycoprotein mediates somatic transgenesis in S. mansoni, and vertical transmission of integrated transgenes in S. mansoni has been demonstrated, leading the establishment of transgenic lines. In addition, MLV transgenes encoding antibiotic resistance allow the selection of MLV-transduced parasites with the appropriate antibiotics. Here we describe detailed methods to produce and quantify pseudotyped MLV particles for use in transducing developmental stages of schistosomes. Approaches to analyze MLV-transduced schistosomes, including qPCR and high throughput approaches to verify and map genome integration of transgenes are also presented. We anticipate these tools should find utility in genetic investigations in other laboratories and for other helminth pathogens of important neglected tropical diseases.

Current State of Knowledge on Blood and Tissue-Based Biomarkers for Opisthorchis viverrini-induced Cholangiocarcinoma: A Review of Prognostic, Predictive, and Diagnostic Markers.

Cholangiocarcinoma (CCA) is a prevalent cancer in Southeast Asia, with Opisthorchis viverrini (O.viverrini) infection being the primary risk factor. Most CCA cases in this region are diagnosed at advanced stages, leading to unfavorable prognoses. The development of stage-specific biomarkers for Opisthorchis viverrini-induced cholangiocarcinoma (Ov-CCA) holds crucial significance, as it facilitates early detection and timely administration of curative interventions, effectively mitigating the high morbidity and mortality rates associated with this disease in the Great Mekong region. Biomarkers are a promising approach for early detection, prognosis, and targeted treatment of CCA. Disease-specific biomarkers facilitate early detection and enable monitoring of therapy effectiveness, allowing for any necessary corrections. This review provides an overview of the potential O. viverrini-specific molecular biomarkers and important markers for diagnosing and monitoring Ov-CCA, discussing their prognostic, predictive, and diagnostic value. Despite the limited research in this domain, several potential biomarkers have been identified, encompassing both worm-induced and host-induced factors. This review offers a thorough examination of historical and contemporary progress in identifying biomarkers through multiomics techniques, along with their potential implications for early detection and treatment.

Identification and functional analysis of mitogenic miRNA of the carcinogenic liver fluke Opisthorchis viverrini.

Opisthorchis viverrini is a pathogenic liver fluke that is known to cause cholangiocarcinoma in chronic infections. The underlying mechanism for this carcinogenesis is believed to be multifactorial, with parasite-derived excretory-secretory (ES) products potentially playing major roles. A recent study on these ES products has identified microRNAs (miRNA) that originate from O. viverrini but their influence on carcinogenesis remains understudied. Hence, we aimed to investigate the role of these miRNAs in the carcinogenesis of O. viverrini-associated cholangiocarcinoma. The mature miRNA sequences were retrieved from published data. Bioinformatics analysis was employed to identify miRNA targets and to identify potentially mitogenic miRNAs. An in vitro study was conducted to test the effects of miRNA on the bile duct epithelial cell lines. The miRNA target prediction analysis revealed that Ov_miRNA_EV_36/ovi-miR-3479a targets cancer-associated pathways. Hence, it was selected and used to assess its effect on the cell proliferation rate of H69 and MMNK-1 cholangiocyte cell lines. The results showed that Ov_miRNA_EV_36/ovi-miR-3479a induced significant cell proliferation in both cell lines when compared to negative controls. These results indicate that Ov_miRNA_EV_36/ovi-miR-3479a may play an essential role in the carcinogenesis of O. viverrini and therefore warrant further investigations.

Potential distribution of malaria vectors in Central Vietnam: A MaxEnt modeling approach.

Background and Aim: In Central Vietnam, Anopheles dirus and Anopheles minimus are the primary malaria vector species. These Anopheles spp.' distribution and prevalence are determined by environmental, climatic, and socioeconomic conditions. This study aimed to predict the potential distribution of these two Anopheles spp. in this region. Materials and Methods: This study was conducted in 15 Central Vietnamese provinces. From 2014 to 2018, we utilized An. dirus and An. minimus presence records. Proxy data from the Google Earth Engine platform for the study area, encompassing environmental, climatic, and socioeconomic factors. MaxEnt software predicted the potential environmental, climatic, and socioeconomic suitability of these two Anopheles spp. in Central Vietnam. Results: The test area under the curve values for An. dirus and An. minimus MaxEnt models averaged 0.801 and 0.806, respectively, showing excellent performance. Minimum air temperature had the greatest impact on the distribution of both species. A negative correlation between precipitation and normalized difference water index influences the occurrence of An. dirus. In the temperature range of 13-19.5°C, An. minimus is most likely to be present, with nighttime light detrimentally influencing its distribution. The Central Highlands region is inhabited by both species, with some presence in North-Central and South-Central Coastal areas. Conclusion: The importance of temperature in determining the presence of both species is emphasized by our findings, with subtle differences in the temperature-related factors shaping their distributions. The results highlight the need for focused malaria vector control and surveillance initiatives in the study area.