Identification of the protease inhibitory domain of Trichinella spiralis novel cystatin (TsCstN).

The Trichinella spiralis novel cystatin (TsCstN) inhibits cathepsin L (CatL) activity and inflammation of macrophages during lipopolysaccharide (LPS) induction. To identify the protease inhibitory region, this study applied an in silico modeling approach to simulate truncation sites of TsCstN (Ts01), which created four truncated forms, including TsCstN∆1-39 (Ts02), TsCstN∆1-71 (Ts03), TsCstN∆1-20, ∆73-117 (Ts04), and TsCstN∆1-20, ∆42-117 (Ts05). The superimposition of these truncates modeled with AlphaFold Colab indicated that their structures were more akin to Ts01 than those modeled with I-TASSER. Moreover, Ts04 exhibited the closest resemblance to the structure of Ts01. The recombinant Ts01 (rTs01) and truncated proteins (rTs02, rTs03, and rTs04) were successfully expressed in a prokaryotic expression system while Ts05 was synthesized, with sizes of approximately 14, 12, 8, 10, and 2.5 kDa, respectively. When determining the inhibition of CatL activity, both rTs01 and rTs04 effectively reduced CatL activity in vitro. Thus, the combination of the α1 and L1 regions may be sufficient to inhibit CatL. This study provides comprehensive insights into TsCstN, particularly regarding its protein function and inhibitory domains against CatL.

Exploring urinary proteomics and peptidomics biomarkers for the diagnosis of mekong schistosomiasis.

Schistosomiasis caused by Schistosoma mekongi is one of the causative agents of human blood fluke infection in the lower Mekong River. Traditionally, the detection of egg morphology in stool samples has served as the prevailing method for diagnosing Schistosoma infection. Nonetheless, this approach exhibits low sensitivity, particularly in early infection detection. Urine has been extensively studied as a noninvasive clinical sample for diagnosing infectious diseases. Despite this, urine proteomic analysis of S. mekongi infection has been less investigated. This study aimed to characterize proteins and peptides present in mouse urine infected with S. mekongi both before infection and at intervals of 1, 2, 4, and 8 weeks post-infection using mass spectrometry-based proteomics. Proteomics analysis revealed 13 up- and only one down-regulated mouse protein consistently found across all time points. Additionally, two S. mekongi uncharacterized proteins were detected throughout the infection period. Using a peptidomics approach, we consistently identified two peptide sequences corresponding to S. mekongi collagen alpha-1(V) in mouse urine across all time points. These findings highlight the potential of these unique proteins, particularly the S. mekongi uncharacterized proteins and collagen alpha-1(V), as potential biomarkers for early detection of S. mekongi infection. Such insights could significantly advance diagnostic strategies for human Mekong schistosomiasis.

A reanalysis and integration of transcriptomics and proteomics datasets unveil novel drug targets for Mekong schistosomiasis.

Schistosomiasis, caused by Schistosoma trematodes, is a significant global health concern, particularly affecting millions in Africa and Southeast Asia. Despite efforts to combat it, the rise of praziquantel (PZQ) resistance underscores the need for new treatment options. Protein kinases (PKs) are vital in cellular signaling and offer potential as drug targets. This study focused on focal adhesion kinase (FAK) as a candidate for anti-schistosomal therapy. Transcriptomic and proteomic analyses of adult S. mekongi worms identified FAK as a promising target due to its upregulation and essential role in cellular processes. Molecular docking simulations assessed the binding energy of FAK inhibitors to Schistosoma FAK versus human FAK. FAK inhibitor 14 and PF-03814735 exhibited strong binding to Schistosoma FAK with minimal binding for human FAK. In vitro assays confirmed significant anti-parasitic activity against S. mekongi, S. mansoni, and S. japonicum, comparable to PZQ, with low toxicity in human cells, indicating potential safety. These findings highlight FAK as a promising target for novel anti-schistosomal therapies. However, further research, including in vivo studies, is necessary to validate efficacy and safety before clinical use. This study offers a hopeful strategy to combat schistosomiasis and reduce its global impact.

