Assessing the efficiency and significance of Methylated DNA Immunoprecipitation (MeDIP) assays in using in vitro methylated genomic DNA

<p>Abstract</p> <p>Background</p> <p>DNA methylation contributes to the regulation of gene expression during development and cellular differentiation. The recently developed Methylated DNA ImmunoPrecipitation (MeDIP) assay allows a comprehensive analysis of this epigenetic mark at the genomic level in normal and disease-derived cells. However, estimating the efficiency of the MeDIP technique is difficult without previous knowledge of the methylation status of a given cell population. Attempts to circumvent this problem have involved the use of <it>in vitro </it>methylated DNA in parallel to the investigated samples. Taking advantage of this stratagem, we sought to improve the sensitivity of the approach and to assess potential biases resulting from DNA amplification and hybridization procedures using MeDIP samples.</p> <p>Findings</p> <p>We performed MeDIP assays using <it>in vitro </it>methylated DNA, with or without previous DNA amplification, and hybridization to a human promoter array. We observed that CpG content at gene promoters indeed correlates strongly with the MeDIP signal obtained using <it>in vitro </it>methylated DNA, even when lowering significantly the amount of starting material. In analyzing MeDIP products that were subjected to whole genome amplification (WGA), we also revealed a strong bias against CpG-rich promoters during this amplification procedure, which may potentially affect the significance of the resulting data.</p> <p>Conclusion</p> <p>We illustrate the use of <it>in vitro </it>methylated DNA to assess the efficiency and accuracy of MeDIP procedures. We report that efficient and reproducible genome-wide data can be obtained via MeDIP experiments using relatively low amount of starting genomic DNA; and emphasize for the precaution that must be taken in data analysis when an additional DNA amplification step is required.</p

Genome-Wide Divergence of DNA Methylation Marks in Cerebral and Cerebellar Cortices

Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function.Using methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum.These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions

Hystricognathy vs Sciurognathy in the Rodent Jaw: A New Morphometric Assessment of Hystricognathy Applied to the Living Fossil Laonastes (Diatomyidae)

While exceptional for an intense diversification of lineages, the evolutionary history of the order Rodentia comprises only a limited number of morphological morphotypes for the mandible. This situation could partly explain the intense debates about the taxonomic position of the latest described member of this clade, the Laotian rock rat Laonastes aenigmamus (Diatomyidae). This discovery has re-launched the debate on the definition of the Hystricognathi suborder identified using the angle of the jaw relative to the plane of the incisors. Our study aims to end this ambiguity. For clarity, it became necessary to revisit the entire morphological diversity of the mandible in extant and extinct rodents. However, current and past rodent diversity brings out the limitations of the qualitative descriptive approach and highlights the need for a quantitative approach. Here, we present the first descriptive comparison of the masticatory apparatus within the Ctenohystrica clade, in combining classic comparative anatomy with morphometrical methods. First, we quantified the shape of the mandible in rodents using 3D landmarks. Then, the analysis of osteological features was compared to myological features in order to understand the biomechanical origin of this morphological diversity. Among the morphological variation observed, the mandible of Laonastes aenigmamus displays an intermediate association of features that could be considered neither as sciurognathous nor as hystricognathous

Canalization of Gene Expression and Domain Shifts in the Drosophila Blastoderm by Dynamical Attractors

The variation in the expression patterns of the gap genes in the blastoderm of the fruit fly Drosophila melanogaster reduces over time as a result of cross regulation between these genes, a fact that we have demonstrated in an accompanying article in PLoS Biology (see Manu et al., doi:10.1371/journal.pbio.1000049). This biologically essential process is an example of the phenomenon known as canalization. It has been suggested that the developmental trajectory of a wild-type organism is inherently stable, and that canalization is a manifestation of this property. Although the role of gap genes in the canalization process was established by correctly predicting the response of the system to particular perturbations, the stability of the developmental trajectory remains to be investigated. For many years, it has been speculated that stability against perturbations during development can be described by dynamical systems having attracting sets that drive reductions of volume in phase space. In this paper, we show that both the reduction in variability of gap gene expression as well as shifts in the position of posterior gap gene domains are the result of the actions of attractors in the gap gene dynamical system. Two biologically distinct dynamical regions exist in the early embryo, separated by a bifurcation at 53% egg length. In the anterior region, reduction in variation occurs because of stability induced by point attractors, while in the posterior, the stability of the developmental trajectory arises from a one-dimensional attracting manifold. This manifold also controls a previously characterized anterior shift of posterior region gap domains. Our analysis shows that the complex phenomena of canalization and pattern formation in the Drosophila blastoderm can be understood in terms of the qualitative features of the dynamical system. The result confirms the idea that attractors are important for developmental stability and shows a richer variety of dynamical attractors in developmental systems than has been previously recognized

