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Functional identification of cancer stem-like cells (CSCs) is an established method to identify and study this cancer subpopulation critical for cancer progression and metastasis. The method is based on the unique capability of single CSCs to survive and grow to tumorspheres in harsh suspension culture environment. Recent advances in microfluidic technology have enabled isolating and culturing thousands of single cells on a chip. However, tumorsphere assay takes a relatively long period of time, limiting the throughput of this assay. In this work, we incorporated machine learning with single-cell analysis to expedite tumorsphere assay. We collected 1,710 single-cell events as the database and trained a convolutional neural network model that predicts whether a single cell could grow to a tumorsphere on Day 14 based on its Day 4 image. With this future-telling model, we precisely estimated the sphere formation rate of SUM159 breast cancer cells to be 17.8% based on Day 4 images. The estimation was close to the ground truth of 17.6% on Day 14. The preliminary work demonstrates not only the feasibility to significantly accelerate tumorsphere assay but also a synergistic combination between single-cell analysis with machine learning, which can be applied to many other biomedical applications.

To identify the core structure of 2-aminoethoxydiphenyl borate (2-APB) responsible for the anti-oxidative and protective effect on the ischemia/reperfusion (I/R)-induced heart injury, various 2-APB analogues were analyzed, and several antioxidant assays were performed. Cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Myocardial infarct size was quantified using triphenyl tetrazolium chloride (TTC) staining. The levels of tumor necrosis factor-alpha (TNF-α) and cleaved-caspase-3 protein were evaluated as an indicator for the anti-inflammatory and anti-apoptotic effect, respectively. Our data show that 2-APB, diphenylborinic anhydride (DPBA) and 3-(diphenylphosphino)-1-propylamine (DP3A) all exerted the anti-oxidative activity, but only 2-APB and DPBA can scavenge H2O2. 2-APB and DPBA can potently inhibit hydrogen peroxide (H2O2)- and hypoxanthine/xanthine oxidase (HX/XOD)-induced increases in intracellular H2O2 and H9c2 cell death. 2-APB and DPBA were able to decrease the I/R-induced adult rat cardiomyocytes death, myocardial infarct size, and the levels of malondialdehyde (MDA) and creatine kinase-MB (CK-MB). Our results suggest that the two benzene rings with a boron atom comprise the core structure of 2-APB responsible for the anti-oxidative effect mediated through the reaction with H2O2 and generation of phenolic compounds, which in turn reduced the I/R-induced oxidative stress and injury in the rat heart.

In this work, we investigate the dynamic buffer response of AlInN/GaN high electron mobility transistors (HEMTs) grown on silicon substrates. Significant improvements in vertical breakdown voltage, reduced buffer conduction and charge trapping effect are observed on the samples with a superlattice layer (SL) in the buffer. The dynamic response of the buffer layer largely depends on the composition, thickness and position of the SL in the stress-mitigating buffer. The vertical breakdown voltage increases consistently with decreasing edge-type dislocations when the density of screw-type dislocations is low. Negative back-gating measurements show the lowest buffer current hysteresis in the sample with the lowest edge-type dislocation density, indicating least charge trapping in the sample. Four terminal positive back-gating measurements in the on-state, further confirm the observed trend. Based on the experimental observations, we propose that the total density of threading dislocations, (acts as vertical leakage path) and the acceptor like edge-type dislocations should be accounted along with the electron injection from the silicon substrate to the buffer. Therefore, resulting in higher vertical breakdown voltage by alleviating serious dynamic buffer response in GaN-based HEMTs.

Migration and invasion of cancer cells constitute fundamental processes in tumor progression and metastasis. Migratory cancer cells commonly upregulate expression of plasminogen activator inhibitor 1 (PAI1), and PAI1 correlates with poor prognosis in breast cancer. However, mechanisms by which PAI1 promotes migration of cancer cells remain incompletely defined. Here we show that increased PAI1 drives rearrangement of the actin cytoskeleton, mitochondrial fragmentation, and glycolytic metabolism in triple-negative breast cancer (TNBC) cells. In two-dimensional environments, both stable expression of PAI1 and treatment with recombinant PAI1 increased migration, which could be blocked with the specific inhibitor tiplaxtinin. PAI1 also promoted invasion into the extracellular matrix from coculture spheroids with human mammary fibroblasts in fibrin gels. Elevated cellular PAI1 enhanced cytoskeletal features associated with migration, actin-rich migratory structures, and reduced actin stress fibers. In orthotopic tumor xenografts, we discovered that TNBC cells with elevated PAI1 show collagen fibers aligned perpendicular to the tumor margin, an established marker of invasive breast tumors. Further studies revealed that PAI1 activates ERK signaling, a central regulator of motility, and promotes mitochondrial fragmentation. Consistent with known effects of mitochondrial fragmentation on metabolism, fluorescence lifetime imaging microscopy of endogenous NADH showed that PAI1 promotes glycolysis in cell-based assays, orthotopic tumor xenografts, and lung metastases. Together, these data demonstrate for the first time that PAI1 regulates cancer cell metabolism and suggest targeting metabolism to block motility and tumor progression. Implications: We identified a novel mechanism through which cancer cells alter their metabolism to promote tumor progression.

Background Low levels of natriuretic peptide may activate the renin-angiotensin-aldosterone system, which may contribute to the development of obesity. Therefore, in study we aim to evaluate the relationship between metabolic syndrome (MetS) and serum N-terminal pro‒B-type natriuretic peptide (NT-proBNP) concentration in kidney transplant recipients. Methods Fasting blood samples were obtained from 66 kidney transplant recipients. MetS and its components were defined using the diagnostic criteria of the International Diabetes Federation. Results A total of 20 patients (30.3%) had MetS. Hypertension, prevalence of diabetes, use of statin or fibrate, body weight, body mass index, waist circumference, body fat mass, and levels of systolic blood pressure, total cholesterol, triglyceride, blood urea nitrogen, insulin, and HOMA-IR were higher, whereas the levels of high-density lipoprotein cholesterol and NT-proBNP were lower in patients with MetS. Logarithmically transformed creatinine and log-HOMA-IR were associated with NT-proBNP levels in a multivariable linear regression analysis. Multivariate logistic regression analysis revealed that NT-proBNP was an independent predictor of MetS in kidney transplant recipients. Conclusion Our study has revealed that fasting level of NT-proBNP was negatively associated with MetS and that serum creatinine and HOMA-IR were independent predictors of serum NT-proBNP level in kidney transplant recipients.

Overexpression of EZH2 in estrogen receptor negative (ER-) breast cancer promotes metastasis. EZH2 has been mainly studied as the catalytic component of the Polycomb Repressive Complex 2 (PRC2) that mediates gene repression by trimethylating histone H3 at lysine 27 (H3K27me3). However, how EZH2 drives metastasis despite the low H3K27me3 levels observed in ER- breast cancer is unknown. Here we show that in human invasive carcinomas and distant metastases, cytoplasmic EZH2 phosphorylated at T367 is significantly associated with ER- disease and low H3K27me3 levels. p38-mediated EZH2 phosphorylation at T367 promotes EZH2 cytoplasmic localization and potentiates EZH2 binding to vinculin and other cytoskeletal regulators of cell migration and invasion. Ectopic expression of a phospho-deficient T367A-EZH2 mutant is sufficient to inhibit EZH2 cytoplasmic expression, disrupt binding to cytoskeletal regulators, and reduce EZH2-mediated adhesion, migration, invasion, and development of spontaneous metastasis. These results point to a PRC2-independent non-canonical mechanism of EZH2 pro-metastatic function., Polycomb group protein EZH2 is overexpressed in ER- breast cancer, promoting metastasis. Here, the authors show that independent of the polycomb group, phosphorylation of EZH2 at T367 by p38 promotes cytoplasmic localization of EZH2, binding to vinculin and other regulators of cell migration and invasion.

