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High-throughput Methods (high-throughput + methods)
Selected AbstractsSystematic Hydrothermal Investigation of Metal Phosphonatobenzenesulfonates by High-Throughput MethodsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 24 2010Palanikumar Maniam Abstract A high-throughput (HT) investigation using the rigid bifunctional ligand 4-phosphonobenzenesulfonic acid, H2O3P-C6H4 -SO3H (H3L), generated five new phosphonatobenzenesulfonates with copper(II) or lead(II) ions. A comprehensive HT study comprising the screenings of different metal ions, metal salt types and the synthesis optimization were conducted whereby the influence of pH and molar ratios M2+/H3L were investigated. The HT-study led to five new compounds Pb2[(O3P-C6H4 -SO3)(OH)] (1), Cu1.5[(O3P-C6H4 -SO3)(H2O)] (2), NaCu(O3P-C6H4 -SO3)(H2O)3 (3), Cu2[(O3P-C6H4 -SO3)(OH)(H2O)] (4) and Cu3[(O3P-C6H4 -SO3)2(H2O)2] (5). Metal ion screening showed lead(II) and copper(II) to be suitable metal ions. The utilization of discovery and focused arrays allowed to determine the optimal formation fields of the respective compounds. The crystal structures were determined from single-crystal X-ray diffraction and revealed the presence of various MOx polyhedra that form clusters, chains or layers which are connected through the organic linker. IR spectra, thermogravimetric studies, magnetic susceptibility measurements and elemental analyses were conducted to further characterize the compounds 1, 3, 4 and 5. [source] Structural MRI biomarkers for preclinical and mild Alzheimer's disease,HUMAN BRAIN MAPPING, Issue 10 2009Christine Fennema-Notestine Abstract Noninvasive MRI biomarkers for Alzheimer's disease (AD) may enable earlier clinical diagnosis and the monitoring of therapeutic effectiveness. To assess potential neuroimaging biomarkers, the Alzheimer's Disease Neuroimaging Initiative is following normal controls (NC) and individuals with mild cognitive impairment (MCI) or AD. We applied high-throughput image analyses procedures to these data to demonstrate the feasibility of detecting subtle structural changes in prodromal AD. Raw DICOM scans (139 NC, 175 MCI, and 84 AD) were downloaded for analysis. Volumetric segmentation and cortical surface reconstruction produced continuous cortical surface maps and region-of-interest (ROI) measures. The MCI cohort was subdivided into single- (SMCI) and multiple-domain MCI (MMCI) based on neuropsychological performance. Repeated measures analyses of covariance were used to examine group and hemispheric effects while controlling for age, sex, and, for volumetric measures, intracranial vault. ROI analyses showed group differences for ventricular, temporal, posterior and rostral anterior cingulate, posterior parietal, and frontal regions. SMCI and NC differed within temporal, rostral posterior cingulate, inferior parietal, precuneus, and caudal midfrontal regions. With MMCI and AD, greater differences were evident in these regions and additional frontal and retrosplenial cortices; evidence for non-AD pathology in MMCI also was suggested. Mesial temporal right-dominant asymmetries were evident and did not interact with diagnosis. Our findings demonstrate that high-throughput methods provide numerous measures to detect subtle effects of prodromal AD, suggesting early and later stages of the preclinical state in this cross-sectional sample. These methods will enable a more complete longitudinal characterization and allow us to identify changes that are predictive of conversion to AD. Hum Brain Mapp 2009. © 2009 Wiley-Liss, Inc. [source] The evolution of mathematical immunologyIMMUNOLOGICAL REVIEWS, Issue 1 2007Yoram Louzoun Summary:, The types of mathematical models used in immunology and their scope have changed drastically in the past 10 years. Classical models were based on ordinary differential equations (ODEs), difference equations, and cellular automata. These models focused on the ,simple' dynamics obtained between a small number of reagent types (e.g. one type of receptor and one type of antigen or two T-cell populations). With the advent of high-throughput methods, genomic data, and unlimited computing power, immunological modeling shifted toward the informatics side. Many current applications of mathematical models in immunology are now focused around the concepts of high-throughput measurements and system immunology (immunomics), as well as the bioinformatics analysis of molecular immunology. The types of models have shifted from mainly ODEs of simple systems to the extensive use of Monte Carlo simulations. The transition to a more molecular and more computer-based attitude is similar to the one occurring over all the fields of complex systems analysis. An interesting additional aspect in theoretical immunology is the transition from