High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison

We report the development of a heteroduplex-based mutation detection method using multicapillary automated sequencers, known as conformation-sensitive capillary electrophoresis (CSCE). Our optimized CSCE protocol detected 93 of 95 known base substitution sequence variants. Since the optimization of the method, we have analyzed 215 Mb of DNA and identified 3397 unique variants. An analysis of this data set indicates that the sensitivity of CSCE is above 95% in the central 56% of the average PCR product. To fully exploit the mutation detection capacity of this method, we have developed software, canplot, which automatically compares normal and test results to prioritize samples that are most likely to contain variants. Using multiple fluorescent dyes, CSCE has the capacity to screen over 2.2 Mb on one ABI3730 each day. Therefore this technique is suitable for projects where a rapid and sensitive DNA mutation detection system is required.     

Relationships among habitat quality and measured condition variables in Gulf of Mexico mangroves

Ecosystem condition assessments were conducted for 12 mangrove sites in the northern Gulf of Mexico. Nine sites were selected randomly; three were selected a priori based on best professional judgment to represent a poor, intermediate and good environmental condition. During single visits a visual assessment generated numerical scores for each site relative to the reference condition range. Relationships were examined among variables to evaluate relevance of visual observations and empirical data of environmental condition. Sites in poorest visual condition possessed low concentrations of soil contaminants as hydrological attributes that restricted contaminant inflow lowered visual scores. Bacterial abundance related to the greatest number of variables, and was the only indicator that correlated with the visually derived score (0.60, p < 0.05). Soil enzymatic activity ratios within classes provided estimates of microbial nutrient status. Low peptidases/phosphatase and glycosidases/phosphatase ratios supported the conclusion reached from the molar ratios of nutrients, that mangrove soils were phosphorous limited. A positive correlation (0.71, p < 0.05) observed between C:N ratios in leaf tissue and soil from all mangrove sites indicated that relative molar ratios of major nutrients in leaf tissue were reflective of limitations in the soil. Principal components analysis guided a reduction in variables retained for analysis, and provided an ordination of the sites which grouped into three clusters and two outliers. Grouping was primarily influenced by soil C:N. In future studies measuring major nutrients in soil, C:N:P, could possibly function as a single, cost effective indicator to validate rapid visual condition assessments of mangroves.     

MIP-1alpha induces activation of phosphatidylinositol-3 kinase that associates with Pyk-2 and is necessary for B-cell migration.

The chemokine macrophage inflammatory protein-1 alpha [MIP-1alpha] causes migration of B cells and also induces changes in antibody secretion. However, the signal transduction pathways leading to these phenotypic changes remain undefined. We have identified a signal transduction pathway initiated by MIP-1alpha in B cells. Here we report that stimulation of tonsil B cells with MIP-1alpha induces phosphatidylinositol 3-kinase [PI3-K] activation. Kinase activity was transient with peak induction occurring within 2.5 to 5 min after stimulation and was dose-dependent. In addition stimulation with MIP-1alpha induces tyrosine phosphorylation of the proline-rich tyrosine kinase Pyk-2. Immunoprecipitation analysis showed a constitutive association between Pyk-2 and PI3-K and pretreatment of MIP-1alpha-stimulated B cells with wortmannin, a specific inhibitor of PI3-K, resulted in a loss of PI3-K activity. The PI3-K inhibitor wortmannin prevented B cells from migrating in response to MIP-1alpha. Hence, PI3-K and Pyk-2 seem to be components of a signal transduction pathway induced by stimulation of B cells with MIP-1alpha, and this pathway may play a role in B-cell migration.     

A generalized model of inversion polymorphism

An earlier mathematical analysis of chromosomal inversion dynamics is extended. A complete list of possible equilibria is given. The various types are compared with those found in the previous work.     

Massive genomic rearrangement acquired in a single catastrophic event during cancer development

Cancer is driven by somatically acquired point mutations and chromosomal rearrangements, conventionally thought to accumulate gradually over time. Using next-generation sequencing, we characterize a phenomenon, which we term chromothripsis, whereby tens to hundreds of genomic rearrangements occur in a one-off cellular crisis. Rearrangements involving one or a few chromosomes crisscross back and forth across involved regions, generating frequent oscillations between two copy number states. These genomic hallmarks are highly improbable if rearrangements accumulate over time and instead imply that nearly all occur during a single cellular catastrophe. The stamp of chromothripsis can be seen in at least 2%-3% of all cancers, across many subtypes, and is present in ～25% of bone cancers. We find that one, or indeed more than one, cancer-causing lesion can emerge out of the genomic crisis. This phenomenon has important implications for the origins of genomic remodeling and temporal emergence of cancer.