Barcode sequence could be a good target for developing a species-specific anti-parasite agent based on CRISPR-Cas9.

Parasitic infections are a severe issue in many regions of the world. We assume that if a chemical can destroy a DNA barcode sequence, then this chemical could be developed as a species-specific parasiticidal agent. To test this hypothesis, we designed sgRNAs that target the sequences of both a DNA barcode (ITS-2) and a control (5.8S rDNA) in Cryptocaryon irritans. In in vivo tests, we found that exposure to Cas9 mRNA mixed with sgRNAs was able to significantly reduce the hatching rate of tomont and the survival rate of theront. Quantitative Real-time PCR demonstrated that the DNAs of tomont and theront exposed to sgRNAs and Cas9 mRNA were significantly disrupted, no matter whether they were exposed to a single sgRNA or a mixture of two sgRNAs. DNA sequencing also suggested the test group that was exposed to a single sgRNA mixed with Cas9-induced mutation at sgRNA targeted fragments and the test group exposed to two sgRNAs combined with Cas9-induced deletion of large pieces. The findings and principles provided by this study contribute to the development of novel nucleic acid therapeutic drugs for cryptocaryoniasis and other parasitic diseases and provide insight into the development of species-specific parasiticidal agents.

Antioxidant and anti-inflammatory activities of ethyl acetate extract of Gynura formosana (Kitam) leaves.

Gynura formosana Kitam (family Compositae) has traditionally been used for the prevention of diabetes, cancer and inflammation in China. However, there are few reports of its anti-inflammatory effects. In the present study, after assessing the in vitro antioxidant activities of extracts from Gynura formosana Kitam leaves, the anti-inflammatory and antioxidant activities of ethyl acetate extract of Gynura formosana Kitam leaves (EAEG) were further investigated in rats using a cotton pellet-induced model of granuloma. EAEG significantly inhibited the formation of cotton pellet-induced granuloma in rats in a dose-dependent manner. Moreover, EAEG treatment significantly decreased the level of plasma C-reactive protein and suppressed the activities of plasma glutamate pyruvate transaminase and lactate dehydrogenase in model groups. Furthermore, EAEG increased the hepatic levels of anti-oxidative enzymes or antioxidants, including catalase, superoxide dismutase and reduced glutathione, and reduced the level of lipid peroxidation in the rat model of inflammation. In addition, EAEG decreased plasma levels of tumor necrosis factor-α and interleukin-1ß in the rat model of inflammation. Therefore, our results indicated that EAEG had potent anti-inflammatory effects, which was achieved at least in part through activating antioxidant enzyme activities and suppressing the production of proinflammatory mediators by macrophages.