Evidence of collective influence in innate sensing using fluidic force microscopy.

The innate immune system initiates early response to infection by sensing molecular patterns of infection through pattern-recognition receptors (PRRs). Previous work on PRR stimulation of macrophages revealed significant heterogeneity in single cell responses, suggesting the importance of individual macrophage stimulation. Current methods either isolate individual macrophages or stimulate a whole culture and measure individual readouts. We probed single cell NF-κB responses to localized stimuli within a naïve culture with Fluidic Force Microscopy (FluidFM). Individual cells stimulated in naïve culture were more sensitive compared to individual cells in uniformly stimulated cultures. In cluster stimulation, NF-κB activation decreased with increased cell density or decreased stimulation time. Our results support the growing body of evidence for cell-to-cell communication in macrophage activation, and limit potential mechanisms. Such a mechanism might be manipulated to tune macrophage sensitivity, and the density-dependent modulation of sensitivity to PRR signals could have relevance to biological situations where macrophage density increases.

Brominated Luciferins Are Versatile Bioluminescent Probes.

We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.

An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein.

The phosphorylation state and corresponding activity of the retinoblastoma tumor suppressor protein (Rb) are modulated by a balance of kinase and phosphatase activities. Here we characterize the association of Rb with the catalytic subunit of protein phosphatase 1 (PP1c). A crystal structure identifies an enzyme docking site in the Rb C-terminal domain that is required for efficient PP1c activity toward Rb. The phosphatase docking site overlaps with the known docking site for cyclin-dependent kinase (Cdk), and PP1 competition with Cdk-cyclins for Rb binding is sufficient to retain Rb activity and block cell-cycle advancement. These results provide the first detailed molecular insights into Rb activation and establish a novel mechanism for Rb regulation in which kinase and phosphatase compete for substrate docking.

Characterization of pHEMA-based hydrogels that exhibit light-induced bactericidal effect via release of NO.

A light-activated NO donor, [Mn(PaPy(3))(NO)]ClO(4) (1a), has been incorporated into HEMA-based polymer hydrogel and the nitrosyl-polymer conjugate materials 1a(x) · HG and 1a(x) · HG(MB) have been characterized. The NO releasing properties and antibacterial capabilities of these materials in conjunction with growth attenuators such as hydrogen peroxide and methylene blue (MB) are reported. Since the nitrosyl releases NO only upon exposure to light, materials like 1a(x) · HG(MB) could be used as wound dressings that deliver NO under controlled conditions.