Designing an efficient, low-cost catalyst is an important objective in the catalytic conversion of methane into value-added products that would be useful in many technological applications. Although IrO2 can activate C–H bonds at mild temperature and have excellent catalytic activity towards C–C coupling reactions, the high cost hinders its deployment. Herein, we propose IrO2 supported on TiO2 surface (IrO2/TiO2) and investigate its catalytic activity towards the methane dehydrogenation and the possibility of C–C coupling reactions with the help of density functional theory (DFT) calculations. Its catalytic performance is compared with that of a pure IrO2 surface. We find that the IrO2/TiO2 surface possesses similar adsorption characteristics of CH4 to the pure IrO2 surface. Our kinetic results indicate that the proposed catalyst exhibits similar enhanced catalytic activity towards methane activation and its successive dehydrogenation and C-C coupling reactions to that of pure IrO2 surface. We believe that methane can be more likely activated over the catalyst of one layer IrO2 supported on TiO2 at low temperatures, and it shows similar performances in thermodynamics like high-cost pure IrO2.

Despite recent advances in cancer treatment, developing better therapeutic reagents remains an essential task for oncologists. To accurately characterize drug efficacy, 3D cell culture holds great promise as opposed to conventional 2D monolayer culture. Due to the advantages of cell manipulation in high-throughput, various microfluidic platforms have been developed for drug screening with 3D models. However, the dissemination of microfluidic technology is overall slow, and one missing part is fast and low-cost assay readout. In this work, we developed a microfluidic chip forming 1920 tumor spheres for drug testing, and the platform is supported by automatic image collection and cropping for analysis. Using conventional LIVE/DEAD staining as the ground truth of sphere viability, we trained a convolutional neural network to estimate sphere viability based on its bright-field image. The estimated sphere viability was highly correlated with the ground truth (R-value > 0.84). In this manner, we precisely estimated drug efficacy of three chemotherapy drugs, doxorubicin, oxaliplatin, and irinotecan. We also cross-validated the trained networks of doxorubicin and oxaliplatin and found common bright-field morphological features indicating sphere viability. The discovery suggests the potential to train a generic network using some representative drugs and apply it to many different drugs in large-scale screening. The bright-field estimation of sphere viability saves LIVE/DEAD staining reagent cost and fluorescence imaging time. More importantly, the presented method allows viability estimation in a label-free and nondestructive manner. In short, with image processing and machine learning, the presented method provides a fast, low-cost, and label-free method to assess tumor sphere viability for large-scale drug screening in microfluidics.

In this work, the role of an $\mathrm{Al}_{\mathrm{x}}\mathrm{Ga}_{1-\mathrm{x}}\mathrm{N}/\mathrm{Al}_{\mathrm{y}}\mathrm{Ga}_{1-\mathrm{y}}\mathrm{N}$ superlattice (SL) layer in the buffer layer on the dislocation density and transport property of AlInN/AIN/GaN heterostructures grown on 150 mm silicon (111) substrate is investigated. It is found that the threading dislocation density in the GaN buffer strongly depends on the composition, thickness and the position of the SL layer. Electron mobility of 1,940 cm2/V-s with two dimensional electron gas density of $\mathrm{1.45}\mathrm{x}\mathrm{10}^{13}\mathrm{cm}^{-2}$ , resulting in a sheet resistance of $221\ \mathrm{ohm}/\square$ , is achieved by using 10 pairs of Al 0.2 Ga 0.8 N/Al 0.8 Ga 0.2 N SL placed just above the thin AIN nucleation layer on Si substrate.

Objectives Diets may alter an individual's metabolism and inflammation, collectively leading to the modulation of cardiovascular health and disease process. The aim of this study was to investigate the effects of diets and diet-associated metabolites on metabolic profiles, inflammatory status, and severity of atherosclerosis. Methods A cross-sectional study was conducted with 81 healthy adults in Taiwan. A food frequency questionnaire was obtained for evaluating dietary intake. Carotid intima-media thickness (CIMT), a relevant marker of subclinical atherosclerosis, was measured by ultrasound. Results Consumption of instant noodles and sugary beverages was associated with worse metabolic profiles. In contrast, the intake of fresh fruit and green vegetables was correlated with better metabolic parameters. Sugary beverages were dose-dependently correlated with higher expressions of toll-like receptor (TLR)2 and TLR4 on monocytes, whereas fresh fruit intake was associated with lower TLRs. Furthermore, consumption of green vegetables, brown rice, and >2000 mL/d of water was inversely correlated with CIMT. The diet-associated metabolites including trimethylamine N-oxide and S-adenosyl- l -homocysteine, were positively associated with CIMT, whereas l -lysine and l -carnitine were associated with decreased CIMT. Interestingly, intake of strict vegetarian foods resulted in lower serum total cholesterol levels without a detectable effect on inflammatory status or CIMT. Conclusions Independent of the pattern of strict vegetarian foods, individuals who consumed more vegetables, fresh fruit, and water showed better cardiovascular health as evidenced by their metabolic and inflammatory status and CIMT results.

Proteolytic degradation of the extracellular matrix represents a key step in cancer dissemination and metastasis. To probe cellular proteolytic activity, fluorescent sensing substrate was developed, yet prior studies focused on average activity of thousands of cells. Considerable evidence suggests a specialized subset of cancer cells are driving metastasis, highlighting the value of single-cell approach to reveal cancer cellular heterogeneity. In addition, when only a small number of cells are available, single-cell analysis is required to draw a statistical conclusion. Here, we present a microfluidic platform that provides high-efficiency cell loading and simple valveless isolation, so the proteolytic activity of a small number (10–100) of cells can be individually characterized. Furthermore, the platform allows monitoring single cells at multiple time points for the investigation of dynamics in proteolytic activity. The presented platform represents a simple and reliable tool for single-cell proteolytic analysis, illuminating the heterogeneous and dynamic nature of cancer cells.

As the molecular precursor of ZnSe, ZnSe·0.5N2H4 inorganic-organic hybrids have received relatively less attention due to the feasibility of their further processing and decomposition into pure-phase ZnSe. Here we demonstrated that ZnSe·0.5N2H4 hybrid nanostructures, which were prepared using a facile hydrazine-assisted hydrothermal method, may practically harvest solar energy for photoconversion applications. By modulating the volume ratio of hydrazine hydrate to deionized water employed in the synthesis, the morphology of the grown ZnSe·0.5N2H4 can be varied, which included nanowires, nanobelts and nanoflakes. With the relatively long exciton lifetime and highly anisotropic structure, ZnSe·0.5N2H4 nanowires performed much better in the photodegradation of rhodamine B than the other two counterpart products. As compared to pure ZnSe nanoparticles and single-phase ZnSe nanowires obtained from further processing ZnSe·0.5N2H4, the ZnSe·0.5N2H4 hybrid nanowires exhibited superior photocatalytic performance under visible light illumination. The hybrid nanowires were further decorated with Au particles to endow them with structural and compositional diversities. Time-resolved photoluminescence spectra suggested that almost 40% of the photoexcited electrons in ZnSe·0.5N2H4 nanowires can be transported to the decorated Au, which enabled a fuller extent of participation of charge carriers in the photocatalytic process and thus conduced to a significant enhancement in the photocatalytic activity. The demonstrations from this work illustrate that ZnSe·0.5N2H4 hybrid nanostructures can serve as a versatile photocatalyst platform for advanced photocatalytic applications.

A facile, green one-pot hydrothermal method has been developed to prepare ZnO-reduced graphene oxide (rGO) composites with controllable rGO content. Time-resolved photoluminescence spectra revealed that an increased electron-transfer rate constant was observed for ZnO–rGO with increasing rGO contents, suggesting that an increased number of photoexcited charge carriers were separated and available for photocatalysis utilization. The photocatalytic properties of the ZnO–rGO composites were investigated by using gaseous acetaldehyde (CH 3 CHO), a typical volatile organic compound (VOC), as the test pollutant. This is the first time that ZnO–rGO composites have been used as photocatalysts for gaseous CH 3 CHO degradation. Compared to pure ZnO, ZnO–rGO composites with suitable rGO contents (1.0 and 3.0 wt%) displayed significantly enhanced photocatalytic activity in both CH 3 CHO degradation and CO 2 generation. This enhancement was ascribed to the rGO support that can promote the carrier utilization efficiency of ZnO by readily accepting the photoexcited electrons from ZnO. As a result, abundant photogenerated holes remained on the ZnO and were available for participation in the CH 3 CHO photodegradation. Furthermore, the photoactivity of ZnO–rGO toward electrolytic water oxidation was evaluated. The results showed that ZnO–rGO composites achieved 50% increase in water oxidation current over pure ZnO under white light illumination. The demonstration from this work may facilitate the use of ZnO–rGO composites in photodegradation of VOCs as well as for photoelectrochemical applications, which is imperative from the environmental and energetic points of view.

