Joon Myong Song

SERS imaging of HER2-overexpressed MCF7 cells using antibody-conjugated gold nanorods

Antibody-conjugated gold nanorods (GNRs) have been used for the targeting and imaging of specific cancer markers expressed on the surface membrane of cancer cells. GNRs with various aspect ratios were fabricated, and their surface-enhanced Raman scattering enhancement effects were evaluated. To attach the GNRs selectively onto the targets in cancer cells, specific antibodies were immobilized on the surface of GNRs using the layer-by-layer deposition method. First, Raman reporter molecules, mercaptopyridine, were attached to the surface of GNRs, and their surface charge was modified using poly(sodium 4-styrene-sulfonate) to make the surface charge negative. Then, anti-rabbit IgGs were immobilized onto the surface of the GNRs by electrostatic interactions. HER2 markers, expressed on the cell surface, were treated with anti-HER2 primary antibodies. Finally, the functionalized nanoprobes, conjugated with secondary antibodies, were attached to the markers on cancer cells by antibody-antibody interactions. In the present study, MCF7 cells overexpressing breast cancer marker HER2 were used as the optical imaging targets. Our experimental results demonstrate the potential feasibility of antibody-conjugated GNRs for the highly sensitive targeting and imaging of biomarkers expressed on the surface membrane of cancer cells.

Highly sensitive trace analysis of paraquat using a surface-enhanced Raman scattering microdroplet sensor

We report a rapid and highly sensitive trace analysis of paraquat (PQ) in water using a surface-enhanced Raman scattering (SERS)-based microdroplet sensor. Aqueous samples of PQ, silver nanoparticles, and NaCl as the aggregation agent were introduced into a microfluidic channel and were encapsulated by a continuous oil phase to form a microdroplet. PQ molecules were adsorbed onto particle surfaces in isolated droplets by passing through the winding part of the channel. Memory effects, caused by the precipitation of nanoparticle aggregates on channel walls, were removed because the aqueous droplets were completely isolated by a continuous oil phase. The limit of detection (LOD) of PQ in water, determined by the SERS-based microdroplet sensor, was estimated to be below 2×10(-9) M, and this low detection limit was enhanced by one to two orders of magnitude compared to conventional analytical methods.

Synthesis of highly antibacterial nanocrystalline trivalent silver polydiguanide.

Highly monodispersed nanoparticles of a trivalent silver polydiguanide complex are synthesized by oxidation of the monovalent silver, followed by stabilization of the oxidized higher-valent metal through complexation with a polydiguanide ligand in a reverse microemulsion at room temperature. The synthesized nanoparticles have excellent photostability and displayed superior antibacterial activity toward Gram-positive and Gram-negative prokaryotes of clinical interest in vitro compared to silver sulfadiazine. These nanoparticles may serve as a new generation antibacterial metallopharmaceutical in wound care.

Interaction of two translational components, lysyl-tRNA synthetase and p40/37LRP, in plasma membrane promotes laminin-dependent cell migration

Although human lysyl‐tRNA synthetase (KRS), an enzyme for protein synthesis, is often highly expressed in various cancer cells, its pathophysiological implications have not been understood. Here we found that KRS induces cancer cell migration through interaction with the 67‐kDa laminin receptor (67LR) that is converted from ribosomal subunit p40. On laminin signal, KRS was phosphorylated at the T52 residue by p38MAPK and dissociated from the cytosolic multi‐tRNA synthetase complex for membrane translocation. The importance of T52 phosphorylation for membrane translocation of KRS was confirmed by site‐directed mutagenesis. In the membrane, turnover of 67LR was controlled by Nedd4‐mediated ubiquitination, and KRS inhibited ubiquitin‐dependent degradation of 67LR, thereby enhancing laminin‐induced cell migration. This work thus unveiled a unique function of KRS in the control of cell migration and its pathological implication in metastasis.—Kim, D. G., Choi, J. W., Lee, J. Y., Kim, H., Oh, Y. S., Lee, J. W., Tak, Y. K., Song, J. M., Razin, E., Yun, S.‐H., Kim, S. Interaction of two translational components, lysyl‐tRNA synthetase and p40/37LRP, in plasma membrane promotes laminin‐dependent cell migration. FASEB J. 26, 4142–4159 (2012). www.fasebj.org

Shape-Dependent Skin Penetration of Silver Nanoparticles: Does It Really Matter?

