Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project

Microarray-based expression profiling experiments typically use either a one-color or a two-color design to measure mRNA abundance. The validity of each approach has been amply demonstrated. Here we provide a simultaneous comparison of results from one- and two-color labeling designs, using two independent RNA samples from the Microarray Quality Control (MAQC) project, tested on each of three different microarray platforms. The data were evaluated in terms of reproducibility, specificity, sensitivity and accuracy to determine if the two approaches provide comparable results. For each of the three microarray platforms tested, the results show good agreement with high correlation coefficients and high concordance of differentially expressed gene lists within each platform. Cumulatively, these comparisons indicate that data quality is essentially equivalent between the one- and two-color approaches and strongly suggest that this variable need not be a primary factor in decisions regarding experimental microarray design.     

Discovery of novel and selective tertiary alcohol containing inhibitors of the norepinephrine transporter

A novel series of tertiary alcohol containing 2-substituted benzyl morpholines have been discovered as potent and selective inhibitors of the norepinephrine transporter. Efficient synthetic routes were developed featuring a highly diastereoselective nucleophilic addition of benzyl Grignard reagents to enantiopure (4-benzylmorpholin-2-yl)phenylmethanone (11) as the key synthetic step. In vitro binding affinity for the norepinephrine, dopamine and serotonin transporters and in vivo examination of a select compound (16) in a pharmacodynamic animal model for norepinephrine reuptake inhibition are presented.     

Structure and Conformation of the Duplex Consensus Acceptor Exon: Intron Junction d [(CpTpApCpApGpGpT) (ApCpCpTpGpTpApG)] deduced from High-Field 1H-NMR of Non-Exchangeable and Imino Protons

Abstract

The complementary consensus acceptor exon:intron junction d(ApCpCpTpGpTpApG) has been synthesized by a modified phosphotriester method. The non self-complementary octamer exists in the random coil form in aqueous buffer at 20°C as evidenced by temperature variable ¹H-NMR and NOE measurements. The non-exchangeable proton assignments were secured using a combination of techniques including two-dimensional COSY, NOESY and ¹H-¹H-INADEQUATE. The octamer was annealed with the primary consensus sequence d(CpTpApCpApGpGpT). Confirmation of complete duplex formation was confirmed by detection and assignment of imino protons in D₂O:H₂O mixtures. Assignment of the nonexchangeable proton signals in the duplex consensus junction was then secured by a combination of two-dimensional COSY correlations, NOESY and NOE experiments. Determination of individual vicinal coupling constants in the component deoxyribose moieties permitted deduction of the population of S conformations in this sequence. It is concluded that the consensus acceptor junction exists in solution in a conformation belonging to the B family, and that the bases are oriented anti. In addition the deoxyribose moieties in the 5′ regions exist predominantly in the S form (2′endo—3′exo) whereas those residues on or adjacent to the junction on the primary strand show more N character (2′exo—3′endo). The contiguous bases A₅-G₆ (adjacent to the junction) and A₁₅-G₁₆ are stacked more closely than the other neighbor bases in this duplex sequence. These subtle structural and conformational differences in the exon:intron junction may serve as recognition signals for these critical sites in the genome.     

Higher Order Structure of Chromatin: Influence of Ionic Strength and Proteolytic Digestion on the Birefringence Properties of Polynucleosomal Fibers

Abstract

Effects of ionic strength and proteolytic digestion on the conformation of chromatin fibers were studied by electric birefringence and relaxation measurements. The results confirm that at low ionic strength chromatin presents structural features reflecting those observed in the presence of cations. Soluble chromatin prepared from rat liver nuclei by brief nuclease digestion exhibits a positive birefringence. As the salt concentration is increased, the transition to a compact solenoidal structure is deduced from changes in electro-optical properties: the positive birefringence gradually decreases and the observed reduction in 40 mM NaCl is nearly 95%; the relaxation time decreases dramatically and the character of the kinetic changes since the decay of birefringence described initially by a spectrum of relaxation times becomes monoexponential.

On digestion with proteases at low ionic strength we observe at first a rapid increase of the positive birefringence concomitant with an increase of the relaxation time. Then the birefringence decreases and becomes negative. Chromatin undergoes two successive transitions: the first transition is explained by a lengthening of nucleosomal chains without modification of the orientation of nucleosomes within the superstructure and the second one by the unwinding of the DNA tails and internucleosomal segments.

When chromatin is digested at 30 mM NaCl we find a single unfolding transition characterized by the decrease of birefringence and a slight increase in the relaxation time. The results imply that the positive birefringence of chromatin does not depend on the presence of whole histone HI and that a salt concentration of 30 mM NaCl is sufficient to modify the initial site or/and the effects of proteolytic attack.     

