Dominique Schneider

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the approximately 18,000 families of orthologous genes, we found approximately 2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.

Epistatic interactions between mutations play a prominent role in evolutionary theories. Many studies have found that epistasis is widespread, but they have rarely considered beneficial mutations. We analyzed the effects of epistasis on fitness for the first five mutations to fix in an experimental population of Escherichia coli. Epistasis depended on the effects of the combined mutations--the larger the expected benefit, the more negative the epistatic effect. Epistasis thus tended to produce diminishing returns with genotype fitness, although interactions involving one particular mutation had the opposite effect. These data support models in which negative epistasis contributes to declining rates of adaptation over time. Sign epistasis was rare in this genome-wide study, in contrast to its prevalence in an earlier study of mutations in a single gene.

Adaptation by natural selection depends on the rates, effects and interactions of many mutations, making it difficult to determine what proportion of mutations in an evolving lineage are beneficial. Here we analysed 264 complete genomes from 12 Escherichia coli populations to characterize their dynamics over 50,000 generations. The populations that retained the ancestral mutation rate support a model in which most fixed mutations are beneficial, the fraction of beneficial mutations declines as fitness rises, and neutral mutations accumulate at a constant rate. We also compared these populations to mutation-accumulation lines evolved under a bottlenecking regime that minimizes selection. Nonsynonymous mutations, intergenic mutations, insertions and deletions are overrepresented in the long-term populations, further supporting the inference that most mutations that reached high frequency were favoured by selection. These results illuminate the shifting balance of forces that govern genome evolution in populations adapting to a new environment.

Allelic exchange experiments allow investigation of the functions of many unknown genes identified during the sequencing of entire genomes. Isogenic strains differing by only specific mutations can be constructed. Among other tools, suicide plasmids are widely used for this task. They present many advantages because they leave no scars on the chromosome, and therefore allow combining several mutations in the same genetic background. While using the previously described pCVD442 suicide plasmid [Infect. Immun. 59 (1991) 4310], we found untargeted recombination events due to the presence of an IS1 element on this plasmid. The plasmid was therefore improved by removal of the IS1 element. We also replaced the bla gene of pCVD442, conferring ampicillin resistance, by the cat gene conferring chloramphenicol resistance, leading to the new suicide plasmid pDS132. The plasmid was entirely sequenced. We demonstrate that this new vector can be easily used to introduce various types of mutations into different genetics backgrounds: removal of IS elements, introduction of point mutations or deletions. It can be introduced into bacterial strains by either transformation or conjugation.

The repeatability of evolutionary change is difficult to quantify because only a single outcome can usually be observed for any precise set of circumstances. In this study, however, we have quantified the frequency of parallel and divergent genetic changes in 12 initially identical populations of Escherichia coli that evolved in identical environments for 20,000 cell generations. Unlike previous analyses in which candidate genes were identified based on parallel phenotypic changes, here we sequenced four loci ( pykF , nadR , pbpA-rodA , and hokB / sokB ) in which mutations of unknown effect had been discovered in one population, and then we compared the substitution pattern in these “blind” candidate genes with the pattern found in 36 randomly chosen genes. Two candidate genes, pykF and nadR , had substitutions in all 11 other populations, and the other 2 in several populations. There were very few cases, however, in which the exact same mutations were substituted, in contrast to the findings from conceptually related work performed with evolving virus populations. No random genes had any substitutions except in four populations that evolved defects in DNA repair. Tests of four different statistical aspects of the pattern of molecular evolution all indicate that adaptation by natural selection drove the parallel changes in these candidate genes.

Mutations are the ultimate source of heritable variation for evolution. Understanding how mutation rates themselves evolve is thus essential for quantitatively understanding many evolutionary processes. According to theory, mutation rates should be minimized for well-adapted populations living in stable environments, whereas hypermutators may evolve if conditions change. However, the long-term fate of hypermutators is unknown. Using a phylogenomic approach, we found that an adapting Escherichia coli population that first evolved a mutT hypermutator phenotype was later invaded by two independent lineages with mutY mutations that reduced genome-wide mutation rates. Applying neutral theory to synonymous substitutions, we dated the emergence of these mutations and inferred that the mutT mutation increased the point-mutation rate by ∼150-fold, whereas the mutY mutations reduced the rate by ∼40-60%, with a corresponding decrease in the genetic load. Thus, the long-term fate of the hypermutators was governed by the selective advantage arising from a reduced mutation rate as the potential for further adaptation declined.

ABSTRACT Twelve populations of Escherichia coli B all lost d -ribose catabolic function during 2,000 generations of evolution in glucose minimal medium. We sought to identify the population genetic processes and molecular genetic events that caused these rapid and parallel losses. Seven independent Rbs − mutants were isolated, and their competitive fitnesses were measured relative to that of their Rbs + progenitor. These Rbs − mutants were all about 1 to 2% more fit than the progenitor. A fluctuation test revealed an unusually high rate, about 5 × 10 −5 per cell generation, of mutation from Rbs + to Rbs − , which contributed to rapid fixation. At the molecular level, the loss of ribose catabolic function involved the deletion of part or all of the ribose operon ( rbs genes). The physical extent of the deletion varied between mutants, but each deletion was associated with an IS 150 element located immediately upstream of the rbs operon. The deletions apparently involved transposition into various locations within the rbs operon; recombination between the new IS 150 copy and the one upstream of the rbs operon then led to the deletion of the intervening sequence. To confirm that the beneficial fitness effect was caused by deletion of the rbs operon (and not some undetected mutation elsewhere), we used P1 transduction to restore the functional rbs operon to two Rbs − mutants, and we constructed another Rbs − strain by gene replacement with a deletion not involving IS 150 . All three of these new constructs confirmed that Rbs − mutants have a competitive advantage relative to their Rbs + counterparts in glucose minimal medium. The rapid and parallel evolutionary losses of ribose catabolic function thus involved both (i) an unusually high mutation rate, such that Rbs − mutants appeared repeatedly in all populations, and (ii) a selective advantage in glucose minimal medium that drove these mutants to fixation.

