METHODS: Anti-SARS-CoV-2 potential of the SKF7® was evaluated in SARS-CoV-2-infected Vero E6 cells and SARS-CoV-2-infected A549 cells by cytopathic effect-based assay and RT-qPCR, respectively. Target based assays were performed on the SKF7® against the S1-ACE2 interaction and 3CL protease activities. Anti-inflammatory activity of the SKF7® was evaluated by nitric oxide inhibitory and TLR2/TLR4 receptor blocker assays.
RESULTS: The SKF7® inhibited wild-type Wuhan (EC50 of 21.99 µg/mL) and omicron (EC50 of 16.29 µg/mL) SARS-CoV-2 infections in Vero-E6 cells. The SKF7® also inhibited the wild-type SARS-CoV-2 infection in A549 cells (EC50 value of 6.31 µg/mL). The SKF7® prominently inhibited 3CL protease activity. The SKF7® inhibited the LPS induced-TLR4 response with the EC50 of 16.19 µg/mL.
CONCLUSIONS: In conclusion, our in vitro study highlighted anti-SARS-CoV-2 and anti-inflammatory potentials of the SKF7®. Future pre-clinical in vivo studies focusing on antiviral and immunomodulatory potentials of the SKF7® in affecting the COVID-19 pathogenesis are warranted.
RESULTS: We show that SYMRK is essential for nodulation and endomycorrhization in Parasponia andersonii. Subsequently, it is revealed that the 5'-intron donor splice site of SYMRK intron 12 is variable and, in most dicotyledon species, doesn't contain the canonical dinucleotide 'GT' signature but the much less common motif 'GC'. Strikingly, in T. orientalis, this motif is converted into a rare non-canonical 5'-intron donor splice site 'GA'. This SYMRK allele, however, is fully functional and spreads in the T. orientalis population of Malaysian Borneo. A further investigation into the occurrence of the non-canonical GA-AG splice sites confirmed that these are extremely rare.
CONCLUSION: SYMRK functioning is highly conserved in legumes, actinorhizal plants, and Parasponia. The gene possesses a non-common 5'-intron GC donor splice site in intron 12, which is converted into a GA in T. orientalis accessions of Malaysian Borneo. The discovery of this functional GA-AG splice site in SYMRK highlights a gap in our understanding of splice donor sites.
METHODS AND RESULTS: In this study, we have developed a simplified, tissue culture-independent protocol to deliver the CRISPR/Cas9 system through in planta transformation in Malaysian rice (Oryza sativa L. subsp. indica cv. MR 219). Sprouting seeds with cut coleoptile were used as the target for the infiltration by Agrobacterium tumefaciens and we achieved 9% transformation efficiency. In brief, the dehusked seeds were surface-sterilised and imbibed, and the coleoptile was cut to expose the apical meristem. Subsequently, the cut coleoptile was inoculated with A. tumefaciens strain EHA105 harbouring CRISPR/Cas9 expression vector. The co-cultivation was conducted for five to six days in a dark room (25 ± 2 °C) followed by rooting, acclimatisation, and growing phases. Two-month-old plant leaves were then subjected to a hygromycin selection, and hygromycin-resistant plants were identified as putative transformants. Further validation through the polymerase chain reaction verified the integration of the Cas9 gene in four putative T0 lines. During the fruiting stage, it was confirmed that the Cas9 gene was still present in three randomly selected tillers from two 4-month-old transformed plants.
CONCLUSION: This protocol provides a rapid method for editing the rice genome, bypassing the need for tissue culture. This article is the first to report the delivery of the CRISPR/Cas9 system for in planta transformation in rice.