Background It has been shown that metabolic syndrome is associated with lower levels of plasma N-terminal pro-B-type natriuretic peptide (Nt-proBNP) in the general population. However, there is no study about the association between Nt-proBNP and metabolic syndrome in hypertensive patients. Aim To elucidate the relationship between Nt-proBNP and components of metabolic syndrome in hypertensive patients. Methods Fasting blood samples were obtained from 74 hypertensive patients in our institution. Plasma levels of Nt-proBNP and other biochemical data were measured. Metabolic syndrome and its components were defined using diagnostic criteria from the International Diabetes Federation. Results Forty-four hypertensive patients met the criteria for metabolic syndrome. We found that plasma Nt-proBNP levels were lower in hypertensive patients with metabolic syndrome attributable to inverse relationships between Nt-proBNP and albumin, triglyceride, insulin, homeostasis model assessment of insulin resistance (HOMA-IR) and pancreatic β-cell function (HOMA-β). We further performed a multivariable linear regression analysis. The result showed that HOMA-IR is the independent predictor of plasma Nt-proBNP levels in hypertensive patients. Conclusions Plasma Nt-proBNP levels are inversely associated with metabolic syndrome in hypertensive patients. HOMA-IR is the independent predictor of Nt-proBNP in hypertensive patients.

Colorectal cancer is a popular cancer type all over the world. Among various treatment regimen, oxaliplatin (Oxa) is approved in combination with 5-fluorouracil (5-FU) and leucovorin (FOLFOX) as the first-line therapy for colorectal cancer of metastatic or advanced stage. MicroRNAs (miRs) may target genes critical for regulating tumor proliferation and invasion. Among different miRs, miR-c has been proved to induce apoptosis, inhibit proliferation and epithelial-mesenchymal transition of cancer cells. Peptide-modified nanoparticles, which are capable of increasing cellular uptake and targeting to specific receptors, are well suitable for delivering Oxa and miR. Moreover, coating pH-sensitive polymers to the outer layer of peptide-modified nanoparticles may enhance specific targeting to the acidic tumor sites. Physicochemical characterization, cellular uptake, cytotoxicity, migration assay, and western blotting studies of these nanoparticle formulations have been performed. Our results indicated that these pH-sensitive and peptide-modified nanoparticles displayed a narrow size distribution. The cellular uptake of peptide-modified nanoparticles was found to be greater than that of naked nanoparticles. Besides, the cellular uptake of pH-sensitive nanoparticles was significantly increased at pH 6.0 compared to that at pH 7.4, suggesting the successful pH-responsive property of these delivery systems. Furthermore, the combined treatment of miR-c and Oxa by peptide-modified nanoparticles increased the cytotoxicity of Oxa to colorectal cancer cells. We demonstrated that the upregulation of apoptosis pathway and the downregulation of β-catenin pathway were accounted for the efficient antitumor effect. In conclusion, the delivery of miR-c and Oxa in pH-sensitive and peptide-modified nanoparticles may provide a novel and potential strategy for colorectal cancer treatment. Citation Format: Yu-Li Lo, Y.C. Chen, C.H. Chang, W.H. Tseng, C.S. Wang. Tumor-targeted and pH-shiftable nanoparticles to encapsulate oxaliplatin and microRNA for colorectal cancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A100. doi:10.1158/1535-7163.TARG-19-A100

A significant improvement in electron mobility has been achieved by several authors on AlInGaN/GaN heterostructures by adding a small amount of Ga to the AlInN alloy. In this study, we propose that thermodynamic stability plays an important role in controlling the electron transport properties of these heterostructures. A quantitative investigation of the thermodynamic stability of the AlInGaN barrier has been carried out analytically, for a wide range of compositions (0.5 ≤ Al ≤ 0.8; In = 0.2, 0.15, 0.1). A slow change in the thermodynamic stability is observed when the Ga atoms replace only the Al atoms. In contrast, a significant improvement in thermodynamic stability is observed when the indium atoms are replaced by the Ga atoms in the same Al0.83In0.17N layer. It is found that the Al content in the range of 65%–70% with 10% In exhibits the highest thermodynamic stability within the calculated composition range owing to the significant reduction in total elastic strain in the barrier. Thereby, it leads to the highest electron mobility, as evidenced by the experimental observations in this work, i.e., electron mobility of 2090 cm2/V s with a sheet carrier density of 1.09 × 1013 cm−2. Therefore, the thermodynamic stability apart from commonly observed scattering mechanisms may at least be partially held to be responsible for the consistent improvement in electron mobility in AlInGaN/GaN heterostructures.

Osteopontin (OPN) is involved in the regulation of vascular calcification processes. The aim of this study was to evaluate the relationship between fasting serum OPN concentration and carotid-femoral pulse wave velocity (cfPWV) in geriatric persons. Fasting blood samples were obtained from 93 geriatric persons. cfPWV were performed by SphygmoCor system. Serum OPN levels were measured using a commercially available enzyme-linked immunosorbent assay. Geriatric adults who had diabetes (P=0.007) or dyslipidemia (P=0.029) had higher cfPWV levels than those without diabetes or dyslipidemia. The univariable linear regression analysis showed that age (P=0.002), waist circumference (P=0.048), body mass index (P=0.004), systolic blood pressure (P=0.001), diastolic blood pressure (P=0.036), pulse pressure (P=0.017), creatinine (P=0.002), and log-OPN level (P=0.001) were positively correlated with cfPWV levels, while the high-density lipoprotein cholesterol (HDL-cholesterol) level (P=0.007) and glomerular filtration rate (P=0.001) were negatively correlated with cfPWV levels among the geriatric adults. Multivariable forward stepwise linear regression analysis of the significant variables also showed that log-OPN (β=0.233,R2=0.123, regression coefficient: 1.868,P=0.011) was still an independent predictor of cfPWV levels in geriatric persons.

Long-acting natriuretic peptide (LANP) is one of the peptide hormones in atrial natriuretic peptide (ANP) prohormone. Its biological properties are blood pressure regulation, maintenance of plasma volume and anticancer effects. The aim of this study was to evaluate the relationship between metabolic syndrome (MetS) and fasting serum LANP concentration in hypertensive patients.Fasting blood samples were obtained from 224 patients with or without hypertension. MetS and its components were defined using diagnostic criteria from the International Diabetes Federation.Eighty-eight hypertensive patients (59.5 %) had MetS. Hypertensive patients with MetS had higher body weight (p = 0.003), waist circumference (p = 0.003), body mass index (p = 0.002), triglyceride concentrations (p = 0.029), insulin levels (p = 0.001), HOMA-IR (p0.003) and HOMA-β (p = 0.049) and lower HDL-C concentrations (p = 0.001), LANP levels (p = 0.012) than those without MetS. The univariable linear regression analysis showed that age (p = 0.038) and the BUN concentration (p = 0.022) were positively correlated with the serum LANP levels, whereas the insulin level (p = 0.001), HOMA-IR (p = 0.004), and HOMA-β (p = 0.001) were negatively correlated with the fasting serum LANP levels among the hypertensive patients. Multivariable forward stepwise linear regression analysis of the significant variables showed that the HOMA-β (β = -0.387, R(2) = 0.141, p0.001) was an independent predictor of fasting serum LANP levels in hypertensive patients.LANP level is significantly reduced in hypertensive patients affected by MetS and is negatively related to pancreatic beta cell function in hypertensive patients.

