Measurement of dielectric properties of cells at single-cell resolution using electrorotation.

Dielectric properties of a cell are biophysical properties of high interest for various applications. However, measuring these properties accurately is not easy, which can be exemplified by the large variations in reported dielectric properties of the same cell types. This paper presents a method for measuring the dielectric properties of cells at high frequency, especially lipid-producing microalgae, at single-cell resolution, by integrating an electrorotation-based dielectric property measurement method with a negative dielectrophoretic (nDEP) force-based single-cell trapping method into a single device. In this method, a four-electrode nDEP structure was used to trap a single cell in an elevated position in the center of another four-electrode structure that can apply electrorotational force. By measuring the speed of cell rotation under different applied electrorotation frequencies and fitting the results into a theoretical core-shell cell model, the dielectric properties of cells, including membrane capacitance and cytoplasm conductivity, could be obtained. This system was applied to measure the dielectric properties of lipid-accumulating microalga Chlamydomonas reinhardtii strain Sta6 by applying an electrorotation signal of up to 100 MHz. By utilizing a broad frequency range and expanding the measurement spectra to a high frequency region, increased accuracy in fitting the dielectric parameters to a theoretical model was possible, especially the cytoplasm conductivity. The developed method can be used in various applications, such as screening microalgae based on their lipid production capabilities, separating cells of different dielectric properties, identifying different cell types, as well as conducting basic biophysical analyses of cellular properties.

Cell Washing and Solution Exchange in Droplet Microfluidic Systems.

Water-in-oil emulsion droplet microfluidic systems have been extensively developed, and currently, almost all cell handling steps can be conducted in this format. An exception is the cell washing and solution exchange step, which is commonly utilized in many conventional cell assays. This paper presents an in-droplet cell washing and solution exchange technology that utilizes dielectrophoretic (DEP) force to move all cells to one side of a droplet, followed by asymmetrical splitting of the droplet to obtain a small daughter droplet that contains all or most of the cells, and then finally merges this cell-concentrated droplet with a new droplet that contains the desired solution. These sequential droplet manipulation steps were integrated into a single platform, where up to 88% of the original solution in the droplet could be exchanged with the new solution while keeping cell loss to less than 5%. Two application examples were demonstrated using the developed technology. In the first example, green microalga Chlamydomonas reinhardtii cells were manipulated using negative DEP force to exchange the regular culture medium with a nitrogen-limited medium to induce lipid production. In the second example, Salmonella enterica cells were manipulated using positive DEP force to replace fluorescent dye that models fluorescent cell stains that contribute to high background noise in fluorescence-based droplet content detection with fresh buffer solution, significantly improving the droplet content detection sensitivity. Since the cell washing step is one of the most frequently utilized steps in many cell biology assays, we expect that the developed technology can significantly broaden the type of assay that can be conducted in droplet microfluidic format.

Phosphonate-Modified UiO-66 Brønsted Acid Catalyst and Its Use in Dehydra-Decyclization of 2-Methyltetrahydrofuran to Pentadienes.

Phosphorus-modified all-silica zeolites exhibit activity and selectivity in certain Brønsted acid catalyzed reactions for biomass conversion. In an effort to achieve similar performance with catalysts having well-defined sites, we report the incorporation of Brønsted acidity to metal-organic frameworks with the UiO-66 topology, achieved by attaching phosphonic acid to the 1,4-benzenedicarboxylate ligand and using it to form UiO-66-PO3 H2 by post-synthesis modification. Characterization reveals that UiO-66-PO3 H2 retains stability similar to UiO-66, and exhibits weak Brønsted acidity, as demonstrated by titrations, alcohol dehydration, and dehydra-decyclization of 2-methyltetrahydrofuran (2-MTHF). For the later reaction, the reported catalyst exhibits site-time yields and selectivity approaching that of phosphoric acid on all-silica zeolites. Using solid-state NMR and deprotonation energy calculations, the chemical environments of P and the corresponding acidities are determined.

Single-Cell Isolation of Circulating Tumor Cells from Whole Blood by Lateral Magnetophoretic Microseparation and Microfluidic Dispensing.