A Criterion for Brittle Failure of Rocks Using the Theory of Critical Distances

This paper presents a new analytical criterion for brittle failure of rocks and heavily overconsolidated soils. Griffith’s model of a randomly oriented defect under a biaxial stress state is used to keep the criterion simple. The Griffith’s criterion is improved because the maximum tensile strength is not evaluated at the boundary of the defect but at a certain distance from the boundary, known as the critical distance. This fracture criterion is known as the Point Method, and is part of the Theory of Critical Distances, which is utilized in fracture mechanics. The proposed failure criterion has two parameters: the inherent tensile strength, ó0, and the ratio of the half-length of the initial crack/flaw to the critical distance, a/L. These parameters are difficult to measure but they may be correlated with the uniaxial compressive and tensile strengths, óc and ót. The proposed criterion is able to reproduce the common range of strength ratios for rocks and heavily overconsolidated soils (óc/ót=3-50) and the influence of several microstructural rock properties, such as texture and porosity. Good agreement with laboratory tests reported in the literature is found for tensile and low confining stresses.The work presented was initiated during a research project on “Structural integrity assessments of notch-type defects", for the Spanish Ministry of Science and Innovation (Ref.: MAT2010-15721)

Combinatorial discovery of polymers resistant to bacterial attachment

Bacterial attachment and subsequent biofilm formation are key challenges to the long term performance of many medical devices. Here, a high throughput approach coupled with the analysis of surface structure-property relationships using a chemometics approach has been developed to simultaneously investigate the interaction of bacteria with hundreds of polymeric materials on a microarray format. Using this system, a new group of materials comprising ester and hydrophobic moieties are identified that dramatically reduce the attachment of clinically relevant, pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus and uropathogenic Escherichia coli). Hit materials coated on silicone catheters resulted in up to a 30 fold reduction in coverage compared to a commercial silver embedded catheter, which has been proven to half the incidence of clinically acquired infection. These polymers represent a new class of materials resistant to bacterial attachment that could not have been predicted from the current understanding of bacteria-surface interactions

Identification of a gene signature for discriminating metastatic from primary melanoma using a molecular interaction network approach

Understanding the biological factors that are characteristic of metastasis in melanoma remains a key approach to improving treatment. In this study, we seek to identify a gene signature of metastatic melanoma. We configured a new network-based computational pipeline, combined with a machine learning method, to mine publicly available transcriptomic data from melanoma patient samples. Our method is unbiased and scans a genome-wide protein-protein interaction network using a novel formulation for network scoring. Using this, we identify the most influential, differentially expressed nodes in metastatic as compared to primary melanoma. We evaluated the shortlisted genes by a machine learning method to rank them by their discriminatory capacities. From this, we identified a panel of 6 genes, ALDH1A1, HSP90AB1, KIT, KRT16, SPRR3 and TMEM45B whose expression values discriminated metastatic from primary melanoma (87% classification accuracy). In an independent transcriptomic data set derived from 703 primary melanomas, we showed that all six genes were significant in predicting melanoma specific survival (MSS) in a univariate analysis, which was also consistent with AJCC staging. Further, 3 of these genes, HSP90AB1, SPRR3 and KRT16 remained significant predictors of MSS in a joint analysis (HR = 2.3, P = 0.03) although, HSP90AB1 (HR = 1.9, P = 2 × 10−4) alone remained predictive after adjusting for clinical predictors