Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

RNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.

Homologous tumor cell membrane vesicles active preferential self-recognition of tumor cells in vitro

Cell membrane vesicles, as delivery carriers of drugs or biological agents in vivo, are an important therapeutic mode in the study of disease treatment. Tumor membrane-derived vesicles have been widely used in tumor therapy because of their good tumor enrichment effect. The most common method is the surface of nanoparticles coated with tumor cell membrane, which can effectively prolong the circulation time of particles in the blood and the enrichment of tumors. In this study, we prepared vesicles of different tumor cell membrane derivate and studied their targeting to tumors detailly. The results showed that homologous vesicles have high targeting to homologous tumor cells. The fluorescence of vesicles in homologous tumor cells was significantly higher than that in other tumor cells. This study will provide a new strategy and guidance for the clinical treatment of cancer based on the tumor cell membrane system. Graphical Abstract

Comparison of antioxidant activity assays in fresh purple waxy corn (Zea mays L.) during grain filling

Purple waxy corn is a good source of antioxidant compounds such as anthocyanins and polyphenols. Promotion of its use requires an appropriate assay to determine antioxidant activity. The aim of this study is to verify compatibility of the antioxidant activity assays by comparing five different assays in daily kernel samples of the fresh purple waxy corn during grain filling. The levels of antioxidants measured by 2,2-diphenyl-1-picrylhydrazyl, ferric reducing antioxidant power, and chemiluminescence showed significant positive correlations with the levels measured by the other assays (r = 0.761–0.893; p < 0.01) and with anthocyanin content (r = 0.798–0.924; p < 0.01). Reducing capacity of 2,2ʹ-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and total phenolic content also showed significant correlations (r = 0.764; p < 0.01). However, during late grain filling, the levels of antioxidants measured by all methods showed strong correlations with each other (r > 0.699; p < 0.05). The inconsistencies among the measurements are largely dependent on the developmental stage of the corn kernel. The combination of at least two assays is required to ensure reliable antioxidant activity estimates, especially for early grain-filling stages. These results will inform efforts to promote fresh purple corn as a source of antioxidants.

Acaricidal properties of 5-methylfurfural identified from Valeriana fauriei and its structural analogues against synanthropic mites and Asian longhorned tick with color alterations

Acaricidal activities and color alterations of 5-methylfurfural derived from Valeriana fauriei essential oil and its structural analogues against Dermatophagoides farinae, D. pteronyssinus, Haemaphysalis longicornis and Tyrophagus putrescentiae were evaluated in the present study. Based on the LD50 values of 5-methylfurfural and its analogues, 4,5-dimethylfurfural showed the highest acaricidal activity (LD50; 9.95, 9.91, and 7.12 μg/cm2), followed by 5-methylfurfural (11.87, 11.00, and 8.59 μg/cm2), furfural (12.94, 13.25, and 10.36 μg/cm2), and V. fauriei essential oil (15.15, 13.64, and 10.14 μg/cm2) against D. farinae, D. pteronyssinus and T. putrescentiae, respectively. However, all tested compounds did not show the acaricidal activities against H. longicornis. Interestingly, the color alterations of the mites and ticks were observed by furfural, 5-methylfurfural, and 4,5-dimethylfurfural from colorless to red brown during the acaricidal experiments. Furthermore, 4,5-dimethylfurfural which exhibited the highest acaricidal activity was formulated as nanoemulsion. The nanoemulsion of 4,5-dimethylfurfural showed higher acaricidal activity than it was emulsified in ethanol. The nanoemulsion was also found to show color changes of the mites and ticks from colorless to red brown. The results suggest that 5-methylfurfural and its analogues could be developed as an effective and easy-to-recognize acaricides to mites and ticks.

Development of a molecular marker based on chloroplast gene for specific identification of Korean Hibiscus (Hibiscus syriacus ‘Simbaek’)

Due to the rise in substitution and adulteration of herbal products, as well as the lack of genetic information on Hibiscus plants, more molecular markers are needed to understand the genetic diversity and avoid their misidentification. There are many allelic variants of the functional genes in Hibiscus and other plants, which control their respective phenotypes and other characteristics. Identifying alleles of the desired trait by determining diversification through gene-typing allele-specific markers for authentication is, therefore, a potent strategy. The purpose of this study was to use insertion/deletion (InDel) markers to identify Hibiscus syriacus cultivars. We developed a novel InDel marker for Korean Hibiscus ‘Simbaek’, based on the trnL-F region of the chloroplast gene. Through this InDel site, a modified specific primer pair and a novel multiplex polymerase chain reaction (PCR) system were developed for specific identification of the Korean Hibiscus Simbaek cultivar. The molecular markers developed in this study were highly specific and accurately authenticated as Simbaek from the five main cultivars of H. syriacus. Taken together, the described method is a potential tool for the identification and selection of germplasm resource of Simbaek cultivar. Graphical Abstract

Potential and beneficial effects of Cinnamomum cassia on gastritis and safety: Literature review and analysis of standard extract