Molecular and biochemical characterizations of a Fasciola gigantica retinoid X receptor-α isoform A (FgRXRα-A).

Fascioliasis is a parasitic infection in animals and humans caused by the parasitic flatworm genus Fasciola, which has two major species, F. hepatica and F. gigantica. A major concern regarding this disease is drug resistance, which is increasingly reported worldwide. Hence, the discovery of a novel drug as well as drug targets is crucially required. Therefore, this study aims to characterize the novel drug target in the adult F. gigantica. In the beginning, we hypothesized that the parasite might interact with some host molecules when it lives inside the liver parenchyma or bile ducts, specifically hormones and hormone-like molecules, through the specific receptors, primarily nuclear receptors (NRs), which are recognized as a major drug target in various diseases. The retinoid X receptor (RXR) is a member of subfamily 2 NRs that plays multitudinous roles in organisms by forming homodimers or heterodimers with other NRs. We obtained the full-length amino acid sequences of F. gigantica retinoid X receptor-alpha (FgRXRα-A) from the transcriptome of F. gigantica that existed in the NCBI database. The FgRXRα-A were computationally predicted for the basic properties, multiple aligned, phylogeny analyzed, and generated of 2D and 3D models. Moreover, FgRXRα-A was molecular cloned and expressed as a recombinant protein (rFgRXRα-A), then used for immunization for specific polyclonal antibodies. The native FgRXRα-A was detected in the parasite extracts and tissues, and the function was investigated by in vitro binding assay. The results demonstrated the conservation of FgRXRα-A to the other RXRs, especially RXRs from the trematodes. Interestingly, the native FgRXRα-A could be detected in the testes of the parasite, where the sex hormones are accumulated. Moreover, the binding assay revealed the interaction of 9-cis retinoic acid and FgRXRα-A, suggesting the function of FgRXRα-A. Our findings suggested that FgRXRα-A will be involved with the sexual reproduction of the parasite by forming heterodimers with other NRs, and it could be the potential target for further drug development of fascioliasis.

Sericin promotes chondrogenic proliferation and differentiation via glycolysis and Smad2/3 TGF-ß signaling inductions and alleviates inflammation in three-dimensional models.

Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-ß signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1ß, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-ß signaling pathways, and exhibiting anti-inflammatory properties.

Identification of trans-genus biomarkers for early diagnosis of intestinal schistosomiasis and progression of gut pathology in a mouse model using metabolomics.

Schistosomiasis is one of the most devastating human diseases worldwide. The disease is caused by six species of Schistosoma blood fluke; five of which cause intestinal granulomatous inflammation and bleeding. The current diagnostic method is inaccurate and delayed, hence, biomarker identification using metabolomics has been applied. However, previous studies only investigated infection caused by one Schistosoma spp., leaving a gap in the use of biomarkers for other species. No study focused on understanding the progression of intestinal disease. Therefore, we aimed to identify early gut biomarkers of infection with three Schistosoma spp. and progression of intestinal pathology. We infected 3 groups of mice, 3 mice each, with Schistosoma mansoni, Schistosoma japonicum or Schistosoma mekongi and collected their feces before and 1, 2, 4 and 8 weeks after infection. Metabolites in feces were extracted and identified using mass spectrometer-based metabolomics. Metabolites were annotated and analyzed with XCMS bioinformatics tool and Metaboanalyst platform. From >36,000 features in all conditions, multivariate analysis found a distinct pattern at each time point for all species. Pathway analysis reported alteration of several lipid metabolism pathways as infection progressed. Disturbance of the glycosaminoglycan degradation pathway was found with the presence of parasite eggs, indicating involvement of this pathway in disease progression. Biomarkers were discovered using a combination of variable importance for projection score cut-off and receiver operating characteristic curve analysis. Five molecules met our criteria and were present in all three species: 25-hydroxyvitamin D2, 1α-hydroxy-2ß-(3-hydroxypropoxy) vitamin D3, Ganoderic acid Md, unidentified feature with m/z 455.3483, and unidentified feature with m/z 456.3516. These molecules were proposed as trans-genus biomarkers of early schistosomiasis. Our findings provide evidence for disease progression in intestinal schistosomiasis and potential biomarkers, which could be beneficial for early detection of this disease.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

