A four-electron O(2)-electroreduction biocatalyst superior to platinum and a biofuel cell operating at 0.88 V

O2 was electroreduced to water, at a true-surface-area-based current density of 0.5 mA cm-2, at 37 degrees C and at pH 5 on a "wired" laccase bioelectrocatalyst-coated carbon fiber cathode. The polarization (potential vs the reversible potential of the O2 /H2O half-cell in the same electrolyte) of the cathode was only -0.07 V, approximately one-fifth of the -0.37 V polarization of a smooth platinum fiber cathode, operating in its optimal electrolyte, 0.5 M H2SO4. The bioelectrocatalyst was formed by "wiring" laccase to carbon through an electron conducting redox hydrogel, its redox functions tethered through long and flexible spacers to its cross-linked and hydrated polymer. Incorporation of the tethers increased the apparent electron diffusion coefficient 100-fold to (7.6 +/- 0.3) x 10-7 cm 2 s-1. A miniature single-compartment glucose-O2 biofuel cell made with the novel cathode operated optimally at 0.88 V, the highest operating voltage for a compartmentless miniature fuel cell.     

A high concentration of genistein down-regulates activin A, Smad3 and other TGF-β pathway genes in human uterine leiomyoma cells

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 µg/ml) in UtLM cells. Downregulation of TGF-β signaling pathway genes, activin A, activin B, Smad3, TGF-β2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real-time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that down-regulation of activin A and Smad3, both members of the TGF-β pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.     

Ritter Reactions between Alcohols and Acetonitrile Mediated by the Conducting Polymer Poly-(3,4-ethylenedioxy Thiophene) (PEDOT)

Herein, we report new reactivity of the conducting polymer, poly-(3,4-ethylenedioxy thiophene) (PEDOT), where PEDOT mediates a Ritter reaction between alcohols and acetonitrile. The yields were variable and in most cases competitive with results obtained using sulfuric acid. Attempts at a stoichiometric reaction between benzonitrile and diphenylmethanol are also reported herein. Finally, described here are preliminary mechanistic studies that suggest PEDOT is behaving as an alcohol-selective or specific Lewis acid.

Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for full experimental and spectral details.     

Irreversible and Reversible Deactivation of Bilirubin Oxidase by Urate

Oxygen is electroreduced to water on a carbon cathode coated with wired bilirubin oxidase in a pH 7.4 0.15 M NaCl phosphate buffer solution at 37 °C at much lesser polarization than it is on a pure platinum cathode in 0.5 M H₂SO₄. While the wired bilirubin oxidase cathode operates for over a week in the aerated or oxygenated buffer solution, it is degraded rapidly when in serum. We reported earlier that in the presence of O₂ an intermediate product of the electrooxidation of urate, which is a normal serum component, irreversibly damages the wired bilirubin oxidase and also reported that the electrocatalyst is irreversibly damaged, in the absence of urate, when it is brought, by disconnecting the electrode, to the O₂/H₂O half cell potential at pH 7.4. Here we report that a) dissolved bilirubin oxidase is irreversibly and rapidly damaged by urate in the presence of O₂; and b) that the immobilized wired bilirubin oxidase electrocatalyst is not only irreversibly deactivated by urate in the presence of O₂ in a few hours, but is initially reversibly deactivated, in 1 min or less, by the urate in the presence of O₂.     

Tailoring the electrochemical charge storage properties of carbonaceous support by redox properties of heteropoly acids: where does the synergy come from?

Journal of Solid State Electrochemistry - The synergistic effects between two Keggin-type heteropoly acids (HPAs) and carbon surface were examined and elucidated. An improved high rate capability...     

Two-stage Off-Gel isoelectric focusing: protein followed by peptide fractionation and application to proteome analysis of human plasma

This paper presents the recently introduced Off-Gel electrophoresis (OGE) technology as a versatile tool to reproducibly fractionate intact proteins and peptides into discrete liquid fractions. The coupling of two stages of OGE, i.e., the separation of intact proteins in a first-stage followed by fractionation of peptides derived from each protein fraction after proteolysis in a second stage, results in an array of 15 x 15 fractions that are directly amenable to additional peptide fractionation like reverse-phase liquid chromatography (RPC). The analysis of all second-stage peptide fractions from only the first-stage protein fraction representing pH 5.0 -5.15 by on-line reverse-phase LC-tandem mass spectrometry resulted in the identification of 53 proteins (337 peptides), of which 10 were on different immunoglobulin (Ig) chains, with an input of only 1.5 mg human blood plasma proteins. Increasing the protein load to approximately 12 mg increased the number of identified proteins in the same protein fraction to 73 proteins (449 peptides), of which 15 were Ig-related. Immunodepletion of six of the most abundant proteins (albumin, transferrin, haptoglobin, IgG, IgA, and alpha-1-antitrypsin) prior to first-stage OGE with an input of 1.5 mg of protein (equivalent to approximately 10 mg nondepleted plasma) resulted in the identification of 81 proteins (660 peptides), of which three were still Ig fragments. The pI-based separation of peptides appears to be nonuniform based on the theoretically determined pI values of identified peptides. This observation specifically accounts for the neutral zone (pI 5-8) and can be accounted for by the physicochemical properties of the peptides given by their amino acid composition. The power of OGE separation of proteins and peptides is discussed with a focus on the use of the knowledge about the pI of proteins and peptides that assist the validation of correct identifications together with the retention time of peptides on RPC.     

cyc-DEP: Cyclic immunofluorescence profiling of particles collected using dielectrophoresis

Cancer is a highly heterogenous disease that requires precise detection tools and active surveillance methods. Liquid biopsy assays provide an agnostic way to follow the complex trajectory of cancer, providing better patient stratification tools for optimized treatment. Here, we present the development of a low-volume liquid biopsy assay called cyc-DEP (cyclic immunofluorescent imaging on dielectrophoretic chip) to profile biomarkers collected on a dielectrophoretic microfluidic chip platform. To enable on-chip cyclic imaging, we optimized a fluorophore quenching method and sequential rounds of on-chip staining with fluorescently conjugated primary antibodies. cyc-DEP allows for the quantification of a multiplex array of proteins using 25 µl of a patient plasma sample. We utilized nanoparticles from a prostate adenocarcinoma (LNCaP) cell line and a panel of six target proteins to develop our proof-of-concept technique. We then used cyc-DEP to quantify blood plasma levels of target proteins from healthy individuals, low-grade and high-grade prostate cancer patients (n = 3 each) in order to demonstrate that our platform is suitable for liquid biopsy analysis in its present form. To ensure accurate quantification of signal intensities and comparisons between different samples, we incorporated a signal intensity normalization method (fluorescent beads) and a custom signal intensity quantification algorithm that account for the distribution of signal across hundreds of collection regions on each chip. Our technique enabled a threefold improvement in multiplicity for detecting proteins associated with fluid samples, opening doors for early detection, and active surveillance through quantification of a multiplex array of biomarkers from low-volume liquid biopsies.