Molluscicide screening and identification of novel targets against Pomacea canaliculata.

BACKGROUND: Owing to the nonavailability of any clear targets for molluscicides against Pomacea canaliculata, target-based screening strategy cannot be employed. In this study, the molluscicidal effects of typical pesticides on P. canaliculata were evaluated to obtain the molluscicide target. A series of arylpyrrole compounds were synthesized based on the discovered target, and their structure-activity relationships explored. A preliminary strategy for screening molluscicides based on specific targets was also developed. RESULTS: A laboratory colony of P. canaliculata was developed, which showed no difference in sensitivity to niclosamide compared with the wild group, while exhibiting a higher stability against pesticide response. Mitochondrial adenosine triphosphate (ATP) synthase inhibitors and mitochondrial membrane potential uncouplers were identified and validated as potential targets for molluscicide screening against P. canaliculata. A series of arylpyrrole compounds were designed and synthesized. The median lethal concentration of 4-bromo-2-(4-chlorophenyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile (Compound 102) was 10-fold lower than that of niclosamide. CONCLUSION: New molluscicide targets were discovered and validated, and preliminary strategies were explored for pesticide screening based on these targets. Compound 102 exhibited a high molluscicidal activity and had a great potential value for exploring a molluscicide to control P. canaliculata. © 2024 Society of Chemical Industry.

B7-H4 reduces the infiltration of CD8+T cells and induces their anti-tumor dysfunction in gliomas.

B7-H4 is a promising immune checkpoint molecule in tumor immunotherapy. Our previous study showed that high B7-H4 expression was strongly correlated with deficiency in tumor infiltrated lymphocytes (TILs) in glioma patients. On this basis, we investigated the impact of B7-H4 on CD8+TILs in gliomas and the associated molecular mechanism here. B7-H4-positive tumor samples (n=129) from our glioma cohort were used to assess B7-H4 expression and CD8+TIL quantification by immunohistochemistry. CD8+TILs from five glioma patients cultured with B7-H4 protein were used to evaluate anti-tumor dysfunction by flow cytometry and ELISpot. An orthotopic murine glioma model was used to investigate the role of B7-H4 in glioma CD8+TILs by immunohisto- chemistry and flow cytometry. CD8+TILs from glioma patients cultured with B7-H4 protein were used to explore the potential molecular mechanism by RNA sequencing and western blot. Our results showed that glioma CD8+TIL density was negatively correlated with B7-H4 expression both in glioma patient cohort (P < 0.05) and orthotopic glioma murine model (P < 0.01). B7-H4 also lowered the expression of CD137 and CD103 (P < 0.05 for both) in glioma CD8+TILs and reduced their secretion of the anti-tumor cytokines IFN-γ and TNF-α (P < 0.01 for both) in a dose-dependent manner. Furthermore, B7-H4 was found to induce early dysfunction of glioma CD8+TILs by downregulating the phosphorylation of AKT and eNOS (P < 0.05 for both). In conclusion, B7-H4 reduced the infiltration of glioma CD8+TILs and induced an anti-tumor dysfunction phenotype. B7-H4 may also impair the anti-tumor function of glioma CD8+TILs via the AKT-eNOS pathway. These results indicated that B7-H4 may serve as a potential target in future glioma immunotherapy.

Anti-Inflammatory Responses Produced with Nippostrongylus brasiliensis-Derived Uridine via the Mitochondrial ATP-Sensitive Potassium Channel and Its Anti-Atherosclerosis Effect in an Apolipoprotein E Gene Knockout Mouse Model.

