Na-Reum Ha

Ultra-sensitive detection of kanamycin for food safety using a reduced graphene oxide-based fluorescent aptasensor

Overuse of antibiotics has caused serious problems, such as appearance of super bacteria, whose accumulation in the human body through the food chain is a concern. Kanamycin is a common antibiotic used to treat diverse infections; however, residual kanamycin can cause many side effects in humans. Thus, development of an ultra-sensitive, precise, and simple detection system for residual kanamycin in food products is urgently needed for food safety. In this study, we identified kanamycin-binding aptamers via a new screening method, and truncated variants were analyzed for optimization of the minimal sequence required for target binding. We found various aptamers with high binding affinity from 34.7 to 669 nanomolar Kdapp values with good specificity against kanamycin. Furthermore, we developed a reduced graphene oxide (RGO)-based fluorescent aptasensor for kanamycin detection. In this system, kanamycin was detected at a concentration as low as 1 pM (582.6 fg/mL). In addition, this method could detect kanamycin accurately in kanamycin-spiked blood serum and milk samples. Consequently, this simple, rapid, and sensitive kanamycin detection system with newly structural and functional analysis aptamer exhibits outstanding detection compared to previous methods and provides a new possibility for point of care testing and food safety.

Development of receptor-based inhibitory RNA aptamers for anthrax toxin neutralization

Anthrax toxin excreted by Bacillus anthracis is the key causative agent of infectious anthrax disease. In the present study, we targeted the binding of PA to the ATR/TEM8 Von Willebrand factor type A (VWA) domain, which we cloned into Escherichia coli and purified to homogeneity under denaturing conditions. To develop an anthrax toxin inhibitor, we selected and identified short single strand RNA aptamers (approximately 30mer) consisting of different sequences of nucleic acids with a high binding affinity in the 100 nanomolar range against the recombinant ATR/TEM8 VWA domain using systematic evolution of ligands by exponential enrichment (SELEX). Five candidate aptamers were further characterized by several techniques including secondary structural analysis. The inhibitor efficiency (IC50) of one of the aptamers toward anthrax toxin was approximately 5μM in macrophage RAW 264.7 cells, as determined from cytotoxicity analysis by MTT assay. We believe that the candidate aptamers should be useful for blocking the binding of PA to its receptor in order to neutralize anthrax toxin.

Biochemical characterization and evaluation of potent inhibitors of the Pseudomonas aeruginosa PA01 acetohydroxyacid synthase

Microbes and plants synthesize essential branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine via a common biosynthetic pathway in which the first reaction is catalyzed by acetohydroxyacid synthase (AHAS, EC 4.1.3.18). Recently, AHAS was identified as a potential anti bacterial target. To help find an effective inhibitor that could act as an antibacterial compound, we cloned and characterized the catalytic subunit (CSU) of Pseudomonas aeruginosa AHAS, and found four potent inhibitors through chemical library screening. The ilvI gene of P. aeruginosa encodes a 65-kDa AHAS protein, consistent with the size of the purified enzyme on SDS-PAGE. Enzyme kinetics showed that the enzyme has a Km of 14.2 mM and a specific activity of 0.12 U/mg. Enzyme activity was optimum at a temperature of 37 °C and a pH of 7.5. The Kd for thiamine diphosphate (ThDP) was 89.92 ± 17.9 μM, as determined by fluorescence quenching. The cofactor activation constants (Ks) for ThDP and (Kc) for Mg(2+) were 0.6 ± 0.1 and 560.8 ± 7.4 μM, respectively. Further, we determined that AVS2087, AVS2093, AVS2236, and AVS2387 compounds are potent inhibitors of the catalytic subunit of P. aeruginosa AHAS. These compounds inhibit nearly 100% of AHAS activity, with IC50 values of 1.19 μM, 5.0 nM, 25 nM, and 13 nM, respectively. Compound AVS2093 showed growth inhibition with a minimal inhibitory concentration (MIC) of 742.9 μg/ml against P. aeruginosa strain ATCC 9027. Furthermore, these findings were supported by molecular docking studies with the AVS compounds against P. aeruginosa AHAS in which AVS2093 showed minimum binding energy (-7.8 kJ/mol) by interacting with the receptor through a single hydrogen bond of 2.873 Å. Correlation of AVS2093 activity with P. aeruginosa AHAS cell growth inhibition suggested that AHAS might serve as a target protein for the development of novel antibacterial therapeutics. Results of the current study provide an impetus to further evaluate the potency of these inhibitors against pathogenic P. aeruginosa strains in vivo and to design more potent antibacterial agents based on these AVS inhibitors.

