Yu Kyung Tak

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.

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.

A multifunctional composite of an antibacterial higher-valent silver metallopharmaceutical and a potent wound healing polypeptide: a combined killing and healing approach to wound care

The present study relates to a combined killing and healing approach for the treatment of infected wounds. Herein we report a multifunctional, including antimicrobial and wound healing, composite containing a conjugate of a bi-valent silver polydiguanide that demonstrated high antibacterial activity in vitro and a potent wound healing polypeptide, histatin-1, for the treatment of infected wounds. The synthesis of silver(II) chlorhexidine [Ag(II)CHX] was accomplished by the oxidation of Ag(I), followed by the complexation of the oxidized metal with chlorhexidine (CHX), whereas the metal complex conjugate of the solid phase-synthesized histatin polypeptide (Hst-1), Hst-1-[Ag(II)CHX], was realized by mixing the starting materials in aqueous solution. The change in the Hst-1 structure upon binding with the silver complex was examined by circular dichroism spectroscopy. The wound healing applicability of the histatin polypeptide and its metal complex conjugate was tested using the synthesized Hst-1 and Hst-1-[Ag(II)CHX] complex on 3T3-L1 preadipocytes in a cell-spreading assay. The antibacterial activity of the silver metal complex and its Hst-1 conjugate was tested against several gram positive and gram negative bacteria, including Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-resistant coagulase negative staphylococcus (MRCNS) by a broth microdilution method. The results of these experiments revealed that the polypeptide and silver(II) polydiguanide complex retained their individual wound healing and antimicrobial activity even in their conjugate. The conjugate of an antibacterial higher-valent silver polydiguanide complex with a potent wound healing polypeptide (Hst-1) showed promise as a new multifunctional therapeutic wherein the killing and healing functions of the constituent materials are preserved together for the development of new-generation wound-care agents.

Knock-down of argonaute 2 (AGO2) induces apoptosis in myeloid leukaemia cells and inhibits siRNA-mediated silencing of transfected oncogenes in HEK-293 cells.

Understanding the role of oncomirs allows new insights into the development of modern therapeutic approaches for the repression of multiple oncomirs in cancer cells. At present, no suitable approach is available to repress the development of multiple oncomirs in cancer cells. Herein, we report that argonaute 2 (AGO2) could be a unique molecule to regulate the development of multiple oncomirs in cancer cells. Knock-down of AGO2 by custom-made AGO2 siRNA resulted in the induction of apoptosis in myeloid leukaemia cells (HL-60). Further investigations revealed that knock-down of AGO2 by custom-made AGO2 siRNA in HEK-293 cells resulted in silencing of the expression of target genes vascular endothelial growth factor A and histone deacetylase 2, which are known to be involved in the development of myeloid leukaemia. From these results, it can be predicted that AGO2 could regulate siRNA-mediated RNAi pathways in cancer cells. Furthermore, we investigated the possible implication of AGO2 in drug-induced apoptosis. Investigations revealed that treatment with the newly synthesized drug analogue SH-03[{(7S,7aR,13aS)-9,10-dimethoxy-3,3-dimethyl-7,7a,13,13atetrahydro-3H-chromeno[3,4-b]pyrano[2,3-h]chromen-7-ol}] could induce AGO2-mediated apoptosis in myeloid leukaemia cells via intrinsic apoptotic pathways independent of Dicer.

Highly sensitive polymerase chain reaction-free quantum dot-based quantification of forensic genomic DNA

Forensic DNA samples can degrade easily due to exposure to light and moisture at the crime scene. In addition, the amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. The accurately quantified extracted genomic DNA is then used as a DNA template in polymerase chain reaction (PCR) amplification for short tandem repeat (STR) human identification. Accordingly, highly sensitive and human-specific quantification of forensic DNA samples is an essential issue in forensic study. In this work, a quantum dot (Qdot)-labeled Alu sequence was developed as a probe to simultaneously satisfy both the high sensitivity and human genome selectivity for quantification of forensic DNA samples. This probe provided PCR-free determination of human genomic DNA and had a 2.5-femtogram detection limit due to the strong emission and photostability of the Qdot. The Qdot-labeled Alu sequence has been used successfully to assess 18 different forensic DNA samples for STR human identification.