an extreme focus on the adaptive immune system (that was considered more interesting from a theoretical point of view) to a more balanced focus taking into account the innate immune system also. We here review the origin and evolution of mathematical modeling in immunology and the contribution of such models to many important immunological concepts. [source] Evolutionary combinatorial chemistry, a novel tool for SAR studies on peptide transport across the blood,brain barrier.JOURNAL OF PEPTIDE SCIENCE, Issue 12 2005Part 2. Abstract The use of high-throughput methods in drug discovery allows the generation and testing of a large number of compounds, but at the price of providing redundant information. Evolutionary combinatorial chemistry combines the selection and synthesis of biologically active compounds with artificial intelligence optimization methods, such as genetic algorithms (GA). Drug candidates for the treatment of central nervous system (CNS) disorders must overcome the blood,brain barrier (BBB). This paper reports a new genetic algorithm that searches for the optimal physicochemical properties for peptide transport across the blood,brain barrier. A first generation of peptides has been generated and synthesized. Due to the high content of N -methyl amino acids present in most of these peptides, their syntheses were especially challenging due to over-incorporations, deletions and DKP formations. Distinct fragmentation patterns during peptide cleavage have been identified. The first generation of peptides has been studied by evaluation techniques such as immobilized artificial membrane chromatography (IAMC), a cell-based assay, log Poctanol/water calculations, etc. Finally, a second generation has been proposed. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd. [source] A combinatorial approach to studying protein complex composition by employing size-exclusion chromatography and proteome analysisJOURNAL OF SEPARATION SCIENCE, JSS, Issue 10 2007Shi-Sheng Li Abstract The genome sequences of numerous organisms are available now, but gene sequences alone do not provide sufficient information to accurately deduce protein functions. Protein function is largely dependent on the association of multiple polypeptide chains into large structures with interacting subunits that regulate and support each other. Therefore, the mapping of protein interaction networks in a physiological context is conducive to deciphering protein functions, including those of hypothetical proteins. Although several high-throughput methods to globally identify protein interactions have been reported in recent years, these approaches often have a high rate of nonspecific or artificial interactions detected. For instance, the fraction of false positives of the protein interactions identified by yeast two-hybrid assay has been predicted to be of the order of 50%. We have developed a strategy to globally map Bacillus subtilis protein,protein interactions in a physiological context by fractionating the cell lysates using size-exclusion chromatography (SEC), followed by proteome analysis. Components of both known and unknown protein complexes, multisubunits and multiproteins, have been identified using this strategy. In one case, the partners of the B. subtilis protein complex have been coexpressed in Escherichia coli, and the formation of the overexpressed protein complex has been further confirmed by a pull-down assay. [source] Quantitative analysis of phosphopeptides in search of the disease biomarker from the hepatocellular carcinoma specimenPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 12 2009Hyoung-Joo Lee Abstract Reversible phosphorylation of proteins is the most common PTM in cell-signaling pathways. Despite this, high-throughput methods for the systematic detection, identification, and quantification of phosphorylated peptides have yet to be developed. In this paper, we describe the establishment of an efficient online titaniuim dioxide (TiO2)-based 3-D LC (strong cationic exchange/TiO2/C18)-MS3 -linear ion trap system, which provides fully automatic and highly efficient identification of phosphorylation sites in complex peptide mixtures. Using this system, low-abundance phosphopeptides were isolated from cell lines, plasma, and tissue of healthy and hepatocellular carcinoma (HCC) patients. Furthermore, the phosphorylation sites were identified and the differences in phosphorylation levels between healthy and HCC patient specimens were quantified by labeling the phosphopeptides with isotopic analogs of amino acids (stable isotope labeling with amino acids in cell culture for HepG2 cells) or water (HO for tissues and plasma). Two examples of potential HCC phospho-biomarkers including plectin-1(phopho-Ser-4253) and alpha-HS-glycoprotein (phospho-Ser 138 and 312) were identified by this