BACKGROUND: Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O. viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O. viverrini infection in field-collected human feces. METHODS: To detect O. viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O. viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. RESULTS: The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O. viverrini DNA. On the basis of the detection limit, the assay could detect O. viverrini DNA at concentrations as low as 10-1 ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1 ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O. viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. CONCLUSIONS: The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O. viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O. viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Evaluation of Immunodiagnostic Performances of Neospora caninum Peroxiredoxin 2 (NcPrx2), Microneme 4 (NcMIC4), and Surface Antigen 1 (NcSAG1) Recombinant Proteins for Bovine Neosporosis.

Bovine neosporosis is among the main causes of abortion in cattle worldwide, causing serious economic losses in the beef and dairy industries. A highly sensitive and specific diagnostic method for the assessment of the epidemiology of the disease, as well as it surveillance and management, is imperative, due to the absence of an effective treatment or vaccine against neosporosis. In the present study, the immunodiagnostic performance of Neospora caninum peroxiredoxin 2 (NcPrx2), microneme 4 (NcMIC4), and surface antigen 1 (NcSAG1) to detect IgG antibodies against N. caninum in cattle were evaluated and compared with that of the indirect fluorescent antibody test (IFAT). The results revealed that NcSAG1 had the highest sensitivity and specificity, with values of 88.4% and 80.7%, respectively, followed by NcPrx2, with a high sensitivity of 87.0% but a low specificity of 67.0%, whereas NcMIC4 showed sensitivity and specificity of 84.1% and 78.9%, respectively, when compared with IFAT. A high degree of agreement was observed for NcSAG1 (k = 0.713) recombinant protein, showing the highest diagnostic capability, followed by NcMIC4 (k = 0.64) and NcPrx2 (k = 0.558). The present study demonstrates that NcSAG1 is helpful as an antigen marker and also demonstrates the potential immunodiagnostic capabilities of NcPrx2 and NcMIC4, which could serve as alternative diagnostic markers for detecting N. caninum infection in cattle. These markers may find utility in future treatment management, surveillance, and risk assessment of neosporosis in livestock or other animal host species. Further research should be directed toward understanding the in vivo immune response differences resulting from immunization with both recombinant proteins.

Anthelmintic efficacy evaluation and mechanism of N-methylbenzo[d]oxazol-2-amine.

Parasitic roundworms cause significant sickness and mortality in animals and humans. In livestock, these nematodes have severe economic impact and result in losses in food production on a global scale. None of the currently available drugs ideally suit all treatment circumstances, and the development of drug-resistant nematode strains has become a challenge to control the infection. There is an urgent need to develop novel anthelmintic compounds. According to our previous report, N-methylbenzo[d]oxazol-2-amine (1) showed anthelmintic activity and lowest cytotoxicity. In this study, in vivo anthelmintic properties were evaluated using Trichinella spiralis infected mice. Toxicity was evaluated using the rats and mode of action using molecular docking and metabolomics approaches. The in vivo results demonstrate that a dose of 250 mg/kg reduced the T. spiralis abundance in the digestive tract by 49%. The 250 mg/kg Albendazole was served as control. The relatively low acute toxicity was categorized into chemical category 5, with an LD50 greater than 2000 mg/kg body. Molecular docking analysis showed the T. spiralis tubulin beta chain and glutamate-gated channels might not be the main targets of compound 1. Metabolomics analysis was used to explain the effects of compound 1 on the T. spiralis adult worm. The results demonstrated that compound 1 significantly up-regulated the metabolism of purine, pyrimidine and down-regulated sphingolipid metabolism. In conclusion, compound 1 could be a potential molecule for anthelmintic development. The bioavailability, pharmacokinetics, and absorption of this compound should be studied further to provide information for its future efficacy improvement.