Molecular methods are used widely to measure genetic diversity within populations and determine relationships among species. However, it is difficult to observe genomic evolution in action because these dynamics are too slow in most organisms. To overcome this limitation, we sampled genomes from populations of Escherichia coli evolving in the laboratory for 10,000 generations. We analyzed the genomes for restriction fragment length polymorphisms (RFLP) using seven insertion sequences (IS) as probes; most polymorphisms detected by this approach reflect rearrangements (including transpositions) rather than point mutations. The evolving genomes became increasingly different from their ancestor over time. Moreover, tremendous diversity accumulated within each population, such that almost every individual had a different genetic fingerprint after 10,000 generations. As has been often suggested, but not previously shown by experiment, the rates of phenotypic and genomic change were discordant, both across replicate populations and over time within a population. Certain pivotal mutations were shared by all descendants in a population, and these are candidates for beneficial mutations, which are rare and difficult to find. More generally, these data show that the genome is highly dynamic even over a time scale that is, from an evolutionary perspective, very brief.

The modeling and simulation of genetic regulatory networks have created the need for tools for model validation. The main challenges of model validation are the achievement of a match between the precision of model predictions and experimental data, as well as the efficient and reliable comparison of the predictions and observations.We present an approach towards the validation of models of genetic regulatory networks addressing the above challenges. It combines a method for qualitative modeling and simulation with techniques for model checking, and is supported by a new version of the computer tool Genetic Network Analyzer (GNA). The model-validation approach has been applied to the analysis of the network controlling the nutritional stress response in Escherichia coli.GNA and the model of the stress response network are available at http://www-helix.inrialpes.fr/gna.

The quantification of spontaneous mutation rates is crucial for a mechanistic understanding of the evolutionary process. In bacteria, traditional estimates using experimental or comparative genetic methods are prone to statistical uncertainty and consequently estimates vary by over one order of magnitude. With the advent of next-generation sequencing, more accurate estimates are now possible. We sequenced 19 Escherichia coli genomes from a 40,000-generation evolution experiment and directly inferred the point-mutation rate based on the accumulation of synonymous substitutions. The resulting estimate was 8.9 × 10(-11) per base-pair per generation, and there was a significant bias toward increased AT-content. We also compared our results with published genome sequence datasets for other bacterial evolution experiments. Given the power of our approach, our estimate represents the most accurate measure of bacterial base-substitution rates available to date.

We review the intersection between two areas of microbial evolution that were research foci of Michel Blot. One focus is the behavior of insertion sequence (IS) elements, including their role in promoting the evolutionary adaptation of their hosts. The other focus is experimental evolution, an approach that allows the dynamics of genomic and phenotypic change to be observed in the laboratory. This review shows that IS elements are useful as markers for detecting genomic change over experimental time scales and, moreover, that IS elements generate some of the beneficial mutations that increase organismal fitness.

Evolution has shaped all living organisms on Earth, although many details of this process are shrouded in time. However, it is possible to see, with one's own eyes, evolution as it happens by performing experiments in defined laboratory conditions with microbes that have suitably fast generations. The longest-running microbial evolution experiment was started in 1988, at which time twelve populations were founded by the same strain of Escherichia coli. Since then, the populations have been serially propagated and have evolved for tens of thousands of generations in the same environment. The populations show numerous parallel phenotypic changes, and such parallelism is a hallmark of adaptive evolution. Many genetic targets of natural selection have been identified, revealing a high level of genetic parallelism as well. Beneficial mutations affect all levels of gene regulation in the cells including individual genes and operons all the way to global regulatory networks. Of particular interest, two highly interconnected networks -- governing DNA superhelicity and the stringent response -- have been demonstrated to be deeply involved in the phenotypic and genetic adaptation of these experimental populations.

BJOG: An International Journal of Obstetrics & GynaecologyVolume 74, Issue 6 p. 799-811 BIOCHEMICAL SUPERVISION OF THE FOETUS DURING LABOUR Byerich Saling, Byerich Saling Unit for Perinatal Medicine and Research, The Municipal Women's Hospital, Berlin-Neukolln, I Berlin 44, Mariendorfer Weg 28–38Search for more papers by this authorDominique Schneider, Dominique Schneider Unit for Perinatal Medicine and Research, The Municipal Women's Hospital, Berlin-Neukolln, I Berlin 44, Mariendorfer Weg 28–38 Guest-Resident from the General Hospital, Münsterlingen/Thurgau, Switzerland.Search for more papers by this author Byerich Saling, Byerich Saling Unit for Perinatal Medicine and Research, The Municipal Women's Hospital, Berlin-Neukolln, I Berlin 44, Mariendorfer Weg 28–38Search for more papers by this authorDominique Schneider, Dominique Schneider Unit for Perinatal Medicine and Research, The Municipal Women's Hospital, Berlin-Neukolln, I Berlin 44, Mariendorfer Weg 28–38 Guest-Resident from the General Hospital, Münsterlingen/Thurgau, Switzerland.Search for more papers by this author First published: December 1967 https://doi.org/10.1111/j.1471-0528.1967.tb15561.xCitations: 118 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume74, Issue6December 1967Pages 799-811 RelatedInformation

Ecological opportunities promote population divergence into coexisting lineages. However, the genetic mechanisms that enable new lineages to exploit these opportunities are poorly understood except in cases of single mutations. We examined how two Escherichia coli lineages diverged from their common ancestor at the outset of a long-term coexistence. By sequencing genomes and reconstructing the genetic history of one lineage, we showed that three mutations together were sufficient to produce the frequency-dependent fitness effects that allowed this lineage to invade and stably coexist with the other. These mutations all affected regulatory genes and collectively caused substantial metabolic changes. Moreover, the particular derived alleles were critical for the initial divergence and invasion, indicating that the establishment of this polymorphism depended on specific epistatic interactions.

