Minseok S. Kim

Potent Antiandrogen and Androgen Receptor Activities of an Angelica gigas–Containing Herbal Formulation: Identification of Decursin as a Novel and Active Compound with Implications for Prevention and Treatment of Prostate Cancer

Androgen and androgen receptor (AR)-mediated signaling are crucial for the development of prostate cancer. Identification of novel and naturally occurring phytochemicals that target androgen and AR signaling from Oriental medicinal herbs holds exciting promises for the chemoprevention of this disease. In this article, we report the discovery of strong and long-lasting antiandrogen and AR activities of the ethanol extract of a herbal formula (termed KMKKT) containing Korean Angelica gigas Nakai (AGN) root and nine other Oriental herbs in the androgen-dependent LNCaP human prostate cancer cell model. The functional biomarkers evaluated included a suppression of the expression of prostate-specific antigen (PSA) mRNA and protein (IC50, approximately 7 microg/mL, 48-hour exposure) and an inhibition of androgen-induced cell proliferation through G1 arrest and of the ability of androgen to suppress neuroendocrine differentiation at exposure concentrations that did not cause apoptosis. Through activity-guided fractionation, we identified decursin from AGN as a novel antiandrogen and AR compound with an IC50 of approximately 0.4 microg/mL (1.3 micromol/L, 48-hour exposure) for suppressing PSA expression. Decursin also recapitulated the neuroendocrine differentiation induction and G1 arrest actions of the AGN and KMKKT extracts. Mechanistically, decursin in its neat form or as a component of AGN or KMKKT extracts inhibited androgen-stimulated AR translocation to the nucleus and down-regulated AR protein abundance without affecting the AR mRNA level. The novel antiandrogen and AR activities of decursin and decursin-containing herbal extracts have significant implications for the chemoprevention and treatment of prostate cancer and other androgen-dependent diseases.

Full Surface Embedding of Gold Clusters on Silicon Nanowires for Efficient Capture and Photothermal Therapy of Circulating Tumor Cells

We report on rapid thermal chemical vapor deposition growth of silicon nanowires (Si NWs) that contain a high density of gold nanoclusters (Au NCs) with a uniform coverage over the entire length of the nanowire sidewalls. The Au NC-coated Si NWs with an antibody-coated surface obtain the unique capability to capture breast cancer cells at twice the highest efficiency currently achievable (~88% at 40 min cell incubation time) from a nanostructured substrate. We also found that irradiation of breast cancer cells captured on Au NC-coated Si NWs with a near-infrared light resulted in a high mortality rate of these cancer cells, raising a fine prospect for simultaneous capture and plasmonic photothermal therapy for circulating tumor cells.

Breast cancer diagnosis using a microfluidic multiplexed immunohistochemistry platform.

Background Biomarkers play a key role in risk assessment, assessing treatment response, and detecting recurrence and the investigation of multiple biomarkers may also prove useful in accurate prediction and prognosis of cancers. Immunohistochemistry (IHC) has been a major diagnostic tool to identify therapeutic biomarkers and to subclassify breast cancer patients. However, there is no suitable IHC platform for multiplex assay toward personalized cancer therapy. Here, we report a microfluidics-based multiplexed IHC (MMIHC) platform that significantly improves IHC performance in reduction of time and tissue consumption, quantification, consistency, sensitivity, specificity and cost-effectiveness. Methodology/Principal Findings By creating a simple and robust interface between the device and human breast tissue samples, we not only applied conventional thin-section tissues into on-chip without any additional modification process, but also attained perfect fluid control for various solutions, without any leakage, bubble formation, or cross-contamination. Four biomarkers, estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR) and Ki-67, were examined simultaneously on breast cancer cells and human breast cancer tissues. The MMIHC method improved immunoreaction, reducing time and reagent consumption. Moreover, it showed the availability of semi-quantitative analysis by comparing Western blot. Concordance study proved strong consensus between conventional whole-section analysis and MMIHC (n = 105, lowest Kendalls coefficient of concordance, 0.90). To demonstrate the suitability of MMIHC for scarce samples, it was also applied successfully to tissues from needle biopsies. Conclusions/Significance The microfluidic system, for the first time, was successfully applied to human clinical tissue samples and histopathological diagnosis was realized for breast cancers. Our results showing substantial agreement indicate that several cancer-related proteins can be simultaneously investigated on a single tumor section, giving clear advantages and technical advances over standard immunohistochemical method. This novel concept will enable histopathological diagnosis using numerous specific biomarkers at a time even for small-sized specimens, thus facilitating the individualization of cancer therapy.

Highly Efficient Assay of Circulating Tumor Cells by Selective Sedimentation with a Density Gradient Medium and Microfiltration from Whole Blood

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.

