Isolimonic acid interferes with Escherichia coli O157:H7 biofilm and TTSS in QseBC and QseA dependent fashion

BACKGROUND: E. coli O157:H7 (EHEC) is an important human pathogen. The antibiotic treatment of EHEC reportedly results in release of Shiga toxin and is therefore discouraged. Consequently, alternative preventive or therapeutic strategies for EHEC are required. The objective of the current study was to investigate the effect of citrus limonoids on cell-cell signaling, biofilm formation and type III secretion system in EHEC. RESULTS: Isolimonic acid and ichangin were the most potent inhibitors of EHEC biofilm (IC(25)=19.7 and 28.3 μM, respectively) and adhesion to Caco-2 cells. The qPCR analysis revealed that isolimonic acid and ichangin repressed LEE encoded genes by ≈3 to 12 fold. In addition, flhDC was repressed by the two limonoids (≈3 to 7 fold). Further studies suggested that isolimonic acid interferes with AI-3/epinephrine activated cell-cell signaling pathway. Loss of biofilm inhibitory activity of isolimonic acid in ΔqseBC mutant, which could be restored upon complementation, suggested a dependence on functional QseBC. Additionally, overexpression of qseBC in wild type EHEC abated the inhibitory effect of isolimonic acid. Furthermore, the isolimonic acid failed to differentially regulate ler in ΔqseA mutant, while plasmid borne expression of qseA in ΔqseA background restored the repressive effect of isolimonic acid. CONCLUSIONS: Altogether, results of study seem to suggest that isolimonic acid and ichangin are potent inhibitors of EHEC biofilm and TTSS. Furthermore, isolimonic acid appears to interfere with AI-3/epinephrine pathway in QseBC and QseA dependent fashion

Antagonistic Regulation, Yet Synergistic Defense: Effect of Bergapten and Protease Inhibitor on Development of Cowpea Bruchid Callosobruchus maculatus

The furanocoumarin compound bergapten is a plant secondary metabolite that has anti-insect function. When incorporated into artificial diet, it retarded cowpea bruchid development, decreased fecundity, and caused mortality at a sufficient dose. cDNA microarray analysis indicated that cowpea bruchid altered expression of 543 midgut genes in response to dietary bergapten. Among these bergapten-regulated genes, 225 have known functions; for instance, those encoding proteins related to nutrient transport and metabolism, development, detoxification, defense and various cellular functions. Such differential gene regulation presumably facilitates the bruchids' countering the negative effect of dietary bergapten. Many genes did not have homology (E-value cutoff 10(−6)) with known genes in a BlastX search (206), or had homology only with genes of unknown function (112). Interestingly, when compared with the transcriptomic profile of cowpea bruchids treated with dietary soybean cysteine protease inhibitor N (scN), 195 out of 200 coregulated midgut genes are oppositely regulated by the two compounds. Simultaneous administration of bergapten and scN attenuated magnitude of change in selected oppositely-regulated genes, as well as led to synergistic delay in insect development. Therefore, targeting insect vulnerable sites that may compromise each other's counter-defensive response has the potential to increase the efficacy of the anti-insect molecules

Optimization of Extraction Solvent and Fast Blue BB Assay for Comparative Analysis of Antioxidant Phenolics from Cucumis melo L.

Melon (Cucumis melo L.) fruits contain multiple health-promoting compounds, including phenolic compounds, which are antioxidants. Accurate measurement of antioxidant activities and total phenolic contents (TPCs) require an efficient solvent extraction. In this study, we evaluated free radical scavenging activity and TPC of melon extracts extracted with 22 different solvent combinations. The DPPH scavenging activities were high in 100% methanolic (39.48 ± 0.36 µg g−1) and 80% methanolic extracts (38.99 ± 0.44 µg g−1). Similarly, the ABTS scavenging activities were high in 100% methanolic (315.11 ± 10.38 µg g−1) and 80% methanol extracts (297.39 ± 14.98 µg g−1). The Folin–Ciocalteu (F–C) assay is typically used to measure TPC but may be affected by interference from sugars and other compounds. Therefore, we optimized an assay for TPC using Fast Blue (FB) salt and developed a standard operating procedure for microplate analysis using FB. Our analysis of standard samples and comparisons with the F–C assay suggested that the optimized FB assay could be used to measure TPC in fruit and juice samples. Moreover, we successfully detected six phenolic compounds in methanol extracts of melon by LC-HR-QTOF/MS