Enzyme microarrays assembled by acoustic dispensing technology

Miniaturizing bioassays to the nanoliter scale for high-throughput screening reduces the consumption of reagents that are expensive or difficult to handle. Through the use of acoustic dispensing technology, nanodroplets containing 10 μM ATP (3 μCi/μL 32P) and reaction buffer in 10% glycerol were positionally dispensed to the surface of glass slides to form 40-nL compartments (100 droplets/slide) for Pim1 (proviral integration site 1) kinase reactions. The reactions were activated by dispensing 4 nL of various levels of a pyridocarbazolo-cyclopentadienyl ruthenium complex Pim1 inhibitor, followed by dispensing 4 nL of a Pim1 kinase and peptide substrate solution to achieve final concentrations of 150 nM enzyme and 10 μM substrate. The microarray was incubated at 30 °C (97% Rh) for 1.5 h. The spots were then blotted to phosphocellulose membranes to capture phosphorylated substrate. With phosphor imaging to quantify the washed membranes, the assay showed that, for doses of inhibitor from 0.75 to 3 μM, Pim1 was increasingly inhibited. Signal-to-background ratios were as high as 165, and average coefficients of variation for the assay were not, vert, ∼20%. Coefficients of variation for dispensing typical working buffers were under 5%. Thus, microarrays assembled by acoustic dispensing are promising as cost-effective tools that can be used in protein assay development

Steady-State Kinetic Modeling Constrains Cellular Resting States and Dynamic Behavior

A defining characteristic of living cells is the ability to respond dynamically to external stimuli while maintaining homeostasis under resting conditions. Capturing both of these features in a single kinetic model is difficult because the model must be able to reproduce both behaviors using the same set of molecular components. Here, we show how combining small, well-defined steady-state networks provides an efficient means of constructing large-scale kinetic models that exhibit realistic resting and dynamic behaviors. By requiring each kinetic module to be homeostatic (at steady state under resting conditions), the method proceeds by (i) computing steady-state solutions to a system of ordinary differential equations for each module, (ii) applying principal component analysis to each set of solutions to capture the steady-state solution space of each module network, and (iii) combining optimal search directions from all modules to form a global steady-state space that is searched for accurate simulation of the time-dependent behavior of the whole system upon perturbation. Importantly, this stepwise approach retains the nonlinear rate expressions that govern each reaction in the system and enforces constraints on the range of allowable concentration states for the full-scale model. These constraints not only reduce the computational cost of fitting experimental time-series data but can also provide insight into limitations on system concentrations and architecture. To demonstrate application of the method, we show how small kinetic perturbations in a modular model of platelet P2Y1 signaling can cause widespread compensatory effects on cellular resting states

Neutrophil String Formation: Hydrodynamic Thresholding and Cellular Deformation during Cell Collisions

Neutrophils unexpectedly display flow-enhanced adhesion (hydrodynamic thresholding) to L-selectin in rolling or aggregation assays. We report that the primary collision efficiency (ε) of flowing neutrophils with preadhered neutrophils on intercellular adhesion molecule-1 (ICAM-1) or fibrinogen also displayed a maximum of ε ~ 0.4–0.45 at a wall shear rate of 100 s-1, an example of thresholding. Primary collision lifetime with no detectable bonding decreased from 130 to 10 ms as wall shear rate increased from 30 to 300 s-1, whereas collision lifetimes with bonding decreased from 300 to 100 ms over this shear range using preadhered neutrophils on ICAM-1, with similar results for fibrinogen. Antibodies against L-selectin, but not against CD11a, CD11b, or CD18, reduced ε at 100 s-1 by \u3e85%. High resolution imaging detected large scale deformation of the flowing neutrophil during the collision at 100 s-1 with the apparent contact area increasing up to ~40 μm2. We observed the formation of long linear string assemblies of neutrophils downstream of neutrophils preadhered to ICAM-1, but not fibrinogen, with a maximum in string formation at 100 s-1. Secondary capture events to the ICAM-1 or fibrinogen coated surfaces after primary collisions were infrequent and short lived, typically lasting from 500 to 3500 ms. Between 5 and 20% of neutrophil interactions with ICAM-1 substrate converted to firm arrest (\u3e3500 ms) and greatly exceeded that observed for fibrinogen, thus defining the root cause of poor string formation on fibrinogen at all shear rates. Additionally, neutrophils mobilized calcium after incorporation into strings. Static adhesion also caused calcium mobilization, as did the subsequent onset of flow. To our knowledge, this is the first report of 1), hydrodynamic thresholding in neutrophil string formation; 2), string formation on ICAM-1 but not on fibrinogen; 3), large cellular deformation due to collisions at a venous shear rate; and 4), mechanosensing through neutrophil β2-integrin/adhesion. The increased contact area during deformation was likely responsible for the hydrodynamic threshold observed in the primary collision efficiency since no increase in primary collision lifetime was detected as shear forces were increased (for either surface coating)

Neutrophil-bead collision assay: Pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime

Visualization of flowing neutrophils colliding with adherent 1-µm-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (ε), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s-1 ε decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, ∠mtether, increased from 0.35 µm to ⋍2.0 µm over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress offrate, Koff(0) = 0.56 s-1and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (± tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3–5 (11%)). For collisions at 100 s-1, pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on ε, but increased ∠mtether by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-ß-cyclodextrin or formaldehyde fixation had no effect on ε, but reduced ∠mtether by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophilbead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes

