Putative null distributions corresponding to tests of differential expression in the Golden Spike dataset are intensity dependent

<p>Abstract</p> <p>Background</p> <p>We provide a re-analysis of the Golden Spike dataset, a first generation "spike-in" control microarray dataset. The original analysis of the Golden Spike dataset was presented in a manuscript by Choe et al. and raised questions concerning the performance of several statistical methods for the control of the false discovery rate (across a set of tests for differential expression). These original findings are now in question as it has been reported that the p-values associated with the tests of differential expression for null probesets (i.e., probesets designed to be fold change 1 across the two arms of the experiment) are not uniformly distributed. Two recent publications have speculated as to the reasons the null distributions are non-uniform. A publication by Dabney and Storey concludes that the non-uniform distributions of null p-values are the direct consequence of an experimental design which requires technical replicates to approximate biological replicates. Irizarry et al. identify four characteristics of the feature level data (three related to experimental design and one artifact). Irizarry et al. argue that the four observed characteristics imply that the assumptions common to most pre-processing algorithms are not satisfied and hence the expression measure methodologies considered by Choe et al. are likely to be flawed.</p> <p>Results</p> <p>We replicate and extend the analyses of Dabney and Storey and present our results in the context of a two stage analysis. We provide evidence that the Stage I pre-processing algorithms considered in Dabney and Storey fail to provide expression values that are adequately centered or scaled. Furthermore, we demonstrate that the distributions of the p-values, test statistics, and probabilities associated with the relative locations and variabilities of the Stage II expression values vary with signal intensity. We provide diagnostic plots and a simple logistic regression based test statistic to detect these intensity related defects in the processed data.</p> <p>Conclusion</p> <p>We agree with Dabney and Storey that the null p-values considered in Choe et al. are indeed non-uniform. We also agree with the conclusion that, given current pre-processing technologies, the Golden Spike dataset should not serve as a reference dataset to evaluate false discovery rate controlling methodologies. However, we disagree with the assessment that the non-uniform p-values are merely the byproduct of testing for differential expression under the incorrect assumption that chip data are approximate to biological replicates. Whereas Dabney and Storey attribute the non-uniform p-values to violations of the Stage II model assumptions, we provide evidence that the non-uniformity can be attributed to the failure of the Stage I analyses to correct for systematic biases in the raw data matrix. Although we do not speculate as to the root cause of these systematic biases, the observations made in Irizarry et al. appear to be consistent with our findings. Whereas Irizarry et al. describe the effect of the experimental design on the feature level data, we consider the effect on the underlying multivariate distribution of putative null p-values. We demonstrate that the putative null distributions corresponding to the pre-processing algorithms considered in Choe et al. are all intensity dependent. This dependence serves to invalidate statistical inference based upon standard two sample test statistics. We identify a flaw in the characterization of the appropriate "null" probesets described in Choe et al. and we provide a corrected analysis which reduces (but does not eliminate) the intensity dependent effects.</p

A New Normalizing Algorithm for BAC CGH Arrays with Quality Control Metrics

The main focus in pin-tip (or print-tip) microarray analysis is determining which probes, genes, or oligonucleotides are differentially expressed. Specifically in array comparative genomic hybridization (aCGH) experiments, researchers search for chromosomal imbalances in the genome. To model this data, scientists apply statistical methods to the structure of the experiment and assume that the data consist of the signal plus random noise. In this paper we propose “SmoothArray”, a new method to preprocess comparative genomic hybridization (CGH) bacterial artificial chromosome (BAC) arrays and we show the effects on a cancer dataset. As part of our R software package “aCGHplus,” this freely available algorithm removes the variation due to the intensity effects, pin/print-tip, the spatial location on the microarray chip, and the relative location from the well plate. removal of this variation improves the downstream analysis and subsequent inferences made on the data. Further, we present measures to evaluate the quality of the dataset according to the arrayer pins, 384-well plates, plate rows, and plate columns. We compare our method against competing methods using several metrics to measure the biological signal. With this novel normalization algorithm and quality control measures, the user can improve their inferences on datasets and pinpoint problems that may arise in their BAC aCGH technology

