Diamond, Scott L
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Publication Enzyme microarrays assembled by acoustic dispensing technology(2008-10-01) Wong, E. Y; Diamond, Scott LMiniaturizing 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.Publication Kinetics of random aggregation-fragmentation processes with multiple components(2003-05-09) Laurenzi, Ian J.; Diamond, Scott LA computationally efficient algorithm is presented for exact simulation of the stochastic time evolution of spatially homogeneous aggregation-fragmentation processes featuring multiple components or conservation laws. The algorithm can predict the average size and composition distributions of aggregating particles as well as their fluctuations, regardless of the functional form (e.g., composition dependence) of the aggregation or fragmentation kernels. Furthermore, it accurately predicts the complete time evolutions of all moments of the size and composition distributions, even for systems that exhibit gel transitions. We demonstrate the robustness and utility of the algorithm in case studies of linear and branched polymerization processes, the last of which is a two-component process. These simulation results provide the stochastic description of these processes and give new insights into their gel transitions, fluctuations, and long-time behavior when deterministic approaches to aggregation kinetics may not be reliable.Publication Hemodynamic Regulation of Inflammation at the Endothelial-Neutrophil Interface(2008-04-01) Ji, Julie Y; Jing, Huiyan; Diamond, Scott LArterial shear stress can regulate endothelial phenotype. The potential for anti-inflammatory effects of shear stress on TNFα-activated endothelium was tested in assays of cytokine expression and neutrophil adhesion. In cultured human aortic endothelial cells (HAEC), arterial shear stress of 10 dyne/cm2 blocked by > 80% the induction by 5 ng/ml TNFα of interleukin-8 (IL-8) and IL-6 secretion (50% and 90% reduction, respectively, in the presence of nitric oxide synthase antagonism with 200 μM nitro-L-arginine methylester, L-NAME). Exposure of TNFα-stimulated HAEC to arterial shear stress for 5 hr also reduced by 60% (P &#; 0.001) the conversion of neutrophil rolling to firm arrest in a venous flow assay conducted at 1 dyne/cm2. Also, neutrophil rolling lengths at 1 dyne/cm2 were longer when TNFα-stimulated HAEC were presheared for 5 hr at arterial stresses. In experiments with a synthetic promoter that provides luciferase induction to detect cis interactions of glucocorticoid receptor (GR) and NFκB, shear stress caused a marked 40-fold induction of luciferase in TNFα-treated cells, suggesting a role for GR pathways in the anti-inflammatory actions of fluid shear stress. Hemodynamic force exerts anti-inflammatory effects on cytokine activated endothelium by attenuation of cytokine expression and neutrophil firm arrest.Publication Kinetics of random aggregation-fragmentation processes with multiple components(2003-05-09) Laurenzi, Ian J; Diamond, Scott LA computationally efficient algorithm is presented for exact simulation of the stochastic time evolution of spatially homogeneous aggregation-fragmentation processes featuring multiple components or conservation laws. The algorithm can predict the average size and composition distributions of aggregating particles as well as their fluctuations, regardless of the functional form (e.g., composition dependence) of the aggregation or fragmentation kernels. Furthermore, it accurately predicts the complete time evolutions of all moments of the size and composition distributions, even for systems that exhibit gel transitions. We demonstrate the robustness and utility of the algorithm in case studies of linear and branched polymerization processes, the last of which is a two-component process. These simulation results provide the stochastic description of these processes and give new insights into their gel transitions, fluctuations, and long-time behavior when deterministic approaches to aggregation kinetics may not be reliable.Publication Harry L. Goldsmith, Ph.D.