A panel of biomarkers in the prediction for early allograft dysfunction and mortality after living donor liver transplantation.

Early allograft dysfunction (EAD) is associated with graft failure and mortality after living donor liver transplantation (LDLT). In this study, we report biomarkers superior to other conventional clinical markers in the prediction of EAD and all-cause in-hospital mortality in LDLT patient cohort. Blood samples of living donor liver transplant recipients were collected on postoperative day 1 and analyzed by liquid chromatography coupled with mass spectrometry (LC-MS). Significant metabolites associated with the prediction of EAD were identified using orthogonal projection to latent structures-discriminant analysis (OPLS-DA). A few lipids, more specifically, lysoPC (16:0), PC (18:0/20:5), betaine and palmitic acid (C16:0) were found to effectively differentiate EAD from non-EAD on postoperative day 1. A combination of these four metabolites showed an AUC of 0.821, which was further improved to 0.846 by the addition of a clinical parameter, total bilirubin. The panel exhibits a high prognostic accuracy in prediction of all-cause in-hospital mortality and mortality within 7 postoperative days with AUCs of 0.843 and 0.954. These results show the combination of metabolomics-derived biomarkers and clinical parameters demonstrates the power of panels in diagnostic and prognostic evaluation of LDLT.

A highly selective and sensitive fluorescence assay for determination of copper(II) and cobalt(II) ions in environmental water and toner samples.

In this study, a highly selective and sensitive fluorescence assay has been proposed for the determination of copper(II) and cobalt(II) ions in environmental water and toner samples. In the presence of hydrogen peroxide, copper(II) reacted with a new fluorescence reagent Amplex® UltraRed (AUR), forming a fluorescence product only at pH 7.0, while the fluorescence product of cobalt(II) with AUR formed only at pH 9.0. The fluorescence signal obtained was linear with respect to the copper(II) concentration over the range of 1.6-12.0 µM (R(2) = 0.988) and was linear with respect to the cobalt(II) concentration over the range of 45.0 nM to 1.0 µM (R(2) = 0.992). The limits of detection (at a signal-to-noise ratio of 3) for copper(II) and cobalt(II) were 0.17 µM and 14.0 nM, respectively. Our present approach is simpler, faster, and more cost-effective than other techniques for the detection of copper(II) and cobalt(II) ions in environmental water samples and that of copper(II) ions in toner samples.

Reversible synthesis of sub-10†nm spherical and icosahedral gold nanoparticles from a covalent Au(CN)2(-) precursor and recycling of cyanide to form ferric ferrocyanide for cell staining.

A solution approach based on Au(CN)(2)(-) chemistry is reported for the formation of nanoparticles. The covalent character of the Au(CN)(2)(-) precursor was exploited in the formation of sub-10 nm nanospheres (≈2.4 nm) and highly monodisperse icosahedral Au nanoparticles (≈8 nm) at room temperature in a one-pot aqueous synthesis. The respective spherical and icosahedral Au morphologies can be controlled by either the absence or presence of the polymer polyvinylpyrrolidone (PVP). Using Au(CN)(2)(-) as a metal ion source, our findings suggest that the addition of citrate ions is necessary to enhance the particle formation rate as well as to generate a more homogeneous colloidal dispersion. Because of the presence of oxygen and the operation of a CN(-) etching process associated with Au(CN)(2)(-) complex formation, an interesting reversible formation-dissolution process was observed, which allowed us to repeatedly prepare spherical and icosahedral Au nanoparticles. Time-dependent TEM images and UV/Vis spectra were carefully acquired to study the reversibility of this formation-dissolution process. In view of the accompanying generation of toxic cyanide anions, we have developed a protocol to recycle cyanide in the presence of citrate ions through ferric ferrocyanide formation. After completion of particle formation, the residual solutions containing citrate ions and cyanide ions were processed to stain iron oxide nanoparticles endocytosized in cells. Additionally, the as-prepared 8 nm Au icosahedra could be isolated and grown to larger 57 nm-sized icosahedra using the seed-mediated growth approach.

Quantitative analysis of multiple urinary biomarkers of carcinoid tumors through gold-nanoparticle-assisted laser desorption/ionization time-of-flight mass spectrometry.