Advancements in nano-structured materials have facilitated several applications of nanoparticles (NPs). Skin penetration of NPs is a crucial factor for designing suitable topical antibacterial agents with low systemic toxicity. Available reports focus on size-dependent skin penetration of NPs, mainly through follicular pathways. Herein, for the first time, we demonstrate a proof-of-concept study that entails variations in skin permeability and diffusion coefficients, penetration rates and depth-of-penetration of differently shaped silver NPs (AgNPs) via intercellular pathways using both in vitro and in vivo models. The antimicrobial activity of AgNPs is known. Different shapes of AgNPs may exhibit diverse antimicrobial activities and skin penetration capabilities depending upon their active metallic facets. Consideration of the shape dependency of AgNPs in antimicrobial formulations could help developing an ideal topical agent with the highest efficacy and low systemic toxicity.

Hair analysis and self-report of methamphetamine use by methamphetamine dependent individuals

The questions of whether the dose of drug that is consumed corresponds to drug concentration levels in hair and how results of hair analyses can be interpreted are still debated. The aim of this study was to investigate (1) whether there is a correlation between doses of Methamphetamine (MA) use and MA concentration levels in hair and (2) whether results of hair analyses can be used to estimate dose, frequency, and patterns of MA use. In this study, segmental hair analysis was performed through consecutive 1cm as well as 1-4 cm (=3 cm) segmental hair lengths. MA dependent individuals (n=9) provided information on doses (0.25-4 g/day) of MA use as well as the frequency of MA use. The concentrations of MA and its metabolite amphetamine (AP) in hair were determined using gas chromatography/mass spectrometry (GC/MS). One-way analysis of variance (ANOVA) test was performed to evaluate whether MA and AP concentrations in consecutive 1cm length segmental hair were consistent with the history of MA use. The cumulative doses of MA use calculated from the daily dose and the frequency during 1-4 months were well correlated to the concentrations of MA and AP in 1-4 cm segmental hair length (correlation coefficient, r=0.87 for MA and r=0.77 for AP). The results from this study show the patterns and histories of MA use from MA dependent individuals and could assist in the interpretation of hair results in forensic toxicology as well as in rehabilitation and treatment programs.

Real-time concurrent monitoring of apoptosis, cytosolic calcium, and mitochondria permeability transition for hypermulticolor high-content screening of drug-induced mitochondrial dysfunction-mediated hepatotoxicity.

A quantitative high-content screening (HCS) was suggested for the real-time monitoring of drug-induced mitochondrial dysfunction-mediated hepatotoxicity. This HCS is very advantageous in that it allows simultaneous observation of drug-induced activations of hepatotoxic pathways using hypermulticolor cellular imaging. The mitochondrial permeability transition (MPT), cytosolic calcium, and caspase-3 were selected as functional markers to verify drug-induced hepatotoxicity and were concurrently monitored in HepG2 cells in a real-time manner. Nefazodone, tolcapone, and troglitazone caused mitochondrial dysfunction and subsequent apoptotic HepG2 cell death in addition to marked cytosolic calcium increase. On the other hand, extrinsic pathway-mediated apoptotic cell death was monitored when HepG2 cells were treated with piroxicam. It was found that piroxicam-treated HepG2 cells showed apoptotic cell death without the MPT formation, while a cytosolic calcium increase was clearly observed. This finding was confirmed by the caspase-8 inhibition assay. These results demonstrated the unique potential of real-time hypermulticolor HCS to screen hepatotoxic drugs at the in vitro stage rather than the later in vivo stage based on an animal model and to ultimately reduce the probability of drug failure.