This paper introduces a single-cell isolation technology for circulating tumor cells (CTCs) using a microfluidic device (the "SIM-Chip"). The SIM-Chip comprises a lateral magnetophoretic microseparator and a microdispenser as a two-step cascade platform. First, CTCs were enriched from whole blood by the lateral magnetophoretic microseparator based on immunomagnetic nanobeads. Next, the enriched CTCs were electrically identified by single-cell impedance cytometer and isolated as single cells using the microshooter. Using 200 µL of whole blood spiked with 50 MCF7 breast cancer cells, the analysis demonstrated that the single-cell isolation efficiency of the SIM-Chip was 82.4%, and the purity of the isolated MCF7 cells with respect to WBCs was 92.45%. The data also showed that the WBC depletion rate of the SIM-Chip was 2.5 × 10(5) (5.4-log). The recovery rates were around 99.78% for spiked MCF7 cells ranging in number from 10 to 90. The isolated single MCF7 cells were intact and could be used for subsequent downstream genetic assays, such as RT-PCR. Single-cell culture evaluation of the proliferation of MCF7 cells isolated by the SIM-Chip showed that 84.1% of cells at least doubled in 5 days. Consequently, the SIM-Chip could be used for single-cell isolation of rare target cells from whole blood with high purity and recovery without cell damage.

Analytical evaluation for somatic mutation detection in circulating tumor cells isolated using a lateral magnetophoretic microseparator.

CTCs are currently in the spotlight because provide comprehensive genetic information that enables monitoring of the evolution of cancer and selection of appropriate therapeutic strategies that cannot be obtained from a single-site tumor biopsy. Despite their importance, current techniques for isolating CTCs are limited in terms of their ability to yield high-quality CTCs from peripheral blood for use in profiling cancer genetic mutations by DNA sequencing technologies. This paper introduces a lateral magnetophoretic microseparator (the 'CTC-µChip') for isolating highly pure CTCs from blood, which facilitates the detection of somatic mutations in isolated CTCs. To isolate CTCs from peripheral blood, nucleated cells were first prepared by red blood cell lysis. Then, CTCs were isolated from nucleated cells within 30 min using the CTC-µChip. Analytical evaluation using 5 mL blood samples spiked with 5-50 MCF7 breast cancer cells demonstrated that the average recovery rate of the CTC-µChip was 99.08 %. The average number of residual white blood cells (WBCs) in isolated samples was 53, meaning that the WBC depletion rate is 472,000-fold (5.67 log), assuming that blood contains 5 × 10(6) WBCs per milliliter. The isolated MCF7 cells had a purity of 6.9 - 67.9 %, depending on the spiked MCF7 concentration. Using next-generation sequencing technology, heterozygous somatic mutations (PIK3CA and APC) of MCF7 cells were evaluated in the isolated samples. The results showed that somatic mutations could be detected in as few as two MCF7 cells per milliliter of blood, indicating that the CTC-µChip facilitates the detection of somatic variants in CTCs.

Electrical Detection Method for Circulating Tumor Cells Using Graphene Nanoplates.

This paper presents a microfluidic device for electrical discrimination of circulating tumor cells (CTCs) using graphene nanoplates (GNPs) as a highly conductive material bound to the cell surface. For two-step cascade discrimination, the microfluidic device is composed of a CTC-enrichment device and an impedance cytometry. Using lateral magnetophoresis, the CTC-enrichment device enriches rare CTCs from millions of background blood cells. Then, the impedance cytometry electrically identifies CTCs from the enriched sample, containing CTCs and persistent residual blood cells, based on the electrical impedance of CTCs modified by the GNPs. GNPs were used as a highly conductive material for modifying surface conductivity of CTCs, thereby improving the accuracy of electrical discrimination. The experimental results showed that a colorectal cancer cell line (DLD-1) spiked into peripheral blood was enriched by nearly 500-fold by the CTC-enrichment device. The phase of the electrical signal measured from DLD-1 cells covered by GNPs shifted by about 100° in comparison with that from normal blood cells, which allows the impedance cytometry to identify CTCs at a rate of 94% from the enriched samples.

Quercetin 3-O-glucoside suppresses epidermal growth factor-induced migration by inhibiting EGFR signaling in pancreatic cancer cells.

