Hybridization thermodynamics of NimbleGen Microarrays

Background While microarrays are the predominant method for gene expression profiling, probe signal variation is still an area of active research. Probe signal is sequence dependent and affected by probe-target binding strength and the competing formation of probe-probe dimers and secondary structures in probes and targets. Results We demonstrate the benefits of an improved model for microarray hybridization and assess the relative contributions of the probe-target binding strength and the different competing structures. Remarkably, specific and unspecific hybridization were apparently driven by different energetic contributions: For unspecific hybridization, the melting temperature Tm was the best predictor of signal variation. For specific hybridization, however, the effective interaction energy that fully considered competing structures was twice as powerful a predictor of probe signal variation. We show that this was largely due to the effects of secondary structures in the probe and target molecules. The predictive power of the strength of these intramolecular structures was already comparable to that of the melting temperature or the free energy of the probe-target duplex. Conclusions This analysis illustrates the importance of considering both the effects of probe-target binding strength and the different competing structures. For specific hybridization, the secondary structures of probe and target molecules turn out to be at least as important as the probe-target binding strength for an understanding of the observed microarray signal intensities. Besides their relevance for the design of new arrays, our results demonstrate the value of improving thermodynamic models for the read-out and interpretation of microarray signals

Connector Inversion Probe Technology: A Powerful One-Primer Multiplex DNA Amplification System for Numerous Scientific Applications

We combined components of a previous assay referred to as Molecular Inversion Probe (MIP) with a complete gap filling strategy, creating a versatile powerful one-primer multiplex amplification system. As a proof-of-concept, this novel method, which employs a Connector Inversion Probe (CIPer), was tested as a genetic tool for pathogen diagnosis, typing, and antibiotic resistance screening with two distinct systems: i) a conserved sequence primer system for genotyping Human Papillomavirus (HPV), a cancer-associated viral agent and ii) screening for antibiotic resistance mutations in the bacterial pathogen Neisseria gonorrhoeae. We also discuss future applications and advances of the CIPer technology such as integration with digital amplification and next-generation sequencing methods. Furthermore, we introduce the concept of two-dimension informational barcodes, i.e. “multiplex multiplexing padlocks” (MMPs). For the readers' convenience, we also provide an on-line tutorial with user-interface software application CIP creator 1.0.1, for custom probe generation from virtually any new or established primer-pairs

Tetradecylthioacetic Acid Increases Hepatic Mitochondrial β-Oxidation and Alters Fatty Acid Composition in a Mouse Model of Chronic Inflammation

The administration of tetradecylthioacetic acid (TTA), a hypolipidemic and anti-inflammatory modified bioactive fatty acid, has in several experiments based on high fat diets been shown to improve lipid transport and utilization. It was suggested that increased mitochondrial and peroxisomal fatty acid oxidation in the liver of Wistar rats results in reduced plasma triacylglycerol (TAG) levels. Here we assessed the potential of TTA to prevent tumor necrosis factor (TNF) α-induced lipid modifications in human TNFα (hTNFα) transgenic mice. These mice are characterized by reduced β-oxidation and changed fatty acid composition in the liver. The effect of dietary treatment with TTA on persistent, low-grade hTNFα overexpression in mice showed a beneficial effect through decreasing TAG plasma concentrations and positively affecting saturated and monounsaturated fatty acid proportions in the liver, leading to an increased anti-inflammatory fatty acid index in this group. We also observed an increase of mitochondrial β-oxidation in the livers of TTA treated mice. Concomitantly, there were enhanced plasma levels of carnitine, acetyl carnitine, propionyl carnitine, and octanoyl carnitine, no changed levels in trimethyllysine and palmitoyl carnitine, and a decreased level of the precursor for carnitine, called γ-butyrobetaine. Nevertheless, TTA administration led to increased hepatic TAG levels that warrant further investigations to ascertain that TTA may be a promising candidate for use in the amelioration of inflammatory disorders characterized by changed lipid metabolism due to raised TNFα levels

Genome-Scale Reconstruction of Escherichia coli's Transcriptional and Translational Machinery: A Knowledge Base, Its Mathematical Formulation, and Its Functional Characterization

Metabolic network reconstructions represent valuable scaffolds for ‘-omics’ data integration and are used to computationally interrogate network properties. However, they do not explicitly account for the synthesis of macromolecules (i.e., proteins and RNA). Here, we present the first genome-scale, fine-grained reconstruction of Escherichia coli's transcriptional and translational machinery, which produces 423 functional gene products in a sequence-specific manner and accounts for all necessary chemical transformations. Legacy data from over 500 publications and three databases were reviewed, and many pathways were considered, including stable RNA maturation and modification, protein complex formation, and iron–sulfur cluster biogenesis. This reconstruction represents the most comprehensive knowledge base for these important cellular functions in E. coli and is unique in its scope. Furthermore, it was converted into a mathematical model and used to: (1) quantitatively integrate gene expression data as reaction constraints and (2) compute functional network states, which were compared to reported experimental data. For example, the model predicted accurately the ribosome production, without any parameterization. Also, in silico rRNA operon deletion suggested that a high RNA polymerase density on the remaining rRNA operons is needed to reproduce the reported experimental ribosome numbers. Moreover, functional protein modules were determined, and many were found to contain gene products from multiple subsystems, highlighting the functional interaction of these proteins. This genome-scale reconstruction of E. coli's transcriptional and translational machinery presents a milestone in systems biology because it will enable quantitative integration of ‘-omics’ datasets and thus the study of the mechanistic principles underlying the genotype–phenotype relationship

Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources