Catalysis of Dioxygen Reduction by <i>Thermus thermophilus</i> Strain HB27 Laccase on Ketjen Black Electrodes

We present electrochemical analyses of the catalysis of dioxygen reduction by <i>Thermus thermophilus</i> strain HB27 laccase on ketjen black substrates. Our cathodes reliably produce 0.56 mA cm^–2 at 0.0 V vs Ag|AgCl reference at 30 °C in air-saturated buffer, under conditions of nonlimiting O₂ flux. We report the electrochemical activity of this laccase as a function of temperature, pH, time, and the efficiency of its conversion of dioxygen to water. We have measured the surface concentration of electrochemically active species, permitting the extraction of electron transfer rates at the enzyme-electrode interface: 1 s^–1 for this process at zero driving force at 30 °C and a limiting rate of 23 s^–1 at 240 mV overpotential at 50 °C

Modeling Dioxygen Reduction at Multicopper Oxidase Cathodes

We report a general kinetics model for catalytic dioxygen reduction on multicopper oxidase (MCO) cathodes. Our rate equation combines Butler–Volmer (BV) electrode kinetics and the Michaelis–Menten (MM) formalism for enzymatic catalysis, with the BV model accounting for interfacial electron transfer (ET) between the electrode surface and the MCO type 1 copper site. Extending the principles of MM kinetics to this system produced an analytical expression incorporating the effects of subsequent intramolecular ET and dioxygen binding to the trinuclear copper cluster into the cumulative model. We employed experimental electrochemical data on Thermus thermophilus laccase as benchmarks to validate our model, which we suggest will aid in the design of more efficient MCO cathodes. In addition, we demonstrate the model’s utility in determining estimates for both the electronic coupling and average distance between the laccase type-1 active site and the cathode substrate

Supramolecular Probes for Assessing Glutamine Uptake Enable Semi-Quantitative Metabolic Models in Single Cells

We describe a supramolecular surface competition assay for quantifying glutamine uptake from single cells. Cy3-labeled cyclodextrins were immobilized on a glass surface as a supramolecular host/FRET donor, and adamantane-BHQ2 conjugates were employed as the guest/quencher. An adamantane-labeled glutamine analog was selected through screening a library of compounds and validated by cell uptake experiments. When integrated onto a single cell barcode chip with a multiplex panel of 15 other metabolites, associated metabolic enzymes, and phosphoproteins, the resultant data provided input for a steady-state model that describes energy potential in single cells and correlates that potential with receptor tyrosine kinase signaling. We utilize this integrated assay to interrogate a dose-dependent response of model brain cancer cells to EGFR inhibition. We find that low-dose (1 μM erlotinib) drugging actually increases cellular energy potential even as glucose uptake and phosphoprotein signaling is repressed. We also identify new interactions between phosphoprotein signaling and cellular energy processes that may help explain the facile resistance exhibited by certain cancer patients to EGFR inhibitors

The Microscopic Structure of Adsorbed Water on Hydrophobic Surfaces under Ambient Conditions

The interaction of water vapor with hydrophobic surfaces is poorly understood. We utilize graphene templating to preserve and visualize the microscopic structures of adsorbed water on hydrophobic surfaces. Three well-defined surfaces [H–Si(111), graphite, and functionalized mica] were investigated, and water was found to adsorb as nanodroplets (∼10–100 nm in size) on all three surfaces under ambient conditions. The adsorbed nanodroplets were closely associated with atomic-scale surface defects and step-edges and wetted all the hydrophobic substrates with contact angles <∼10°, resulting in total water adsorption that was similar to what is found for hydrophilic surfaces. These results point to the significant differences between surface processes at the atomic/nanometer scales and in the macroscopic world

Visualizing Local Doping Effects of Individual Water Clusters on Gold(111)-Supported Graphene

The local charge carrier density of graphene can exhibit significant and highly localized variations that arise from the interaction between graphene and the local environment, such as adsorbed water, or a supporting substrate. However, it has been difficult to correlate such spatial variations with individual impurity sites. By trapping (under graphene) nanometer-sized water clusters on the atomically well-defined Au(111) substrate, we utilize scanning tunneling microscopy and spectroscopy to characterize the local doping influence of individual water clusters on graphene. We find that water clusters, predominantly nucleated at the atomic steps of Au(111), induce strong and highly localized electron doping in graphene. A positive correlation is observed between the water cluster size and the local doping level, in support of the recently proposed electrostatic-field-mediated doping mechanism. Our findings quantitatively demonstrate the importance of substrate-adsorbed water on the electronic properties of graphene

Allosteric Inhibitor of KRas Identified Using a Barcoded Assay Microchip Platform