Large-scale rearrangements may be important in evolution because they can alter chromosome organization and gene expression in ways not possible through point mutations. In a long-term evolution experiment, twelve Escherichia coli populations have been propagated in a glucose-limited environment for over 25 years. We used whole-genome mapping (optical mapping) combined with genome sequencing and PCR analysis to identify the large-scale chromosomal rearrangements in clones from each population after 40,000 generations. A total of 110 rearrangement events were detected, including 82 deletions, 19 inversions, and 9 duplications, with lineages having between 5 and 20 events. In three populations, successive rearrangements impacted particular regions. In five populations, rearrangements affected over a third of the chromosome. Most rearrangements involved recombination between insertion sequence (IS) elements, illustrating their importance in mediating genome plasticity. Two lines of evidence suggest that at least some of these rearrangements conferred higher fitness. First, parallel changes were observed across the independent populations, with ~65% of the rearrangements affecting the same loci in at least two populations. For example, the ribose-utilization operon and the manB-cpsG region were deleted in 12 and 10 populations, respectively, suggesting positive selection, and this inference was previously confirmed for the former case. Second, optical maps from clones sampled over time from one population showed that most rearrangements occurred early in the experiment, when fitness was increasing most rapidly. However, some rearrangements likely occur at high frequency and may have simply hitchhiked to fixation. In any case, large-scale rearrangements clearly influenced genomic evolution in these populations.Bacterial chromosomes are dynamic structures shaped by long histories of evolution. Among genomic changes, large-scale DNA rearrangements can have important effects on the presence, order, and expression of genes. Whole-genome sequencing that relies on short DNA reads cannot identify all large-scale rearrangements. Therefore, deciphering changes in the overall organization of genomes requires alternative methods, such as optical mapping. We analyzed the longest-running microbial evolution experiment (more than 25 years of evolution in the laboratory) by optical mapping, genome sequencing, and PCR analyses. We found multiple large genome rearrangements in all 12 independently evolving populations. In most cases, it is unclear whether these changes were beneficial themselves or, alternatively, hitchhiked to fixation with other beneficial mutations. In any case, many genome rearrangements accumulated over decades of evolution, providing these populations with genetic plasticity reminiscent of that observed in some pathogenic bacteria.

Natural transformation by competence is a major mechanism of horizontal gene transfer in bacteria. Competence is defined as the genetically programmed physiological state that enables bacteria to actively take up DNA from the environment. The conditions that signal competence development are multiple and elusive, complicating the understanding of its evolutionary significance. We used expression of the competence gene comEA as a reporter of competence development and screened several hundred molecules for their ability to induce competence in the freshwater living pathogen Legionella pneumophila. We found that comEA expression is induced by chronic exposure to genotoxic molecules such as mitomycin C and antibiotics of the fluoroquinolone family. These results indicated that, in L. pneumophila, competence may be a response to genotoxic stress. Sunlight-emitted UV light represents a major source of genotoxic stress in the environment and we found that exposure to UV radiation effectively induces competence development. For the first time, we show that genetic exchanges by natural transformation occur within an UV-stressed population. Genotoxic stress induces the RecA-dependent SOS response in many bacteria. However, genetic and phenotypic evidence suggest that L. pneumophila lacks a prototypic SOS response and competence development in response to genotoxic stress is RecA independent. Our results strengthen the hypothesis that competence may have evolved as a DNA damage response in SOS-deficient bacteria. This parasexual response to DNA damage may have enabled L. pneumophila to acquire and propagate foreign genes, contributing to the emergence of this human pathogen.

Closely related organisms usually occupy similar ecological niches, leading to intense competition and even extinction. Such competition also can promote rapid phenotypic evolution and ecological divergence. This process may end with the stable occupation of distinct niches or, alternatively, may entail repeated bouts of evolution. Here we examine two Escherichia coli lineages, called L and S, that coexisted for more than 30,000 generations after diverging from a common ancestor. Both lineages underwent sustained phenotypic evolution based on global transcription and resource utilization profiles, with L seeming to encroach over time on the catabolic profile of S. Reciprocal invasion experiments with L and S clones from the same or different generations revealed evolutionary changes in their interaction, including an asymmetry that confirmed the encroachment by L on the niche of the S lineage. In general, L and S clones from the same generation showed negative frequency-dependent effects, consistent with stable coexistence. However, L clones could invade S clones from both earlier and later generations, whereas S clones could invade only L clones from earlier generations. In this system, the long-term coexistence of competing lineages evidently depended on successive rounds of evolution, rather than on initial divergence followed by a static equilibrium.

