In this study, the presence of microRNAs in coconut water was identified by real-time polymerase chain reaction (PCR) based on the results of high-throughput small RNA sequencing. In addition, the differences in microRNA content between immature and mature coconut water were compared. A total of 47 known microRNAs belonging to 25 families and 14 new microRNAs were identified in coconut endosperm. Through analysis using a target gene prediction software, potential microRNA target genes were identified in the human genome. Real-time PCR showed that the level of most microRNAs was higher in mature coconut water than in immature coconut water. Then, exosome-like nanoparticles were isolated from coconut water. After ultracentrifugation, some particle structures were seen in coconut water samples using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate fluorescence staining. Subsequent scanning electron microscopy observation and dynamic light scattering analysis also revealed some exosome-like nanoparticles in coconut water, and the mean diameters of the particles detected by the two methods were 13.16 and 59.72 nm, respectively. In conclusion, there are extracellular microRNAs in coconut water, and their levels are higher in mature coconut water than in immature coconut water. Some exosome-like nanoparticles were isolated from coconut water, and the diameter of these particles was smaller than that of animal-derived exosomes.
L-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of L-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure L-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for L-theanine production. The CFPS expressed L-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce L-theanine at a concentration of 11.31 μM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for L-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce L-theanine at the highest concentration of 302.96 μM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step L-theanine biosynthetic pathway consisting of the L-alanine decarboxylase from C. sinensis (CsAD) and multiple L-theanine synthases. Among them, the combination of CsAD and the L-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize L-theanine at the highest concentration of 13.42 μM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the L-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert L-alanine and L-glutamate in the medium to L-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the L-theanine through the two-step L-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.
This study demonstrates the effects of microwaves on the microRNA (miRNA) content of milk and milk extracellular vesicles (EV). We determined the miRNA concentration in milk subjected to different treatments using real-time PCR and a spectrophotometer. The miRNA expression and total RNA content of the microwaved milk samples were lower when compared with untreated milk. We measured the microstructure and the size distribution by scanning electron microscopy and dynamic light scattering to verify the loss of miRNA in microwaved milk due to damage to the EV. The results revealed that 2 different-sized EV were present and had an average size of 147.50 and 22.14 nm, respectively. Furthermore, acridine orange staining showed that the total RNA content in microwaved milk EV was lower than that in cow milk. These results suggest that EV may confer the protection and the stability of the miRNA in milk.
Extracellular vesicle-mediated transfer of microRNAs is a novel mode of cell-to-cell genetic transmission. Extracellular vesicles produced by microbes have been shown to contain significant quantities of physiologically active molecules such as proteins, lipids, and RNA, which could be transported to host cells and play a key role in both inter-kingdom signaling and physiological responses. In this study, we identified sRNAs by sequencing small RNAs (sRNAs) from Lactobacillus plantarum-derived extracellular vesicles (LDEVs) and detected the expression levels of vesicular sRNAs using quantitative reverse transcription-polymerase chain reaction (RT-PCR), which demonstrated the presence of microRNA-sized RNAs (msRNAs) within these vesicles. We chose sRNA71, a highly expressed msRNA, for further investigation, predicted its potential target genes for the human genome, and indicated that it could be translocated into mammalian cells. The biological functions of this sRNA71 were subsequently explored through cellular proteomics, western blot, and luciferase reporter assay. According to the findings, transfection with synthetic sRNA71 mimics substantially reduced Tp53 expression in HEK293T cells and suppressed the gene expression through binding to the 3′ UTR of Tp53 mRNA. In conclusion, it is hypothesized that microbial-derived extracellular vesicles serve as carriers of functional molecules such as sRNAs, which play an essential role in regulating microbial-host communication.
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