Surprisal Analysis of Glioblastoma Multiform (GBM) MicroRNA Dynamics Unveils Tumor Specific Phenotype

Gliomablastoma multiform (GBM) is the most fatal form of all brain cancers in humans. Currently there are limited diagnostic tools for GBM detection. Here, we applied surprisal analysis, a theory grounded in thermodynamics, to unveil how biomolecule energetics, specifically a redistribution of free energy amongst microRNAs (miRNAs), results in a system deviating from a non-cancer state to the GBM cancer –specific phenotypic state. Utilizing global miRNA microarray expression data of normal and GBM patients tumors, surprisal analysis characterizes a miRNA system response capable of distinguishing GBM samples from normal tissue biopsy samples. We indicate that the miRNAs contributing to this system behavior is a disease phenotypic state specific to GBM and is therefore a unique GBM-specific thermodynamic signature. MiRNAs implicated in the regulation of stochastic signaling processes crucial in the hallmarks of human cancer, dominate this GBM-cancer phenotypic state. With this theory, we were able to distinguish with high fidelity GBM patients solely by monitoring the dynamics of miRNAs present in patients'' biopsy samples. We anticipate that the GBM-specific thermodynamic signature will provide a critical translational tool in better characterizing cancer types and in the development of future therapeutics for GBM.     

Comparative analysis of 8-oxoG:C, 8-oxoG:A,A:C and C:C DNA repair in extracts from wild type or 8-oxoG DNA glycosylase deficient mammalian and bacterial cells

We have investigated repair of DNA containing 8-oxoguanine and certain mismatches in cell-free extracts from mouse embryonic fibroblasts (MEFs) using a plasmid substrate with a single lesion at a defined position. Repair synthesis was monitored in a small restriction fragment with different size single strands in order to follow the fate of repair reactions in both strands at the same time. An important part of the study was to assess the role of OGG1 in various repair reactions and the experiments were carried out with extracts from mouse embryonic fibroblasts diploid for a mogg1 deletion (Ogg1(-/-)) as well as wild type. In wild type, DNA containing 8-oxoG:C was repaired in the expected fashion predominantly through short-patch repair. Overall repair was reduced to 20% in the Ogg1(-/-) extracts and to 40% if only long-patch repair was considered. The 8-oxoG:A pair was processed similarly in wild type and Ogg1(-/-) extracts and repair synthesis at A as well as at 8-oxoG could be demonstrated, however, to the same extent in Ogg1(-/-) and wild type for both strands. Extracts from Ogg1(-/-) behaved normally in the correction of A:C and C:C mismatches, with a strong bias for correction of A for A:C and no significant strand discrimination for C:C. Similar experiments with extracts from Escherichia coli showed a 50% reduction in the repair of 8-oxoG:C in fpg extracts and an increase to 50% above wild type in mutY. These results show that the mouse OGG1 is the major enzyme for 8-oxoG repair in the MEF cells and does not participate in mismatch repair of A:C or C:C. Furthermore, 8-oxoG opposite A appears to be repaired by a two-step repair pathway with sequential removal of A and 8-oxoG mediated by enzymes different from OGG1.     

Correlation of diffusion and metabolic alterations in different clinical forms of multiple sclerosis

Diffusion tensor imaging (DTI) and MR spectroscopic imaging (MRSI) provide greater sensitivity than conventional MRI to detect diffuse alterations in normal appearing white matter (NAWM) of Multiple Sclerosis (MS) patients with different clinical forms. Therefore, the goal of this study is to combine DTI and MRSI measurements to analyze the relation between diffusion and metabolic markers, T2-weighted lesion load (T2-LL) and the patients clinical status. The sensitivity and specificity of both methods were then compared in terms of MS clinical forms differentiation. MR examination was performed on 71 MS patients (27 relapsing remitting (RR), 26 secondary progressive (SP) and 18 primary progressive (PP)) and 24 control subjects. DTI and MRSI measurements were obtained from two identical regions of interest selected in left and right centrum semioval (CSO) WM. DTI metrics and metabolic contents were significantly altered in MS patients with the exception of N-acetyl-aspartate (NAA) and NAA/Choline (Cho) ratio in RR patients. Significant correlations were observed between diffusion and metabolic measures to various degrees in every MS patients group. Most DTI metrics were significantly correlated with the T2-LL while only NAA/Cr ratio was correlated in RR patients. A comparison analysis of MR methods efficiency demonstrated a better sensitivity/specificity of DTI over MRSI. Nevertheless, NAA/Cr ratio could distinguish all MS and SP patients groups from controls, while NAA/Cho ratio differentiated PP patients from controls. This study demonstrated that diffusivity changes related to microstructural alterations were correlated with metabolic changes and provided a better sensitivity to detect early changes, particularly in RR patients who are more subject to inflammatory processes. In contrast, the better specificity of metabolic ratios to detect axonal damage and demyelination may provide a better index for identification of PP patients.