Understanding the extreme variation among bacterial genomes remains an unsolved challenge in evolutionary biology, despite long-standing debate about the relative importance of natural selection, mutation, and random drift. A potentially important confounding factor is the variation in mutation rates between lineages and over evolutionary history, which has been documented in several species. Mutation accumulation experiments have shown that hypermutability can erode genomes over short timescales. These results, however, were obtained under conditions of extremely weak selection, casting doubt on their general relevance. Here, we circumvent this limitation by analyzing genomes from mutator populations that arose during a long-term experiment with Escherichia coli, in which populations have been adaptively evolving for >50,000 generations. We develop an analytical framework to quantify the relative contributions of mutation and selection in shaping genomic characteristics, and we validate it using genomes evolved under regimes of high mutation rates with weak selection (mutation accumulation experiments) and low mutation rates with strong selection (natural isolates). Our results show that, despite sustained adaptive evolution in the long-term experiment, the signature of selection is much weaker than that of mutational biases in mutator genomes. This finding suggests that relatively brief periods of hypermutability can play an outsized role in shaping extant bacterial genomes. Overall, these results highlight the importance of genomic draft, in which strong linkage limits the ability of selection to purge deleterious mutations. These insights are also relevant to other biological systems evolving under strong linkage and high mutation rates, including viruses and cancer cells.

Predicting adaptive trajectories is a major goal of evolutionary biology and useful for practical applications. Systems biology has enabled the development of genome-scale metabolic models. However, analysing these models via flux balance analysis (FBA) cannot predict many evolutionary outcomes including adaptive diversification, whereby an ancestral lineage diverges to fill multiple niches. Here we combine in silico evolution with FBA and apply this modelling framework, evoFBA, to a long-term evolution experiment with Escherichia coli.Simulations predicted the adaptive diversification that occurred in one experimental population and generated hypotheses about the mechanisms that promoted coexistence of the diverged lineages. We experimentally tested and, on balance, verified these mechanisms, showing that diversification involved niche construction and character displacement through differential nutrient uptake and altered metabolic regulation.The evoFBA framework represents a promising new way to model biochemical evolution, one that can generate testable predictions about evolutionary and ecosystem-level outcomes.

Abstract Insertion sequences (IS) are ubiquitous bacterial mobile genetic elements, and the mutations they cause can be deleterious, neutral, or beneficial. The long-term dynamics of IS elements and their effects on bacteria are poorly understood, including whether they are primarily genomic parasites or important drivers of adaptation by natural selection. Here, we investigate the dynamics of IS elements and their contribution to genomic evolution and fitness during a long-term experiment with Escherichia coli . IS elements account for ~35% of the mutations that reached high frequency through 50,000 generations in those populations that retained the ancestral point-mutation rate. In mutator populations, IS-mediated mutations are only half as frequent in absolute numbers. In one population, an exceptionally high ~8-fold increase in IS 150 copy number is associated with the beneficial effects of early insertion mutations; however, this expansion later slowed down owing to reduced IS 150 activity. This population also achieves the lowest fitness, suggesting that some avenues for further adaptation are precluded by the IS 150 -mediated mutations. More generally, across all populations, we find that higher IS activity becomes detrimental to adaptation over evolutionary time. Therefore, IS-mediated mutations can both promote and constrain evolvability.

Abstract DNA supercoiling acts as a global transcriptional regulator in bacteria, that plays an important role in adapting their expression programme to environmental changes, but for which no quantitative or even qualitative regulatory model is available. Here, we focus on spatial supercoiling heterogeneities caused by the transcription process itself, which strongly contribute to this regulation mode. We propose a new mechanistic modeling of the transcription-supercoiling dynamical coupling along a genome, which allows simulating and quantitatively reproducing in vitro and in vivo transcription assays, and highlights the role of genes’ local orientation in their supercoiling sensitivity. Consistently with predictions, we show that chromosomal relaxation artificially induced by gyrase inhibitors selectively activates convergent genes in several enterobacteria, while conversely, an increase in DNA supercoiling naturally selected in a long-term evolution experiment with Escherichia coli favours divergent genes. Simulations show that these global expression responses to changes in DNA supercoiling result from fundamental mechanical constraints imposed by transcription, independently from more specific regulation of each promoter. These constraints underpin a significant and predictable contribution to the complex rules by which bacteria use DNA supercoiling as a global but fine-tuned transcriptional regulator.

Abstract As part of a long-term evolution experiment, two populations of Escherichia coli B adapted to a glucose minimal medium for 10,000 generations. In both populations, multiple IS-associated mutations arose that then went to fixation. We identify the affected genetic loci and characterize the molecular events that produced nine of these mutations. All nine were IS-mediated events, including simple insertions as well as recombination between homologous elements that generated inversions and deletions. Sequencing DNA adjacent to the insertions indicates that the affected genes are involved in central metabolism (knockouts of pykF and nadR), cell wall synthesis (adjacent to the promoter of pbpA-rodA), and ill-defined functions (knockouts of hokB-sokB and yfcU). These genes are candidates for manipulation and competition experiments to determine whether the mutations were beneficial or merely hitchhiked to fixation.

Abstract The genetic bases of adaptation are being investigated in 12 populations of Escherichia coli, founded from a common ancestor and serially propagated for 20,000 generations, during which time they achieved substantial fitness gains. Each day, populations alternated between active growth and nutrient exhaustion. DNA supercoiling in bacteria is influenced by nutritional state, and DNA topology helps coordinate the overall pattern of gene expression in response to environmental changes. We therefore examined whether the genetic controls over supercoiling might have changed during the evolution experiment. Parallel changes in topology occurred in most populations, with the level of DNA supercoiling increasing, usually in the first 2000 generations. Two mutations in the topA and fis genes that control supercoiling were discovered in a population that served as the focus for further investigation. Moving the mutations, alone and in combination, into the ancestral background had an additive effect on supercoiling, and together they reproduced the net change in DNA topology observed in this population. Moreover, both mutations were beneficial in competition experiments. Clonal interference involving other beneficial DNA topology mutations was also detected. These findings define a new class of fitness-enhancing mutations and indicate that the control of DNA supercoiling can be a key target of selection in evolving bacterial populations.