Development of potent chemical antituberculosis agents targeting Mycobacterium tuberculosis acetohydroxyacid synthase

Mycobacterium tuberculosis acetohydroxyacid synthase (MTB-AHAS) has been suggested as a crucial target for antibacterial agents. High-throughput screening of a chemical library was performed to identify potent new inhibitors of MTB-AHAS. Among the 6800 tested compounds, 15 were identified as potent inhibitors, exhibiting >80-90% inhibition of in vitro MTB-AHAS activity at a fixed concentration of 20 µM. Five compounds belonging to the triazolopyrimidine structural class showed greater inhibition potency, with a half-maximum inhibition concentration (IC50 value) in the low micromolar range (0.4-1.24 µM). Furthermore, potent inhibitors demonstrated non-competitive, uncompetitive or mixed-competitive inhibition. Molecular docking experiments with these potent chemicals using a homology model of MTB-AHAS indicated hydrophobic and hydrogen bond interactions with some key herbicide binding site residues with binding energies (ΔG) of -8.04 to -10.68 Kcal/mol, respectively. The binding modes were consistent with inhibition mechanisms, as the chemicals were oriented outside the active site. Importantly, these potent inhibitors demonstrated significant growth inhibition of various clinically isolated multidrug-resistant and extensively drug-resistant M. tuberculosis strains, with 50% minimum inhibitory concentrations (MIC50 values) ranging from 0.2 µg/mL to 0.8 µg/mL, which resemble the MICs of conventional drugs for tuberculosis (isoniazid, 0.1 µg/mL; rifampicin, 0.4 µg/mL). Thus, the identified potent inhibitors show potential as scaffolds for further in vivo studies and might provide an impetus for the development of strong antituberculosis agents targeting MTB-AHAS.

A novel peptide-based recognition probe for the sensitive detection of CD44 on breast cancer stem cells

Metastasis and recurrence of breast cancer remain significant clinical problems. The expression level of CD44 protein is higher in breast cancer-initiating cancer stem cells; therefore, the early detection of CD44 using a sensitive diagnostic probe is important for breast cancer diagnosis and therapeutic purposes. In this study, we fabricated a polyvalent directed peptide polymer (PDPP) that specifically recognized the CD44 biomarker, as confirmed by immunocytochemistry tests and fluorescence-activated cell sorting assessment. Our results indicate that PDPP is useful as a novel tool for the sensitive detection of breast cancer stem cells.

Sensitive fluorescent imaging of Salmonella enteritidis and Salmonella typhimurium using a polyvalent directed peptide polymer

AbstractThe authors describe three fluorescein-conjugated peptides generated by cell-phage display for use as a diagnostic probes for fluorescent detection and imaging of Salmonella enteritidis and Salmonella typhimurium. The authors also designed a polyvalent-directed peptide polymer synthesized with poly-D-lysine and bifunctional succinimidyl 3-(2-pyridyldithio)propionate with an affinity and sensitivity that is higher by more than an order of magnitude compared to single peptides due to multiple binding site interactions. In order to establish a diagnostic system for food poisoning, imaging analysis was performed using fluorescence microscopy. The limit of detection of the diagnostic system based on polyvalent directed peptide interaction is 102 colony-forming units per mL for Salmonella. Graphical abstractSchematic of a fluorescent method for detection and imaging of Salmonella enteritidis and Salmonella typhimurium by using a fluorescein labeled polyvalent-directed peptide polymer (PDPP) with a high affinity and sensitivity as a diagnostic probe. The system uses a microplate reader and was applied to the detection of food poisoning.

Mutation analysis of the interactions between Mycobacterium tuberculosis caseinolytic protease C1 (ClpC1) and ecumicin

Ecumicin is a well-known and potent inhibitor of Mycobacterium tuberculosis. Although the target of ecumicin is caseinolytic protease C1 (ClpC1), the exact mechanism by which ecumicin inhibits ClpC1 has not been identified. To analyze ecumicins action on ClpC1, site-directed mutagenesis was performed on its binding site. The estimated binding residues within ClpC1 to ecumicin were selected via in silico analysis using molecular docking. The selected residues were mutated by site-directed mutagenesis and the effects on ecumicin binding were analyzed. Mutation at the R83 residue, especially the R83A mutation, in ClpC1 resulted in strong resistance to ATPase activation and inhibition of proteolytic activity. In addition, binding of ecumicin to the R83A ClpC1 N-terminal domain (residues 1-145) was not observed in native gel analysis. These results reveal that the R83 residue plays an important role in the binding of ecumicin. This result provides a basis for the development of an anti-tuberculosis agent based on ecumicin derivatives.

Development of a ssDNA aptamer system with reduced graphene oxide (rGO) to detect nonylphenol ethoxylate in domestic detergent

Endocrine‐disrupting chemicals are a major public health problem throughout the world. In the human body, these compounds functionalize the same as sexual hormones, inducing precocious puberty, gynecomastia, etc. To help prevent this occurrence, a simple detection system is needed. In this study, a nonylphenol ethoxylate (NPE)‐specific aptamer was selected by reduced graphene oxide‐systematic evolution of ligands by exponential enrichment. A random ssDNA library was incubated with rGO for adsorption, followed by elution with the target molecule. As a result of screening, a DNA aptamer was found that specifically bounds to the target with high binding affinity (Kd = 100.9 ± 13.2 nM) and had a low limit of detection (LOD = 696 pM). Furthermore, this NPE‐binding aptamer bounds selectively to the target. Characterization of the aptamer was confirmed by measuring the fluorescence signal recovery from rGO. In addition, detection of NPE was performed with several water samples, and the detection accuracy was 100 ± 10%. From these results, we expect that this aptamer could be applied to an on‐site detection system for NPE in industrial sites or domestic fields.

Inhibition of anthrax lethal factor by ssDNA aptamers

Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a Kd value of 11.0 ± 2.7 nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC50 value of 15 ± 1.5 μM and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF.