• Molecules and Cells
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• v.39 no.11
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• pp.807-813
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• 2016

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2'F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of $1.3{\times}10^6CFU/ml$. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate ($5{\times}10^4CFU/ml$). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of $2{\times}10^4CFU/mL$ of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

• Journal of Ginseng Research
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• v.44 no.3
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• pp.461-474
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• 2020

Background: Ginseng effectively reduces fatigue in both animal models and clinical trials. However, the mechanism of action is not completely understood, and its molecular targets remain largely unknown. Methods: By screening for proteins that interact with the primary components of ginseng (ginsenosides) in an affinity chromatography assay, we have identified muscle-type creatine kinase (CK-MM) as a potential target in skeletal muscle tissues. Results: Biolayer interferometry analysis showed that ginsenoside metabolites, instead of parent ginsenosides, had direct interaction with recombinant human CK-MM. Subsequently, 20(S)-protopanaxadiol (PPD), which is a ginsenoside metabolite and displayed the strongest interaction with CK-MM in the study, was selected as a representative to confirm direct binding and its biological importance. Biolayer interferometry kinetics analysis and isothermal titration calorimetry assay demonstrated that PPD specifically bound to human CK-MM. Moreover, the mutation of key amino acids predicted by molecular docking decreased the affinity between PPD and CK-MM. The direct binding activated CK-MM activity in vitro and in vivo, which increased the levels of tissue phosphocreatine and strengthened the function of the creatine kinase/phosphocreatine system in skeletal muscle, thus buffering cellular ATP, delaying exercise-induced lactate accumulation, and improving exercise performance in mice. Conclusion: Our results suggest a cellular target and an initiating molecular event by which ginseng reduces fatigue. All these findings indicate PPD as a small molecular activator of CK-MM, which can help in further developing better CK-MM activators based on the dammarane-type triterpenoid structure.

• Journal of Ginseng Research
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• v.45 no.4
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• pp.465-472
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• 2021

Background: Ginseng can help regulate brain excitability, promote learning and memory, and resist cerebral ischemia in the central nervous system. Ginsenosides are the major effective compounds of Ginseng, but their protein targets in the brain have not been determined. Methods: We screened proteins that interact with the main components of ginseng (ginsenosides) by affinity chromatography and identified the 14-3-3 ζ protein as a potential target of ginsenosides in brain tissues. Results: Biolayer interferometry (BLI) analysis showed that 20(S)-protopanaxadiol (PPD), a ginseng saponin metabolite, exhibited the highest direct interaction to the 14-3-3 ζ protein. Subsequently, BLI kinetics analysis and isothermal titration calorimetry (ITC) assay showed that PPD specifically bound to the 14-3-3 ζ protein. The cocrystal structure of the 14-3-3 ζ protein-PPD complex showed that the main interactions occurred between the residues R56, R127, and Y128 of the 14-3-3 ζ protein and a portion of PPD. Moreover, mutating any of the above residues resulted in a significant decrease of affinity between PPD and the 14-3-3 ζ protein. Conclusion: Our results indicate the 14-3-3 ζ protein is the target of PPD, a ginsenoside metabolite. Crystallographic and mutagenesis studies suggest a direct interaction between PPD and the 14-3-3 ζ protein. This finding can help in the development of small-molecular compounds that bind to the 14-3-3 ζ protein on the basis of the structure of dammarane-type triterpenoid.

• Journal of Ginseng Research
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• v.46 no.5
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• pp.666-674
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• 2022