Bacterial populations can evolve and adapt to become diverse niche specialists, even in seemingly homogeneous environments. One source of this diversity arises from newly 'constructed' niches that result from the activities of the bacteria themselves. Ecotypes specialized to exploit these distinct niches can subsequently coexist via frequency-dependent interactions. Here, we describe a novel form of niche construction that is based upon differential death and cannibalism, and which evolved during 20 000 generations of experimental evolution in Escherichia coli in a seasonal environment with alternating growth and starvation. In one of 12 populations, two monophyletic ecotypes, S and L, evolved that stably coexist with one another. When grown and then starved in monoculture, the death rate of S exceeds that of L, whereas the reverse is observed in mixed cultures. As shown by experiments and numerical simulations, the competitive advantage of S cells is increased by extending the period of starvation, and this advantage results from their cannibalization of the debris of lysed L cells, which allows the S cells to increase both their growth rate and total cell density. At the molecular level, the polymorphism is associated with divergence in the activity of the alternative sigma factor RpoS, with S cells displaying no detectable activity, while L cells show increased activity relative to the ancestral genotype. Our results extend the repertoire of known cross-feeding mechanisms in microbes to include cannibalism during starvation, and confirm the central roles for niche construction and seasonality in the maintenance of microbial polymorphisms.

In case of nutritional stress, like carbon starvation, Escherichia coli cells abandon their exponential-growth state to enter a more resistant, non-growth state called stationary phase. This growth-phase transition is controlled by a genetic regulatory network integrating various environmental signals. Although E. coli is a paradigm of the bacterial world, it is little understood how its response to carbon starvation conditions emerges from the interactions between the different components of the regulatory network. Using a qualitative method that is able to overcome the current lack of quantitative data on kinetic parameters and molecular concentrations, we model the carbon starvation response network and simulate the response of E. coli cells to carbon deprivation. This allows us to identify essential features of the transition between exponential and stationary phase and to make new predictions on the qualitative system behavior following a carbon upshift.

Abstract Twelve populations of Escherichia coli evolved in and adapted to a glucose-limited environment from a common ancestor. We used two-dimensional protein electrophoresis to compare two evolved clones, isolated from independently derived populations after 20,000 generations. Exceptional parallelism was detected. We compared the observed changes in protein expression profiles with previously characterized global transcription profiles of the same clones; this is the first time such a comparison has been made in an evolutionary context where these changes are often quite subtle. The two methodologies exhibited some remarkable similarities that highlighted two different levels of parallel regulatory changes that were beneficial during the evolution experiment. First, at the higher level, both methods revealed extensive parallel changes in the same global regulatory network, reflecting the involvement of beneficial mutations in genes that control the ppGpp regulon. Second, both methods detected expression changes of identical gene sets that reflected parallel changes at a lower level of gene regulation. The protein profiles led to the discovery of beneficial mutations affecting the malT gene, with strong genetic parallelism across independently evolved populations. Functional and evolutionary analyses of these mutations revealed parallel phenotypic decreases in the maltose regulon expression and a high level of polymorphism at this locus in the evolved populations.

We conducted a cross-sectional study of Chagas disease in five endemic areas in Argentina, Bolivia, Honduras, and México to estimate the prevalence of Trypanosoma cruzi–specific antibodies in pregnant women, and to assess the use of a rapid test (Chagas Stat-Pak) to screen for T. cruzi infection at the time of delivery. The prevalence of antibodies to T. cruzi measured by enzyme-linked immunosorbent assay (ELISA) in maternal blood was 5.5% (a range of 0.8–28.8% among the countries) in 2,495 women enrolled. Compared with ELISA in maternal blood samples, the Chagas Stat-Pak rapid test sensitivity and specificity in umbilical cord blood were 94.6% and 99.0%, respectively. These results show the ability for a rapid determination of the presence of T. cruzi–specific antibodies in umbilical cord blood as a pragmatic strategy to screen for infection in pregnant women.

s S (RpoS), the master regulator of the general stress response in Escherichia coli, is a model system for regulated proteolysis in bacteria.s S turnover requires ClpXP and the response regulator RssB, whose phosphorylated form exhibits high af®nity for s S .Here, we demonstrate that recognition by the RssB/ClpXP system involves two distinct regions in s S .Region 2.5 of s S (a long a-helix) is suf®cient for binding of phosphorylated RssB.However, this interaction alone is not suf®cient to trigger proteolysis.A second region located in the N-terminal part of s S , which is exposed only upon RssB±s S interaction, serves as a binding site for the ClpX chaperone.Binding of the ClpX hexameric ring to s S -derived reporter proteins carrying the ClpX-binding site (but not the RssB-binding site) is also not suf®cient to commit the protein to degradation.Our data indicate that RssB plays a second role in the initiation of s S proteolysis that goes beyond targeting of s S to ClpX, and suggest a model for the sequence of events in the initiation of s S proteolysis.

Vector control has led to a drastic decrease in the prevalence of acquired Chagas disease in Latin America, thus redirecting attention to congenital Chagas disease. We report results of a longitudinal study of 359 pregnant women in Yacuiba in southern Bolivia, of whom 147 (40.9%) were infected with Trypanosoma cruzi, to evaluate the relationship between the patency period of the parasitemia and the risk of congenital infection. Maternal infection was assessed by using T. cruzi-specific serologic tests, and parasitemia in mothers and newborns was diagnosed by using microscopic examination of blood in heparinized microhematocrit tubes. Parasitemia was present in 28.6% of the infected women. Its prevalence increased during the third trimester, then decreased at delivery. The likelihood of congenital infection was significantly correlated with the parasite density in the mother's blood. The risk of transmission increased during the third trimester of pregnancy and could explain premature births or low-weight newborns for infected mothers.

The extent and nature of epistatic interactions between mutations are issues of fundamental importance in evolutionary biology. However, they are difficult to study and their influence on adaptation remains poorly understood. Here, we use a systems-level approach to examine epistatic interactions that arose during the evolution of Escherichia coli in a defined environment. We used expression arrays to compare the effect on global patterns of gene expression of deleting a central regulatory gene, crp. Effects were measured in two lineages that had independently evolved for 20,000 generations and in their common ancestor. We found that deleting crp had a much more dramatic effect on the expression profile of the two evolved lines than on the ancestor. Because the sequence of the crp gene was unchanged during evolution, these differences indicate epistatic interactions between crp and mutations at other loci that accumulated during evolution. Moreover, a striking degree of parallelism was observed between the two independently evolved lines; 115 genes that were not crp-dependent in the ancestor became dependent on crp in both evolved lines. An analysis of changes in crp dependence of well-characterized regulons identified a number of regulatory genes as candidates for harboring beneficial mutations that could account for these parallel expression changes. Mutations within three of these genes have previously been found and shown to contribute to fitness. Overall, these findings indicate that epistasis has been important in the adaptive evolution of these lines, and they provide new insight into the types of genetic changes through which epistasis can evolve. More generally, we demonstrate that expression profiles can be profitably used to investigate epistatic interactions.

ABSTRACT Peptidoglycan is the major component of the bacterial cell wall and is involved in osmotic protection and in determining cell shape. Cell shape potentially influences many processes, including nutrient uptake as well as cell survival and growth. Peptidoglycan is a dynamic structure that changes during the growth cycle. Penicillin-binding proteins (PBPs) catalyze the final stages of peptidoglycan synthesis. Although PBPs are biochemically and physiologically well characterized, their broader effects, especially their effects on organismal fitness, are not well understood. In a long-term experiment, 12 populations of Escherichia coli having a common ancestor were allowed to evolve for more than 40,000 generations in a defined environment. We previously identified mutations in the pbpA operon in one-half of these populations; this operon encodes PBP2 and RodA proteins that are involved in cell wall elongation. In this study, we characterized the effects of two of these mutations on competitive fitness and other phenotypes. By constructing and performing competition experiments with strains that are isogenic except for the pbpA alleles, we showed that both mutations that evolved were beneficial in the environment used for the long-term experiment and that these mutations caused parallel phenotypic changes. In particular, they reduced the cellular concentration of PBP2, thereby generating spherical cells with an increased volume. In contrast to their fitness-enhancing effect in the environment where they evolved, both mutations decreased cellular resistance to osmotic stress. Moreover, one mutation reduced fitness during prolonged stationary phase. Therefore, alteration of the PBP2 concentration contributed to physiological trade-offs and ecological specialization during experimental evolution.

DNA supercoiling is the master function that interconnects chromosome structure and global gene transcription. This function has recently been shown to be under strong selection in Escherichia coli. During the evolution of 12 initially identical populations propagated in a defined environment for 20,000 generations, parallel increases in DNA supercoiling were observed in ten populations. The genetic changes associated with the increased supercoiling were examined in one population, and beneficial mutations in the genes topA (encoding topoisomerase I) and fis (encoding a histone-like protein) were identified. To elucidate the molecular basis and impact of these changes, we quantified the level of genetic, phenotypic, and molecular parallelism linked to DNA supercoiling in all 12 evolving populations. First, sequence determination of DNA topology-related loci revealed strong genetic parallelism, with mutations concentrated in three genes (topA, fis, and dusB), although the populations had different alleles at each locus. Statistical analyses of these polymorphisms implied the action of positive selection and, moreover, suggested that fis and dusB, which belong to the same operon, have related functions. Indeed, we demonstrated that DusB regulates the expression of fis by both experimental and phylogenetic analyses. Second, molecular analyses of five mutations in fis and dusB affecting the transcription, translation, and protein activity of Fis also revealed strong parallelism in the resulting phenotypic effects. Third, artificially increasing DNA supercoiling in one of the two populations that lacked DNA topology changes led to a significant fitness increase. The high levels of molecular and genetic parallelism, targeting a small subset of the many genes involved in DNA supercoiling, indicate that changes in DNA superhelicity have been important in the evolution of these populations. Surprisingly, however, most of the evolved alleles we tested had either no detectable or slightly deleterious effects on fitness, despite these signatures of positive selection.

Francisella tularensis, a CDC class A potential bioterrorism agent, is a Gram-negative bacterium responsible for tularaemia. Understanding the mechanisms of resistance to antibiotics used as first-line treatment is of major security relevance. We propagated the three parental reference strains Francisella tularensis subsp. holarctica live vaccine strain, Francisella novicida and Francisella philomiragia with increasing concentrations of ciprofloxacin, a fluoroquinolone used as curative and prophylactic treatment for tularaemia. This evolution procedure provided us with high-level ciprofloxacin-resistant mutants and all evolutionary intermediates towards high-level resistance. We determined the resistance levels to other fluoroquinolones (levofloxacin and moxifloxacin) and other antibiotic families (aminoglycosides, tetracyclines and macrolides) and characterized the genetic changes in the fluoroquinolone target genes encoding DNA gyrase and topoisomerase IV. All high-level resistant mutants shared cross-resistance to the tested fluoroquinolones, while some also revealed striking levels of cross-resistance to other clinically relevant antibiotic classes. High-level resistant mutants carried one to three mutations, including some not previously reported. We mapped all mutations onto known topoisomerase three-dimensional structures. Along the pathways towards high-level resistance, we identified complex evolutionary trajectories including polymorphic states and additional resistance mechanisms likely to be associated with efflux processes. Our data demonstrated the efficiency and speed of in vitro production of mutants highly resistant to fluoroquinolones in Francisella species. They emphasize the urgent need to identify all antibiotic resistance mechanisms in these species, develop molecular tools for their detection and design new therapeutic alternatives for tularaemia.

