Richard Waugh - Academia.edu (original) (raw)

Papers by Richard Waugh

Biophysical Journal, 2004

Neutrophil capture and recruitment from the circulation requires the formation of specific recept... more Neutrophil capture and recruitment from the circulation requires the formation of specific receptor/ligand bonds under hydrodynamic forces. In the present study we examine bond formation between b 2-integrins on neutrophils and immobilized ICAM-1 while using micropipettes to control the force of contact between the cell and substrate. Magnesium was used to induce the high affinity conformation of the integrins, and bond formation was assessed by measuring the probability of adhesion during repeated contacts. Increasing the impingement force caused an increase in the contact area and led to a proportional increase in adhesion probability (from ;20 to 50%) over the range of forces tested (50-350 pN). In addition, different-sized beads were used to change the force per unit area in the contact zone (contact stress). We conclude that for a given contact stress, the rate of bond formation increases linearly with contact area, but that increasing contact stress results in higher intrinsic rates of bond formation.

Biophysical Journal, 1998

Journal of Laboratory …, 1997

Changes in the surface area and volume of reticulocytes were measured in vivo during late stage m... more Changes in the surface area and volume of reticulocytes were measured in vivo during late stage maturation. Baboons were treated with erythropoietin to produce mild reticulocytosis. Reticulocyte-rich cohorts of cells were obtained from whole blood by density gradient ...

Blood

Two distinct waves of hematopoietic progenitors originate in the yolk sac of the mammalian embryo... more Two distinct waves of hematopoietic progenitors originate in the yolk sac of the mammalian embryo. The first “primitive” wave contains primitive erythroid and macrophage progenitors that generate the embryo’s first red cells (Palis, et al. Development126:5073, 1999). The second “definitive” wave consists of definitive erythroid (BFU-E) and multiple myeloid progenitors that arise later in the yolk sac and are subsequently found in the fetal liver and postnatal marrow. While megakaryocyte progenitors have been detected in the yolk sac, the ontogeny of the megakaryocyte lineage is poorly understood. Furthermore, it is not been determined when platelets first enter the bloodstream of the mouse embryo. The presence and size of platelets in the embryonic bloodstream were examined by microscopy after staining with anti-GPV antibodies. Rare platelets were identified in 2 of 4 litters of mice at E10.5. These platelets were very large with a diameter of 4.2 ± 0.4 (mean ± SEM) microns. Platele...

American Journal of Physiology-Cell Physiology

The consequences of lost membrane area for long-term erythrocyte survival in the circulation were... more The consequences of lost membrane area for long-term erythrocyte survival in the circulation were investigated. Mouse red blood cells were treated with lysophosphatidylcholine to reduce membrane area, labeled fluorescently, reinfused into recipient mice, and then sampled periodically for 35 days. The circulating fraction of the modified cells decreased on an approximately exponential time course, with time constants ranging from 2 to 14 days. The ratio of volume to surface area of the surviving cells, measured using micropipettes, decreased rapidly over the first 5 days after infusion to within 5% of normal. This occurred by both preferential removal of the most spherical cells and modification of others, possibly due to membrane stress developed during transient trapping of cells in the microvasculature. After 5 days, the cell area decreased with time in the circulation, but the ratio of volume to surface area remained essentially constant. These results demonstrate that the ratio ...

Journal of biomedical materials research. Part A, Apr 1, 2017

Poly(ethylene glycol) (PEG) hydrogels provide a versatile platform to develop cell instructive ma... more Poly(ethylene glycol) (PEG) hydrogels provide a versatile platform to develop cell instructive materials through incorporation of a variety of cell adhesive ligands and degradable chemistries. Synthesis of PEG gels can be accomplished via two mechanisms: chain and step growth polymerizations. The mechanism dramatically impacts hydrogel nanostructure, whereby chain polymerized hydrogels are highly heterogeneous and step growth networks exhibit more uniform structures. Underpinning these alterations in nanostructure of chain polymerized hydrogels are densely-packed hydrophobic poly(methyl methacrylate) or poly(acrylate) kinetic chains between hydrophilic PEG crosslinkers. As cell-material interactions, such as those mediated by integrins, occur at the nanoscale and affect cell behavior, it is important to understand how different modes of polymerization translate into nanoscale mechanical and hydrophobic heterogeneities of hydrogels. Therefore, chain- and step-growth polymerized PEG h...