Segmental Hair Analysis for 11-Nor-Δ9-Tetrahydrocannabinol-9-Carboxylic Acid and the Patterns of Cannabis Use

Cannabis is the most widely abused drug in the world. The purpose of this study is to detect 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol (THCCOOH) in segmental hair and to evaluate the patterns of cannabis use. We investigated the relationship between the concentrations of THCCOOH in hair and the self-reported use data and the route of administration. For this purpose, the hair samples were washed, digested with 1 mL of 1 M NaOH at 85°C for 30 min along with the internal standard, THCCOOH-d₃ (2.5 pg/mg) and extracted in 2 mL of n-hexane-ethyl acetate (9:1) twice after adding 1 mL of 0.1N sodium acetate buffer (pH = 4.5) and 200 µL of acetic acid. The organic extract was transferred and evaporated and the mixture was derivatized with 50 µL of pentafluoropropionic anhydride and 25 µL of pentafluoropropanol for 30 min at 70°C. Reconstituted final extract was injected into the gas chromatography-tandem mass spectrometer operating in the negative chemical ionization mode. In segmental hair analysis, the concentrations of THCCOOH decreased from the proximal to distal segments. The concentrations of THCCOOH in hair and the self-reported dose and frequency of administration from cannabis users were not well correlated because of the low accuracy and reliability of the self-reported data. However, this study provides preliminary information on the dose and frequency of administration among cannabis users in our country.

Detection of bacterial pathogen DNA using an integrated complementary metal oxide semiconductor microchip system with capillary array electrophoresis

In this paper, we show an integrated complementary metal oxide semiconductor (CMOS)-based microchip system with capillary array electrophoresis (CAE) for the detection of bacterial pathogen amplified by polymerase chain reaction (PCR). In order to demonstrate the efficacy of PCR reaction for the heat-labile toxin producing enterotoxigenic Escherichia coli (E. coli), which causes cholera-like diarrhea, 100 bp DNA ladders were injected along with the PCR product. Poly(vinylpyrrolidone) (PVP) was used as the separation medium and provided separation resolution which was adequate for the identification of PCR product. The miniaturized integrated CMOS microchip system with CAE has excellent advantages over conventional instrumental systems for analysis of bacterial pathogens such as compactness, low cost, high speed, and multiplex capability. Furthermore, the miniaturized integrated CMOS microchip system should be compatible with a variety of microfabricated devices that aim at more rapid and high-throughput analysis.

Simultaneous analysis of Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-tetrahydrocannabinol in hair without different sample preparation and derivatization by gas chromatography–tandem mass spectrometry

The present study describes a gas chromatography/tandem mass spectrometry-negative ion chemical ionization assay (GC/MS/MS-NCI) for simultaneous analysis of Δ(9)-tetrahydrocannabinol (THC) and 11-nor-9-carboxy-tetrahydrocannabinol (THCCOOH) in hair. Each hair sample, of approximately 20mg, was weighed and the sample was dissolved in 1ml of 1M sodium hydroxide (30min at 85°C) in the presence of THC-d(3) and THCCOOH-d(3). For the analysis of THC, hair samples were extracted with n-hexane:ethyl acetate (9:1) two times; acetic acid and sodium acetate buffer were added for the analysis of THCCOOH, and hair samples were re-extracted with n-hexane:ethyl acetate (9:1) two times. The extracts were then derivatized with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH). This method allowed the analysis of THC and THCCOOH using the GC/MS/MS-NCI assay. This method was also fully validated and applied to hair specimens (n=54) collected from known cannabis users whose urine test results were positive. The concentrations of THC and THCCOOH in hair ranged from 7.52 to 60.41ng/mg and from 0.10 to 11.68pg/mg, respectively. In this paper, we simultaneously measured THC and THCCOOH in human hair using GC/MS/MS-NCI without requiring different sample preparation and derivatization procedures. The analytical sensitivity for THCCOOH in hair was good, while that for THC in hair needs to be improved in further study.