Infectious diseases are the leading cause of human morbidity and mortality worldwide. One dramatic issue is the emergence of microbial resistance to antibiotics which is a major public health concern. Surprisingly however, such in vivo adaptive ability has not been reported yet for many intracellular human bacterial pathogens such as Legionella pneumophila.We examined 82 unrelated patients with Legionnaire's disease from which 139 respiratory specimens were sampled during hospitalization and antibiotic therapy. We both developed a real time PCR assay and used deep-sequencing approaches to detect antibiotic resistance mutations in L. pneumophila and follow their selection and fate in these samples.We identified the in vivo selection of fluoroquinolone resistance mutations in L. pneumophila in two infected patients treated with these antibiotics. By investigating the mutational dynamics in patients, we showed that antibiotic resistance occurred during hospitalization most likely after fluoroquinolone treatment.In vivo selection of antibiotic resistances in L. pneumophila may be associated with treatment failures and poor prognosis. This hidden resistance must be carefully considered in the therapeutic management of legionellosis patients and in the control of the gradual loss of effectiveness of antibiotics.

The ubiquitous ATP synthase uses an electrochemical gradient to synthesize cellular energy in the form of ATP. The production of this electrochemical gradient relies on liposoluble proton carriers like ubiquinone (UQ), which is used in the respiratory chains of eukaryotes and proteobacteria. The biosynthesis of UQ requires three hydroxylation reactions on contiguous positions of an aromatic ring. In Escherichia coli, each of three UQ flavin monooxygenases (FMOs), called UbiF, UbiH, and UbiI, modifies a single position of the aromatic ring. This pattern of three hydroxylation reactions/three proteins has been accepted as a paradigm in UQ biology. Using a phylogenetic analysis, we found that UbiF, UbiH, and UbiI are detected only in a small fraction of proteobacteria, and we identified two new types of UQ FMOs: UbiM, which is distributed in members of the alpha, beta, and gamma classes of proteobacteria, and UbiL, which is restricted to members of the alphaproteobacteria. Remarkably, the ubiL and ubiM genes were found in genomes with fewer than three UQ hydroxylase-encoding genes. We demonstrated, using biochemical approaches, that UbiL from Rhodospirillum rubrum and UbiM from Neisseria meningitidis hydroxylate, respectively, two and three positions of the aromatic ring during UQ biosynthesis. We conclude that bacteria have evolved a large repertoire of hydroxylase combinations for UQ biosynthesis, including pathways with either three specialist enzymes or pathways with one or two generalist enzymes of broader regioselectivity. The emergence of the latter is potentially related to genome reduction events. IMPORTANCE UQ, a key molecule for cellular bioenergetics that is conserved from proteobacteria to humans, appeared in an ancestral proteobacterium more than 2 billion years ago. UQ biosynthesis has been studied only in a few model organisms, and thus, the diversity of UQ biosynthesis pathways is largely unknown. In the work reported here, we conducted a phylogenomic analysis of hydroxylases involved in UQ biosynthesis. Our results support the existence of at least two UQ hydroxylases in the proteobacterial ancestor, and yet, we show that their number varies from one to four in extant proteobacterial species. Our biochemical experiments demonstrated that bacteria containing only one or two UQ hydroxylases have developed generalist enzymes that are able to catalyze several steps of UQ biosynthesis. Our study documents a rare case where evolution favored the broadening of an enzyme's regioselectivity, which resulted in gene loss in several proteobacterial species with small genomes.

To assess the impact of a daily oral iron supplementation on hematological status, cell-mediated immunity and susceptibility to infections in children living in an environment where iron deficiency, malaria and other infections are frequent.Randomized, double-blind iron supplementation including a placebo group.A village in Togo, West Africa.Of the 229 6-36-month-old children of both sexes recruited, 197 with hemoglobin concentration >/=80 g/l were included and 163 completed the study.Children received daily a placebo (n=79) or a dose of 2-3 mg of elemental iron per kg of body weight (n=84) for 3 months. Hematological, nutritional and immune status were assessed at the beginning and at the end of the supplementation period, and 6 months later. Morbidity was recorded throughout the study.Iron supplementation had a significant and positive effect on iron status of children and no impact on the incidence of infections, especially malaria. Its probable effect on immune status was masked by interference of infections and their treatment, which contributed to improve hematological and immune status in both groups.According to the negative consequences of anemia and iron deficiency on global child development, control of iron deficiency by oral iron supplementation in young children has to be conducted, associated with prophylaxis and treatment of malaria and repeated deworming.Program supported by IRD. European Journal of Clinical Nutrition (2000) 54, 29-35

This study aims to typify the Trypanosoma cruzi (sub)lineage(s) in umbilical cord blood of congenitally infected Bolivian newborns, using PCR amplifications of "Region Markers", mini-exon or kDNA fragments followed by hybridization or sequencing. New probes were also designed to distinguish three variants within the TcIId sublineage. The IIb, IId, or IIe T. cruzi sublineages, as well as different variants of the IId sublineage, were detected in infected neonates, whereas mixed infections were not found. The frequencies of the IId sublineage were similar in neonates (95.1%) and adults of the same area (94.1%). The IId-infected newborns displayed either asymptomatic, or severe and fatal clinical forms of congenital Chagas disease, as well as low or high parasitemia. Altogether these data show that T. cruzi DNA polymorphism, based on the presently available markers, is not associated with the occurrence of congenital infection or the development of severe clinical forms of congenital Chagas disease.

