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Jeffrey S Urbach

Publications and source records attributed to Jeffrey S Urbach.

9 recordsLinked to original sources
PubMed2006

Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria.

We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.

Animals↗
PubMed2006

Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 2. Altered vacuolar volume regulation in drug resistant malaria.

In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3(Dd2) (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.

Animals↗
PubMed2006

Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope.

We develop an extension of fluorescence correlation spectroscopy (FCS) using a spinning disk confocal microscope. This approach can spatially map diffusion coefficients or flow velocities at up to approximately 10(5) independent locations simultaneously. Commercially available cameras with frame rates of 1000 Hz allow FCS measurements of systems with diffusion coefficients D~10(-7) cm(2)/s or smaller. This speed is adequate to measure small microspheres (200-nm diameter) diffusing in water, or hindered diffusion of macromolecules in complex media (e.g., tumors, cell nuclei, or the extracellular matrix). There have been a number of recent extensions to FCS based on laser scanning microscopy. Spinning disk confocal microscopy, however, has the potential for significantly higher speed at high spatial resolution. We show how to account for a pixel size effect encountered with spinning disk confocal FCS that is not present in standard or scanning FCS, and we introduce a new method to correct for photobleaching. Finally, we apply spinning disk confocal FCS to microspheres diffusing in Type I collagen, which show complex spatially varying diffusion caused by hydrodynamic and steric interactions with the collagen matrix.

Animals↗
PubMed2005

Adaptation is not required to explain the long-term response of axons to molecular gradients.

It has been suggested that growth cones navigating through the developing nervous system might display adaptation, so that their response to gradient signals is conserved over wide variations in ligand concentration. Recently however, a new chemotaxis assay that allows the effect of gradient parameters on axonal trajectories to be finely varied has revealed a decline in gradient sensitivity on either side of an optimal concentration. We show that this behavior can be quantitatively reproduced with a computational model of axonal chemotaxis that does not employ explicit adaptation. Two crucial components of this model required to reproduce the observed sensitivity are spatial and temporal averaging. These can be interpreted as corresponding, respectively, to the spatial spread of signaling effects downstream from receptor binding, and to the finite time over which these signaling effects decay. For spatial averaging, the model predicts that an effective range of roughly one-third of the extent of the growth cone is optimal for detecting small gradient signals. For temporal decay, a timescale of about 3 minutes is required for the model to reproduce the experimentally observed sensitivity.

Axons↗
PubMed2004

Nonequilibrium two-phase coexistence in a confined granular layer.

We report the observation of the homogenous nucleation of crystals in a dense layer of steel spheres confined between two horizontal plates vibrated vertically. Above a critical vibration amplitude, two-layer crystals with square symmetry were found to coexist in steady state with a surrounding granular liquid. By analogy to equilibrium hard-sphere systems, the phase behavior may be explained through entropy maximization. However, dramatic nonequilibrium effects are present, including a significant difference in the granular temperatures of the two phases.

Journal Article↗
PubMed2004

A new chemotaxis assay shows the extreme sensitivity of axons to molecular gradients.

Axonal chemotaxis is believed to be important in wiring up the developing and regenerating nervous system, but little is known about how axons actually respond to molecular gradients. We report a new quantitative assay that allows the long-term response of axons to gradients of known and controllable shape to be examined in a three-dimensional gel. Using this assay, we show that axons may be nature's most-sensitive gradient detectors, but this sensitivity exists only within a narrow range of ligand concentrations. This assay should also be applicable to other biological processes that are controlled by molecular gradients, such as cell migration and morphogenesis.

Animals↗
PubMed2004

Predicting axonal response to molecular gradients with a computational model of filopodial dynamics.

Axons are often guided to their targets in the developing nervous system by attractive or repulsive molecular concentration gradients. We propose a computational model for gradient sensing and directed movement of the growth cone mediated by filopodia. We show that relatively simple mechanisms are sufficient to generate realistic trajectories for both the short-term response of axons to steep gradients and the long-term response of axons to shallow gradients. The model makes testable predictions for axonal response to attractive and repulsive gradients of different concentrations and steepness, the size of the intracellular amplification of the gradient signal, and the differences in intracellular signaling required for repulsive versus attractive turning.

Algorithms↗
PubMed2002

Forcing and velocity correlations in a vibrated granular monolayer.

The role of forcing on the dynamics of a vertically shaken granular monolayer is investigated. Using a flat plate, surprising negative velocity correlations are measured. A mechanism for this anticorrelation is proposed with support from both experimental results and molecular dynamics simulations. Using a rough plate, velocity correlations are positive, and the velocity distribution evolves from a Gaussian at very low densities to a broader distribution at high densities. These results are interpreted as a balance between stochastic forcing, interparticle collisions, and friction with the plate.

Journal Article↗