Pancreatic cancer is one of the most dangerous cancers and is associated with a grave prognosis. Despite increased knowledge of the complex signaling networks responsible for progression of pancreatic cancer, many challenging therapies have fallen short of expectations. In this study, we examined the anti-migratory effect of quercetin 3-O-glucoside in epidermal growth factor-induced cell migration by inhibiting EGF receptor (EGFR) signaling in several human pancreatic cancer cell lines. Treatment with quercetin, quercetin 3-O-glucoside, and quercetin 7-O-glucoside differentially suppressed epidermal growth factor-induced migration activity of human pancreatic cancer cells. In particular, quercetin 3-O-glucoside strongly inhibited the infiltration activity of pancreatic cancer cells in a dose-dependent manner. Furthermore, quercetin 3-O-glucoside exerted the anti-migratory effect even at a relatively low dose compared with other forms of quercetin. The anti-tumor effects of quercetin 3-O-glucoside were mediated by selectively inhibiting the EGFR-mediated FAK, AKT, MEK1/2, and ERK1/2 signaling pathway. Combinatorial treatment with quercetin 3-O-glucoside plus gemcitabine showed the synergistic anti-migratory effect on epidermal growth factor-induced cell migration in human pancreatic cancer cell lines. These results suggest that quercetin 3-O-glucoside has potential for anti-metastatic therapy in human pancreatic cancer.

Direct dynamic nuclear polarization targeting catalytically active (27)Al sites.

Here we present a systematic study of direct (27)Al Dynamic Nuclear Polarization (DNP) as induced by three different mono-radical probes with side groups of varying charge states. By employing 4-amino TEMPO that adsorbs to negatively charged surface sites of Al-SBA-15, we achieve a (27)Al signal enhancement factor of ∼13 compared to a signal enhancement factor of ∼3-4 from mono-radicals that do not adsorb as strongly to the surfaces of Al-SBA-15, here 4-carboxy- and 4-hydroxy-TEMPO. By performing Electron Spin Echo Envelope Modulation (ESEEM) experiments and continuous wave (cw) Electron Paramagnetic Resonance (EPR) lineshape analysis using various nitroxide probes imbibed in Al-SBA-15, we find that direct (27)Al DNP enhancements achieved with different spin probes can be attributed to proximity and local concentration of the spin probes to aluminum on the surface of mesoporous alumina-silica.

Probing water density and dynamics in the chaperonin GroEL cavity.

ATP-dependent binding of the chaperonin GroEL to its cofactor GroES forms a cavity in which encapsulated substrate proteins can fold in isolation from bulk solution. It has been suggested that folding in the cavity may differ from that in bulk solution owing to steric confinement, interactions with the cavity walls, and differences between the properties of cavity-confined and bulk water. However, experimental data regarding the cavity-confined water are lacking. Here, we report measurements of water density and diffusion dynamics in the vicinity of a spin label attached to a cysteine in the Tyr71 → Cys GroES mutant obtained using two magnetic resonance techniques: electron-spin echo envelope modulation and Overhauser dynamic nuclear polarization. Residue 71 in GroES is fully exposed to bulk water in free GroES and to confined water within the cavity of the GroEL-GroES complex. Our data show that water density and translational dynamics in the vicinity of the label do not change upon complex formation, thus indicating that bulk water-exposed and cavity-confined GroES surface water share similar properties. Interestingly, the diffusion dynamics of water near the GroES surface are found to be unusually fast relative to other protein surfaces studied. The implications of these findings for chaperonin-assisted folding mechanisms are discussed.

Effect of electron spin dynamics on solid-state dynamic nuclear polarization performance.

For the broadest dissemination of solid-state dynamic nuclear polarization (ssDNP) enhanced NMR as a material characterization tool, the ability to employ generic mono-nitroxide radicals as spin probes is critical. A better understanding of the factors contributing to ssDNP efficiency is needed to rationally optimize the experimental condition for the practically accessible spin probes at hand. This study seeks to advance the mechanistic understanding of ssDNP by examining the effect of electron spin dynamics on ssDNP performance at liquid helium temperatures (4-40 K). The key observation is that bi-radicals and mono-radicals can generate comparable nuclear spin polarization at 4 K and 7 T, which is in contrast to the observation for ssDNP at liquid nitrogen temperatures (80-150 K) that finds bi-radicals to clearly outperform mono-radicals. To rationalize this observation, we analyze the change in the DNP-induced nuclear spin polarization (Pn) and the characteristic ssDNP signal buildup time as a function of electron spin relaxation rates that are modulated by the mono- and bi-radical spin concentration. Changes in Pn are consistent with a systematic variation in the product of the electron spin-lattice relaxation time and the electron spin flip-flop rate that constitutes an integral saturation factor of an inhomogeneously broadened EPR spectrum. We show that the comparable Pn achieved with both radical species can be reconciled with a comparable integral EPR saturation factor. Surprisingly, the largest Pn is observed at an intermediate spin concentration for both mono- and bi-radicals. At the highest radical concentration, the stronger inter-electron spin dipolar coupling favors ssDNP, while oversaturation diminishes Pn, as experimentally verified by the observation of a maximum Pn at an intermediate, not the maximum, microwave (µw) power. At the maximum µw power, oversaturation reduces the electron spin population differential that must be upheld between electron spins that span a frequency difference matching the (1)H NMR frequency-characteristic of the cross effect DNP. This new mechanistic insight allows us to rationalize experimental conditions where generic mono-nitroxide probes can offer competitive ssDNP performance to that of custom designed bi-radicals, and thus helps to vastly expand the application scope of ssDNP for the study of functional materials and solids.