Amelioration of ovalbumin-induced lung inflammation in a mouse model by Trichinella spiralis novel cystatin.

Background and Aims: Asthma, a chronic disease affecting humans and animals, has recently become increasingly prevalent and steadily widespread. The alternative treatment of asthma using helminth infections or helminth-derived immunomodulatory molecules (IMs) has been evaluated and demonstrated significant amelioration of disease severity index in vitro and in vivo. Trichinella spiralis, a parasitic nematode and its IMs, elicits a potential to relieve asthma and other immune-related disorders. In this study, we investigated the immunomodulatory function of recombinant T. spiralis novel cystatin (rTsCstN) in ameliorating acute inflammatory asthma disorders in a murine model. Materials and Methods: Female BALB/c mice were sensitized using intraperitoneal injection of ovalbumin (OVA)/alum and subsequently challenged with intranasal administration of OVA alone or OVA + rTsCstN for 3 consecutive days, producing OVA-induced allergic asthma models. To evaluate the therapeutic efficacy of rTsCstN, the inflammatory cells and cytokines in bronchoalveolar lavage fluid (BALF) and OVA-specific immunoglobulin E levels in serum were assessed. Histological alterations in the lung tissues were determined by hematoxylin and eosin (H&E) staining and eventually scored for the extent of inflammatory cell infiltration. Results: The asthmatic mouse models challenged with OVA + rTsCstN demonstrated a significant reduction of eosinophils (p < 0.01), macrophages (p < 0.05), and cytokines tumor necrosis factor-α (p < 0.05) and interferon (IFN)-γ (p < 0.05) in BALF when compared with the mice challenged with OVA alone. However, the levels of interleukin (IL)-4 and IL-10 remained unchanged. Histological examination revealed that mice administered OVA + rTsCstN were less likely to have inflammatory cell infiltration in their perivascular and peribronchial lung tissues than those administered OVA alone. Conclusion: Recombinant T. spiralis novel cystatin demonstrated immunomodulatory effects to reduce severe pathogenic alterations in asthma mouse models, encouraging a viable alternative treatment for asthma and other immunoregulatory disorders in humans and animals in the future.

Metabolite profiling of Trichinella spiralis adult worms and muscle larvae identifies their excretory and secretory products.

Human trichinellosis is a parasitic infection caused by roundworms belonging to the genus Trichinella, especially Trichinella spiralis. Early and accurate clinical diagnoses of trichinellosis are required for efficacious prognosis and treatment. Current drug therapies are limited by antiparasitic resistance, poor absorption, and an inability to kill the encapsulating muscle-stage larvae. Therefore, reliable biomarkers and drug targets for novel diagnostic approaches and anthelmintic drugs are required. In this study, metabolite profiles of T. spiralis adult worms and muscle larvae were obtained using mass spectrometry-based metabolomics. In addition, metabolite-based biomarkers of T. spiralis excretory-secretory products and their related metabolic pathways were characterized. The metabolic profiling identified major, related metabolic pathways involving adenosine monophosphate (AMP)-dependent synthetase/ligase and glycolysis/gluconeogenesis in T. spiralis adult worms and muscle larvae, respectively. These pathways are potential drug targets for the treatment of the intestinal and muscular phases of infection. The metabolome of larva excretory-secretory products was characterized, with amino acid permease and carbohydrate kinase being identified as key metabolic pathways. Among six metabolites, decanoyl-l-carnitine and 2,3-dinor-6-keto prostaglandin F1α-d9 were identified as potential metabolite-based biomarkers that might be related to the host inflammatory processes. In summary, this study compared the relationships between the metabolic profiles of two T. spiralis growth stages. Importantly, the main metabolites and metabolic pathways identified may aid the development of novel clinical diagnostics and therapeutics for human trichinellosis and other related helminthic infections.

Human gnathostomiasis: A review on the biology of the parasite with special reference on the current therapeutic management.