Introduction to Homogenous Catalysis with Ruthenium-Catalyzed Oxidation of Alcohols: An Experiment for Undergraduate Advanced Inorganic Chemistry Students

A three-week laboratory experiment, which introduces students in an advanced inorganic chemistry course to air-sensitive chemistry and catalysis, is described. During the first week, the students synthesize RuCl2(PPh3)3. During the second and third weeks, the students characterize the formed coordination compound and use it as a precatalyst for the oxidation of 1-phenylethanol to acetophenone. The synthesized RuCl2(PPh3)3 is characterized using 1H and 31P NMR spectroscopy, IR spectroscopy, and magnetic susceptibility measurements. The students run catalytic and control reactions and determine the percent yield of the product using 1H NMR. The synthesis and catalytic conditions are modified from previously published research articles. The RuCl2(PPh3)3 complex is air sensitive and is prepared under a nitrogen gas atmosphere and worked up in an inert atmosphere glovebox. The catalytic and control reactions are set up in the inert atmosphere glovebox and carried out at reflux outside of the glovebox under a nitrogen gas atmosphere. In this laboratory, the students learn how to set up and run a reaction under a nitrogen atmosphere, how to work in a glovebox, and how to set up and characterize catalytic and control reactions

Synthesis, Characterization, Density Functional Theory Calculations, and Activity of a Thione-Containing NNN-Zinc Pincer Complex Based on a Bis-triazole Precursor

A novel ambidentate tridentate pincer ligand based on a bis-triazole precursor, was prepared, characterized, and metallated with ZnCl2 to give a new tridentate NNN-bound pincer zinc(II) pincer complex: dichloro(η3-N,N,N)-[2,6-bis(3-[N-butyl]triazol-5-thione-1-yl)]pyridinezinc(II), [(NNN)ZnCl2]. This compound has pseudo-trigonal bipyramidal geometry at the zinc(II) center and exhibits metal–ligand binding that contrasts with our previously reported SNS-bound systems despite the availability of these same donor atoms in the current ligand set. The zinc complex was characterized with single crystal X-ray diffraction, 1H, 13C, and HSQC NMR spectroscopies, and electrospray mass spectrometry. The ligand precursors were characterized with 1H, 13C, and HSQC NMR spectroscopies, and cyclic voltammetry, and were found to be redox active. Density functional calculations, which investigate and support the nature of the NNN binding suggest that the experimentally observed oxidation and reduction waves are not the result of a simple one-electron process. The zinc complex was screened for the reduction of electron-poor aldehydes in the presence of a hydrogen donor, 1-benzyl-1,4-dihydronicotinamide (BNAH), and it was determined that they enhance the reduction of 4-nitrobenzaldehyde. Quantitative stoichiometric conversion was seen for the reduction of pyridine-2-carboxaldehyde

Syntheses, characterization, density functional theory calculations, and activity of tridentate SNS zinc pincer complexes based on bis-imidazole or bis-triazole precursors