A simple technique for quantitative analysis of four urinary biomarkers, tryptophan (TRP), 5-hydroxytryptophan (5-HTP), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) of carcinoid tumors is developed using gold nanoparticles as the assisted matrix in surface-assisted laser desorption/ionization time-of-flight mass spectrometry (SALDI-TOF MS). The optimal SALDI conditions for the efficient ionization of those biomarkers are systematically explored by the adjustments of the concentrations of gold nanoparticles and internal standards. The mass spectra with strong signals and minimal background noise are obtained using 1-naphthaleneacetamide (NAD) as the internal standard. The calibration curves of the biomarker concentrations are determined using SALDI-TOF MS and the high linearity is obtained in all samples. For future clinical testing, multiplexed detection of those biomarkers in the urine samples of healthy males is performed. The successful quantitative detections of TRP, 5-HTP, 5-HT and 5-HIAA indicate that our technique provided great potentials to be developed a simple and rapid platform for the tumor biomarker detections.

In vitro and in vivo study of phloretin-induced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter.

Phloretin (Ph), a natural product found in apples and pears with glucose transporter (GLUT) inhibitory activity, exerts antitumor effects. However, little is known about its effects on human liver cancer. The purpose of this study is to test the cytotoxic effects of Ph on HepG2 cells and to identify the underlying molecular pathways. Human hepatocellular carcinoma specimens and HepG2 show a high level of GLUT2 transporter activity in the cell membrane. Real-time PCR and MTT assays demonstrate that Ph-induced cytotoxicity correlates with the expression of GLUT2. Flow cytometry and DNA fragmentation studies show that 200 microM Ph induces apoptosis in HepG2, which was reversed by glucose pretreatment. GLUT2 siRNA knockdown induced HepG2 apoptosis, which was not reversed by glucose. Western blot analysis demonstrates that both intrinsic and extrinsic apoptotic pathways in addition to Akt and Bcl-2 family signaling pathways are involved in Ph-induced cell death in HepG2 cells. Furthermore, using flow cytometry analysis, a mitochondrial membrane potential assay and Western blot analysis, we show that cytochalasin B, a glucose transport inhibitor, enhances the Ph-induced apoptotic effect on HepG2 cells, which was reversed by pretreatment with glucose. Furthermore, we found significant antitumor effects in vivo by administering Ph at 10 mg/kg intraperitoneally to severe combined immune deficiency mice carrying a HepG2 xenograft. A microPET study in the HepG2 tumor-bearing mice showed a 10-fold decrease in (18)F-FDG uptake in Ph-treated tumors compared to controls. Taken together, these results suggest that Ph-induced apoptosis in HepG2 cells involves inhibition of GLUT2 glucose transport mechanisms.

Apple polyphenol phloretin potentiates the anticancer actions of paclitaxel through induction of apoptosis in human hep G2 cells.

Phloretin (Ph), which can be obtained from apples, apple juice, and cider, is a known inhibitor of the type II glucose transporter (GLUT2). In this study, real-time PCR analysis of laser-capture microdissected (LCM) human hepatoma cells showed elevated expression (>5-fold) of GLUT2 mRNA in comparison with nonmalignant hepatocytes. In vitro and in vivo studies were performed to assess Ph antitumor activity when combined with paclitaxel (PTX) for treatment of human liver cancer cells. Inhibition of GLUT2 by Ph potentiated the anticancer effects of PTX, resensitizing human liver cancer cells to drugs. These results demonstrate that 50-150 microM Ph significantly potentiates DNA laddering induced in Hep G2 cells by 10 nM PTX. Activity assays showed that caspases 3, 8, and 9 are involved in this apoptosis. The antitumor therapeutic efficacy of Ph (10 mg/kg body weight) was determined in cells of the SCID mouse model that were treated in parallel with PTX (1 mg/kg body weight). The Hep G2-xenografted tumor volume was reduced more than fivefold in the Ph + PTX-treated mice compared to the PTX-treated group. These results suggest that Ph may be useful for cancer chemotherapy and chemoprevention.