Gnathostoma is a parasitic nematode that can infect a wide range of animal species, but human populations have become accidental hosts because of their habit of eating raw or undercooked meat from a wide variety of intermediate hosts. While gnathostomiasis is considered an endemic disease, cases of human gnathostomiasis have been increasing over time, most notably in nonendemic areas. There are several complexities to this parasitic disease, and this review provides an update on human gnathostomiasis, including the life cycle, diagnosis, treatment, and treatment strategies used to combat drug resistance. Even now, a definitive diagnosis of gnathostomiasis is still challenging because it is difficult to isolate larvae for parasitological confirmation. Another reason is the varying clinical symptoms recorded in reported cases. Clinical cases can be confirmed by immunodiagnosis. For Gnathosotoma spinigerum, the detection of IgG against a specific antigenic band with a molecular weight of 24 kDa from G. spinigerum advanced third-stage larvae (aL3), while for other species of Gnathostoma including G. binucleatum, the 33-kDa antigen protein is being used. This review also discusses cases of recurrence of gnathostomiasis and resistance mechanisms to two effective chemotherapeutics (albendazole and ivermectin) used against gnathostomiasis. This is significant, especially when planning strategies to combat anthelmintic resistance. Lastly, while no new chemotherapeutics against gnathostomiasis have been made available, we describe the management of recurrent gnathostomiasis using albendazole and ivermectin combinations or extensions of drug treatment plans.

Evaluation of Kato-Katz and multiplex quantitative polymerase chain reaction performance for clinical helminth infections in Thailand using a latent class analysis.

Using an appropriate diagnostic tool is essential to soil-transmitted helminth control and elimination efforts. Kato-Katz (KK) is the most commonly used diagnostic, but recently other tools, such as real-time quantitative polymerase chain reaction (multiplex qPCR), are starting to be employed more. Here, we evaluated the performance of these two diagnostic tools for five helminth species in Thailand. In the absence of a gold standard, diagnostic performance can be evaluated using latent class analysis. Our results suggest that in moderate to high prevalence settings above 2% multiplex qPCR could be more sensitive than KK, this was particularly apparent for Opisthorchis viverrini in the northeastern provinces. However, for low prevalence, both diagnostics suffered from low sensitivity. Specificity of both diagnostics was estimated to be high (above 70%) across all settings. For some specific helminth infection such as O. viverrini, multiplex qPCR is still a preferable choice of diagnostic test. KK performed equally well in detecting Ascaris lumbricoides and Taenia solium when the prevalence is moderate to high (above 2%). Neither test performed well when the prevalence of infection is low (below 2%), and certainly in the case for hookworm and Trichuris trichiura. Combination of two or more diagnostic tests can improve the performance although the cost would be high. Development of new methods for helminth surveillance at the pre-elimination phase is therefore very important. This article is part of the theme issue 'Challenges and opportunities in the fight against neglected tropical diseases: a decade from the London Declaration on NTDs'.

Venom-gland transcriptomics of the Malayan pit viper (Calloselasma rhodostoma) for identification, classification, and characterization of venom proteins.

The Malayan pit viper (Calloselasma rhodostoma) is a hemotoxic snake widely found in Southeast Asia and is responsible for the majority of poisoning cases in this region, including Thailand. However, a comprehensive knowledge of the venom protein profile and classification, as well as novel venom proteins, of this viper is still limited. Recently, the detailed composition of several snake venoms has been discovered through the use of transcriptome analysis. Therefore, the aim of this study was to employ a next-generation sequencing platform and bioinformatics analysis to undertake venom-gland de novo transcriptomics of Malayan pit vipers. Furthermore, 21,272 functional coding genes were identified from 36,577 transcripts, of which 314 transcripts were identified as toxin proteins, accounting for 61.41% of total FPKM, which can be categorized into 22 toxin gene families. The most abundant are snake venom metalloproteinase kistomin (P0CB14) and zinc metalloproteinase/disintegrin (P30403), which account for 60.47% of total toxin FPKM and belong to the SVMP toxin family, followed by snake venom serine protease 1 (O13059) and Snaclec rhodocetin subunit beta (P81398), which account for 6.84% and 5.50% of total toxin FPKM and belong to the snake venom serine protease (SVSP) and Snaclec toxin family, respectively. Amino acid sequences of the aforementioned toxins were compared with those identified in other important medical hemotoxic snakes from Southeast Asia, including the Siamese Russell's viper (Daboia siamensis) and green pit viper (Trimeresurus albolabris), in order to analyze their protein homology. The results demonstrated that ranges of 58%-62%, 31%-60%, and 48%-59% identity was observed among the SVMP, Snaclec, and SVSP toxin families, respectively. Understanding the venom protein profile and classification is essential in interpreting clinical symptoms during human envenomation and developing potential therapeutic applications. Moreover, the variability of toxin families and amino acid sequences among related hemotoxic snakes found in this study suggests the use and development of universal antivenom for the treatment of envenomating patients is still challenging.