Diffusion-based and long-range concentration gradients of multiple chemicals for bacterial chemotaxis assays.

We present a diffusion-driven and long-range concentration gradient generator that uses hydrogel as a porous membrane to prevent convection flows but allow the diffusion of cell signaling molecules for the study of bacterial chemotaxis in a microfluidic device. Using this device, we characterized the critical concentrations associated with the chemotactic responses of cells that initially created a population band and then migrated in bands in the presence of multiconcentration gradients. In addition, this device can be used to study the preferential chemotaxis of bacterial cells toward different carbon sources: glucose, galactose, and mannose were preferred over arabinose and xylose, in this order. This was possible since the device is able to simultaneously produce long-range concentration gradients of different chemicals as well. The method presented in this study is easy to perform and the device is cheap to fabricate, so that we believe that these characteristics not only make this device a very useful tool to study the chemotaxis of various, motile microorganisms but also permit parallel experimentation and reduce the time and effort needed in characterizing bacterial responses to various chemicals.

A microfluidic in vitro cultivation system for mechanical stimulation of bovine embryos.

This work demonstrates a novel microfluidic in vitro cultivation system for embryos that improves their development using a partially constricted channel that mimics peristaltic muscle contraction. Conventional photolithography and a PDMS replica molding process were used to make straight or constricted microchannels. To investigate the effects of constriction geometry on embryonic development, different constriction widths of the channel were designed. Bovine embryos were loaded and incubated by simply placing them on a tilting machine to provide embryo movement via gravity. The fertilized embryos were cultivated on the microfluidic in vitro cultivation system until the blastocyst, hatching, or hatched blastocyst stages. To confirm the quality of blastocysts in the microfluidic channel, double staining was performed and compared with bovine embryos cultivated by the conventional droplet method. The proportion of eight‐cell development among total embryos in the constricted channel (56.7±13.7%; mean±SD) was superior to that in the straight channel (23.9±11.0%). This suggests that the effect of constriction is vital for the early development of bovine embryos in assisted‐reproduction research.

Efficient Isolation and Accurate In Situ Analysis of Circulating Tumor Cells Using Detachable Beads and a High-Pore-Density Filter**

Circulating tumor cells (CTCs) in the bloodstream of cancer patients may indicate the likelihood and severity of metastatic progression. Identification, enumeration, and characterization of CTCs may provide a minimally invasive method for assessing cancer status of patients and prescribing personalized anticancer therapy. However, examination of CTCs requires isolation of these cells from whole blood of patients, which is difficult owing to their low quantity (around one CTC per 10 non-cancerous hematopoietic cells in patient blood). Many techniques are used to isolate CTCs, including density gradient centrifugation and dielectrophoresis, but methods using either size-based exclusion or affinity-based enrichment are common. Size-based exclusion assumes that CTCs are larger than most hematopoietic cells and removes all cells smaller than a pre-determined size threshold. Affinity-based enrichment relies on the expression of surface proteins specific to cancer cells and absent in hematopoietic cells. These methods generally use antibody-conjugated magnetic beads and enrich for CTCs by magnetic separation, such as the CellSearch system. Owing to their heterogeneous nature, it may be practically impossible to isolate CTCs with high isolation efficiency using the aforementioned methods. Some CTCs are reported to be nearly identical in size or even smaller than leukocytes, making them difficult to discriminate by size. As for protein expression, epithelial markers, such as EpCAM (epithelial cell adhesion molecule), are downregulated during epithelialto-mesenchymal transition (EMT). Furthermore, the type and expression levels of surface proteins may vary greatly depending on cancer histological subtype. Considering these variations, finding the right antibody or combination of antibodies that consistently captures all CTCs may prove to be difficult. To maximize isolation efficiency, we devised a dual-mode isolation strategy that combines affinity-based enrichment and size-based exclusion. By using microbeads conjugated with CTC-specific antibodies, the size of CTCs can be augmented to enable better discrimination against leukocytes. Subsequent size filtration isolates bead-bound CTCs, allowing the recovery of even smaller-sized CTCs. However, all beadbased capture methods have the inherent limitation of prohibiting accurate image analysis, which is due to optical distortion created by the presence of beads attached to cells. The attached beads not only impede observation of cellular morphology but can actually alter fluorescence signal intensities (Figure 1), demonstrating the incompatibility of in situ quantitative analysis with bead-based capture methods (see the Supporting Information). Accurate quantification of protein expression can lead to better clinical management, particularly in regards to personalized therapy. The expression levels of predictive biomarkers, such as HER2 and EGFR, are commonly used to match patients with appropriate treatment strategies and predict the effectiveness of anticancer drugs. Therefore, it is important to accurately characterize CTCs, and removal of beads from the surface of CTCs prior to imaging is necessary. Thus, we have developed a new method to detach beads from beadbound cells: By inserting a photocleavable linker between the bead surface and conjugated antibodies, it is possible to remove the attached beads from cells by light irradiation without affecting cell viability. Herein, we demonstrate a novel approach for isolation and subsequent in situ protein-expression analysis of CTCs using detachable beads termed RIA (reversible bead attachment for cell isolation and analysis). Scheme 1 illustrates the entire RIA process. Detachable beads conjugated to CTC-specific antibodies bind to CTCs in whole blood of patients. After incubation, the entire sample is filtered through a high-pore-density membrane filter chip, which contains a maximal number of uniform-sized (pore diameter 8 mm) evenly spaced circular pores (distance between pores 5 mm). This step eliminates almost all hematopoietic cells, while CTCs remain on the filter surface. The [*] H. J. Lee, Dr. J. H. Oh, J. M. Oh, J. M. Park, Dr. J. G. Lee, Dr. M. S. Kim, Dr. Y. J. Kim, Dr. H. J. Kang, Dr. S. S. Lee, Dr. N. Huh In Vitro Diagnostics Lab, Bio Research Center, Samsung Biomedical Research Institute, Samsung Advanced Institute of Technology Gyeonggi-do 446-712 (Korea) E-mail: soosuk@samsung.com namhuh@samsung.com H. J. Lee, Prof. J. W. Choi Interdisciplinary Program of Integrated Biotechnology, Department of Chemical & Biomolecular Engineering Sogang University, Seoul 121-742, Korea E-mail: jwchoi@sogang.ac.kr