Circulating tumor cell microseparator based on lateral magnetophoresis and immunomagnetic nanobeads.

This paper presents a circulating tumor cell (CTC) microseparator for isolation of CTCs from human peripheral blood using immunomagnetic nanobeads with bound antiepithelial cell adhesive molecule (EpCAM) antibodies that specifically bind to epithelial cancer cells. The isolation is performed through lateral magnetophoresis, which is induced by high-gradient magnetic separation technology, involving a ferromagnetic wire array inlaid in the bottom substrate of a microchannel. Experimental results showed that the CTC microseparator isolates about 90% of spiked CTCs in human peripheral blood at a flow rate of up to 5 mL/h and purifies to approximately 97%. The overall isolation procedure was completed within 15 min for 200 µL of peripheral blood. CTCs from peripheral blood of patients with breast and lung cancers were isolated with the CTC microseparator, and the results were compared with those of healthy donors. Using a fluorescence-based viability assay, the viability of CTCs isolated from peripheral blood of patients with cancer was observed. In addition, the usefulness of the CTC microseparator for subsequent genetic assay was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of cancer-specific genes using CTCs isolated from patients with cancer.

An electrorotation technique for measuring the dielectric properties of cells with simultaneous use of negative quadrupolar dielectrophoresis and electrorotation.

This paper presents an effective electrorotation technique for measuring the dielectric properties of cells using a superposed electrical signal, which can simultaneously generate negative quadrupolar dielectrophoretic (nQDEP) force and electrorotational (ROT) torque. The proposed technique involves a three-dimensional (3D) octode, which includes four electrodes arranged in a crisscross pattern on the top and bottom of a microchannel, respectively. A single cell was trapped in the center of the 3D octode by the nQDEP force and simultaneously rotated by the ROT torque. Using the proposed electrorotation technique, ROT spectra of human leukocyte subpopulations (T and B lymphocytes, granulocytes, and monocytes) and metastatic human breast (SkBr3) and lung (A549) cancer cell lines were accurately measured without any disturbance. Torque on the cells generated by the ROT signal was analyzed theoretically based on the single-shell dielectric model for the cells. Furthermore, the dielectric properties of the cells, such as area-specific membrane capacitance and cytoplasm conductivity, were extracted using the measured ROT spectra and the analyzed torque.

Tutorial on Lateral Dielectrophoretic Manipulations in Microfluidic Systems.

Microfluidic lab-on-a-chip systems can offer cost- and time-efficient biological assays by providing high-throughput analysis at very small volume scale. Among these extremely broad ranges of assays, accurate and specific cell and reagent control is considered one of the most important functions. Dielectrophoretic (DEP)-based manipulation technologies have been extensively developed for these purposes due to their label-free and high selectivity natures as well as due to their simple microstructures. Here, we provide a tutorial on how to develop DEP-based microfluidic systems, including a detailed walkthrough of dielectrophoresis theory, instruction on how to conduct simulation and calculation of electric field and generated DEP force, followed with guidance on microfabricating two forms of DEP microfluidic systems, namely lateral DEP and droplet DEP, and how best to conduct experiments in such systems. Finally, we summarize most recent DEP-based microfluidic technologies and applications, including systems for blood diagnoses, pathogenicity studies, in-droplet content manipulations, droplet manipulations and merging, to name a few. We conclude by suggesting possible future directions on how DEP-based technologies can be utilized to overcome current challenges and improve the current status in microfluidic lab-on-a-chip systems.

