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High-throughput Manner (high-throughput + manner)
Selected AbstractsCell microarray platform for anticancer drug development,DRUG DEVELOPMENT RESEARCH, Issue 5 2007Min-Jung Lee Abstract Pharmacodynamic assessment of whether a drug has interacted with and modified its target is an essential component of molecularly targeted clinical trials. Although many trials are written with the intent to assess tumor biopsies, if available, thus far the great majority of early drug trials have used peripheral blood mononuclear cells (PBMC) as a tumor surrogate. Typically, PBMC are studied by low-throughput techniques such as Western blot. We present the use of a cell-based tissue microarray for assessment of anticancer drug activity in vivo. We demonstrate the utility of this technique for analysis of protein hyperacetylation in response to treatment with the histone deacetylase inhibitor, SNDX-275 in PBMC treated in vitro and in PBMC and bone marrow aspirates from patients in Phase I clinical trials with SNDX-275. We demonstrate that the cell microarray can be used to measure drug response in a high-throughput manner, allowing analysis of an entire trial on one or two glass slides. The cell microarray technique brings the advantages of the tissue microarray platform to the pharmacodynamic assessment of single cells, such as those isolated from bone marrow aspirates, fine needle aspirates, or malignant effusions, and to analysis of PBMC, the most commonly studied surrogate in oncology trials. Drug Dev Res 68:226,234, 2007. Published 2007 Wiley-Liss, Inc. [source] "Reverse degradomics", monitoring of proteolytic trimming by multi-CE and confocal detection of fluorescent substrates and reaction productsELECTROPHORESIS, Issue 13 2009Helene Piccard Abstract A platform for profiling of multiple proteolytic activities acting on one specific substrate, based on the use of a 96-channel capillary DNA sequencer with CE-LIF of labeled substrate peptides and reaction products is introduced. The approach consists of synthesis of a substrate peptide of interest, fluorescent labeling of the substrate, either aminoterminally by chemical coupling, or carboxyterminally by transglutaminase reaction, proteolysis by a biological mixture of proteases in the absence or presence of protease inhibitors, multi-channel analysis of substrate and reaction products, and data collection and processing. Intact substrate and reaction products, even when varying by only one amino acid, can be relatively semi-quantified in a high-throughput manner, yielding information on proteases acting in complex biological mixtures and without prepurification. Monitoring, classification and inhibition of multiple proteolytic activities are demonstrated on a model substrate, the aminoterminus of the mouse granulocyte chemotactic protein-2. In view of extensive processing of chemokines into various natural forms with different specific biological activities, and of the fragmentary knowledge of processing proteases, examples of processing by neutrophil degranulate, tumor cell culture fluids and plasma are provided. An example of selection and comparison of inhibitory mAbs illustrates that the platform is suitable for inhibitor screening. Whereas classical degradomics technologies analyze the substrate repertoire of one specific protease, here the complementary concept, namely the study of all proteases acting, in a biological context, on one specific substrate, is developed and tuned to identify key proteases and protease inhibitors for the processing of any biological substrate of interest. [source] Rapid Generation of Biologically Relevant Hydrogels Containing Long-Range Chemical GradientsADVANCED FUNCTIONAL MATERIALS, Issue 1 2010Jiankang He Abstract Many biological processes are regulated by gradients of bioactive chemicals. Thus, the generation of materials with embedded chemical gradients may be beneficial for understanding biological phenomena and generating tissue-mimetic constructs. Here a simple and versatile method to rapidly generate materials containing centimeter-long gradients of chemical properties in a microfluidic channel is described. The formation of a chemical gradient is initiated by a passive-pump-induced forward flow and further developed during an evaporation-induced backward flow. The gradient is spatially controlled by the backward flow time and the hydrogel material containing the gradient is synthesized via photopolymerization. Gradients of a cell-adhesion ligand, Arg-Gly-Asp-Ser (RGDS), are incorporated in poly(ethylene glycol)-diacrylate (PEG-DA) hydrogels to test the response of endothelial cells. The cells attach and spread along the hydrogel material in a manner consistent with the RGDS-gradient profile. A hydrogel containing a PEG-DA concentration gradient and constant RGDS concentration is also shown. The morphology of cells cultured on such hydrogel changes