16S rDNA pyrosequencing analysis of bacterial community in heavy metals polluted soils.

Soil contamination with heavy metals is a widespread problem, especially prominent on grounds lying in the vicinity of mines, smelters, and other industrial facilities. Many such areas are located in Southern Poland; they are polluted mainly with Pb, Zn, Cd, or Cu, and locally also with Cr. As for now, little is known about most bacterial species thriving in such soils and even less about a core bacterial community--a set of taxa common to polluted soils. Therefore, we wanted to answer the question if such a set could be found in samples differing physicochemically and phytosociologically. To answer the question, we analyzed bacterial communities in three soil samples contaminated with Pb and Zn and two contaminated with Cr and lower levels of Pb and Zn. The communities were assessed with 16S rRNA gene fragments pyrosequencing. It was found that the samples differed significantly and Zn decreased both diversity and species richness at species and family levels, while plant species richness did not correlate with bacterial diversity. In spite of the differences between the samples, they shared many operational taxonomic units (OTUs) and it was possible to delineate the core microbiome of our sample set. The core set of OTUs comprised members of such taxa as Sphingomonas, Candidatus Solibacter, or Flexibacter showing that particular genera might be shared among sites ~40 km distant.

Molecular evolution in court: analysis of a large hepatitis C virus outbreak from an evolving source.

BACKGROUND: Molecular phylogenetic analyses are used increasingly in the epidemiological investigation of outbreaks and transmission cases involving rapidly evolving RNA viruses. Here, we present the results of such an analysis that contributed to the conviction of an anesthetist as being responsible for the infection of 275 of his patients with hepatitis C virus. RESULTS: We obtained sequences of the NS5B and E1-E2 regions in the viral genome for 322 patients suspected to have been infected by the doctor, and for 44 local, unrelated controls. The analysis of 4,184 cloned sequences of the E1-E2 region allowed us to exclude 47 patients from the outbreak. A subset of patients had known dates of infection. We used these data to calibrate a relaxed molecular clock and to determine a rough estimate of the time of infection for each patient. A similar analysis led to an estimate for the time of infection of the source. The date turned out to be 10 years before the detection of the outbreak. The number of patients infected was small at first, but it increased substantially in the months before the detection of the outbreak. CONCLUSIONS: We have developed a procedure to integrate molecular phylogenetic reconstructions of rapidly evolving viral populations into a forensic setting adequate for molecular epidemiological analysis of outbreaks and transmission events. We applied this procedure to a large outbreak of hepatitis C virus caused by a single source and the results obtained played a key role in the trial that led to the conviction of the suspected source.

Biostasis: A Roadmap for Research in Preservation and Potential Revival of Humans.

Human biostasis, the preservation of a human when all other contemporary options for extension of quality life are exhausted, offers the speculative potential for survival via continuation of life in the future. While provably reversible preservation, also known as suspended animation, is not yet possible for humans, the primary justification for contemporary biostasis is the preservation of the brain, which is broadly considered the seat of memories, personality, and identity. By preserving the information contained within the brain's structures, it may be possible to resuscitate a healthy whole individual using advanced future technologies. There are numerous challenges in biostasis, including inadequacies in current preservation techniques, methods to evaluate the quality of preservation, and potential future revival technologies. In this report, we describe a roadmap that attempts to delineate research directions that could improve the field of biostasis, focusing on optimizing preservation protocols and establishing metrics for querying preservation quality, as well as pre- and post-cardiac arrest factors, stabilization strategies, and methods for long-term preservation. We acknowledge the highly theoretical nature of future revival technologies and the importance of achieving high-fidelity brain preservation to maximize the potential of future repair technologies. We plan to update the research roadmap biennially. Our goal is to encourage multidisciplinary communication and collaboration in this field.

Influence of the Escherichia coli oxyR gene function on lambda prophage maintenance.