A series of tridentate pincer ligands, each possessing two sulfur- and one nitrogen-donor functionalities (SNS), based on bis-imidazole or bis-triazole salts were metallated with ZnCl2 to give new tridentate SNS pincer zinc(II) complexes [(SNS)ZnCl]+. The zinc complexes serve as models for the zinc active site in liver alcohol dehydrogenase (LADH) and were characterized with single crystal X-ray diffraction, 1H, 13C, and HSQC NMR spectroscopies, electrospray mass spectrometry, and elemental analysis. The zinc complexes feature SNS donor atoms and pseudotetrahedral geometry about the zinc center, as is seen for liver alcohol dehydrogenase. The bond lengths and bond angles of the zinc complexes correlate well to those in horse LADH. The SNS ligand precursors were characterized with 1H, 13C, and HSQC NMR spectroscopies, elemental analysis, and cyclic voltammetry, and were found to be redox active. Gaussian calculations were performed and agree with the experimentally observed oxidation potentials for the pincer ligand precursors. The zinc complexes were screened for the reduction of electron-poor aldehydes in the presence of a hydrogen donor, 1-benzyl-1,4-dihydronicotinamide (BNAH), and it was determined that they enhance the reduction of electron-poor aldehydes. The SNS zinc pincer complexes with bis-triazole ligand precursors exhibit higher activity for the reduction of 4-nitrobenzaldehyde than do SNS zinc pincer complexes with bis-imidazole ligand precursors. Quantitative stoichiometric conversion was seen for the reduction of pyridine-2-carboxaldehyde via SNS zinc pincer complexes with either bis-imidazole or bis-triazole ligand precursors

Microelectrode-based probing of charge propagation and redox transitions in concentrated polyoxometallate electrolyte of potential utility for redox flow battery

Concentrated solutions of Keggin-type silicotungstic acid, as well as the system's single crystals (H4SiW12O40*31H2O) and their colloidal suspensions have been tested using the microelectrode methodology to determine mass-transport, electron self-exchange and apparent (effective) diffusion-type coefficients for charge propagation and homogeneous (electron self-exchange) rates of electron transfers. Silicotungstic acid facilitates proton conductivity, and undergoes fast, reversible, multi-electron transfers leading to the formation of highly conducting, mixed-valence (tungsten(VI,V) heteropoly blue) compounds. To develop useful electroanalytical diagnostic criteria, electroanalytical approaches utilizing microdisk electrodes have been adapted to characterize redox transitions of the system and to determine kinetic parameters. Combination of microelectrode-based experiments performed in two distinct diffusional regimes: radial (long-term experiment; e.g., slow scan rate voltammetry or long-pulse chronoamperometry) and linear (short-term experiment; e.g., fast scan rate voltammetry or short-pulse chronocoulometry) permits absolute determination of such parameters as effective concentration of redox centers (C0) and apparent transport (diffusion) coefficient (Dapp). The knowledge of these parameters, in particular of [Dapp1/2 C0] seems to be of importance to the evaluation of utility of redox electrolytes for charge storage. For the colloidal suspension of silicotungstic acid (H4SiW12O40) crystals in the saturated solution, the following values have been obtained: Dapp = 1.8*10-6 cm2 s−1 and C0 = 1.1 mol dm−3, as well as the [Dapp1/2 C0] diagnostic parameter has reached the value as high as 6*10-3 mol/dm−3 cm s−1/2, provided that four electrons are involved in the H4SiW12O40 redox transitions. In this respect, the fact that crystals (dispersed solids) are characterized by high electron self-exchange rate (kex = 1.1*108 dm3 mol−1 s−1) and low activation energy (EA = 18.7 kJ mol−1) facilitating electron transfers between immobilized WVI and WV redox sites is also advantageous

Copper (I) SNS Pincer Complexes: Impact of Ligand Design and Solvent Coordination on Conformer Interconversion from Spectroscopic and Computational Studies

The syntheses and detailed characterizations (X-ray crystallography, NMR spectroscopy, cyclic voltammetry, infrared spectroscopy, electrospray mass spectrometry, and elemental analyses) of two new Cu(I) pincer complexes are reported. The pincer ligand coordinates through one nitrogen and two sulfur donor atoms and is based on bis-imidazole or bis-triazole precursors. These tridentate SNS ligands incorporate pyridine and thione-substituted imidazole or triazole functionalities with connecting methylene units that provide flexibility to the ligand backbone and enable high bite-angle binding. Variable temperature 1H NMR analysis of these complexes and of a similar zinc(II) SNS system shows that all are fluxional in solution and permits the determination of ΔGexp‡ and ΔSexp‡. DFT calculations are used to model the fluxionality of these complexes and indicate that a coordinating solvent molecule can promote hemilability of the SNS ligand by lowering the energy barrier involved in the partial rotation of the methylene units

