Open access to tree genomes: the path to a better forest

An open-access culture and a well-developed comparative-genomics infrastructure must be developed in forest trees to derive the full potential of genome sequencing in this diverse group of plants that are the dominant species in much of the earth''s terrestrial ecosystems.     

Epitope Predictions Indicate the Presence of Two Distinct Types of Epitope-Antibody-Reactivities Determined by Epitope Profiling of Intravenous Immunoglobulins

Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.     

Enduring Effects of Early Life Stress on Firing Patterns of Hippocampal and Thalamocortical Neurons in Rats: Implications for Limbic Epilepsy

Early life stress results in an enduring vulnerability to kindling-induced epileptogenesis in rats, but the underlying mechanisms are not well understood. Recent studies indicate the involvement of thalamocortical neuronal circuits in the progression of kindling epileptogenesis. Therefore, we sought to determine in vivo the effects of early life stress and amygdala kindling on the firing pattern of hippocampus as well as thalamic and cortical neurons. Eight week old male Wistar rats, previously exposed to maternal separation (MS) early life stress or early handling (EH), underwent amygdala kindling (or sham kindling). Once fully kindled, in vivo juxtacellular recordings in hippocampal, thalamic and cortical regions were performed under neuroleptic analgesia. In the thalamic reticular nucleus cells both kindling and MS independently lowered firing frequency and enhanced burst firing. Further, burst firing in the thalamic reticular nucleus was significantly increased in kindled MS rats compared to kindled EH rats (p<0.05). In addition, MS enhanced burst firing of hippocampal pyramidal neurons. Following a stimulation-induced seizure, somatosensory cortical neurons exhibited a more pronounced increase in burst firing in MS rats than in EH rats. These data demonstrate changes in firing patterns in thalamocortical and hippocampal regions resulting from both MS and amygdala kindling, which may reflect cellular changes underlying the enhanced vulnerability to kindling in rats that have been exposed to early life stress.     

Biological and Structural Characterization of Glycosylation on Ephrin-A1, a Preferred Ligand for EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase is overexpressed in a number of malignancies and is activated by ephrin ligands, most commonly by ephrin-A1. The crystal structure of the ligand-receptor complex revealed a glycosylation on the Asn-26 of ephrin-A1. Here we report for the first time the significance of the glycosylation in the biology of EphA2 and ephrin-A1. Ephrin-A1 was enzymatically deglycosylated, and its activity was evaluated in several assays using glioblastoma (GBM) cells and recombinant EphA2. We found that deglycosylated ephrin-A1 does not efficiently induce EphA2 receptor internalization and degradation, and does not activate the downstream signaling pathways involved in cell migration and proliferation. Data obtained by surface plasmon resonance confirms that deglycosylated ephrin-A1 does not bind EphA2 with high affinity. Mutations in the glycosylation site on ephrin-A1 result in protein aggregation and mislocalization. Analysis of Eph/ephrin crystal structures reveals an interaction between the ligand''s carbohydrates and two residues of EphA2: Asp-78 and Lys-136. These findings suggest that the glycosylation on ephrin-A1 plays a critical role in the binding and activation of the EphA2 receptor.     

Comprehensive DNA signature discovery and validation

DNA signatures are nucleotide sequences that can be used to detect the presence of an organism and to distinguish that organism from all other species. Here we describe Insignia, a new, comprehensive system for the rapid identification of signatures in the genomes of bacteria and viruses. With the availability of hundreds of complete bacterial and viral genome sequences, it is now possible to use computational methods to identify signature sequences in all of these species, and to use these signatures as the basis for diagnostic assays to detect and genotype microbes in both environmental and clinical samples. The success of such assays critically depends on the methods used to identify signatures that properly differentiate between the target genomes and the sample background. We have used Insignia to compute accurate signatures for most bacterial genomes and made them available through our Web site. A sample of these signatures has been successfully tested on a set of 46 Vibrio cholerae strains, and the results indicate that the signatures are highly sensitive for detection as well as specific for discrimination between these strains and their near relatives. Our approach, whereby the entire genomic complement of organisms are compared to identify probe targets, is a promising method for diagnostic assay development, and it provides assay designers with the flexibility to choose probes from the most relevant genes or genomic regions. The Insignia system is freely accessible via a Web interface and has been released as open source software at: http://insignia.cbcb.umd.edu.     

Optimized multiplex PCR: efficiently closing a whole-genome shotgun sequencing project

A new method has been developed for rapidly closing a large number of gaps in a whole-genome shotgun sequencing project. The method employs multiplex PCR and a novel pooling strategy to minimize the number of laboratory procedures required to sequence the unknown DNA that falls in between contiguous sequences. Multiplex sequencing, a novel procedure in which multiple PCR primers are used in a single sequencing reaction, is used to interpret the multiplex PCR results. Two protocols are presented, one that minimizes pipetting and another that minimizes the number of reactions. The pipette optimized multiplex PCR method has been employed in the final phases of closing the Streptococcus pneumoniae genome sequence, with excellent results.     

Evolutionary dynamics of cellular automata-based self-replicators in hostile environments

In this paper we investigate population dynamics, genealogy and complexity-increase of locally interacting populations of cellular automata-based evolving self-replicating loops (evoloops). We outline experiments indicating that the evolutionary growth in complexity, known to be achievable in principle given the complete genetic accessibility granted by universal construction, may be achievable in practice using much simpler replicating structures. By introducing evoloop populations to hostile environments, we demonstrate that selection pressures toward smaller species can be mediated to enable evolutionary accessibility to larger species, which themselves roam a much more vast portion of genetic state-space. We show that this growth in size results from intrinsically biased genealogy inherent in the rules of the evoloop CA, normally suppressed by selection pressures from direct competition favouring the smallest species. This shows that, in populations of simple self-replicating structures, a limited form of complexity-increase may result from a process which is driven by biased genealogical connectivity--a purely emergent property arising out of bottom-up evolutionary dynamics--and not just by adaptation . Implications of this result are discussed and contrasted with other self-replication studies in Artificial Life and Biology.