In Escherichia coli hosts, hydrogen peroxide is one of the factors that may cause induction of lambda prophage. Here, we demonstrate that H2O2-mediated lambda prophage induction is significantly enhanced in the oxyR mutant host. The mRNA levels for cI gene expression were increased in a lambda lysogen in the presence of H2O2. On the other hand, stimulation of the p(M) promoter by cI857 overproduced from a multicopy plasmid was decreased in the DeltaoxyR mutant in the presence of H2O2 but not under normal growth conditions. The purified OxyR protein did bind specifically to the p(M) promoter region. This binding impaired efficiency of interaction of the cI protein with the OR3 site, while stimulating such a binding to OR2 and OR1 sites, in the regulatory region of the p(M) promoter. We propose that changes in cI gene expression, perhaps in combination with moderately induced SOS response, may be responsible for enhanced lambda prophage induction by hydrogen peroxide in the oxyR mutant. Therefore, OxyR seems to be a factor stimulating lambda prophage maintenance under conditions of oxidative stress. This proposal is discussed in the light of efficiency of induction of lambdoid prophages bearing genes coding for Shiga toxins.

Using evolutionary tools to refine the new hypervariable region 3 within the envelope 2 protein of hepatitis C virus.

The envelope 2 protein of hepatitis C virus (HCV) presents three hypervariable regions, named HVR1, HVR2 and HVR3, in which the presence of antigenic sites has been described. Genetic variability in these regions may reflect the generation of escape mutants as a consequence of the immune response. Therefore, these regions would tend to accumulate amino acid changes along the infection process, an effect that could be accelerated by antiviral treatments. In this study, we have analyzed the E1-E2 region of 23 HCV patients non-responders to antiviral treatment, 7 of which were infected with subtype 1a, 15 with subtype 1b, and 1 with a new HCV-1 subtype, before and after 6 and/or 12 months of peg-interferon+ribavirin treatment. We have sequenced about 100 clones from each sample, analyzing a total of 4906 sequences. A detailed analysis of the evolutionary forces acting along the genome region studied confirmed the existence of the three hypervariable regions, characterized by significant changes in amino acid composition between samples taken at different times from the same patient and a high number of sites evolving under positive selection. Moreover, for the recently described HVR3, our results suggest that its location could be restricted to residues 434-450, instead of the originally postulated 431-466.

Statistical measures of uncertainty for branches in phylogenetic trees inferred from molecular sequences by using model-based methods.

In recent years, the emphasis of theoretical work on phylogenetic inference has shifted from the development of new tree inference methods to the development of methods to measure the statistical support for the topologies. This paper reviews 3 approaches to assign support values to branches in trees obtained in the analysis of molecular sequences: the bootstrap, the Bayesian posterior probabilities for clades, and the interior branch tests. In some circumstances, these methods give different answers. It should not be surprising: their assumptions are different. Thus the interior branch tests assume that a given topology is true and only consider if a particular branch length is longer than zero. If a tree is incorrect, a wrong branch (a low bootstrap or Bayesian support may be an indication) may have a non-zero length. If the substitution model is oversimplified, the length of a branch may be overestimated, and the Bayesian support for the branch may be inflated. The bootstrap, on the other hand, approximates the variance of the data under the real model of sequence evolution, because it involves direct resampling from this data. Thus the discrepancy between the Bayesian support and the bootstrap support may signal model inaccuracy. In practical application, use of all 3 methods is recommended, and if discrepancies are observed, then a careful analysis of their potential origins should be made.

Topology testing of phylogenies using least squares methods.

BACKGROUND: The least squares (LS) method for constructing confidence sets of trees is closely related to LS tree building methods, in which the goodness of fit of the distances measured on the tree (patristic distances) to the observed distances between taxa is the criterion used for selecting the best topology. The generalized LS (GLS) method for topology testing is often frustrated by the computational difficulties in calculating the covariance matrix and its inverse, which in practice requires approximations. The weighted LS (WLS) allows for a more efficient albeit approximate calculation of the test statistic by ignoring the covariances between the distances. RESULTS: The goal of this paper is to assess the applicability of the LS approach for constructing confidence sets of trees. We show that the approximations inherent to the WLS method did not affect negatively the accuracy and reliability of the test both in the analysis of biological sequences and DNA-DNA hybridization data (for which character-based testing methods cannot be used). On the other hand, we report several problems for the GLS method, at least for the available implementation. For many data sets of biological sequences, the GLS statistic could not be calculated. For some data sets for which it could, the GLS method included all the possible trees in the confidence set despite a strong phylogenetic signal in the data. Finally, contrary to WLS, for simulated sequences GLS showed undercoverage (frequent non-inclusion of the true tree in the confidence set). CONCLUSION: The WLS method provides a computationally efficient approximation to the GLS useful especially in exploratory analyses of confidence sets of trees, when assessing the phylogenetic signal in the data, and when other methods are not available.

