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Lichtheimia corymbifera is considered as one of the most frequent agents of mucormycosis. The lack of efficient genetic manipulation tools hampers the characterization of the pathomechanisms and virulence factors of this opportunistic pathogenic fungus. Although such techniques have been described for certain species, the performance of targeted mutagenesis and the construction of stable transformants have remained a great challenge in Mucorales fungi. In the present study, a plasmid-free CRISPR-Cas9 system was applied to carry out a targeted gene disruption in L. corymbifera. The described method is based on the non-homologous end-joining repair of the double-strand break caused by the Cas9 enzyme. Using this method, short, one-to-five nucleotide long-targeted deletions could be induced in the orotidine 5’-phosphate decarboxylase gene (pyrG) and, as a result, uracil auxotrophic strains were constructed. These strains are applicable as recipient strains in future gene manipulation studies. As we know, this is the first genetic modification of this clinically relevant fungus.

Anastatica hierochuntica L. (Brassicaceae) has been used in folk medicine to treat diseases related to inflammation and pain. This study investigated the antinociceptive and antiinflammatory effects of the aqueous extract and the chloroform fraction of A. hierochuntica plant and possible mechanism of action in Swiss albino mice and Wistar rats. The antinociceptive activity was evaluated using the hot-plate, tail immersion, writhing and formalin-induced paw liking tests, while antiinflammatory action was investigated using the carrageenan-induced paw edema in over a period of 5 h in the dose range of 3.16 to 1000 mg/kg, i.p. These effects were compared to those of the vehicle, morphine, indomethacin and the influence of naloxone. Aqueous extract and the chloroform fraction of A. hierochuntica produced significant increase in the pain latency time in the hot-plate and tail-immersion tests (ED50s, 31.0 and 26.3 mg/kg for aqueous extract of A. hierochuntica, 15.0 and 10.1 mg/kg for A. hierochuntica chloroform fraction, respectively). In the acetic acid-induced writhing and formalin test, administration of the aqueous extract and the chloroform fraction of A. hierochuntica significantly reduced the number of writhings (ED50, 28.6 and 8.7 mg/kg, respectively) and time of licking in early (ED50, 18.2 and 10.6 mg/kg, respectively) and late phase (ED50, 18.9 and 11.9 mg/kg, respectively) of formalin-induced pain. Pretreatment of animals with naloxone significantly reversed the antinociceptive effects of the aqueous extract and the chloroform fraction of A. hierochuntica when assessed against hot-plate and formalin test. The aqueous extract and the chloroform fraction of A. hierochuntica significantly inhibited edema formation in carrageenan-induced edema assay (ED50, 29.1 and 6.5 mg/kg, respectively). These results demonstrated that A. hierochuntica has potential central and peripheral antinociceptive effects as well as antiinflammatory activity.