Long-distance dispersal: a framework for hypothesis testing

Tests of hypotheses about the biogeographical consequences of long-distance dispersal have long eluded biologists, largely because of the rarity and presumed unpredictability of such events. Here, we examine data for terrestrial (including littoral) organisms in the Pacific to show that knowledge of dispersal by wind, birds and oceanic drift or rafting, coupled with information about the natural environment and biology of the organisms, can be used to generate broad biogeographic predictions. We then examine the predictions in the context of the origin, frequency of arrival and location of establishment of dispersed organisms, as well as subsequent patterns of endemism and diversification on remote islands. The predicted patterns are being increasingly supported by phylogenetic data for both terrestrial and littoral organisms.     

Statistics for proteomics: a review of tools for analyzing experimental data

Most proteomics experiments make use of 'high throughput' technologies such as 2-DE, MS or protein arrays to measure simultaneously the expression levels of thousands of proteins. Such experiments yield large, high-dimensional data sets which usually reflect not only the biological but also technical and experimental factors. Statistical tools are essential for evaluating these data and preventing false conclusions. Here, an overview is given of some typical statistical tools for proteomics experiments. In particular, we present methods for data preprocessing (e.g. calibration, missing values estimation and outlier detection), comparison of protein expression in different groups (e.g. detection of differentially expressed proteins or classification of new observations) as well as the detection of dependencies between proteins (e.g. protein clusters or networks). We also discuss questions of sample size planning for some of these methods.     

18O labeling over a coffee break: a rapid strategy for quantitative proteomics

Proteomics-based quantification methods for differential protein expression measurements are among the most important and challenging techniques in the field of mass spectrometry. Though numerous quantification methods have been established, no method meets all the demands for measuring accurate protein expression levels. Of the various relative quantification methods by isotopic labeling, (18)O labeling method has been shown to be simple, specific, cost-effective and applicable to a wide range of analyses. However, some researchers refrain from using the method due to long incubation periods required during the labeling process. To address this problem, we demonstrate a method by which the labeling procedure can be completed in 15 min. We digested and labeled samples using immobilized trypsin on micro-spin columns to speed up the enzyme-mediated oxygen substitution, thereby completing the labeling process within 15 min with high labeling efficiency. We demonstrate the efficiency and accuracy of the method using a four protein mixture and whole cell lysate from rat vascular endothelial cells.     

Multiple hypothesis testing to detect lineages under positive selection that affects only a few sites

Detection of positive Darwinian selection has become ever more important with the rapid growth of genomic data sets. Recent branch-site models of codon substitution account for variation of selective pressure over branches on the tree and across sites in the sequence and provide a means to detect short episodes of molecular adaptation affecting just a few sites. In likelihood ratio tests based on such models, the branches to be tested for positive selection have to be specified a priori. In the absence of a biological hypothesis to designate so-called foreground branches, one may test many branches, but a correction for multiple testing becomes necessary. In this paper, we employ computer simulation to evaluate the performance of 6 multiple test correction procedures when the branch-site models are used to test every branch on the phylogeny for positive selection. Four of the methods control the familywise error rates (FWERs), whereas the other 2 control the false discovery rate (FDR). We found that all correction procedures achieved acceptable FWER except for extremely divergent sequences and serious model violations, when the test may become unreliable. The power of the test to detect positive selection is influenced by the strength of selection and the sequence divergence, with the highest power observed at intermediate divergences. The 4 correction procedures that control the FWER had similar power. We recommend Rom's procedure for its slightly higher power, but the simple Bonferroni correction is useable as well. The 2 correction procedures that control the FDR had slightly more power and also higher FWER. We demonstrate the multiple test procedures by analyzing gene sequences from the extracellular domain of the cluster of differentiation 2 (CD2) gene from 10 mammalian species. Both our simulation and real data analysis suggest that the multiple test procedures are useful when multiple branches have to be tested on the same data set.     

Two-dimensional target decoy strategy for shotgun proteomics

The target-decoy approach to estimating and controlling false discovery rate (FDR) has become a de facto standard in shotgun proteomics, and it has been applied at both the peptide-to-spectrum match (PSM) and protein levels. Current bioinformatics methods control either the PSM- or the protein-level FDR, but not both. In order to obtain the most reliable information from their data, users must employ one method when the number of tandem mass spectra exceeds the number of proteins in the database and another method when the reverse is true. Here we propose a simple variation of the standard target-decoy strategy that estimates and controls PSM and protein FDRs simultaneously, regardless of the relative numbers of spectra and proteins. We demonstrate that even if the final goal is a list of PSMs with a fixed low FDR and not a list of protein identifications, the proposed two-dimensional strategy offers advantages over a pure PSM-level strategy.     