Ethanol-Extracted Acorn Induces Anti-Inflammatory Effects in Human Keratinocyte and Production of Hyaluronic Acid in Human Fibroblasts.

Acorn (Quercus acutissima CARR.) has been used in traditional food and medicinal ethnopharmacology in Asia, and it has shown multifarious functions such as antidementia, antiobesity, and antiasthma functions. However, there is limited scientific evidence about the efficacy of acorn for ameliorating skin problems. Treatment with ethanol-extracted acorns (EeA's) ablated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX2), monocyte chemoattractant protein-1 (MCP-1), and interleukin (IL)-8 stimulated by tumor necrosis factor (TNF)-α in human adult low calcium high temperature (HaCaT) cells under sublethal dosages. In addition, treatment with EeA dose dependently inhibited the ex vivo hyper keratin formation induced by TNF-α in HaCaT cells in conjunction with the blockade of cytokeratin-1 (CK-1) and cytokeratin-5 (CK-5) expression. Moreover, EeA treatment stimulated the expression of hyaluronic acid (HA) expression in human fibroblasts in a dose-dependent manner. Linoleamide was identified as the functional component of EeA using preparative high-performance liquid chromatography and ultra high performance liquid chromatography-mass spectrometry-mass spectrometry analysis, and the anti-inflammatory features and enhanced HA expression were verified. Collectively, these results suggest the efficacy of EeA supplementation in improving skin problems via anti-inflammation and upregulating HA production.

Identification of Microorganisms that Bind Specifically to Target Materials of Interest Using a Magnetophoretic Microfluidic Platform.

Discovery of microorganisms and their relevant surface peptides that specifically bind to target materials of interest can be achieved through iterative biopanning-based screening of cellular libraries having high diversity. Recently, microfluidics-based biopanning methods have been developed and exploited to overcome the limitations of conventional methods where controlling the shear stress applied to remove cells that do not bind or only weakly bind to target surfaces is difficult and the overall experimental procedure is labor-intensive. Despite the advantages of such microfluidic methods and successful demonstration of their utility, these methods still require several rounds of iterative biopanning. In this work, a magnetophoretic microfluidic biopanning platform was developed to isolate microorganisms that bind to target materials of interest, which is gold in this case. To achieve this, gold-coated magnetic nanobeads, which only attached to microorganisms that exhibit high affinity to gold, were used. The platform was first utilized to screen a bacterial peptide display library, where only the cells with surface peptides that specifically bind to gold could be isolated by the high-gradient magnetic field generated within the microchannel, resulting in enrichment and isolation of many isolates with high affinity and high specificity toward gold even after only a single round of separation. The amino acid profile of the resulting isolates was analyzed to provide a better understanding of the distinctive attributes of peptides that contribute to their specific material-binding capabilities. Next, the microfluidic system was utilized to screen soil microbes, a rich source of extremely diverse microorganisms, successfully isolating many naturally occurring microorganisms that show strong and specific binding to gold. The results show that the developed microfluidic platform is a powerful screening tool for identifying microorganisms that specifically bind to a target material surface of interest, which can greatly accelerate the development of new peptide-driven biological materials and hybrid organic-inorganic materials.

ZrO2/ZnO/TiO2 Nanocomposite Coatings on Stainless Steel for Improved Corrosion Resistance, Biocompatibility, and Antimicrobial Activity.

The ultrathin nanocomposite coatings made of zirconium oxide (ZrO2), zinc oxide (ZnO), and titanium oxide (TiO2) on stainless steel (SS) were prepared by the radio frequency sputtering method, and the effects of the nanocomposite coating on corrosion protection and antibacterial activities of nanocomposite coated SS were investigated. Scanning electron microscopy was conducted to observe surface morphology of nanocomposite coatings with distinct distribution of grains with the formation on SS substrate. From the electrochemical impedance spectroscopy results, ZrO2/ZnO/TiO2 nanocomposite coating showed excellent corrosion protection performance at 37 °C during immersion in simulated body fluid and saliva solution for 12 and 4 weeks, respectively. The impedance of ZrO2/ZnO/TiO2 (40/10/50) nanocomposite coated SS exhibited values about 5 orders of magnitude higher than that of uncoated SS with polarization at the low-frequency region. Cell viability of ZrO2/ZnO/TiO2 nanocomposite coated SS was examined under mouse fibroblasts culture (L929), and it was observed that the nanocomposite coating improves proliferation through effective cellular attachment compared to uncoated SS. From the antimicrobial activity results, ZrO2/ZnO/TiO2 nanocomposite-coated SS showed killing efficiency of 81.2% and 72.4% against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus, respectively.

Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins.

Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine's ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine's ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems.

Eriodictyol Attenuates Cholangiocarcinoma Malignancy by Regulating HMOX1 Expression: An In Vitro Study.

BACKGROUND/AIM: Cholangiocarcinoma remains one of the most dangerous types of cancer. Eriodictyol is a well-known flavonoid having effective bioactivity against various malignant tumor types. However, the anticancer effect of eriodictyol against cholangiocarcinoma remains ambiguous. Thus, the aim of the present study was to investigate the effects of eriodictyol on human cholangiocarcinoma. MATERIALS AND METHODS: The biological effects of eriodictyol were validated by viability assay, colony formation and western blot analysis. The significance of heme oxygenase 1 (HMOX1) expression in cholangio-carcinoma was demonstrated using bioinformatics analysis and knockdown of HMOX1 by transfection with short interfering (si)-RNA. RESULTS: Eriodictyol highly reduced the in vitro viability of SNU-308, SNU-478, SNU-1079, and SNU-1196 cholangiocarcinoma cells compared with that of 293T cells, in a dose-dependent manner. The anticancer effect of eriodictyol was achieved by caspase-3-mediated apoptosis. In particular, eriodictyol increased HMOX1 expression, which resulted in attenuation of cholangiocarcinoma cell proliferation. In contrast, ablating HMOX1 expression by si-RNA transfection against HMOX1 made cholangiocarcinoma cells insensitive to the antiproliferative effect of eriodictyol treatment. CONCLUSION: These results collectively indicate that eriodictyol acts as an anticancer agent via regulation of HMOX1 expression against human cholangiocarcinoma.

Development of single-cell-level microfluidic technology for long-term growth visualization of living cultures of Mycobacterium smegmatis.

Analysis of growth and death kinetics at single-cell resolution is a key step in understanding the complexity of the nonreplicating growth phenotype of the bacterial pathogen Mycobacterium tuberculosis. Here, we developed a single-cell-resolution microfluidic mycobacterial culture device that allows time-lapse microscopy-based long-term phenotypic visualization of the live replication dynamics of mycobacteria. This technology was successfully applied to monitor the real-time growth dynamics of the fast-growing model strain Mycobacterium smegmatis (M. smegmatis) while subjected to drug treatment regimens during continuous culture for 48 h inside the microfluidic device. A clear morphological change leading to significant swelling at the poles of the bacterial membrane was observed during drug treatment. In addition, a small subpopulation of cells surviving treatment by frontline antibiotics was observed to recover and achieve robust replicative growth once regular culture media was provided, suggesting the possibility of identifying and isolating nonreplicative mycobacteria. This device is a simple, easy-to-use, and low-cost solution for studying the single-cell phenotype and growth dynamics of mycobacteria, especially during drug treatment.

Lapathoside A Isolated from Fagopyrum esculentum Induces Apoptosis in Human Pancreatic Cancer Cells.

BACKGROUND/AIM: Lapathoside A, a phenylpropanoid ester, was isolated from the roots of buckwheat by searching for bioactive compounds against human pancreatic cancer cells. MATERIALS AND METHODS: Buckwheat root extracts, prepared by 70% ethanol, were separated into n-hexane, methylene chloride, ethyl acetate, n-butanol, and water fraction by solvent partitioning. Seven fractions were obtained from the ethyl acetate fraction by liquid chromatography, and fraction No. 6 contained lapathoside A. The effects of lapathoside A on Panc-1 and SNU-213 human pancreatic cancer cell lines were examined. RESULTS: The structure of lapathoside A was determined by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and nuclear magnetic resonance analysis. Next, we investigated whether lapathoside A has anticancer activity in human pancreatic cancer cell lines (PANC-1 and SNU-213). After treatment with 25 µM lapathoside A, viability of PANC-1 and SNU-213 cells decreased to about 40 and 27%, respectively. In addition, lapathoside A treatment also increased apoptosis while affecting the expression levels of apoptotic proteins. CONCLUSION: The effect of lapathoside A on apoptosis was confirmed in pancreatic cancer cell lines, supporting the application of lapathoside A in the treatment of pancreatic cancer.