from round in the lower PEG-DA concentration regions to well-spread in the higher PEG-DA concentration regions. This approach is expected to be a valuable tool to investigate the cell,material interactions in a simple and high-throughput manner and to design graded biomimetic materials for tissue engineering applications. [source] A High-Throughput Assay of Cell-Surface Interactions using Topographical and Chemical Gradients,ADVANCED MATERIALS, Issue 3 2009Jing Yang Orthogonal wettability and topographical gradients in a combinatorial sample format are fabricated using plasma-polymer-coated microgrooved surfaces. Preferred cell proliferation is found on specific combinations of topography and chemistry. This proof-of-concept study demonstrates the potential applications of this sample format for investigating the relationship between multiple surface properties on cellular response in a high-throughput manner. [source] Parallel implementation of AutoDockJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 3 2007Prashant Khodade Computational docking of ligands to protein structures is a key step in structure-based drug design. Currently, the time required for each docking run is high and thus limits the use of docking in a high-throughput manner, warranting parallelization of docking algorithms. AutoDock, a widely used tool, has been chosen for parallelization. Near-linear increases in speed were observed with 96 processors, reducing the time required for docking ligands to HIV-protease from 81,min, as an example, on a single IBM Power-5 processor (1.65,GHz), to about 1,min on an IBM cluster, with 96 such processors. This implementation would make it feasible to perform virtual ligand screening using AutoDock. [source] A novel on-line solid-phase extraction approach integrated with a monolithic column and tandem mass spectrometry for direct plasma analysis of multiple drugs and metabolitesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 22 2005Xu Zang An on-line solid-phase extraction liquid chromatography/tandem mass spectrometry (SPE LC/MS/MS) assay using a newly developed SPE column and a monolithic column was developed and validated for direct analysis of plasma samples containing multiple analytes. This assay was developed in an effort to increase bioanalysis throughput and reduce the complexity of on-line SPE LC/MS/MS systems. A simple column-switching configuration that requires only one six-port valve and one HPLC pumping system was employed for on-line plasma sample preparation and subsequent gradient chromatographic separation. The resulting analytical method couples the desired sensitivity with ease of use. The method was found to perform satisfactorily for direct plasma analysis with respect to assay linearity, specificity, sensitivity, precision, accuracy, carryover, and short-term stability of an eight-analyte mixture in plasma. A gradient LC condition was applied to separate the eight analytes that cannot be distinctly differentiated by MS/MS. With a run time for every injection of 2.8,min, a minimum of 300 direct plasma injections were made on one on-line SPE column without noticeable changes in system performance. Due to the ruggedness and simplicity of this system, generic methods can be easily developed and applied to analyze a wide variety of compounds in a high-throughput manner without laborious off-line sample preparation. Copyright © 2005 John Wiley & Sons, Ltd. [source] Binding modules alter the activity of chimeric cellulases: Effects of biomass pretreatment and enzyme source,BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Tae-Wan Kim Abstract Improving the catalytic activity of cellulases requires screening variants against solid substrates. Expressing cellulases in microbial hosts is time-consuming, can be cellulase specific, and often leads to inactive forms and/or low yields. These limitations have been obstacles for improving cellulases in a high-throughput manner. We have developed a cell-free expression system and used it to express 54 chimeric bacterial and archaeal endoglucanases (EGs), with and without cellulose binding modules (CBMs) at either the N- or C-terminus, in active enzyme yields of 100,350,µg/mL. The platform was employed to systematically study the role of CBMs in cellulose hydrolysis toward a variety of natural and pretreated solid substrates, including ionic-liquid pretreated Miscanthus and AFEX-pretreated corn stover. Adding a CBM generally increased activity against crystalline Avicel, whereas for pretreated substrates the effect of CBM addition depended on the source of cellulase. The cell-free expression platform can thus provide insights into cellulase structure-function relationships for any substrate, and constitutes a powerful discovery tool for evaluating or engineering cellulolytic enzymes for biofuels production. Biotechnol. Bioeng. 2010;107:601,611. © 2010 Wiley Periodicals, Inc. [source] | ||||||||||||||||