Atherosclerosis (AS) has become the leading cause of cardiovascular disease worldwide. Our previous study had observed that Nippostrongylus brasiliensis (Nb) infection or its derived products could inhibit AS development by inducing an anti-inflammatory response. We performed a metabolic analysis to screen Nb-derived metabolites with anti-inflammation activity and evaluated the AS-prevention effect. We observed that the metabolite uridine had higher expression levels in mice infected with the Nb and ES (excretory-secretory) products and could be selected as a key metabolite. ES and uridine interventions could reduce the pro-inflammatory responses and increase the anti-inflammatory responses in vitro and in vivo. The apolipoprotein E gene knockout (ApoE-/-) mice were fed with a high-fat diet for the AS modeling. Following the in vivo intervention, ES products or uridine significantly reduced serum and liver lipid levels, alleviated the formation of atherosclerosis, and reduced the pro-inflammatory responses in serum or plaques, while the anti-inflammatory responses showed opposite trends. After blocking with 5-HD (5-hydroxydecanoate sodium) in vitro, the mRNA levels of M2 markers were significantly reduced. When blocked with 5-HD in vivo, the degree of atherosclerosis was worsened, the pro-inflammatory responses were increased compared to the uridine group, while the anti-inflammatory responses decreased accordingly. Uridine, a key metabolite from Nippostrongylus brasiliensis, showed anti-inflammatory and anti-atherosclerotic effects in vitro and in vivo, which depend on the activation of the mitochondrial ATP-sensitive potassium channel.

Evaluation of the molluscicidal activities of arylpyrrole on Oncomelania hupensis, the intermediate host of Schistosoma japonicum.

The snail Oncomelania hupensis is the only intermediate host of the highly invasive parasite Schistosoma japonicum. Molluscicide is often used to curb transmission of S. japonicum. Niclosamide, the only World Health Organization (WHO) recognized molluscicide, presents major drawbacks, including high cost and toxicity towards aquatic animals. In the present study, a number of aryl pyrrole derivatives (ADs) were synthesized to serve as potential molluscicides and were tested on O. hupensis. To uncover the underlying mechanisms, adenosine triphosphate (ATP) and adenosine diphosphate (ADP) levels were assessed in the soft body of ADs-exposed O. hupensis, using high performance liquid chromatography (HPLC). The effect of C6 on key points of energy metabolism (the activities of complexes I, III, IV and the membrane potential) was determined. We demonstrated that the Compound 6 (C6, 4-bromo-1-(bromomethyl)-2-(4-chlorophenyl)-5-(trifluoromethyl)-1H-pyrrole-3-carbonitrile) exerted the strongest molluscicidal activity against adult O. hupensis at LC50 of 0.27, 0.19, and 0.13 mg/L for 24, 48, and 72 h respectively. Moreover, we found that the bromide on the pyrrole ring of C6 was essential for molluscicidal activity. Furthermore, the ATP content reduced from 194.46 to 139.75 µg/g after exposure to 1/2 LC50, and reduced to 93.06 µg/g after exposure to LC50. ADP, on the other hand, remained the same level before and after C6 exposure. We found that C6, at 1/2 LC50, reduced the membrane potential of O. hupensis, while no significant changes were observed in the activities of complexes I, III, and IV. C6 was identified with excellent activities on O. hupensis. The obtained structure-activity relationship and action mechanism study results should be useful for further compound design and development.

Discovery and Validation of a Novel Target of Molluscicides against Oncomelania hupensis, the Intermediate Host of Schistosoma japonicum.

In this study, 196 strains of actinomycetes isolated from marshland soil samples were tested for molluscicidal activity against Oncomelania hupensis. Five strains demonstrated molluscicidal activity, of which the molluscicidal efficiency of Actinomycetes strain A183 was the maximum. After the fermentation supernatant of actinomycetes A183 was extracted with ethyl acetate (EWEA), the LC50 of the EWEA after leaching for 48 h and 72 h were 0.2688 and 0.2195 mg/L, respectively. The effect of EWEA on the key points of energy metabolism was determined. We noted that 1 mg/L of EWEA (A813) significantly reduced the activity of mitochondrial respiratory chain complex I (P < 0.05), while no significant changes were observed in the activities of complexes II, III, and IV. In addition, EWEA (A813) could decrease the membrane potential of O. hupensis purified mitochondria in vitro. The LC50 of the 3 uncoupler (FCCP, DNP, and Tyrphostin A9) after immersion for 24 h were 0.065, 0.135, and 0.110 mg/L, respectively; LC50 after 48 h treatment was 0.064, 0.124, and 0.082 mg/L, respectively; LC50 after 72 h treatment was 0.063, 0.129, and 0.061 mg/L, respectively, and all uncoupler showed strong molluscicidal activities, demonstrating that the mitochondrial membrane potential uncoupling is a potential target for molluscicides against O. hupensis. Moreover, the molluscicidal active substance of strain A183 needs to be further isolated, purified, and structurally characterized considering its promising potential applications.