analysis. Our results suggest that this comprehensive TiO2 -based online-3-D LC-MS3 -linear ion trap system with high-throughput potential will be useful for the global profiling and quantification of the phosphoproteome and the identification of disease biomarkers. [source] The analysis of Neisseria meningitidis proteomes: Reference maps and their applications,PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 16 2007Giulia Bernardini Abstract Neisseria meningitidis is an encapsulated Gram-negative bacterium responsible for significant morbidity and mortality worldwide. The availability of meningococcal genome sequences in combination with the rapid growth of proteomic techniques and other high-throughput methods, provided new approaches to the analysis of bacterial system biology. This review considers the meningococcal reference maps so far published as a starting point aimed to elucidate bacterial physiology and pathogenicity, paying particular attention to proteins with potential vaccine and diagnostic applications. [source] An automatable screen for the rapid identification of proteins amenable to refoldingPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 6 2006Nathan P. Cowieson Dr. Abstract Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15,proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism. [source] A modified tandem affinity purification strategy identifies cofactors of the Drosophila nuclear receptor,dHNF4PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 3 2006Ping Yang Abstract With the completion of numerous genome projects, new high-throughput methods are required to ascribe gene function and interactions. A method proven successful in yeast for protein interaction studies is tandem affinity purification (TAP) of native protein complexes followed by MS. Here, we show that TAP, using Protein,A and CBP tags, is not generally suitable for the purification and identification of proteins from tissues. A head-to-head comparison of tags shows that two others, FLAG and His, provide protein yields from Drosophila tissues that are an order of magnitude higher than Protein,A and CBP. FLAG-His purification worked sufficiently well so that two cofactors of the Drosophila nuclear receptor protein,dHNF4 could be purified from whole animals. These proteins, Hsc70 and Hsp83, are important chaperones and cofactors of other nuclear receptor proteins. However, this is the first time that they have been shown to interact with a non-steroid binding nuclear receptor. We show that the two proteins increase the ability of dHNF4 to bind DNA in,vitro and to function in,vivo. The tags and approaches developed here will help facilitate the routine purification of proteins from complex cells, tissues and whole organisms. [source] Genomic mutation rates: what high-throughput methods can tell usBIOESSAYS, Issue 9 2009Koodali T. Nishant Abstract High-throughput DNA analyses are increasingly being used to detect rare mutations in moderately sized genomes. These methods have yielded genome mutation rates that are markedly higher than those obtained using pre-genomic strategies. Recent work in a variety of organisms has shown that mutation rate is strongly affected by sequence context and genome position. These observations suggest that high-throughput DNA analyses will ultimately allow researchers to identify trans -acting factors and cis sequences that underlie mutation rate variation. Such work should provide insights on how mutation rate variability can impact genome organization and disease progression. [source] How to kill a mocking bug?CELLULAR MICROBIOLOGY, Issue 4 2006Vitor B. Pinheiro Summary All metazoans have evolved means to protect themselves from threats present in the environment: injuries, viruses, fungi, bacteria and other parasites. Insect protection includes innate physical barriers and both cellular and humoral responses. The insect innate immune response, best characterized in Drosophila melanogaster, is a rapid broad response, triggered by pathogen-associated molecular patterns (PAMPs) recognition, which produces a limited range of effectors that does not alter upon continued pathogen exposure and lacks immunological memory. The Drosophila response, particularly its humoral response, has been investigated by both low and high-throughput methods. Three signalling pathways conserved between insects and mammals have been implicated in this response: Toll (equivalent to mammalian TLR), Imd (equivalent to TNF,) and Hop (equivalent to JAK/STAT). This review provides an entry point to the insect immune system literature outlining the main themes in D. melanogaster bacterial pathogen detection and humoral and cellular immune responses. The Drosophila immune response is compared with other insects and the mammalian immune system. [source] | |||||||||||||