Untargeted serum metabolomics analysis of Trichinella spiralis-infected mouse.

BACKGROUND: Trichinellosis, caused by a parasitic nematode of the genus Trichinella, is a zoonosis that affects people worldwide. After ingesting raw meat containing Trichinella spp. larvae, patients show signs of myalgia, headaches, and facial and periorbital edema, and severe cases may die from myocarditis and heart failure. The molecular mechanisms of trichinellosis are unclear, and the sensitivity of the diagnostic methods used for this disease are unsatisfactory. Metabolomics is an excellent tool for studying disease progression and biomarkers; however, it has never been applied to trichinellosis. We aimed to elucidate the impacts of Trichinella infection on the host body and identify potential biomarkers using metabolomics. METHODOLOGY/PRINCIPAL FINDINGS: Mice were infected with T. spiralis larvae, and sera were collected before and 2, 4, and 8 weeks after infection. Metabolites in the sera were extracted and identified using untargeted mass spectrometry. Metabolomic data were annotated via the XCMS online platform and analyzed with Metaboanalyst version 5.0. A total of 10,221 metabolomic features were identified, and the levels of 566, 330, and 418 features were significantly changed at 2-, 4-, and 8-weeks post-infection, respectively. The altered metabolites were used for further pathway analysis and biomarker selection. A major pathway affected by Trichinella infection was glycerophospholipid metabolism, and glycerophospholipids comprised the main metabolite class identified. Receiver operating characteristic revealed 244 molecules with diagnostic power for trichinellosis, with phosphatidylserines (PS) being the primary lipid class. Some lipid molecules, e.g., PS (18:0/19:0)[U] and PA (O-16:0/21:0), were not present in metabolome databases of humans and mice, thus they may have been secreted by the parasites. CONCLUSIONS/SIGNIFICANCE: Our study highlighted glycerophospholipid metabolism as the major pathway affected by trichinellosis, hence glycerophospholipid species are potential markers of trichinellosis. The findings of this study represent the initial steps in biomarker discovery that may benefit future trichinellosis diagnosis.

Discovery of N-methylbenzo[d]oxazol-2-amine as new anthelmintic agent through scalable protocol for the synthesis of N-alkylbenzo[d]oxazol-2-amine and N-alkylbenzo[d]thiazol-2-amine derivatives.

We discovered a lead compound, N-methylbenzo[d]oxazol-2-amine (2a), which had comparable potency to albendazole, an orally administered anthelminticdrug, against Gnathostoma spinigerum, Caenorhabditis elegans and Trichinella spiralis. Compound 2a showed about 10 times lower cytotoxicity towards normal human cell line (HEK293) than albendazole. Moreover, we have developed new processes for the synthesis of N-alkylbenzo[d]oxazol-2-amine and N-alkylbenzo[d]thiazol-2-amine derivatives via metal-free conditions. This protocol could serve as a robust and scalable method, especially, to synthesize N-methylbenzo[d]oxazol-2-amine and N-methylbenzo[d]thiazol-2-amine derivatives which were difficult to prepare using other metal-free conditions. The method employed benzoxazole-2-thiol or benzothiazole-2-thiol as the substrate. The reaction was triggered by methylation of the thiol functional group to form the methyl sulfide intermediate, a crucial tactic, which facilitated in a smooth nucleophilic addition-elimination reaction with gaseous methylamine generated in situ from N-methylformamide. In addition, the proteomic analysis of compound 2a was also studied in this work.