Biomedical microdevices, Jan 5, 2018

Silicon nanomembranes are ultrathin, highly permeable, optically transparent and biocompatible su... more Silicon nanomembranes are ultrathin, highly permeable, optically transparent and biocompatible substrates for the construction of barrier tissue models. Trans-epithelial/endothelial electrical resistance (TEER) is often used as a non-invasive, sensitive and quantitative technique to assess barrier function. The current study characterizes the electrical behavior of devices featuring silicon nanomembranes to facilitate their application in TEER studies. In conventional practice with commercial systems, raw resistance values are multiplied by the area of the membrane supporting cell growth to normalize TEER measurements. We demonstrate that under most circumstances, this multiplication does not 'normalize' TEER values as is assumed, and that the assumption is worse if applied to nanomembrane chips with a limited active area. To compare the TEER values from nanomembrane devices to those obtained from conventional polymer track-etched (TE) membranes, we develop finite element mo...

Scientific Reports

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. ... more Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation. However, little is known about the formation and composition of the membrane skeleton in primitive erythroblasts, which progressively mature while circulating in the embryonic bloodstream. We found that primary primitive erythroblasts express the major membrane skeleton genes present in similarly staged definitive erythroblasts, suggesting that the composition and formation of this membrane network is conserved in maturing primitive and definitive erythroblasts despite their respective intravascular and extravascular locations. Membrane deformability and stability of primitive erythroblasts, assayed by microfluidic studies and fluorescence imaged microdeformation, respectively, significantly increase prior to enucleation. These functional changes coincide with protein 4.1 R isoform switching and protein 4.1R-null primitive erythroblasts fail to establish normal membrane stability and deformability. We conclude that maturing primitive erythroblasts initially navigate the embryonic vasculature prior to establishing a deformable cytoskeleton, which is ultimately formed prior to enucleation. Formation of an erythroid-specific, protein 4.1R-dependent membrane skeleton is an important feature not only of definitive, but also of primitive, erythropoiesis in mammals.

Journal of Dental Research

ABSTRACT Candida albicans cells are often detected with Streptococcus mutans in plaque biofilms f... more ABSTRACT Candida albicans cells are often detected with Streptococcus mutans in plaque biofilms from children affected with early childhood caries. The coadhesion between these 2 organisms appears to be largely mediated by the S. mutans-derived exoenzyme glucosyltransferase B (GtfB); GtfB readily binds to C. albicans cells in an active form, producing glucans locally that provide enhanced binding sites for S. mutans. However, knowledge is limited about the mechanisms by which the bacterial exoenzyme binds to and functions on the fungal surface to promote this unique cross-kingdom interaction. In this study, we use atomic force microscopy to understand the strength and binding dynamics modulating GtfB-C. albicans adhesive interactions in situ. Single-molecule force spectroscopy with GtfB-functionalized atomic force microscopy tips demonstrated that the enzyme binds with remarkable strength to the C. albicans cell surface (~2 nN) and showed a low dissociation rate, suggesting a highly stable bond. Strikingly, the binding strength of GtfB to the C. albicans surface was ~2.5-fold higher and the binding stability, ~20 times higher, as compared with the enzyme adhesion to S. mutans. Furthermore, adhesion force maps showed an intriguing pattern of GtfB binding. GtfB adhered heterogeneously on the surface of C. albicans, showing a higher frequency of adhesion failure but large sections of remarkably strong binding forces, suggesting the presence of GtfB binding domains unevenly distributed on the fungal surface. In contrast, GtfB bound uniformly across the S. mutans cell surface with less adhesion failure and a narrower range of binding forces (vs. the C. albicans surface). The data provide the first insights into the mechanisms underlying the adhesive and mechanical properties governing GtfB interactions with C. albicans. The strong and highly stable GtfB binding to C. albicans could explain, at least in part, why this bacterially derived exoenzyme effectively modulates this virulent cross-kingdom interaction. © International & American Associations for Dental Research 2015.

Journal of microscopy, Jun 1, 2016

When investigating the interaction of cells with surfaces, it is becoming increasingly important ... more When investigating the interaction of cells with surfaces, it is becoming increasingly important to perform quantitative measurements of surface protein density to understand reaction kinetics. Previously, to calibrate a surface for an experiment one would have to use a radiometric assay or strip the surface with acid and perform a mass quantification. Although both of these methodologies have been proven to be effective measurement techniques for surface quantification, they can be time consuming and require substantial amounts of material. The latter is particularly problematic when working with specialized molecules or constructs that may be expensive to produce and/or only available in small quantities. Here we present a simple method to measure the intensity and penetration depth of an evanescent wave, and use this information to quantify the density of surface molecules in a microscopic region of a transparent surface.