Background Hypertension is a risk factor for peripheral arterial disease (PAD). Subjects with PAD are at increased risk of future cardiovascular (CV) events. Resistin is involved in the pathological processes of CV diseases. The aim of this study is to investigate whether resistin level is correlated with PAD in hypertensive patients. Methods One hundred and twenty-four hypertensive patients were enrolled in this study. Ankle-brachial index (ABI) values were measured using the automated oscillometric method. An ABI value

Recent research suggests that cancer stem-like cells (CSCs) are the key subpopulation for tumor relapse and metastasis. Due to cancer plasticity in surface antigen and enzymatic activity markers, functional tumorsphere assays are promising alternatives for CSC identification. To reliably quantify rare CSCs (1–5%), thousands of single-cell suspension cultures are required. While microfluidics is a powerful tool in handling single cells, previous works provide limited throughput and lack automatic data analysis capability required for high-throughput studies. In this study, we present the scaling and automation of high-throughput single-cell-derived tumor sphere assay chips, facilitating the tracking of up to ∼10 000 cells on a chip with ∼76.5% capture rate. The presented cell capture scheme guarantees sampling a representative population from the bulk cells. To analyze thousands of single-cells with a variety of fluorescent intensities, a highly adaptable analysis program was developed for cell/sphere counting and size measurement. Using a Pluronic® F108 (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)) coating on polydimethylsiloxane (PDMS), a suspension culture environment was created to test a controversial hypothesis: whether larger or smaller cells are more stem-like defined by the capability to form single-cell-derived spheres. Different cell lines showed different correlations between sphere formation rate and initial cell size, suggesting heterogeneity in pathway regulation among breast cancer cell lines. More interestingly, by monitoring hundreds of spheres, we identified heterogeneity in sphere growth dynamics, indicating the cellular heterogeneity even within CSCs. These preliminary results highlight the power of unprecedented high-throughput and automation in CSC studies.

Background Atherosclerosis is a chronic inflammatory disease of the vessel wall associated with oxidized low-density lipoprotein (oxLDL)-induced apoptosis of endothelial cells. Coenzyme Q10 (CoQ10), a potent antioxidant and a critical intermediate of the electron transport chain, has been reported to inhibit LDL oxidation and thus the progression of atherosclerosis. However, its molecular mechanisms on endothelial cells remain still unclarified. Methods In this study, primary human umbilical vein endothelial cell cultures treated with oxLDL were used to explore the protective effects of CoQ10. Results Our results showed that CoQ10 attenuated the oxLDL-induced generation of reactive oxygen species and improved the antioxidant capacity. CoQ10 also attenuated the oxLDL-mediated down-regulation of endothelial nitric oxide synthase (eNOS) and up-regulation of inducible nitric oxide synthase (iNOS). In addition, CoQ10 suppressed oxLDL-activated NF-κB and downstream inflammatory mediators, including expression of adhesion molecules, release of proinflammatory cytokines and the adherence of monocytic THP-1 cells. Moreover, CoQ10 attenuated oxLDL-altered proapoptotic responses. The inhibitor of eNOS ( l -NIO 10 μM) and iNOS (1400W 10 μM) as well as NO enhancer (SNP 10 μM) were used to clean up the mechanism. Conclusion These results provide new insight into the possible molecular mechanisms by which CoQ10 protects against atherogenesis by NO-related pathways.

We demonstrated for the first time that Na-intercalated H2Ti3O7 (NaxH2−xTi3O7) NBs, prepared in the typical alkaline hydrothermal process, can effectively absorb visible light to carry out photocatalytic reactions. With the capability of effective light absorption in visible range, NaxH2−xTi3O7 NBs performed much better in the photodegradation of thionine than the other three counterpart products including H2Ti3O7, mixed Na2Ti6O13/TiO2 and anatase TiO2 under visible-light irradiations. As compared to the relevant commercial products like P-25 TiO2 and Na2Ti3O7 powders, the as-synthesized NaxH2−xTi3O7 NBs exhibited superior photocatalytic efficiency under UV illumination, demonstrating their potential as an efficient photocatalyst in relevant redox reactions. A further enhancement in the photocatalytic activity can be achieved for NaxH2−xTi3O7 NBs when Au nanoparticles of suitable amount were deposited on their surfaces. This improvement is due to the band offsets between NaxH2−xTi3O7 and Au, which may promote charge carrier separation to favor the photocatalysis. The recycling test reveals that Au-decorated NaxH2−xTi3O7 NBs could be promisingly utilized in the long-term course of photocatalysis. Furthermore, the result of performance evaluation under natural sunlight shows that the current Au-decorated NaxH2−xTi3O7 NBs can be used as highly efficient photocatalysts which may practically harvest energy from sunlight.

Retinal stem cells bear potency of proliferation, self-renewal, and differentiation into many retinal cells. Utilizing appropriate sensors one can effectively detect the self-renewal and aging process abilities. Silencing information regulator (SirT1), a member of the sirtuin family, is a NAD-dependent histone deacetylase and an essential mediator for longevity in normal cells by calorie restriction. We firstly investigate the SirT1 mRNA expression in retinal stem cells from rats and 19 human eyes of different ages. Results revealed that SirT1 expression was significantly decreased in in vivo aged eyes, associated with poor self-renewal abilities. Additionally, SirT1 mRNA levels were dose-dependently increased in resveratrol- treated retinal stem cells. The expression of SirT1 on oxidative stress-induced damage was significantly decreased, negatively correlated with the level of intracellular reactive oxygen species production. Treatment with resveratrol could effectively further reduce oxidative stress induced by H(2)O(2) treatment in retinal stem cells. Importantly, the anti-oxidant effects of resveratrol in H(2)O(2)-treated retinal stem cells were significantly abolished by knockdown of SirT1 expression (sh-SirT1). SirT1 expression provides a feasible sensor in assessing self-renewal and aging process in retinal stem cells. Resveratrol can prevent reactive oxygen species-induced damages via increased retinal SirT1 expression.

Objective Hypoadiponectemia was observed to correlate with the prevalence or extent of coronary artery disease (CAD), the relationship between metabolic syndrome and fasting serum adiponectin concentration in CAD patients are not well elucidated. Patients and Methods Fasting blood samples were obtained from 98 CAD patients. Metabolic syndrome and its components were defined using the diagnostic criteria of the International Diabetes Federation. Adiponectin concentrations were measured using a commercial enzyme immunosassay kit. Results Fifty patients with CAD (51.0%) had metabolic syndrome. For this group of patients, fasting adiponectin concentrations were found to correlate inversely with metabolic syndrome (p=0.009). Fasting adiponectin values for these subjects also tended to decrease as the number of diagnostic criteria for metabolic syndrome increased (p=0.024). CAD patients with hyperlipidemia (p=0.002), obesity (p=0.030) or receiving statin therapy (p=0.005) had lower serum adiponectin values. By univariate linear regression analysis, fasting serum adiponectin values were positively correlated with age (r=0.242; p=0.017) and high density lipoprotein-cholesterol concentration (HDL-cholesterol; r=0.267; p=0.008) but were negatively correlated with triglyceride concentration (TG; r=-0.251; p=0.013). Multivariate forward stepwise linear regression analysis of the significant variables revealed that HDL-cholesterol concentration (R square=0.071, p=0.008) and age (R square=0.039, p=0.044) are the independent predictors of fasting serum adiponectin concentration for patients with CAD. Conclusion Serum adiponectin concentration is inversely correlated with metabolic syndrome and the number of metabolic syndrome criteria in patients with CAD. For these patients HDL-cholesterol concentration and age are independent predictors of the serum adiponectin value.