Protein catalyzed capture agents (PCCs) are synthetic antibody surrogates that can target a wide variety of biologically relevant proteins. As a step toward developing a high-throughput PCC pipeline, we report on the preparation of a barcoded rapid assay platform for the analysis of hits from PCC library screens. The platform is constructed by first surface patterning a micrometer scale barcode composed of orthogonal ssDNA strands onto a glass slide. The slide is then partitioned into microwells, each of which contains multiple copies of the full barcode. Biotinylated candidate PCCs from a click screen are assembled onto the barcode stripes using a complementary ssDNA-encoded cysteine-modified streptavidin library. This platform was employed to evaluate candidate PCC ligands identified from an epitope targeted in situ click screen against the two conserved allosteric switch regions of the Kirsten rat sarcoma (KRas) protein. A single microchip was utilized for the simultaneous evaluation of 15 PCC candidate fractions under more than a dozen different assay conditions. The platform also permitted more than a 10-fold savings in time and a more than 100-fold reduction in biological and chemical reagents relative to traditional multiwell plate assays. The best ligand was shown to exhibit an in vitro inhibition constant (IC₅₀) of ∼24 μM

Structures of peptide ligands in PCC Agent cocktail.

<p>Acetylene-presenting anchor peptides (black) were derived from the immunogenic epitope of HIV-1 gp41 (residues 600–612). A22-nindG (<b>i</b>) and A21-hnpfk (<b>ii</b>) were evolved from the original epitope appended with Pra at the C-terminus whereas A22-eihny (<b>iii</b>) utilizes the “substituted” anchor where residue Leu-607 is replaced with Pra. Secondary ligand branches (colored) were identified from the <i>in situ</i> click screen of a 5-mer OBOC library presenting an azide functionality. Biligands (<b>i</b>) and (<b>ii</b>) were raised against the target anti-HIV antibody 3D6, and the biligand (<b>iii</b>) was raised against the antibody 4B3.</p

Screening strategy for selecting capture agents against anti-HIV antibodies 3D6 and 4B3.

<p>The flow chart represents the use of the A21 and A22 cyclic peptides as anchor ligands for separate in situ click screens against a large OBOC azide-presenting peptide library.</p

A kinetic investigation of interacting, stimulated T cells identifies conditions for rapid functional enhancement, minimal phenotype differentiation, and improved adoptive cell transfer tumor eradication

<div><p>For adoptive cell transfer (ACT) immunotherapy of tumor-reactive T cells, an effective therapeutic outcome depends upon cell dose, cell expansion <i>in vivo</i> through a minimally differentiated phenotype, long term persistence, and strong cytolytic effector function. An incomplete understanding of the biological coupling between T cell expansion, differentiation, and response to stimulation hinders the co-optimization of these factors. We report on a biophysical investigation of how the short-term kinetics of T cell functional activation, through molecular stimulation and cell-cell interactions, competes with phenotype differentiation. T cells receive molecular stimulation for a few minutes to a few hours in bulk culture. Following this priming period, the cells are then analyzed at the transcriptional level, or isolated as single cells, with continuing molecular stimulation, within microchambers for analysis via 11-plex secreted protein assays. We resolve a rapid feedback mechanism, promoted by T cell—T cell contact interactions, which strongly amplifies T cell functional performance while yielding only minimal phenotype differentiation. When tested in mouse models of ACT, optimally primed T cells lead to complete tumor eradication. A similar kinetic process is identified in CD8⁺ and CD4⁺ T cells collected from a patient with metastatic melanoma.</p></div

Correlations of T cell aggregation and T cell functionality increase during the T₁ conditioning period, and a proposed associated mechanism.

<p><b>A</b>. Micrographs showing the kinetics of the aggregation of OT-1 T cells following tetramer stimulation, over the course of T₁, for cell densities of 2×10⁵ and 5×10⁵ cells/cm³. For the higher density culture, small, 2D cell aggregates are observed after T₁ = 5 hours (arrow), while 3D aggregates are observed by T₁ = 16 hours (arrow). At the lower cell density, 2D aggregates are observed by T₁ = 16 hours (arrow). Scale bar = 200 μm. <b>B</b>. Dynamics of the production of the CCL3 and IL2 cytokines following tetramer stimulation of OT-1 T cells, as cell density is varied. In both cases, CCL3 production precedes IL2 production, and the production of both proteins ramps up more quickly at higher cell density. <b>C</b>. Fluorescent micrographs showing the staining of CD25 (the IL2 receptor, in red) for non-stimulated (n.s.) (left column) and tetramer (and CD28) stimulated OT-1 T cells (right column), after T₁ = 16 hours. The cells were co-stained with the DAPI nuclear stain. Scale bar = 20 μm. <b>D</b>. Quantitation of the CD25 staining assays, measured in fluorescence intensity per cell (mean values ± s.e.m), using a threshold (*** <i>P</i> < 0.001, * <i>P</i> < 0.05). Statistics are based on 70–150 cells per condition. <b>E</b>. Drawing illustrating the dynamics of functional activation. The initial molecular stimulation promotes cell motility, via prompting CCL3 and CCL4 secretion. This, in turn, promotes increased contact interactions between T cells. Those interactions amplify the stimulation effects, leading to enhanced cell motility and additional contacts, in a fashion similar to a positive feedback loop. The feedback loop is established within the first one or two hours following molecular stimulation.</p