The prevalence of gastritis in South Korea is rapidly increasing owing to the prevalence of Helicobacter pylori infection and fast eating habit. The usual treatment for acute gastritis following a long intake of non-steroidal anti-inflammatory drugs (NSAIDs) or alcohol is to stop the causal factors. Metronidazole and lansoprazole are recommended for the treatment of H. pylori infection gastritis. Omeprazole a proton pump inhibitor, is used to decrease gastric acid production. However, owing to the side effects and refractoriness of the drug, a safe and efficient treatment is required. Plant-derived phytochemicals have emerged as novel agents against chronic disorders. In this study, firstly, to explore the potential of pharmacological activities, including efficacy and mechanisms of Cinnamomum cassia against gastritis, a literature review was performed based on 20 studies out of a total of 749 records obtained using a search strategy. From the literature review, the therapeutic targets of C. cassia extract and cinnamaldehyde, a compound of C. cassia, were found to be related with NFκB activity, and their signaling pathway were verified by experiments. C. cassia extract plays a role in protection of gastric ulcers induced in four ways (immersion stress-induced, ethanol-induced, hydrochloric acid-induced, or NSAIDs-induced ulcer). None of the clinical studies on C. cassia extracts or compounds met our criteria. When the standardized extract of C. cassia (ECC) was orally administered repeatedly to Beagle Dog for 4 weeks, no toxicologically harmful changes were observed. Therefore, under the test condition, the no observed adverse effect level (NOAEL) of ECC was judged to be 1000 mg/kg/day for both sexes, and no toxic target organ was observed. Administration of ECC in the Sprague–Dawley rat model of acute gastric injury caused by indomethacin administration significantly increased gastric mucus volume. Administration of ECC in the acute gastric injury model caused by indomethacin administration is considered effective in improving gastric injury. However, research and efforts to develop a reliable ‘standardization of natural drugs’ by establishing the best quality evaluation system are limited. Despite the pharmacological potential of ECC, further well-designed experimental studies such as in vitro, in vivo, and clinical trials are required to validate these findings and the underlying mechanisms of ECC.

Phenylpropanoid metabolism enzyme activities and gene expression in postharvest melons inoculated with Alternaria alternata

This study explored the mechanism of melon resistance to Alternaria alternata (A. alternata) infection in Jiashi and 86-1 melons. Melons were inoculated with A.alternata and the change in lesion diameter was measured. The changes in cinnamic acid-4-hydroxylase (C4H), phenylalanine ammonia lyase (PAL), and 4-coumaric acid coenzyme A ligase (4CL) activity and gene expression were studied in the pericarp tissues of Jiashi and 86-1 melons. The lesion diameter was smaller in Jiashi melon than in 86-1 melon, and the pericarp lesions were smaller than pulp lesions, indicating that Jiashi melon can resist A. alternata infection better than 86-1 melon. After inoculation with A. alternata, the C4H, PAL, and 4CL activities of Jiashi and 86-1 melons peaked in the middle and late storage period, and the peak was higher in Jiashi melons. The gene expression changes were consistent with the enzyme activity. The C4H, PAL, and 4CL gene expression was significantly higher in Jiashi melon pericarp than in 86-1 melon, and the C4H, PAL, and 4CL activities in Jiashi melon were positively correlated with their gene expression, confirming the role of phenylpropanoid metabolism enzymes in resistance to A. alternata.

Influence of genetic and environmental factors on the contents of carotenoids and phenolic acids in red pepper fruits (Capsicum annuum L.)

Red pepper is enriched in antioxidant components, such as carotenoids, phenolic compounds, and vitamins. In this study, we investigated the natural variability in the content of carotenoids and phenolic acids in 11 red pepper cultivars grown in two locations in South Korea during 2016, 2017, and 2018. Seven carotenoids and six phenolic acids, including soluble and insoluble forms, were detected in the red fruit pericarps. The major carotenoids were β-carotene (40%) and capsanthin (20%). The content of insoluble phenolic acids was higher than that of soluble phenolic acids because of the large amount of insoluble p-coumaric acid. The statistical analysis of combined data showed significant differences among varieties, locations, and years for most of the measured components. The results from variance component analysis indicated that the effects of location, year and the interaction of location and year mainly accounted for the variation in carotenoids, whereas variations in phenolic acid content were attributed to year and variety. In addition, the results of principal component analysis and orthogonal partial least-squares discriminant showed that carotenoids were well discriminated by location and year, whereas phenolic acids were distinctively separated only by year. The data from this study could explain the natural variation in the content of carotenoids and phenolic acids in red pepper fruits by genotype and environment.

Probiotic effect of Lactococcus lactis subsp. cremoris RPG-HL-0136 on intestinal mucosal immunity in mice

Lactococcus lactis subsp. cremoris is a lactic acid bacterium commonly used in the cheese manufacturing industry. It is known to produce antibacterial peptides and has recently received attention for its role as a probiotic strain. Here, we report the isolation of a new strain, Lactococcus lactis subsp. cremoris RPG-HL-0136 (RPG0136) from dried compost, which exhibits strong antibacterial activity. When RPG0136 was fed to mice, it increased the intestinal population of two beneficial bacteria, Lactobacillus and Bifidobacterium, whereas it decreased the intestinal population of two harmful bacteria, Bacteroides and Enterobacter. In addition, it increased the concentration of short-chain fatty acids, including acetic acid, propionic acid, and butyric acid, with a simultaneous decrease in pH, and accelerated the catabolic degradation of proteins, lipids, and starch. Lastly, RPG0136 increased the plasma IgG and intestinal mucosal SIgA concentrations and upregulated Reg3r, MUC1, and MUC2 expression to improve the intestinal mucosal immune function. The results of this study suggest that RPG0136 is a potential probiotic strain that supports the growth of a beneficial microbiome by promoting the synthesis of organic acids and enhancing intestinal immune function.