Background Monitoring the partial pressure of oxygen in brain tissue (PbtO2) is an important tool for traumatic brain injury (TBI) but is invasive and inconvenient for real time monitoring. Near-infrared spectroscopy (NIRS), which can monitor hemoglobin parameters in the brain tissue, has been used widely as a noninvasive tool for assessing cerebral ischemia and hypoxia. Therefore, it may have the potential as a noninvasive tool for estimating the change of PbtO2. In this study, a novel wireless NIRS system was designed to monitor hemoglobin parameters of rat brains under different impact strengths and was used to estimate the change of PbtO2 noninvasively in TBI. Materials and methods The proposed wireless NIRS system and a PbtO2 monitoring system were used to monitor the oxygenation of rat brains under different impact strengths. Rats were randomly assigned to four different impact strength groups (sham, 1.6 atm, 2.0 atm, and 2.4 atm; n = 6 per group), and the relationships of concentration changes in oxyhemoglobin (HbO2), deoxyhemoglobin (HbR), and total hemoglobin (HbT), and PbtO2 during and after TBI with different impact strengths were investigated. Triphenyltetrazolium chloride (TTC) staining was also used to evaluate infarction volume. Results Concentration changes in HbO2, HbR, and HbT dropped immediately after the impact, increased gradually, and then became stable. Changes in PbtO2 had a similar tendency with the hemoglobin parameters. There was significant correlation between changes in PbtO2 and HbO2 (correlation = 0.76) but not with changes in HbR (correlation = 0.06). In triphenyltetrazolium chloride staining, the infarction volume was highly but negatively associated with oxygen-related parameters like PbtO2 and HbO2. Conclusions Changes in HbO2 under TBI was highly and positively correlated with changes in PbtO2. By using the relative changes in HbO2 as a reference parameter, the proposed wireless NIRS system may be developed as a noninvasive tool for estimating the change of PbtO2 in brain tissue after TBI.

The interleukin-6 (IL-6) gene promoter G-174C polymorphism has been associated with insulin resistance, hypertension, and coronary artery disease; however, its relationship with plasma PAI-1 level has not yet been studied.The G-174C genotypes and plasma PAI-1 antigen and activity were determined in 424 Chinese subjects, 207 with hypertension and 217 without, to study the possible effects of IL-6 genotypes on the regulation of PAI-1 and blood pressure.Hypertensive patients showed significantly greater percentage of IL-6 GG genotype (51.7% v 33.2%, P.001) and G allele frequency (71.7% v 59%, P.001) than normotensive subjects. The GG genotypic group had significantly higher plasma PAI-1 activity (16.1 +/- 9.8 v 12.3 +/- 7.5 IU/mL, P = .03) and antigen (32.4 +/- 23.2 v 23.2 +/- 13.5 ng/mL, P = .01) than the CC genotypic group, with intermediate values in the GC genotypic group (15.9 +/- 9.0 IU/mL and 29.1 +/- 17.5 ng/mL). Multiple linear regression analysis in all study subjects and in normotensive subjects documented an independent dominant effect of IL-6 G-174C gene polymorphism on plasma levels of PAI-1 activity (P = .02 and .01) and antigen (P = .02 and .03) after log transformation and adjustment for confounding factors.The present study showed a positive association of the IL-6 GG genotype with hypertension and with elevated plasma PAI-1 level in normotensive individuals in a Chinese population in Taiwan. Our findings suggest that the IL-6 gene promoter G-174C polymorphism may affect the regulation of PAI-1 and blood pressure through an inflammatory mechanism.

Human retinal pigment epithelium (HRPE) cells are important in maintaining the normal physiology within the neurosensory retina and photoreceptors. Recently, transplantation of HRPE has become a possible therapeutic approach for retinal degeneration. By negative immunoselection (CD45 and glycophorin A), in this study, we have isolated and cultivated adult human bone marrow stem cells (BMSCs) with multilineage differentiation potential. After a 2- to 4-week culture under chondrogenic, osteogenic, adipogenic, and hepatogenic induction medium, these BMSCs were found to differentiate into cartilage, bone, adipocyte, and hepatocyte-like cells, respectively. We also showed that these BMSCs could differentiate into neural precursor cells (nestin-positive) and mature neurons (MAP-2 and Tuj1-positive) following treatment of neural selection and induction medium for 1 month. Furthermore, the plasticity of BMSCs was confirmed by initiating their differentiation into retinal cells and photoreceptor lineages by co-culturing with HRPE cells. The latter system provides an ex vivo expansion model of culturing photoreceptors for the treatment of retinal degeneration diseases.

Considerable evidence suggests that cancer-stem-cells (CSCs) play critical roles in tumorigenesis and metastasis .Although surface marker expression or enzymatic activity of CSC has proven useful, variability of expression highlights the need for the development of high throughput functional CSC assays. One such approach utilizes the capability of CSC’s to form single-cell-derived spheres in suspension environments (functional CSCs). Microfluidic tools are suitable for handling and monitoring a large number of single cells in suspension as well as for examining the interaction of these cells with those in the tumor micro-environment including cancer associated fibroblasts (CAF’s).In order to accomplish this, we have developed a high-throughput (10,000-well), simple-to-use, vertical platform for CSC/CAF co-culture. The platform consists of a non-adherent substrate having 10,000 micro wells for single-cell-derived sphere formation and a porous trans well for CAF adherent co-culture. As predicted by Poisson’s distribution, approximately 30% of micro wells capture single cells when T47D breast cancer cells are loaded at a concentration of 200k/ml. After cell seeding, trans wells containing CAFs are placed onto the substrate. 0.4µm pores on the trans well allow for secretion-based cellular interaction while preventing direct CSC/CAF cell contact. This cell separation facilitates sample cell retrieval without cell cross contamination. Utilizing this device, we determined that co-culture of tumor cells with CAF’s doubled the frequency of sphere formation. Spheres derived from individual CSCs were then retrieved and dissociated for single-cell transcriptome analysis utilizing RNA barcoding followed by RNAseq. Molecular analysis revealed significant cellular heterogeneity among sphere derived cells. In addition, principal component analysis of single cell RNAseq, reveled significant differences in gene expression between mono-cultured and CAF co-cultured cells isolated from tumor spheres. These genes represent potential therapeutic targets for interfering with CSC stromal cell interactions in the tumor microenvironment. The vertical adhesion suspension co-culture platform provides a system to identify and evaluate novel CSC targeted therapeutics. Citation Format: Yu-Chih Chen, Zhixiong Zhang, Yu-Heng Cheng, Euisik Yoon, Max S. Wicha. Microfluidic vertical adhesion-suspension co-culture platform for studying cancer stem cell-fibroblast interactions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5909. doi:10.1158/1538-7445.AM2017-5909

AIM Arterial stiffness is recognized to be an independent risk factor for cardiovascular morbidity and mortality. Recent studies have found that osteoprotegerin (OPG) is associated with increased pulse wave velocity and may reflect endothelial dysfunction. The aim of this study was to evaluate the relationship between the serum OPG level and arterial stiffness in hypertensive patients using the cardio-ankle vascular index (CAVI). METHODS Fasting blood samples were obtained from 115 hypertensive patients and 52 healthy participants. The CAVI value was derived using the waveform device (CAVI-VaSera VS-1000). The serum OPG levels were measured using a commercially available enzyme-linked immunosorbent assay. A CAVI value of ≥9 defined the high arterial stiffness group. RESULTS Sixty-five hypertensive patients (56.5%) were included in the high arterial stiffness group. Diabetes (p=0.032), smoking (p=0.044), age (p < 0.001), systolic blood pressure (p=0.001), diastolic blood pressure (p=0.024), pulse pressure (p=0.046) and the creatinine (p=0.013) and serum OPG (p < 0.001) levels were higher in the high arterial stiffness group than in the low arterial stiffness group, while the glomerular filtration rate (p=0.003) was lower in the high arterial stiffness group than in the low arterial stiffness group among the hypertensive patients. The results of the Spearman's rank correlation coefficient test also indicated a strong positive correlation between the OPG and CAVI values (r=0.484, p < 0.001) in the hypertensive patients. In addition, a multivariate logistic regression analysis showed that age (odds ratio: 1.162, 95% confidence interval (CI): 1.070-1.263, p < 0.001), diastolic blood pressure (odds ratio: 1.109, 95% CI: 1.033-1.190, p=0.004), and serum OPG level (odds ratio: 1.275, 95% CI: 1.030-1.580, p=0.026) were independent predictors of arterial stiffness in hypertensive patients. CONCLUSIONS The serum OPG level is positively associated with arterial stiffness in hypertensive patients.