Syntheses, Characterization, Density Functional Theory Calculations, and Activity of Tridentate SNS Zinc Pincer Complexes Based on Bis-Imidazole or Bis-Triazole Precursors

A series of tridentate pincer ligands, each possessing two sulfur- and one nitrogen-donor functionalities (SNS), based on bis-imidazole or bis-triazole salts were metallated with ZnCl2 to give new tridentate SNS pincer zinc(II) complexes [(SNS)ZnCl]+. The zinc complexes serve as models for the zinc active site in liver alcohol dehydrogenase (LADH) and were characterized with single crystal X-ray diffraction, 1H, 13C, and HSQC NMR spectroscopies, electrospray mass spectrometry, and elemental analysis. The zinc complexes feature SNS donor atoms and pseudotetrahedral geometry about the zinc center, as is seen for liver alcohol dehydrogenase. The bond lengths and bond angles of the zinc complexes correlate well to those in horse LADH. The SNS ligand precursors were characterized with 1H, 13C, and HSQC NMR spectroscopies, elemental analysis, and cyclic voltammetry, and were found to be redox active. Gaussian calculations were performed and agree with the experimentally observed oxidation potentials for the pincer ligand precursors. The zinc complexes were screened for the reduction of electron-poor aldehydes in the presence of a hydrogen donor, 1-benzyl-1,4-dihydronicotinamide (BNAH), and it was determined that they enhance the reduction of electron-poor aldehydes. The SNS zinc pincer complexes with bis-triazole ligand precursors exhibit higher activity for the reduction of 4-nitrobenzaldehyde than do SNS zinc pincer complexes with bis-imidazole ligand precursors. Quantitative stoichiometric conversion was seen for the reduction of pyridine-2-carboxaldehyde via SNS zinc pincer complexes with either bis-imidazole or bis-triazole ligand precursors

Syntheses, characterization, density functional theory calculations, and activity of tridentate SNS zinc pincer complexes

A series of tridentate SNS ligand precursors were metallated with ZnCl2 to give new tridentate SNS pincer zinc complexes. The zinc complexes serve as models for the zinc active site in liver alcohol dehydrogenase (LADH) and were characterized with single crystal X-ray diffraction, 1H, 13C, and HSQC NMR spectroscopies and electrospray mass spectrometry. The bond lengths and bond angles of the zinc complexes correlate well to those in horse LADH. The zinc complexes feature SNS donor atoms and pseudotetrahedral geometry about the zinc center, as is seen for liver alcohol dehydrogenase. The SNS ligand precursors were characterized with 1H, 13C, and HSQC NMR spectroscopies and cyclic voltammetry, and were found to be redox active. Gaussian calculations were performed and agree quite well with the experimentally observed oxidation potential for the pincer ligand. The zinc complexes were screened for the reduction of electron poor aldehydes in the presence of a hydrogen donor, 1-benzyl-1,4-dihydronicotinamide (BNAH). The zinc complexes enhance the reduction of electron poor aldehydes. Density functional theory calculations were performed to better understand why the geometry about the zinc center is pseudo-tetrahedral rather than pseudo-square planar, which is seen for most pincer complexes. For the SNS tridentate pincer complexes, the data indicate that the pseudo-tetrahedral geometry was 43.8 kcal/mol more stable than the pseudo-square planar geometry. Density functional theory calculations were also performed on zinc complexes with monodentate ligands and the data indicate that the pseudo-tetrahedral geometry was 30.6 kcal/mol more stable than pseudo-square planar geometry. Overall, the relative stabilities of the pseudo-tetrahedral and pseudo-square planar systems are the same for this coordination environment whether the ligand set is a single tridentate SNS system or is broken into three separate units. The preference of a d10 Zn center to attain a tetrahedral local environment trumps any stabilization gained by removal of constraints within the ligand set