HyPhy: hypothesis testing using phylogenies

SUMMARY: The HyPhypackage is designed to provide a flexible and unified platform for carrying out likelihood-based analyses on multiple alignments of molecular sequence data, with the emphasis on studies of rates and patterns of sequence evolution. AVAILABILITY: http://www.hyphy.org CONTACT: muse@stat.ncsu.edu SUPPLEMENTARY INFORMATION: HyPhydocumentation and tutorials are available at http://www.hyphy.org.     

Development of a new magnetic beads-based immunoprecipitation strategy for proteomics analysis

Sample pre-treatment is a critical step for an efficient and reliable analysis and it is highly dependent on the complexity of the matrix. This work shows an example of application of an immunoprecipitation approach using a new magnetic beads-based format, which allows a selective/specific extraction of potential biomarkers from metastatic prostate cancer. Results obtained on the development of this method, and its application for the extraction and pre-concentration of certain biomarkers present in metastatic cell lines of prostate cancer, are presented and discussed. It is concluded that the efficiency of the immunoprecipitation step is clearly compromised by the crosslinking conditions and it is highly dependent on the specificity of selected antibodies. The epoxy magnetic beads used in this work allowed an effective crosslinking of the antibodies contributing to an increased efficiency of the immunoprecipitation step. The optimized conditions for the application of these epoxy magnetic beads for the immunoprecipitation of anti-TUBA3C in metastatic prostate cancer cell line (PC3) are discussed here, as an example of application of the immnuprecipitation approach developed, which resulted in a very efficient tool for a specific extraction and pre-concentration of the targeted protein and, therefore, contributing to the efficiency of further analysis.     

PatternLab for proteomics: a tool for differential shotgun proteomics

Background  

A goal of proteomics is to distinguish between states of a biological system by identifying protein expression differences. Liu et al. demonstrated a method to perform semi-relative protein quantitation in shotgun proteomics data by correlating the number of tandem mass spectra obtained for each protein, or "spectral count", with its abundance in a mixture; however, two issues have remained open: how to normalize spectral counting data and how to efficiently pinpoint differences between profiles. Moreover, Chen et al. recently showed how to increase the number of identified proteins in shotgun proteomics by analyzing samples with different MS-compatible detergents while performing proteolytic digestion. The latter introduced new challenges as seen from the data analysis perspective, since replicate readings are not acquired.     

The challenges of developing a sound proteomics strategy

This paper will review the challenges of developing a proteomics strategy. A key issue is the integration of the two-dimensional (2-D) gel platform with mass spectrometry measurements. The use of both matrix-assisted laser/desorption ionization (on off-line coupling) and electrospray (on-line) ionization are complementary. While the use of one-dimensional and 2-D gels are essential to many aspects of proteomics research (sample preparation, preliminary fractionation and quantitation, storage of protein components), the emergence of shotgun sequencing based on high performance liquid chromatography and tandem mass spectrometry offers a powerful new approach. The latter has particular utility in the characterization of low level samples and complex post-translational modifications. The development of capillary columns, such as 75 to 150 micron, that can be packed in a reproducible manner has been a key step in the development of high sensitivity liquid chromatography/mass spectrometry analysis.     

Integrative hypothesis for Huntington's disease: a brief review of experimental evidence

Huntington's disease (HD) is a demential, neurodegenerative inheritable disease affecting middle-aged patients. HD is characterized by uncontrolled choreiform movements, psychiatric symptoms and cognitive decline. Histopathological changes in HD brains reveal a considerable damage to basal ganglia, particularly affecting middle-sized spiny neurons from the caudate-putamen region. Neurochemical changes are specifically oriented to deplete GABAergic and cholinergic systems, while molecular alterations include an increased expression of CAG trinucleotide at exon 1 from the huntingtin (htt) gene, as well as aggregation of mutant htt. Although several hypotheses regarding the mechanisms by which neurotoxicity is triggered in HD brains have been suggested on the basis of experimental evidence, so far it remains not clear which of them are predominant or whether they are complementary. Recent experimental evidence through transgenic mice models reveal an interesting interaction between expanded CAG triplets, mutant htt, and the increase in toxic metabolites from the kynurenine pathway. Further evidence supports the assumption that different toxic mechanisms (i.e. excitotoxicity, energy metabolism impairment, inflammatory events, oxidative stress, etc.) are confluent and depend on each other. In this review we will briefly summarize some of those findings and propose a final integrative hypothesis for HD.     