Cancer–stromal interaction is a critical process in tumorigenesis. Conventional dish-based co-culture assays simply mix two cell types in the same dish; thus, they are deficient in controlling cell locations and precisely tracking single cell behavior from heterogeneous cell populations. Microfluidic technology can provide a good spatial–temporal control of microenvironments, but the control has been typically realized by using external pumps, making long-term cultures cumbersome and bulky. In this work, we have presented a cell–cell interaction microfluidic platform that can accurately control the co-culture microenvironment by using a novel electrolytic cell isolation scheme without using any valves or pneumatic pumps. The proposed microfluidic platform can also precisely control the number of interacting cells and pairing ratios to emulate cancer niches. More than 80% of the chambers captured the desired number of cells. The duration of cell isolation can be adjusted by electrolytic bubble generation and removal. We have verified that the electrolytic process has a negligible effect on cell viability and proliferation in our platform. To the best of our knowledge, this work is the first attempt to incorporate electrolytic bubble generation as a cell isolation method in microfluidics. For proof of feasibility, we have performed cell–cell interaction assays between prostate cancer (PC3) cells and myoblast (C2C12) cells. The preliminary results demonstrated the potential of using electrolysis for micro-environmental control during cell culture. Also, the ratio controlled cell–cell interaction assays were successfully performed which showed that the cell pairing ratios of PC3 to C2C12 affected the proliferation rate of myoblast cells due to increased secretion of growth factors from prostate cancer cells.

This work is aimed at engineering photosensitizer embedded nanoparticles (NPs) that produce optimal amount of reactive oxygen species (ROS) for photodynamic therapy (PDT). A revised synthetic approach, coupled with improved analytical tools, resulted in more efficient PDT. Specifically, methylene blue (MB) conjugated polyacrylamide nanoparticles (PAA NPs), with a polyethylene glycol dimethacrylate (PEGDMA, Mn 550) cross-linker, were synthesized so as to improve the efficacy of cancer PDT. The long cross-linker chain, PEGDMA, increases the distance between the conjugated MB molecules so as to avoid self-quenching of the excited states or species, and also enhances the oxygen permeability of the NP matrix, when compared to the previously used shorter cross-linker. The overall ROS production from the MB-PEGDMA PAA NPs was evaluated using the traditional way of monitoring the oxidation rate kinetics of anthracence-9,10-dipropionic acid (ADPA). We also applied singlet oxygen sensor green (SOSG) so as to selectively derive the singlet oxygen (1O2) production rate. This analysis enabled us to investigate the ROS composition mix based on varied MB loading. To effectively obtain the correlation between the ROS productivity and the cell killing efficacy, a microfluidic chip device was employed to provide homogeneous light illumination from an LED for rapid PDT efficacy tests, enabling simultaneous multiple measurements while using only small amounts of NPs sample. This provided multiplexed, comprehensive PDT efficacy assays, leading to the determination of a near optimal loading of MB in a PAA matrix for high PDT efficacy by measuring the light-dose-dependent cell killing effects of the various MB-PEGDMA PAA NPs using C6 glioma cancer cells.

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteoblasts was explored by using fura-2 as a Ca(2+) indicator. DES at concentrations between 5--20 microM induced an immediate increase in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 70%. Pretreatment with 50 microM La(3+) or 10 microM of nifedipine, verapamil and diltiazem did not change 20 microM DES-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 20 microM DES in Ca(2+)-free medium. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store partly inhibited 20 microM DES-induced Ca(2+) release, but addition of carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin together abolished DES-induced Ca(2+) release. Conversely, pretreatment with 20 microM DES abrogated CCCP- and thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 20 microM DES-induced Ca2+ release. Another estrogen 17beta-estradiol also increased [Ca(2+)](i) in a concentration-dependent manner with an EC50 of 7 microM. Together, the data indicate that in human osteoblasts, DES increased [Ca(2+)](i) via causing Ca(2+) release from both mitochondria and the endoplasmic reticulum in a phospholipase C-independent manner, and by causing Ca(2+) influx.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca2+ dye. Histamine increased ([Ca2+](i)) in a concentration-dependent fashion with an EC(50) value of 0.5 microM. Extracellular Ca2+ removal inhibited the ([Ca2+](i)) signals. Histamine failed to increase ([Ca2+](i)) in Ca2+-free medium after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Addition of Ca2+ induced concentration-dependent ([Ca2+](i)) increases after preincubation with histamine in Ca2+-free medium. Histamine-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The ([Ca2+](i)) increase induced by histamine in Ca2+ medium was abolished by cimetidine, but was not altered by pyrilamine, nifedipine, verapamil, and La(3+). Together, this study shows that histamine increased in ([Ca2+](i)) in osteosarcoma cells by stimulating H2 histamine receptors. The Ca2+ signal was caused by Ca2+ release from the endoplasmic reticulum in a phospholipase C-dependent manner. The Ca2+ release was accompanied by Ca(2+) influx.

Background: The effect of bradykinin on intracellular free Ca2+ levels ([Ca2+]i) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca2+ dye. Methods/Results: Bradykinin (0.1 nM–1 µM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.5 nM. The [Ca2+]i signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca2+ removal inhibited the peak [Ca2+]i signals by 35 ± 3%. Bradykinin (1 nM) failed to increase [Ca2+]i in the absence of extracellular Ca2+ after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor; 1 µM). Bradykinin (1 nM)-induced intracellular Ca2+ release was nearly abolished by inhibiting phospholipase C with 2 µM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca2+]i increase induced by 1 nM bradykinin in Ca2+- free medium was abolished by 1 nM HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 nM Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 pM pertussis toxin for 5 h in Ca2+ medium inhibited 30 ± 3% of 1 nM bradykinin-induced peak [Ca2+]i increase. Conclusions: Together, this study shows that bradykinin induced [Ca2+]i increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca2+ from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca2+ influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.

In obstructive sleep apnea (OSA), recurrent obstruction of the upper airway leads to intermittent hypoxia (IH) during sleep, which can result in impairment of cardiac function. Although exercise can have beneficial effects against IH-induced cardiac dysfunction, the mechanism remains unclear. This study aimed to investigate the interactions of zinc and exercise on IH-triggered left ventricular dysfunction in a rat model that mimics IH in OSA patients. Nine-week-old male Sprague-Dawley rats were randomly assigned to either a control group (CON) or to a group receiving 10 weeks of exercise training (EXE). During weeks 9 and 10, half the rats in each group were subjected to IH for 8 h per day for 14 days (IHCON, IHEXE), whereas the remainder continued to breathe room air. Rats within each of the CON, IHCON, EXE, and IHEXE groups were further randomly assigned to receive intraperitoneal injections of either zinc chloride, the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN), or injection vehicle only. IH induced a lower left ventricular fractional shortening, reduced ejection fraction, higher myocardial levels of inflammatory factors, increased levels oxidative stress, and lower levels of antioxidative capacity, all of which were abolished by zinc treatment. IHEXE rats exhibited higher levels of cardiac function and antioxidant capacity and lower levels of inflammatory factors and oxidative stress than IHCON rats; however, IHEXE rats receiving TPEN did not exhibit these better outcomes. In conclusion, zinc is required for protecting against IH-induced LV functional impairment and likely plays a critical role in exercise-induced cardioprotection by exerting a dual antioxidant and anti-inflammatory effect.

Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis. Though functional CSCs can be identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in a dish-based format. Tumor-stromal interaction is another key regulator of cancer micro-environments, affecting tumorigenesis and metastasis. To enable tumorsphere assays that incorporate interaction with stromal cells, we developed a microfluidic dual adherent/suspension co-culture device. This device combined a suspension environment for single-cell tumorsphere assay and an adherent environment for co-culture of stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allowed the use of small samples ( Citation Format: Yu-Chih Chen, Shamileh Fouladdel, Zhixiong Zhang, Ebrahim Azizi, Euisik Yoon, Max S. Wicha. Single cell dual adherent-suspension co-culture microenvironment for studying tumor-stromal interactions in breast cancer stem cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4076.