(2008-04-01) Diamond, Scott L; Lawrence, Michael B; McIntyre, Larry V; Neelamegham, SriramIn honor of Dr. Harry L. Goldsmith's 75th birthday, we present a collection of articles from his collaborators and colleagues to commemorate Harry's outstanding contributions to the field of Biorheology. On any particular day, bioengineers around the world may find themselves fortunate enough to peer through a microscope to observe molecular or cellular level phenomena manifested before their eyes. Such observations of single molecule mechanics or blood flows or cellular deformation remind us of the power of clever experimental design and rigorous theoretical constructs as well as the complex beauty of dynamical systems in nature. In this spirit, the investigations reported in this issue of the Annals entitled Cellular Biorheology and Biomechanics have followed down many of the research paths pioneered by Dr. Harry Goldsmith.Publication Neutrophil-bead collision assay: Pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime(2005-11-01) Edmondson, Kathryn E; Diamond, Scott L; Denney, William SVisualization 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.Publication High Throughput Screening Using Enzyme Assay Microarrays(2002-10-23) Gosalia, Dhaval N.; Diamond, Scott LWe report a new slide based microarray platform for assaying multiple enzyme activities using fluorogenic substrates. The method enables us to achieve the microfluidic requirements for rapid reaction assembly and compartmentalization. We can thus determine enzymatic activities in individually controlled reaction environments containing cofactors, inhibitors and activators. Fluorogenic substrates in glycerol were arrayed onto glass slides with reaction volumes < 5 nL and feature sizes of < 150 μm. Our method allowed rapid multiple sample deliveries onto the slide (< 3 nL/spot) with no cross contamination between array positions. It enabled us to detect the activation of the fibrinolytic and coagulation proteases namely, thrombin, plasmin, factor Xa, tPa and kallikrein in human plasma. Enzyme-substrate-inhibitor assays using ten caspases were also performed. With over 400 spots/cm2, combinatorial substrate libraries with different proteases can now be rapidly profiled. An assay to detect the dose response of a thrombin inhibitor benzamidine was performed. The inhibitor was arrayed in replicates onto selected positions on the chip. After sequential subnanoliter delivery of the reaction components, the result from the array was analyzed. The expected dose response from benzamidine was seen. A CV of 5.26% was achieved for 232 positions on the array not spiked with the inhibitor. Thus, with potentially several thousand compounds per slide, using rapid sub-nanoliter delivery of components and standard equipment, the true potential of the method is in the field of high throughput screening.Publication Identification and synthesis of a unique thiocarbazate cathepsin L inhibitor(2008-01-01) Myers, Michael C; Diamond, Scott L; Shah, Parag P; Smith, Amos B; Huryn, Donna MLibrary samples containing 2,5-disubstituted oxadiazoles were identified as potent hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) directed at discovering inhibitors of cathepsin L. However, when synthesized in pure form, the putative actives were found to be devoid of biological activity. Analyses by LC–MS of original library samples indicated the presence of a number of impurities, in addition to the oxadiazoles. Synthesis and bioassay of the probable impurities led to the identification of a thiocarbazate that likely originated via ring opening of the oxadiazole. Previously unknown, thiocarbazates (-)-11 and (-)-12 were independently synthesized as single enantiomers and found to inhibit cathepsin 20 L in the low nanomolar range.Publication Identification of Novel Inhibitors of Dietary Lipid Absorption using Zebrafish(2010-08-25) Clifton, Justin D; Lucumi, Edinson; Smith, Amos B; Huryn, Donna M; Myers, Michael C; Diamond, Scott L; Napper, Andrew; Pack, Michael; Hama, Kotaro; Farber, Steven APharmacological inhibition of dietary lipid absorption induces favorable changes in serum lipoprotein levels in patients that are at risk for cardiovascular disease and is considered an adjuvant or alternative treatment with HMG-CoA reductase inhibitors (statins). Here we demonstrate the feasibility of identifying novel inhibitors of intestinal lipid absorption using the zebrafish system. A pilot screen of an unbiased chemical library identified novel compounds that inhibited processing of fluorescent lipid analogues in live zebrafish larvae. Secondary assays identified those compounds suitable for testing in mammals and provided insight into mechanism of action, which for several compounds could be distinguished from ezetimibe, a drug used to inhibit cholesterol absorption in humans that broadly inhibited lipid absorption in zebrafish larvae. These findings support the utility of zebrafish screening assays to identify novel compounds that target complex physiological processes.Publication Neutrophil String Formation: Hydrodynamic Thresholding and Cellular Deformation during Cell Collisions(2004-06-01) Kadash, K. E.; Lawrence, M. B.; Diamond, Scott LNeutrophils 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 >85%. 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 (>3500 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).