Molecular Cloning and Characterization of a Fasciola gigantica Nuclear Receptor Subfamily 1 (FgNR1).

Fasciola gigantica, a giant liver fluke, causes tremendous loss to the livestock economy in several regions throughout the world. The situation of drug resistance has been emerging increasingly; therefore, novel drugs and drug targets need to be discovered. The adult F. gigantica inhabits the major bile ducts where bile salts accumulate­these are steroid-like molecules that mediate several physiological processes in organisms through interacting with their specific nuclear receptors. However, the molecular mechanism of the interaction in the parasitic organisms have not been clearly understood. In this study, putative nuclear receptor subfamily 1 of F. gigantica (FgNR1) was identified. Nucleotide and amino acid sequences of the FgNR1 homolog were obtained from the transcriptome of F. gigantica and predicted for properties and functions using bioinformatics. The full-length cDNA was cloned and expressed in the bacterial expression system and then used for immunization. Western analysis and immunolocalization suggested that FgNR1 could be detected in the crude worm antigens and was highly expressed in the caeca and testes of the adult parasite. Moreover, the bile could significantly activate the expression of FgNR1 in cultured parasites. Our results indicated that FgNR1 has high potential for the development of a novel anthelminthic drug in the future.

Transcriptome profiling of male and female Ascaris lumbricoides reproductive tissues.

BACKGROUND: Ascaris lumbricoides causes human ascariasis, the most prevalent helminth disease, infecting approximately 1 billion individuals globally. In 2019 the global disease burden was estimated to be 754,000 DALYs and resulted in 2090 deaths. In the absence of a vaccination strategy, treatment of ascariasis has relied on anthelminthic chemotherapy, but drug resistance is a concern. The propensity for reinfection is also a major challenge to disease control; female worms lay up to 200,000 eggs daily, which contaminate surrounding environments and remain viable for years, resulting in high transmission rates. Understanding the molecular mechanisms of reproductive processes, including control of egg production, spermatogenesis, oogenesis and embryogenesis, will drive the development of new drugs and/or vaccine targets for future ascariasis control. METHODS: Transcriptome profiles of discrete reproductive and somatic tissue samples were generated from adult male and female worms using Illumina HiSeq with 2 × 150 bp paired-end sequencing. Male tissues included: testis germinal zone, testis part of vas deferens, seminal vesicle and somatic tissue. Female tissues included: ovary germinal zone, ovary part of the oviduct, uterus and somatic tissue. Differentially expressed genes (DEGs) were identified from the fragments per kilobases per million reads (FPKM) profiles. Hierarchical analysis was performed to identify tissue-specific genes. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were employed to identify significant terms and pathways for the DEGs. RESULTS: DEGs involved in protein phosphorylation and adhesion molecules were indicated to play a crucial role in spermatogenesis and fertilization, respectively. Those genes associated with the G-protein-coupled receptor (GPCR) signaling pathway and small GTPase-mediated signal transduction pathway play an essential role in cytoskeleton organization during oogenesis. Additionally, DEGs associated with the SMA genes and TGF-ß signaling pathway are crucial in adult female embryogenesis. Some genes associated with particular biological processes and pathways that were identified in this study have been linked to defects in germline development, embryogenesis and reproductive behavior. In the enriched KEGG pathway analysis, Hippo signaling, oxytocin signaling and tight junction pathways were identified to play a role in Ascaris male and female reproductive systems. CONCLUSIONS: This study has provided comprehensive transcriptome profiles of discrete A. lumbricoides reproductive tissue samples, revealing the molecular basis of these functionally important tissues. The data generated from this study will provide fundamental knowledge on the reproductive biology of Ascaris and will inform future target identification for anti-ascariasis drugs and/or vaccines.