Aim: Mesenchymal stem cells (MSCs) are a multipotent cell type that can differentiate into non-hematopoietic cells, such as adipocytes. Adipocyte tissue is central to the regulation of energy balance. Two functionally different types of fat are present in mammals. White adipose tissue is the primary site for triglyceride storage, while brown adipose tissue is specialized in energy expenditure. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) controls several aspects of mitochondrial biogenesis. In this study, we hypothesized that PGC-1α plays a role in brown fat differentiation of MSCs.Methods: Immortalized human MSCs were infected with adenovirus carrying PGC-1α cDNA to create PGC-1α-expressing MSCs.Results: The genetic profiling of PGC-1α-expressing MSCs shows the significant increase of genes related to mitochondrial functions and lipid metabolism compared to that of MSCs. When expressed in MSCs, PGC-1α activates robust mitochondrial biogenesis and respiration. The increase of oxygen consumption and reactive oxygen species represents a cellular readout of increased activity of the respiratory chain. The expression of thermogenic markers, such as cytochrome C and complex II, was significantly increased in MSCs with treatment of adenovirus expressing PGC-1α. Moreover, PGC-1α markedly inhibited the osteogenesis of MSCs under osteogenic induction. During adipogenesis, PGC-1α-expressing MSCs showed a significant increase in brown fat markers and a decrease in white fat markers. Notably, PGC-1α knockdown inhibited adipocyte differentiation of MSCs.Conclusions: In summary, our data reveal an important role of PGC-1α in promoting brown fat differentiation of MSCs, and provide a new therapeutic approach for the treatment of obesity.

BO-1051 is an N-mustard derivative that is conjugated with DNA-affinic 9-anilinoacridine. Since BO-1051 was reported to have strong anticancer activity, we investigated the effect and underlying mechanism of BO-1051 in human glioma cell lines.Human glioma cell lines U251MG and U87MG were studied with BO-1051 or the combination of BO-1051 and autophagic inhibitors. Growth inhibition was assessed by MTT assay. Apoptosis was measured by annexin V staining followed by flow cytometry and immunoblotting for apoptosis-related molecules. Induction of autophagy was detected by acridine orange labeling, electron microscopy, LC3 localization and its conversion. Transfection of shRNA was used to determine the involvement of Beclin1 in apoptotic cell death.MTT assay showed that BO-1051 suppressed the viability of four glioma cell lines (U251MG, U87MG, GBM-3 and DBTRG-05MG) in a dose-dependent manner. The IC(50) values of BO-1051 for the glioma cells were significantly lower than the values for primary neurons cultures and normal fibroblast cells. Moreover, BO-1051 not only induced apoptotic cell death, but also enhanced autophagic flux via inhibition of Akt/mTOR and activation of Erk1/2. Importantly, suppression of autophagy by 3-methyladenine or bafilomycin A1 significantly increased BO-1051-induced apoptotic cell death in U251MG and U87MG cells. In addition, the proportion of apoptotic cells after BO-1051 treatment was enhanced by co-treatment with shRNA against Beclin1.BO-1051 induced both apoptosis and autophagy, and inhibition of autophagy significantly augmented the cytotoxic effect of BO-1051. Thus, a combination of BO-1051 and autophagic inhibitors offers a potentially new therapeutic modality for the treatment of malignant glioma.

This study is aimed to evaluate the engineered properties of cement-based composites which comprise polyolefin fibers and silica fume in the mixes. Material variables include water-cementitious ratio, dosage of silica fume, polyolefin fiber length and dosage. Compressive strength, splitting tensile strength, direct tensile strength, resistivity, rapid chloride penetration test and microscopic observation were conducted. Test results indicate that the specimens containing silica fume have higher compressive strength than fiber and control specimens. The specimens with polyolefin fiber and silica fume have much higher tensile strength and ductility than the control and silica fume specimens. The specimens containing silica fume and polyolefin fiber demonstrate better resistance in chloride penetration than polyolefin fiber composites or silica fume composites. For a given volume fraction, short polyolefin fiber performs better than long polyolefin fiber in improving concrete properties. The specimen containing silica fume significantly increases in resistivity and decreases in total charge passed. SEM microscopy illustrates that the polyolefin fiber acts as the crack arresters in concrete, which can inhibit internal crack propagation.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the degeneration of dopaminergic (DA) neurons in the midbrain. Induced pluripotent stem (iPS) cells have shown potential for differentiation and may become a resource of functional neurons for the treatment of PD. However, teratoma formation is a major concern for transplantation-based therapies. This study examined whether functional neurons could be efficiently generated from iPS cells using a five-step induction procedure combined with docosahexaenoic acid (DHA) treatment. We demonstrated that DHA, a ligand for the RXR/Nurr1 heterodimer, significantly activated expression of the Nurr1 gene and the Nurr1-related pathway in iPS cells. DHA treatment facilitated iPS differentiation into tyrosine hydroxylase (TH)-positive neurons in vitro and in vivo and functionally increased dopamine release in transplanted grafts in PD-like animals. Furthermore, DHA dramatically upregulated the endogenous expression levels of neuroprotective genes ( Bcl-2, Bcl-xl, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor) and protected against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced apoptosis in iPS-derived neuronal precursor cells. DHA-treated iPS cells significantly improved the behavior of 6-hydroxydopamine (6-OHDA)-treated PD-like rats compared to control or eicosapentaenoic acid-treated group. Importantly, the in vivo experiment suggests that DHA induces the differentiation of functional dopaminergic precursors and improves the abnormal behavior of 6-OHDA-treated PD-like rats by 4 months after transplantation. Furthermore, we found that DHA treatment in iPS cell-grafted rats significantly downregulated the mRNA expression of embryonic stem cell-specific genes (Oct-4 and c-Myc) in the graft and effectively blocked teratoma formation. Importantly, 3 Tesla-magnetic resonance imaging and ex vivo green fluorescence protein imaging revealed that no teratomas were present in transplanted grafts of DHA-treated iPS-derived DA neurons 4 months after implantation. Therefore, our data suggest that DHA plays a crucial role in iPS differentiation into functional DA neurons and that this approach could provide a novel therapeutic approach for PD treatment.

Scope: The lectin-like oxidized low-density lipoprotein receptor (LOX-1) is one pivot receptor for oxidized low-density lipoprotein (oxLDL) in human endothelial cells. Co-enzyme Q10 (Co Q10) has been widely used in clinical intervention. However, the molecular mechanisms underlying its protective effects against oxidative stress in endothelial cells are still largely unknown. This study was designed to test the hypothesis that Co Q10 mitigates oxLDL-induced endothelial oxidative injuries via modulation of LOX-1-mediated reactive oxygen species (ROS) generation and explored the role of AMP-activated protein kinase (AMPK), a negative regulator of NADPH oxidase. Methods and results: Human umbilical vein endothelial cells (HUVECs) were pretreated with Co Q10 and then incubated with oxLDL for 24 h. Co Q10 attenuated oxLDL-elicited LOX-1 expression and ROS generation by suppression of NADPH oxidase activation. Co Q10 rescued dephosphorylation of AMPK caused by oxLDL that in turn led to an activation of NADPH oxidase by PKC. The results were confirmed using AMPK siRNA. Moreover, oxLDL-suppressed Akt/eNOS and enhanced p38 phosphorylation, which in turn activated NF-κB pathway. These detrimental events were ameliorated by Co Q10. Conclusion: These results provide new highlight onto the possible molecular mechanisms of how Q10 suppresses oxLDL-induced endothelial oxidative injuries by the modulation of LOX-1-mediated ROS generation via the AMPK/PKC/NADPH oxidase signaling pathway.