Subtle modification of isotope ratio proteomics; an integrated strategy for expression proteomics

Use of minor modification of isotope ratio to code samples for expression proteomics is being investigated. Alteration of (13)C abundance to approximately 2% yields a measurable effect on peptide isotopic distribution and inferred isotope ratio. Elevation of (13)C abundance to 4% leads to extension of isotopic distribution and background peaks across every unit of the mass range. Assessment of isotope ratio measurement variability suggests substantial contributions from natural measurement variability. A better understanding of this variable will allow assessment of the contribution of sequence dependence. Both variables must be understood before meaningful mixing experiments for relative expression proteomics are performed. Subtle modification of isotope ratio ( approximately 1-2% increase in (13)C) had no effect upon either the ability of data-dependent acquisition software or database searching software to trigger tandem mass spectrometry or match MSMS data to peptide sequences. More severe modification of isotope ratio caused a significant drop in performance of both functionalities. Development of software for deconvolution of isotope ratio concomitant with protein identification using LC-MSMS, or any other proteomics strategy, is underway (Isosolv). The identified peptide sequence is then be used to provide elemental composition for accurate isotope ratio decoding and the potential to control for specific amino acid biases should these prove significant. It is suggested that subtle modification of isotope ratio proteomics (SMIRP) offers a convenient approach to in vivo isotope coding of plants and might ultimately be extended to mammals including humans.     

Protease-mediated arsenic prodrug strategy in cancer and infectious diseases: a hypothesis for targeted activation

A strategy for the selective in vivo activation of prodrugs by proteases is presented. The approach is based on the design of polythiol peptides able to neutralize the toxicity of As(III) through chelation, and contemporarily to be recognized as substrates of a disease-linked specific protease. Enzyme digestion implies conversion of such polythiol peptides into monothiol fragments with irreversible loss of the ability to chelate the metalloid, thus triggering the release in its free and pharmacologically effective form. The proteases whose activity appears dramatically up-regulated in various pathologies, ranging from cancer to infectious diseases, can be conveniently employed as prodrug activators in the disease microenvironment. The design of the representative peptide shown here has been assisted by molecular modeling in order to fulfill the dual characteristic to be an efficient As(III) chelator and simultaneously a substrate of the matrix metalloproteinase-9 (MMP-9) whose activity results dramatically increased at the surface of cells affected by several pathologies.     

Sexual dimorphism in the human pelvis: testing a new hypothesis.

Sexual dimorphism in the human pelvis is inferentially related to parturition. Investigators disagree about the identification and obstetric significance of pelvic dimorphism. Benefiting from a large sample of complete skeletons from the Coimbra Identified Skeletal Collection, we show that the dimensions of the true pelvis (birth canal) that are most sexually dimorphic (that is, the dimensions of females are greater than males) are those which are related to biparietal deformation, which often leads to the death of the human neonate. These dimensions are: the anteroposterior diameter of the inlet (index of dimorphism = 108.41), the transverse diameter of the bispinous midplane (index of dimorphism = 117.13) and the transverse diameter of the outlet (index of dimorphism = 112.3). Therefore, sexual dimorphism in the human pelvis is a reflection of differential selection on the two sexes. These results may stimulate further studies with a fresh approach regarding the fossil and comparative evidence for when and how the modern pattern of birth has evolved.     

Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization

We describe a protocol for selective extraction of the amino (N)-terminal-most peptide of a protein or a mixture of proteins after proteolysis. The first stage of the protocol blocks the free amino groups alpha and epsilon (the latter being lysyl residues) on the intact proteins by acetylation. In the second stage, proteolysis of the acetylated proteins yields a mixture of N-terminally acetylated (true N-terminal) and non-acetylated (internal and carboxy-terminal) peptides. Affinity capture of peptides bearing free amino groups using an immobilized amine-reactive reagent removes internal peptides from the mixture. The unbound fraction is highly enriched in N-terminal peptides, which can be analyzed without further treatment. This method is compatible with a range of proteolytic enzymes and fragmentation methods, and should take 2 d to complete. The N-terminal peptides can then be analyzed by mass spectrometry. This low cost, rapid method is readily adopted using off the shelf reagents.     

Species-pool hypothesis: Limits to its testing

The species richness of a community depends both on the pool of available species and on biotic mechanisms that lead to the exclusion of some of the species from a community. The method suggested byPärtel et al.,Oikos 75: 111–117, 1996 to test the effect of species-pool size on the species richness of a community is discussed in this paper. This method is based on the calculation of a correlation between the actual species richness of a community and the actual species-pool size, and compares the correlation found in the data with that simulated by a null model. In the null model, the species richness has a uniform distribution between zero and the size of the species pool. A correlation significantly higher than that in the null model is interpreted as evidence of the greater role of species pool than of local interactions in formation of community species richness (Zobel,Folia Geobot. 36: 3–8, 2001). It is shown that the interpretation of discrepancies between the null model and reality is difficult or impossible, because: (1) a null model with a uniform distribution of species richness is unrealistic, (2) both models based on the random selection of species from a species pool and models that include competitive interactions in the community predict a higher positive correlation of species richness and size of the species pool than the null model, and (3) local species richness might be affected by species-pool size, but a large species-pool size can also be a result of high local species richness. Caution is urged when interpreting the analyses based on the size of the filtered species-pool size.     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.