Single particle tracking reveals spatial and dynamic organization of the Escherichia coli biofilm matrix

Biofilms are communities of surface-adherent bacteria surrounded by secreted polymers known as the extracellular polymeric substance. Biofilms are harmful in many industries, and thus it is of great interest to understand their mechanical properties and structure to determine ways to destabilize them. By performing single particle tracking with beads of varying surface functionalization it was found that charge interactions play a key role in mediating mobility within biofilms. With a combination of single particle tracking and microrheological concepts, it was found that Escherichia coli biofilms display height dependent charge density that evolves over time. Statistical analyses of bead trajectories and confocal microscopy showed inter-connecting micron scale channels that penetrate throughout the biofilm, which may be important for nutrient transfer through the system. This methodology provides significant insight into a particular biofilm system and can be applied to many others to provide comparisons of biofilm structure. The elucidation of structure provides evidence for the permeability of biofilms to microscale objects, and the ability of a biofilm to mature and change properties over time.National Science Foundation (U.S.) (CBET-1335938)Cystic Fibrosis Foundation (HANES07XX0)Massachusetts Institute of Technology (Charles E. Reed Faculty Initiative Fund)Burroughs Wellcome Fund (Preterm Birth Research Grant)National Institute of Allergy and Infectious Diseases (U.S.) (F30 Fellowship 1F30AI110053-01)National Institute of Allergy and Infectious Diseases (U.S.) (Training Grant in Toxicology 5 T32 ES7020-37

Discovery of a Probable CH Star in the Globular Cluster M14 and Implications for the Evolution of Binaries in Clusters

We report the discovery of a probable CH star in the core of the Galactic globular cluster M14, identified from an integrated-light spectrum of the cluster obtained with the MOS spectrograph on the CFHT. From a high- resolution echelle spectrum of the same star obtained with the Hydra fiber positioner and bench spectrograph on the WIYN telescope, we measure a radial velocity of $-53.0\pm1.2$ km s$^{-1}$. Although this velocity is inconsistent with published estimates of the systemic radial velocity of M14 (eg, ${\bar {v_r}} \approx -123$ km s$^{-1}$), we use high-precision Hydra velocities for 20 stars in the central 2.6 arcminutes of M14 to calculate improved values for the cluster mean velocity and one-dimensional velocity dispersion: $-59.5\pm1.9$ km s$^{-1}$ and $8.2\pm1.4$ km s$^{-1}$, respectively. Both the star's location near the tip of the red giant branch in the cluster color magnitude diagram and its radial velocity therefore argue for membership in M14. Since the intermediate-resolution MOS spectrum shows not only enhanced CH absorption but also strong Swan bands of C$_2$, M14 joins Omega Cen as the only globular clusters known to contain classical CH stars. Although evidence for its duplicity must await additional radial velocity measurements, the CH star in M14 is probably, like all field CH stars, a spectroscopic binary with a degenerate (white dwarf) secondary. The candidate and confirmed CH stars in M14 and Omega Cen, and in a number of Galactic dSph galaxies, may then owe their existence to the long timescales for the shrinking and coalescence of hard binaries in low-concentration environments.Comment: Accepted to the Astrophysical Journal Letters. 13 pages, AAS LaTeX and three postscript figures (numbers 2,3,4). Entire paper (including Figure 1) available at http://www.hia.nrc.ca/DAO/SCIENCE/science.htm

Dynamics of the Globular Cluster System Associated with M87 (NGC 4486). II. Analysis

We present a dynamical analysis of the globular cluster system associated with M87 (= NGC 4486), the cD galaxy near the dynamical center of the Virgo cluster. The analysis utilizes a new spectroscopic and photometric database which is described in a companion paper (Hanes et al. 2001). Using a sample of 278 globular clusters with measured radial velocities and metallicities, and new surface density profiles based on wide-field Washington photometry, we study the dynamics of the M87 globular cluster system both globally --- for the entire cluster sample --- and separately --- for the metal-rich and metal-poor globular cluster samples. This constitutes the largest sample of radial velocities for pure Population II tracers yet assembled for any galaxy. We discuss the implications of our findings for models for the formation of giant elliptical galaxies, globular cluster systems, and the Virgo cluster. (ABRIDGED)Comment: 28 pages, 19 postscript figures, 1 jpeg image. See http://www.physics.rutgers.edu/ast/ast-rap.html to download the manuscript with higher quality figures. Accepted for publication in the Astrophysical Journa

Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design

Engulfing tumors with synthetic extracellular matrices for cancer

available in PMC 2010 June 1.Local immunotherapies are under investigation for the treatment of unresectable tumors and sites of solid tumor resection to prevent local recurrence. Successful local therapy could also theoretically elicit systemic immune responses against cancer. Here we explored the delivery of therapeutic dendritic cells (DCs), cytokines, or other immunostimulatory factors to tumors via the use of ‘self-gelling’ hydrogels based on the polysaccharide alginate, injected peritumorally around established melanoma lesions. Peritumoral injection of alginate matrices loaded with DCs and/or an interleukin-15 superagonist (IL-15SA) around 14-day established ova-expressing B16F0 murine melanoma tumors promoted immune cell accumulation in the peritumoral matrix, and matrix infiltration correlated with tumor infiltration by leukocytes. Single injections of IL-15SA-carrying gels concentrated the cytokine in the tumor site ∼40-fold compared to systemic injection and enabled a majority of treated animals to suppress tumor growth for a week or more. Further, we found that single injections of alginate matrices loaded with IL-15SA and the Toll-like receptor ligand CpG or two injections of gels carrying IL-15SA alone could elicit comparable anti-tumor activity without the need for exogenous DCs. Thus, injectable alginate gels offer an attractive platform for local tumor immunotherapy, and facilitate combinatorial treatments designed to promote immune responses locally at a tumor site while limiting systemic exposure to potent immunomodulatory factors.United States. Defense Advanced Research Projects Agency ( (contract # W81XWH-04-C-0139)National Institutes of Health (U.S.) (NIH Grant EB007280)National Institutes of Health (U.S.) (NIH Grant U54-CA126515)National Institutes of Health (U.S.) (NIH Grant U54-CA112967)National Science Foundation (U.S.) (award 0348259

Age-related delay in information accrual for faces: Evidence from a parametric, single-trial EEG approach

Background: In this study, we quantified age-related changes in the time-course of face processing by means of an innovative single-trial ERP approach. Unlike analyses used in previous studies, our approach does not rely on peak measurements and can provide a more sensitive measure of processing delays. Young and old adults (mean ages 22 and 70 years) performed a non-speeded discrimination task between two faces. The phase spectrum of these faces was manipulated parametrically to create pictures that ranged between pure noise (0% phase information) and the undistorted signal (100% phase information), with five intermediate steps. Results: Behavioural 75% correct thresholds were on average lower, and maximum accuracy was higher, in younger than older observers. ERPs from each subject were entered into a single-trial general linear regression model to identify variations in neural activity statistically associated with changes in image structure. The earliest age-related ERP differences occurred in the time window of the N170. Older observers had a significantly stronger N170 in response to noise, but this age difference decreased with increasing phase information. Overall, manipulating image phase information had a greater effect on ERPs from younger observers, which was quantified using a hierarchical modelling approach. Importantly, visual activity was modulated by the same stimulus parameters in younger and older subjects. The fit of the model, indexed by R2, was computed at multiple post-stimulus time points. The time-course of the R2 function showed a significantly slower processing in older observers starting around 120 ms after stimulus onset. This age-related delay increased over time to reach a maximum around 190 ms, at which latency younger observers had around 50 ms time lead over older observers. Conclusion: Using a component-free ERP analysis that provides a precise timing of the visual system sensitivity to image structure, the current study demonstrates that older observers accumulate face information more slowly than younger subjects. Additionally, the N170 appears to be less face-sensitive in older observers

CD4-Specific Designed Ankyrin Repeat Proteins Are Novel Potent HIV Entry Inhibitors with Unique Characteristics

Here, we describe the generation of a novel type of HIV entry inhibitor using the recently developed Designed Ankyrin Repeat Protein (DARPin) technology. DARPin proteins specific for human CD4 were selected from a DARPin DNA library using ribosome display. Selected pool members interacted specifically with CD4 and competed with gp120 for binding to CD4. DARPin proteins derived in the initial selection series inhibited HIV in a dose-dependent manner, but showed a relatively high variability in their capacity to block replication of patient isolates on primary CD4 T cells. In consequence, a second series of CD4-specific DARPins with improved affinity for CD4 was generated. These 2nd series DARPins potently inhibit infection of genetically divergent (subtype B and C) HIV isolates in the low nanomolar range, independent of coreceptor usage. Importantly, the actions of the CD4 binding DARPins were highly specific: no effect on cell viability or activation, CD4 memory cell function, or interference with CD4-independent virus entry was observed. These novel CD4 targeting molecules described here combine the unique characteristics of DARPins—high physical stability, specificity and low production costs—with the capacity to potently block HIV entry, rendering them promising candidates for microbicide development