High-Voltage Analog System for a Mobile NAND Flash

High-voltage analog circuits, including a novel high-voltage regulation scheme, are presented with emphasis on low supply voltage, low power consumption, low area overhead, and low noise, which are key design metrics for implementing NAND Flash memory in a mobile handset. Regulated high voltage generation at low supply voltage is achieved with optimized oscillator, high-voltage charge pump, and voltage regulator circuits. We developed a design methodology for a high-voltage charge pump to minimize silicon area, noise, and power consumption of the circuit without degrading the high-voltage output drive capability. Novel circuit techniques are proposed for low supply voltage operation. Both the oscillator and the regulator circuits achieve 1.5 V operation, while the regulator includes a ripple suppression circuit that is simple and robust. Through the paper, theoretical analysis of the proposed circuits is provided along with Spice simulations. A mobile NAND Flash device is realized with an advanced 63 nm technology to verify the operation of the proposed circuits. Extensive measurements show agreement with the results predicted by both analysis and simulation.

A microchip filter device incorporating slit arrays and 3-D flow for detection of circulating tumor cells using CAV1-EpCAM conjugated microbeads

Circulating tumor cells (CTCs) are rare cells and the presence of these cells may indicate a poor prognosis and a high potential for metastasis. Despite highly promising clinical applications, CTCs have not been investigated thoroughly, due to many technical limitations faced in their isolation and identification. Current CTC detection techniques mostly take the epithelial marker epithelial cell adhesion molecule (EpCAM), however, accumulating evidence suggests that CTCs show heterogeneous EpCAM expression due to the epithelial-to-mesenchymal transition (EMT). In this study, we report that a microchip filter device incorporating slit arrays and 3-dimensional flow that can separate heterogeneous population of cells with marker for CTCs. To select target we cultured breast cancer cells under prolonged mammosphere culture conditions which induced EMT phenotype. Under these conditions, cells show upregulation of caveolin1 (CAV1) but down-regulation of EpCAM expression. The proposed device which contains CAV1-EpCAM conjugated bead has several tens of times increased throughput. More importantly, this platform enables the enhanced capture yield from metastatic breast cancer patients and obtained cells that expressed various EMT markers. Further understanding of these EMT-related phenotypes will lead to improved detection techniques and may provide an opportunity to develop therapeutic strategies for effective treatment and prevention of cancer metastasis.

A 7MB/s 64Gb 3-bit/cell DDR NAND flash memory in 20nm-node technology

Recently, the demand for 3b/cell NAND flash has been increasing due to a strong market shift from 2b/cell to 3b/cell in NAND flash applications, such as USB disk drives, memory cards, MP3 players and digital still cameras that require cost-effective flash memory. To further expand the 3b/cell market, high write and read performances are essential [1]. Moreover, the device reliability requirements for these applications is a challenge due to continuing NAND scaling to sub-30nm pitches that increases cell-to-cell interference and disturbance. We present a high reliability 64Gb 3b/cell NAND flash with 7MB/s write rate and 200Mb/s asynchronous DDR interface in a 20m-node technology that helps to meet the expanding market demand and application requirement.