Green fluorescent protein (GFP) as a direct biosensor for mutation detection: Elimination of false-negative errors in target gene expression

A new method was developed to determine the mutagenic efficacy of a suspected mutagen by employing green fluorescent protein (GFP) as a direct biosensor for mutation detection. Alterations in target gene (AcGFP1) expression after mutagen [(+/-)-7p,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE)] treatment were measured to detect the mutagenic efficacy of the carcinogen. In contrast to mutagen treatment of the entire plasmid or cell culture, the target AcGFP1gene devoid of the plasmid backbone was exposed to BPDE (10-500 microM) to eliminate the need for an additional fusion gene. Shuttle vectors (pAcGFP-N1) were religated to the AcGFP1 gene with BPDE adducts (0-8.59 microM) and replicated in the eukaryotic host. This approach eliminated false-negative errors in target gene expression that arose from BPDE adduct formation in the residual plasmid backbone rather than in the AcGFP1 gene. Determination of the BPDE-AcGFP1 adducts allowed the quantitative mutagenic effect of the BPDE adducts on AcGFP1 gene expression to be monitored. The results obtained with flow cytometry and confocal microscopy validate our method and demonstrate efficient and direct use of GFP as a biosensor for mutation detection.

Early stage high-content HIV diagnosis based on concurrent monitoring of actin cytoskeleton, CD3, CD4, and CD8.

Antiretroviral treatment can reduce the death rate of human immunodeficiency virus (HIV) infection, and its effectiveness is maximized at the early stage of HIV infection. The present study demonstrates an early stage high-content HIV diagnosis based on multicolor concurrent monitoring of CD4, CD8, and CD3 coreceptors and F-actin cytoskeleton using quantum dot (Qdot)-antibody conjugates at the single cell level. Artificial HIV infection of peripheral blood mononuclear cells (PBMCs) was achieved by the treatment of PBMCs with gp120 glycoproteins. Using the present system, it was determined that the CD4/CD8 ratios of normal PBMCs obtained from the blood samples of 11 adults were in the range of 1.04 to 1.52 as a result of the quantitative counting of single PBMCs while the CD4/CD8 ratios of artificial HIV-infected PBMCs were from 0.045 to 0.63. In addition, the structural changes of actin filament alignments in PBMCs bound to gp120 proteins were clearly observed by the multicolor single cell imaging system. This approach suggests a new model of accurate early stage HIV diagnosis simultaneously providing information on actin cytoskeleton and subtypes of PBMCs as well as their CD4/CD8 ratios.

VEGF inhibitor (Iressa) arrests histone deacetylase expression: Single‐cell cotransfection imaging cytometry for multi‐target‐multi‐drug analysis

Multi‐target‐multi‐drug approaches are needed to accelerate the process of drug discovery screening and to design efficient therapeutic strategies against diseases that involve alterations in multiple cellular targets. Herein we report single‐cell cotransfection imaging cytometry to quantitatively screen drug‐induced off‐target effects. Vascular endothelial growth factor (VEGF) and histone deacetylase (HDAC) genes amplified from the genomic DNA were cloned in fluorescently tagged gene constructs (RFP‐HDAC/YFP‐VEGF). These gene constructs were cotransfected in HEK‐293 cells to explore the possibility of off‐target effects of 4‐phenylbutyrate and Iressa on the expression of VEGF and HDAC through single‐cell imaging cytometry. Iressa (10 µM) treatment at the time of cotransfection or 48 h after cotransfection of RFP‐HDAC/YFP‐VEGF plasmids in HEK‐293 cells resulted in off‐target effects on HDAC expression. These results suggest possible applications of Iressa in the treatment of diseases in which expression of both HDAC and VEGF should be inhibited. 4‐Phenylbutyrate (2.0 mM) did not show any off‐target effects on VEGF expression. The developed quantitative multicolor live single‐cell cotransfection imaging can be employed to select better drug combinations for faster screening and greater accuracy in multi‐target‐multi‐drug analysis by increasing the on‐target/desired off‐target effects and eliminating the undesirable off‐target effects. J. Cell. Physiol. 226: 2115–2122, 2011.