Effects of Roudoukou-8 San Extract on Hydrogen Peroxide Induced Cardiomyocyte Injury

[Objectives] To study the effects of Roudoukou-8 San extract on hydrogen peroxide( H_2O_2) induced cardiomyocyte injury of rats and explore its action mechanism. [Methods] The cardiomyocytes of neonatal rats were isolated and cultured,and the H_2O_2 induced cardiomyocyte injury model was established. Methyl thiazolyl tetrazolium( MTT) assay was used to detect the protective effects of Roudoukou-8 San extract on H_2O_2 induced cardiomyocyte. The effects of Roudoukou-8 San on cardiomyocyte morphology were observed under inverted microscope. The contents of lactate dehydrogenase( LDH),creatine kinase( CK) and aspartate aminotransferase( AST) in cell culture medium were determined by automatic biochemical instrument; the levels of malondialdehyde( MDA),superoxide dismutase( SOD) and nitric oxide( NO) in the cells were detected by kit method. The apoptotic morphology of cardiomyocytes was observed by Hoechst fluorescence staining.Cell apoptosis were measured by Annexin V and PI double staining and flow cytometer. [Results]100 μmol/L of H_2O_2 acting 2 h could cause about 50% of cardiomyocyte injury. H_2O_2 model group showed increased cell gap,decreased cell count,cell cytoplasmic vacuoles and other obvious damages. Compared with H_2O_2 model group,in Roudoukou-8 San extract dose groups,cardiomyocyte morphology showed different degrees of improvement,Roudoukou-8 San extract can significantly reduce the content of LDH,CK and AST in H_2O_2 injured myocardial cell culture solution,significantly reduce content of MDA and NO in H_2O_2 injured myocardial cells,increase the SOD activity,and significantly inhibit the apoptosis of H_2O_2 injured myocardial cells. [Conclusions]Through improving the cell survival status,increasing the cell viability,reducing oxidative stress injury,inhibiting inflammatory responses,and inhibiting apoptosis,Roudoukou-8 San extract can improve the state of H_2O_2 induced cardiomyocyte injury,so as to protect H_2O_2 injured myocardial cells.

Efficient genome editing in rice with miniature Cas12f variants

Genome editing,particularly using the CRISPR/Cas system,has revolutionized biological research and crop improvement.Despite the widespread use of CRISPR/Cas9,it faces limitations such as PAM sequence requirements and challenges in delivering its large protein into plant cells.The hypercompact Cas12f,derived from Acidibacillus sulfuroxidans(AsCas12f),stands out due to its small size of only 422 amino acids and its preference for a T-rich motif,presenting advantageous features over SpCas9.However,its editing efficiency is extremely low in plants.Recent studies have generated two AsCas12f variants,AsCas12f-YHAM and AsCas12f-HKRA,demonstrating higher editing efficiencies in mammalian cells,yet their performance in plants remains unexplored.In this study,through a systematic investigation of genome cleavage activity in rice,we unveiled a substantial enhancement in editing efficiency for both AsCas12f variants,particularly for AsCas12f-HKRA,which achieved an editing efficiency of up to 53%.Furthermore,our analysis revealed that AsCas12f predominantly induces deletion in the target DNA,displaying a unique deletion pattern primarily concentrated at positions 12,13,23,and 24,resulting in deletion size mainly of 10 and 11 bp,suggesting significant potential for targeted DNA deletion using AsCas12f.These findings expand the toolbox for efficient genome editing in plants,offering promising prospects for precise genetic modifications in agriculture.

Engineer and split an efficient hypercompact CRISPR–CasΦ genome editor in plants

The programmable CRISPR-Cas genome editing technology,adopted from prokaryotic adaptive immune systems,has revolutionized genome engineering in plants(Liu et al.,2022a).Many efforts have been made to improve the activity,specificity,and protospacer adjacent motif(PAM)variants of Class 2 Cas nucleases,such as Cas9,Cas12a,and Cas12b(Liu et al.,2022a).However,their large size(∼1000–1400 amino acids)poses a challenge in scenarios requiring a compact Cas nuclease,particularly in urgent situations like plant virus-induced genome editing(Cheuk and Houde,2018;Li et al.,2021;Varanda et al.,2021).

Rice FLOURY ENDOSPERM22, encoding a pentatricopeptide repeat protein, is involved in both mitochondrial RNA splicing and editing and is crucial for endosperm development

Most of the reported P-type pentatricopeptide repeat(PPR) proteins play roles in organelle RNA stabilization and splicing. However, P-type PPRs involved in both RNA splicing and editing have rarely been reported, and their underlying mechanism remains largely unknown. Here, we report a rice floury endosperm22(flo22) mutant with delayed amyloplast development in endosperm cells. Map-based cloning and complementation tests demonstrated that FLO22 encodes a mitochondrion-localized P-type PPR protein.Mutation of FLO22 resulting in defective transsplicing of mitochondrial nad1 intron 1 and perhaps causing instability of mature transcripts affected assembly and activity of complex Ⅰ, and mitochondrial morphology and function. RNA-seq analysis showed that expression levels of many genes involved in starch and sucrose metabolism were significantly down-regulated in the flo22mutant compared with the wild type, whereas genes related to oxidative phosphorylation and the tricarboxylic acid cycle were significantly upregulated. In addition to involvement in splicing as a P-type PPR protein, we found that FLO22 interacted with DYW3, a DYW-type PPR protein, and they may function synergistically in mitochondrial RNA editing. The present work indicated that FLO22 plays an important role in endosperm development and plant growth by participating in nad1 maturation and multi-site editing of mitochondrial messager RNA.

BRITTLE PLANT1 is required for normal cell wall composition and mechanical strength in rice

A series of nucleotide sugar interconversion enzymes(NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDPglucose 4-epimerases(UGEs) are NSEs that function in the interconversion of UDP-glucose(UDP-Glc) and UDP-galactose(UDP-Gal). The roles of UDP-glucose 4-epimerases in monocots remain unclear due to redundancy in the pathways. Here, we report a brittle plant(bp1) rice mutant that exhibits brittle leaves and culms at all growth stages. The mutant culms had reduced levels of rhamnogalacturonan I, homogalacturonan, and arabinogalactan proteins.Moreover, the mutant had altered contents of uronic acids, neutral noncellulosic monosaccharides, and cellulose. Map-based cloning demonstrated that OsBP1 encodes a UDPglucose 4-epimerase(OsUGE2), a cytosolic protein. We also show that BP1 can form homoand hetero-protein complexes with other UGE family members and with UDP-galactose transporters 2(OsUGT2) and 3(OsUGT3), which may facilitate the channeling of Gal to polysaccharides and proteoglycans. Our results demonstrate that BP1 participates in regulating the sugar composition and structure of rice cell walls.