Aim: Silencing information regulator (SirT1), a NAD-dependent histone deacetylase, is an essential mediator of longevity in normal cells by calorie restriction. SirT1 has many biological functions, including transcription regulation, cell differentiation inhibition, cell cycle regulation, and anti-apoptosis. Resveratrol (RV)-induced SirT1 activation also improves endothelial dysfunction and suppresses vascular inflammation. In this study, we investigated the roles of RV-induced SirT1 activation in endothelial cells under oxidative stress.Methods: SirT1 mRNA expression levels were examined in the endothelium layer (endothelial cells) of cardiac coronary vessels from patients receiving coronary artery bypass graft surgery (CABG) surgery and aged rats using reverse transcriptase polymerase chain reaction (RT-PCR). To further explore the effect of SirT1 activation on oxidative stress-induced aging, senescence-associated β-galactosidase (SA-β-gal) expression in RV-treated human umbilical vein endothelial cells (HUVECs) with or without H2O2 treatment was evaluated.Results: SirT1 expression was decreased in aged and atherosclerotic vessels in vivo, and significantly reduced in endothelial cells purified from vessel tissues. Furthermore, SirT1 levels were dose-dependently increased in RV-treated HUVECs. The SA-β gal assay showed that RV inhibited the senescent phenotype of H2O2-treated HUVECs. Reactive oxygen species (ROS) production and the percentage of cells positive for SA-β gal were significantly increased in siRNA-SirT1 (knockdown of SirT1 expression)-treated HUVEC cells. Importantly, the treatment effect of RV was significantly abolished in the oxidative effects of H2O2-treated HUVECs by siRNA-SirT1.Conclusion: Our data suggested that SirT1 could be a crucial factor involved in the endothelial cells of atherosclerotic CAGB patients and aging rats. RV is a potential candidate for preventing oxidative stress-induced aging in endothelial cells. RV may also prevent ROS-induced damage via increased endothelial SirT1 expression.

Ageing, which all creatures must encounter, is a challenge to every living organism. In the human body, it is estimated that cell division and metabolism occurs exuberantly until about 25 years of age. Beyond this age, subsidiary products of metabolism and cell damage accumulate, and the phenotypes of ageing appear, causing disease formation. Among these age-related diseases, neurodegenerative diseases have drawn a lot of attention due to their irreversibility, lack of effective treatment, and accompanied social and economical burdens. In seeking to ameliorate ageing and age-related diseases, the search for anti-ageing drugs has been of much interest. Numerous studies have shown that the plant polyphenol, resveratrol (3,5,4'-trihydroxystilbene), extends the lifespan of several species, prevents age-related diseases, and possesses anti-inflammatory, and anti-cancer properties. The beneficial effects of resveratrol are believed to be associated with the activation of a longevity gene, SirT1. In this review, we discuss the pathogenesis of age-related neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and cerebrovascular disease. The therapeutic potential of resveratrol, diet and the roles of stem cell therapy are discussed to provide a better understanding of the ageing mystery.

Vibrio parahaemolyticus, a common enteropathogen in tropical and subtropical coastal regions, exhibits significant adaptive acid tolerance response and heat-shock response, and the envelope proteins induced by stresses are suggested to be associated with virulence. This work examined the heat-shock proteins located in the envelope of V. parahaemolyticus by two rapid methods; namely, the immunoblotting and biotin-labeling methods. The bacterial cells were cultured at 25 C and heat shocked at 37 or 42 C for 1 or 2 hr. The cells were first lysed, then proteins were separated by gel electrophoresis and probed with antiserum raised against heat-shocked cells. Next, the heat-shocked cells were examined by labeling with water soluble sulfo-NHS-LC-biotin. Proteins of 33, 61, 66, 71, 78, 92 and 101 kDa were induced, while 55, 86, 102, 120 and 160 kDa proteins were markedly enhanced in the envelope of the heat-shocked V. parahaemolyticus cells. The biotin tagged envelope proteins were purified using a monomeric avidin column, and the N-terminal sequence was determined and compared with other high identity protein sequences. The sequence results suggest that Vph1 (55 kDa), Vph2 (46 kDa) and Vph3 (42 kDa) are de novo synthesized heat-shock proteins located in the envelope of this pathogen, and the functions of these proteins in stress protection and virulence have yet to be determined.

Typically, an orthodontic metal bracket is made from stainless steel. It has been shown that such metal may corrode in an acid- and chloride-rich environment. The purpose of the current study was to investigate a titanium nitride (TiN) ion-plated stainless steel orthodontic bracket's anticorrosion properties and compare its biocompatibility with that of non-TiN-plated brackets. The stainless-steel brackets studied here were tested in acidic artificial saliva. The plated metal bracket was produced by the titanium nitride (TiN) ion-plating method. The TiN-plating on the bracket surface was demonstrated to be successful by EDX analysis. The quantity of metallic-ion release under test immersion solutions was analyzed by atomic absorption spectrophotometry. Both TiN- and non-TiN-plated brackets may release detectable ions into the test solution, including nickel, chromium, manganese, copper, and iron (ferric). The anticorrosion ability of the plated bracket was analyzed by means of inductively coupled plasma atomic emission spectroscopy. The results revealed that the TiN-plated metal bracket did not increase the anticorrosion ability of the standard bracket. The biocompatibility of the TiN plating versus the standard bracket material resulting from bracket immersion in the test solution revealed no toxicity on U2OS cells using a methylthiazole tetrazolium (MTT) colorimetric assay. Clearly, the search for an improved technique for enhancing the anticorrosion ability of the normal metal orthodontic bracket should be continued.

The effect of lindane (y-hexachlorocyclohexane), an organochlorine pesticide, on Ca 2+ mobilization in Madin-Darby canine kidney cells was examined by fluorimetry using fura-2 as a Ca 2+ indicator. Lindane (5-200 μM) increased [Ca 2+ ] i concentration-dependently. The [Ca 2+ ] i signal comprised an immediate initial rise followed by a persistent phase. Ca 2+ removal inhibited the [Ca 2+ ] i signal by reducing both the initial rise and the sustained phase. This implies lindane-triggered Ca 2+ influx and Ca 2+ release. In Ca 2+ -free medium. 0.15 mM lindane increased [Ca 2+ ] i after pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and two endoplasmic reticulum Ca 2+ pump inhibitors, thapsigargin and cyclopiazonic acid. Conversely, pretreatment with lindane abolished CCCP- and thapsigargin-induced Ca 2+ release. This suggests that 0.15 mM lindane released Ca 2+ from the endoplasmic reticulum, mitochondria and other stores. La 3+ (1 mM) partly inhibited 0.1 mM lindane-induced [Ca 2+ ] i increase, confirming that lindane induced Ca 2+ influx. Addition of 3 mM Ca 2+ increased [Ca 2+ ] i after pretreatment with 0.15 mM lindane for 750 sec. in Ca 2+ -free medium, which indicates lindane-induced capacitative Ca 2+ entry. Lindane (0.15 mM)-induced Ca 2+ release was not reduced by inhibiting phospholipase C with 2 μM U73122, but was inhibited by 70% by the phospholipase A 2 inhibitor aristolochic acid (40 μM).

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a widely used calmodulin inhibitor, on intracellular free Ca(2+)levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca(2+)probe. W-7 (20-1000 micro m) induced an increase in [Ca(2+)](i)in a dose-dependent manner, with an EC(50)of 100 microm. The [Ca(2+)](i)signal comprised an initial rise and a sustained plateau without significant decay within 5 min. External Ca(2+)removal decreased the Ca(2+)signals by reducing the peak and sustained phase, indicating W-7-activated intracellular Ca(2+)release and extracellular Ca(2+)influx. W-7 (500 microm) failed to induce a [Ca(2+)](i)increase in a Ca(2+)-free medium after pre-treatment with thapsigargin (1 microm), an endoplasmic reticulum Ca(2+)pump inhibitor. Conversely, W-7 pre-treatment abolished the Ca(2+)release induced by thapsigargin. This suggests that W-7 (500 microm ) released internal Ca(2+)mainly from the endoplasmic reticulum. The addition of 3 mm Ca(2+)increased [Ca(2+)](i)dose-dependently after preincubation with 20-1000 microm W-7 in a Ca(2+)-free medium, implying that W-7 induced capacitative Ca(2+)entry. W-7-induced Ca(2+)release was not altered by inhibiting phospholipase C with 2 microm 1-(6-((17 beta - 3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (U73122). Tryphan blue assay demonstrated that W-7 (200 microm) caused gradual cell death within 30 min of the initial drug exposure. Together, it was found that W-7 induced [Ca(2+)](i)increases in human osteosarcoma cells by releasing internal Ca(2+)from the endoplasmic reticulum, and also by triggering Ca(2+)influx. W-7 may be cytotoxic to osteosarcoma cells.