Bioelectrochemistry, May 1, 2004

Red blood cell (RBC) shape, behaviour and deformability can be consistently accounted for by a mo... more Red blood cell (RBC) shape, behaviour and deformability can be consistently accounted for by a model for the elastic properties of the RBC membrane that includes the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending. The role of the corresponding energy terms in different RBC shape and deformation situations is analyzed. RBC shape transformations are compared to the shape transformations of phospholipid vesicles that are driven by the difference between the equilibrium areas of the bilayer leaflets (DA 0). It is deduced that the skeleton energy contributions play a crucial role in the formation of an echinocyte. The effect of a transformation of the natural biconcave RBC shape into an echinocyte on its resistance to entry into capillary-sized cylindrical tubes is analyzed. It is shown that, during the aspiration of an echinocyte into a pipette, there are two competing skeleton deformation effects, which arise due to skeleton density changes, one due to spicule formation and the other due to deformation induced by micropipette aspiration. Furthermore, the shift of the observed dependence of the projection length on the aspiration pressure of more crenated cells towards higher aspiration pressures can be accounted for by an increase of the equilibrium area difference DA 0 and consequent modification of the nonlocal contribution to the cell elastic energy.

Annals of Biomedical Engineering, Sep 20, 2006

Changing affinity of beta(2)-integrins on neutrophils for their ligands on endothelium is a criti... more Changing affinity of beta(2)-integrins on neutrophils for their ligands on endothelium is a critical, regulated step in the inflammatory response. In this report, the dynamics of the neutrophil response to the inflammatory chemokine interleukin-8 (IL-8) is examined. Human IL-8 was immobilized on beads and brought into contact with neutrophils selected from whole blood samples. Resulting changes in cellular adhesion were assessed by measuring the adhesion frequency between a human neutrophil and a bead coated with the endothelial ligand ICAM-1 (intercellular adhesion molecule-1). Cells engulfed the IL-8 coated beads within a few tens of seconds, and most of the cells exhibited an increase in adhesion to ICAM-1 after approximately 5 to 10 min of contact with IL-8 at room temperature (3 to 5 min at 37 degrees C). Neither monocyte chemotactic protein-1 (MCP-1) nor anti-CD45-coated beads caused any changes in adhesion to ICAM-1. IL-8 induced adhesion was blocked by antibody against CD18. At lower surface density of chemokine, approximately 20% of IL-8 coated beads adhered but were not engulfed by the cells, although the increase in adhesion for ICAM-1 was still effected. Heterogeneity in the cellular response and variability between donors was also noted.

The Faseb Journal, Apr 1, 2007

Journal of Biomechanical Engineering, Aug 1, 1986

The elastic properties of marginal band, a microtubular structure isolated from the newt (notopht... more The elastic properties of marginal band, a microtubular structure isolated from the newt (notophthalmus viridescens) have been measured. Bands were isolated using Triton X-100 and pepsin at pH 6.8 according to the method of Cohen (1978). Isolated bands were manipulated with a glass microhook in a buffer-filled chamber under the microscope. Single bands were stretched between the hook and a thin glass fiber. The fiber was calibrated so that the force on the band could be calculated from the displacement of the fiber. The data pairs of force versus band deflection were analyzed according to the theoretical work of Libai and Simmonds (1983) to obtain the flexural and extensional rigidities of the band. Band dimensions calculated from the data were consistent with microscopically determined values. The average flexural rigidity of the bands (EI) was 5.3 × 10−13 dyn•cm2 and the average extensional rigidity (EA) was 0.017 dyn. Compared to the cell membrane, the marginal band is nearly inextensible and has a much greater resistance to bending, indicating that the band makes an important contribution to the deformability of the circulating cell.

Langmuir : the ACS journal of surfaces and colloids, Jan 25, 2015

The nanoscale topography of adhesive surfaces is known to be an important factor governing cellul... more The nanoscale topography of adhesive surfaces is known to be an important factor governing cellular behavior. Previous work has shown that surface coatings composed of halloysite nanotubes enhances the adhesion, and therefore capture, of rare target cells such as circulating tumor cells. Here, we demonstrate a unique feature of these coatings in its ability to reduce the adhesion of leukocytes and prevent leukocyte spreading. Surfaces were prepared with coatings of halloysite nanotubes and functionalized for leukocyte adhesion with E-selectin, and the dilution of nanotube concentration revealed a threshold concentration below which cell spreading became comparable with smooth surfaces. Evaluation of surface roughness characteristics determined that the average distance between discrete surface features correlated with adhesion metrics, with a separation distance of approximately 2 μm identified as the critical threshold. Computational modeling of the interaction of leukocytes with h...