Biodiversity of bacteriophages: morphological and biological properties of a large group of phages isolated from urban sewage.

A large scale analysis presented in this article focuses on biological and physiological variety of bacteriophages. A collection of 83 bacteriophages, isolated from urban sewage and able to propagate in cells of different bacterial hosts, has been obtained (60 infecting Escherichia coli, 10 infecting Pseudomonas aeruginosa, 4 infecting Salmonella enterica, 3 infecting Staphylococcus sciuri, and 6 infecting Enterococcus faecalis). High biological diversity of the collection is indicated by its characteristics, both morphological (electron microscopic analyses) and biological (host range, plaque size and morphology, growth at various temperatures, thermal inactivation, sensitivity to low and high pH, sensitivity to osmotic stress, survivability upon treatment with organic solvents and detergents), and further supported by hierarchical cluster analysis. By the end of the research no larger collection of phages from a single environmental source investigated by these means had been found. The finding was confirmed by whole genome analysis of 7 selected bacteriophages. Moreover, particular bacteriophages revealed unusual biological features, like the ability to form plaques at low temperature (4 °C), resist high temperature (62 °C or 95 °C) or survive in the presence of an organic solvents (ethanol, acetone, DMSO, chloroform) or detergent (SDS, CTAB, sarkosyl) making them potentially interesting in the context of biotechnological applications.

Pseudorandomness of gene expression: a new evo-devo theory of ageing.

In contrast to the first part of life (development), ageing appears to be under less strict genetic control. The precise timing of events so characteristic of development seems to loosen its grasp, while stochastic and environmental factors seem to become the dominant force. Evolutionary theories put forward a decreasing evolutionary pressure over the course of life as the reason behind this pattern, yet dissenting views on ageing as a genetically programmed process linger. In this paper we address this dissent by presenting insights from an artificial evolutionary-developmental system, ET, and propose a new evo-devo theory of ageing-a theory that sees ageing as a continuation of development in the postreproductive period. In this theory both development and ageing are under genetic control. Nonetheless, while gene expression patterns that drive development are optimised by evolution, patterns that drive ageing are not optimised, because evolutionary pressure decreases with age. For these reasons, during ageing the changes orchestrated by genes are "pseudorandom"- deterministic but erratic-and their effects on an individual's health are more likely to be detrimental than beneficial. As such, they contribute to the continuous deterioration of bodily functions that characterise ageing.

Embryogenesis, morphogens and cancer stem cells: putting the puzzle together.

This paper describes a model which puts together three key elements of cancer theory: the analogies between embryogenesis and carcinogenesis, the role played in both processes by morphogens and related pathways, and the recently emerged paradigm of cancer stem cells. The model is called Epigenetic Tracking. Originally conceived as a model of embryonic development, it was later extended to interpret other aspects of biology, such as the presence of junk DNA, the phenomenon of ageing and the process of cancer formation. In this work we deepen our vision of carcinogenesis, and propose a novel hypothesis on the role of morphogen-processing pathways. According to the hypothesis, the interplay of these pathways leads in stem cells to the production of new transcription factors, which act as drivers of cellular differentiation. The disruption of these pathways, caused by mutations in specific genes, would represent the first and most distinctive event in the carcinogenic process. Our hypothesis allows us to make testable predictions on patterns of gene mutations involved in carcinogenesis. Our hypothesis also suggests that cancer stem cells can stay dormant until they are activated in a process that resembles activation of stem cells during tissue repair or at a specific time during development.