Sensor node localisation using a stereo camera rig

In this paper, we use stereo vision processing techniques to detect and localise sensors used for monitoring simulated environmental events within an experimental sensor network testbed. Our sensor nodes communicate to the camera through patterns emitted by light emitting diodes (LEDs). Ultimately, we envisage the use of very low-cost, low-power, compact microcontroller-based sensing nodes that employ LED communication rather than power hungry RF to transmit data that is gathered via existing CCTV infrastructure. To facilitate our research, we have constructed a controlled environment where nodes and cameras can be deployed and potentially hazardous chemical or physical plumes can be introduced to simulate environmental pollution events in a controlled manner. In this paper we show how 3D spatial localisation of sensors becomes a straightforward task when a stereo camera rig is used rather than a more usual 2D CCTV camera

Using Physiology to Predict the Responses of Ants to Climatic Warming

Physiological intolerance of high temperatures places limits on organismal responses to the temperature increases associated with global climatic change. Because ants are geographically widespread, ecologically diverse, and thermophilic, they are an ideal system for exploring the extent to which physiological tolerance can predict responses to environmental change. Here we expand on simple models that use thermal tolerance to predict the responses of ants to climatic warming. We investigated the degree to which changes in the abundance of ants under warming reflect reductions in the thermal niche space for their foraging. In an eastern deciduous forest system in the United States with ~40 ant species, we found that for some species, the loss of thermal niche space for foraging was related to decreases in abundance with increasing experimental climatic warming. However, many ant species exhibited no loss of thermal niche space. For one well-studied species, Temnothorax curvispinosus, we examined both survival of workers and growth of colonies (a correlate of reproductive output) as functions of temperature in the laboratory, and found that the range of thermal tolerances for colony growth was much narrower than for survival of workers. We evaluated these functions in the context of experimental climatic warming and found that the difference in the responses of these two attributes to temperature generates differences in the means and especially the variances of expected fitness under warming. The expected mean growth of colonies was optimized at intermediate levels of warming (2 – 4 °C above ambient), yet the expected variance monotonically increased with warming. In contrast, the expected mean and variance of the survival of workers decreased when warming exceeded 4°C above ambient. Together, these results for T. curvispinosus emphasize the importance of measuring reproduction (colony growth) in context of climatic change: indeed, our examination of the loss of thermal niche space with the larger species pool could be missing much of the warming impact due to these analyses being based on survival rather than reproduction. We suggest that while physiological tolerance of temperature can be a useful predictive tool for modeling responses to climatic change, future efforts should be devoted to understanding the causes and consequences of variability in models of tolerance calibrated with different metrics of performance and fitness.Organismic and Evolutionary BiologyOther Research Uni

The opposites task: Using general rules to test cognitive flexibility in preschoolers

A brief narrative description of the journal article, document, or resource. Executive functions play an important role in cognitive development, and during the preschool years especially, children's performance is limited in tasks that demand flexibility in their behavior. We asked whether preschoolers would exhibit limitations when they are required to apply a general rule in the context of novel stimuli on every trial (the "opposites" task). Two types of inhibitory processing were measured: response interference (resistance to interference from a competing response) and proactive interference (resistance to interference from a previously relevant rule). Group data show 3-year-olds have difficulty inhibiting prepotent tendencies under these conditions, whereas 5-year-olds' accuracy is near ceiling in the task. (Contains 4 footnotes and 1 table.

Nonlinear shock acceleration beyond the Bohm limit

We suggest a physical mechanism whereby the acceleration time of cosmic rays by shock waves can be significantly reduced. This creates the possibility of particle acceleration beyond the knee energy at ~10^15eV. The acceleration results from a nonlinear modification of the flow ahead of the shock supported by particles already accelerated to the knee momentum at p ~ p_*. The particles gain energy by bouncing off converging magnetic irregularities frozen into the flow in the shock precursor and not so much by re-crossing the shock itself. The acceleration rate is thus determined by the gradient of the flow velocity and turns out to be formally independent of the particle mean free path (m.f.p.). The velocity gradient is, in turn, set by the knee-particles at p ~ p_* as having the dominant contribution to the CR pressure. Since it is independent of the m.f.p., the acceleration rate of particles above the knee does not decrease with energy, unlike in the linear acceleration regime. The reason for the knee formation at p ~ p_* is that particles with $p > p_*$ are effectively confined to the shock precursor only while they are within limited domains in the momentum space, while other particles fall into ``loss-islands'', similar to the ``loss-cone'' of magnetic traps. This structure of the momentum space is due to the character of the scattering magnetic irregularities. They are formed by a train of shock waves that naturally emerge from unstably growing and steepening magnetosonic waves or as a result of acoustic instability of the CR precursor. These losses steepen the spectrum above the knee, which also prevents the shock width from increasing with the maximum particle energy.Comment: aastex, 13 eps figure

Axial Symmetry and Rotation in the SiO Maser Shell of IK Tauri

We observed v=1, J=1-0 43-GHz SiO maser emission toward the Mira variable IK Tauri (IK Tau) using the Very Long Baseline Array (VLBA). The images resulting from these observations show that SiO masers form a highly elliptical ring of emission approximately 58 x 32 mas with an axial ratio of 1.8:1. The major axis of this elliptical distribution is oriented at position angle of ~59 deg. The line-of-sight velocity structure of the SiO masers has an apparent axis of symmetry consistent with the elongation axis of the maser distribution. Relative to the assumed stellar velocity of 35 km/s, the blue- and red-shifted masers were found to lie to the northwest and southeast of this symmetry axis respectively. This velocity structure suggests a NW-SE rotation of the SiO maser shell with an equatorial velocity, which we determine to be ~3.6 km/s. Such a NW-SE rotation is in agreement with a circumstellar envelope geometry invoked to explain previous water and OH maser observations. In this geometry, water and OH masers are preferentially created in a region of enhanced density along the NE-SW equator orthogonal to the rotation/polar axis suggested by the SiO maser velocities.Comment: 17 Pages, 4 figures (2 color); accepted for publication in Ap