Background: Ginsenosides and their metabolites have antidepressant-like effects, but the underlying mechanisms remain unclear. We previously identified 14-3-3 ζ as one of the target proteins of 20 (S)-protopanaxadiol (PPD), a fully deglycosylated ginsenoside metabolite. Methods: Corticosterone (CORT) was administered repeatedly to induce the depression model, and PPD was given concurrently. The tail suspension test (TST) and the forced swimming test (FST) were used for behavioral evaluation. All mice were sacrificed. Golgi-cox staining, GSK 3β activity assay, and Western blot analysis were performed. In vitro, the kinetic binding analysis with the Biolayer Interferometry (BLI) was used to determine the molecular interactions. Results: TST and FST both revealed that PPD reversed CORT-induced behavioral deficits. PPD also ameliorated the CORT-induced expression alterations of hippocampal Ser9 phosphorylated glycogen synthase kinase 3β (p-Ser9 GSK 3β), Ser133 phosphorylated cAMP response element-binding protein (p-Ser133 CREB), and brain-derived neurotrophic factor (BDNF). Moreover, PPD attenuated the CORT-induced increase in GSK 3β activity and decrease in dendritic spine density in the hippocampus. In vitro, 14-3-3 ζ protein specifically bound to p-Ser9 GSK 3β polypeptide. PPD promoted the binding and subsequently decreased GSK 3β activity. Conclusion: These findings demonstrated the antidepressant-like effects of PPD on the CORT-induced mouse depression model and indicated a possible target-based mechanism. The combination of PPD with the 14-3-3 ζ protein may promote the binding of 14-3-3 ζ to p-GSK 3β (Ser9) and enhance the inhibition of Ser9 phosphorylation on GSK 3β kinase activity, thereby activating the plasticity-related CREBeBDNF signaling pathway.

• Journal of Ginseng Research
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• v.48 no.2
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• pp.202-210
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• 2024

Background: Panax ginseng Meyer polysaccharides exhibit various biological functions, like antagonizing galectin-3-mediated cell adhesion and migration. Galectin-8 (Gal-8), with its linker-joined N- and C-terminal carbohydrate recognition domains (CRDs), is also crucial to these biological processes, and thus plays a role in various pathological disorders. Yet the effect of ginseng-derived polysaccharides in modulating Gal-8 function has remained unclear. Methods: P. ginseng-derived pectin was chromatographically isolated and enzymatically digested to obtain a series of polysaccharides. Biolayer Interferometry (BLI) quantified their binding affinity to Gal-8, and their inhibitory effects on Gal-8 was assessed by hemagglutination, cell migration and T-cell apoptosis. Results: Our ginseng-derived pectin polysaccharides consist mostly of rhamnogalacturonan-I (RG-I) and homogalacturonan (HG). BLI shows that Gal-8 binding rests primarily in RG-I and its β-1,4-galactan side chains, with sub-micromolar K_D values. Both N- and C-terminal Gal-8 CRDs bind RG-I, with binding correlated with Gal-8-mediated function. Conclusion: P. ginseng RG-I pectin β-1,4-galactan side chains are crucial to binding Gal-8 and antagonizing its function. This study enhances our understanding of galectin-sugar interactions, information that may be used in the development of pharmaceutical agents targeting Gal-8.

• Journal of Ginseng Research
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• v.47 no.5
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• pp.662-671
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• 2023

Background: 20(S)-protopanaxadiol (PPD), a ginsenoside metabolite, has prominent benefits for the central nervous system, especially in improving learning and memory. However, its transcriptional targets in brain tissue remain unknown. Methods: In this study, we first used mass spectrometry-based drug affinity responsive target stability (DARTS) to identify the potential proteins of ginsenosides and intersected them with the transcription factor library. Second, the transcription factor PURA was confirmed as a target of PPD by biolayer interferometry (BLI) and molecular docking. Next, the effect of PPD on the transcriptional levels of target genes of PURA in brain tissues was determined by qRT-PCR. Finally, bioinformatics analysis was used to analyze the potential biological features of these target proteins. Results: The results showed three overlapping transcription factors between the proteomics of DARTS and transcription factor library. BLI analysis further showed that PPD had a higher direct interaction with PURA than parent ginsenosides. Subsequently, BLI kinetic analysis, molecular docking, and mutations in key amino acids of PURA indicated that PPD specifically bound to PURA. The results of qRT-PCR showed that PPD could increase the transcription levels of PURA target genes in brain. Finally, bioinformatics analysis showed that these target proteins were involved in learning and memory function. Conclusion: The above-mentioned findings indicate that PURA is a transcription target of PPD in brain, and PPD upregulate the transcription levels of target genes related to cognitive dysfunction by binding PURA, which could provide a chemical and biological basis for the study of treating cognitive impairment by targeting PURA.