Summary Background Comparing two surveys performed in Bolivia in 1992–1994 and 1999–2001, we reported a significant decrease in the proportions of severe and mortal forms of congenital Chagas disease. This might be due to a reduction of vectorial density (VD) in maternal residence area, raising the question of a possible causal relationship between such VD, maternal parasitaemia and prognosis of congenital infection with Trypanosoma cruzi. Method Comparisons of haematological and parasitological data obtained from Bolivian mothers infected with T. cruzi, and of clinical and biological data obtained from their infected and uninfected newborns, stratified according to VD in the area of maternal residence. Results i) Blood hematocrit rates or hemoglobin amounts were within the normal ranges and similar in all the maternal groups, whatever the VD in their areas of residence; ii) mothers living in high VD areas displayed a higher frequency of hemocultures positive for T. cruzi; iii) newborns congenitally infected with T. cruzi, but not uninfected babies born from infected mothers, displayed higher frequencies of very low Apgar scores, low birth weights, prematurity, respiratory distress syndrome or anasarca, as well as higher mortality rates when their mothers lived in areas of high VD. Conclusion Frequent bites of blood sucking Reduvidae during pregnancy do not induce maternal anaemia, but, likely through multiple maternal re‐infections with T. cruzi, increase maternal parasitemia and worsen congenital Chagas disease. Maternal dwelling in areas of high VD is associated with a serious increased risk of severe and mortal congenital Chagas disease.

Summary Objective To determine the risk factors of congenital Chagas disease and the consequences of the disease in newborns. Methods Study of 2712 pregnant women and 2742 newborns in Yacuiba, south Bolivia. Chagas infection was determined serologically in mothers and parasitologically in newborns. Consequences of congenital Chagas disease were assessed clinically. Results The prevalence of Chagas disease in pregnant women was 42.2%. Congenital transmission was estimated at 6% of infected mothers leading to an incidence rate of 2.6% among newborns. Main risk factors of congenital transmission were mothers’ seropositivity and maternal Trypanosoma cruzi parasitaemia. Parity was higher in infected than in non‐infected mothers, but it was not associated with the risk of congenital transmission. The rate of congenital infection was significantly higher in newborns from multiple pregnancies than in singletons. However, we did not observe statistically significant consequences of Chagas disease in newborns from single pregnancies or among twins. Conclusions The main risk factors for congenital transmission were infection and parasitaemia of mothers. Consequences of the disease seemed mild in newborns from single pregnancies and perhaps more important in multiple births.

Radiotherapy remains the foundation of current treatment for patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN). It has been shown that the addition of concurrent chemotherapy to radiotherapy (chemoradiotherapy, CRT, or chemotherapy-enhanced radiation therapy, CERT) results in improved clinical outcome in terms of both locoregional control and overall survival in some groups of patients. However, CRT is associated with severe, dose-limiting acute toxicities and, in some patients, a higher proportion of late toxicities. In addition, most CRT regimens are platinum-based and there is evidence that the maximum tolerable toxicity has been reached with the dose intensities currently used in bolus cisplatin regimens. Therefore, if we are to further improve outcomes through increased treatment compliance, more effective and more tolerable regimens are needed. Recent results from a phase III randomised study demonstrate that the epidermal growth factor receptor (EGFR) inhibitor cetuximab (Erbitux)given concomitantly with radiotherapy yields a significant clinical benefit over radiotherapy alone without any increase in radiotherapy-associated toxicity. In this review, we explore the question of the degree to which adding cetuximab improves the efficacy of radiotherapy in locally advanced SCCHN and how the benefits of cetuximab plus radiotherapy compare with those achievable with CRT.

The dynamics of host susceptibility to parasites are often influenced by trade-offs between the costs and benefits of resistance. We assayed changes in the resistance to three viruses in six lines of Escherichia coli that had been evolving for almost 45,000 generations in their absence. The common ancestor of these lines was completely resistant to T6, partially resistant to T6* (a mutant of T6 with altered host range), and sensitive to λ. None of the populations changed with respect to resistance to T6, whereas all six evolved increased susceptibility to T6*, probably ameliorating a cost of resistance. More surprisingly, however, the majority of lines evolved complete resistance to λ, despite not encountering that virus during this period. By coupling our results with previous work, we infer that resistance to λ evolved as a pleiotropic effect of a beneficial mutation that downregulated an unused metabolic pathway. The strong parallelism between the lines implies that selection had almost deterministic effects on the evolution of these patterns of host resistance. The opposite outcomes for resistance to T6* and λ demonstrate that the evolution of host resistance under relaxed selection cannot be fully predicted by simple trade-off models.

The authors carried out a 1-year study of a population of pregnant women delivering at Bermejo hospital, South Bolivia. In this area, vectorial transmission of Trypanosoma cruzi is negligible and women infect themselves during displacements in close endemic areas. The prevalence of T. cruzi in 508 pregnant women, diagnosed by several serological tests, was 33.9%. In eight infants, we observed T. cruzi in the umbilical cord (congenital transmission rate of 5.2%). The means of birth weights, lengths and hemoglobin rates were similar in the children from both seronegative and seropositive women, and in children infected or not by T. cruzi. This study could confirm a less severity of the congenital disease of Chagas in the absence of re-infestation of the mother during pregnancy. Serological screening of pregnant women by rapid diagnostic tests and examination of babies born from seropositive mothers by microhematocrit method at birth is a suitable strategy to detect and prevent congenital Chagas disease in non-endemic areas.

We investigated the interactions between snowpack chemistry, mercury (Hg) contamination and microbial community structure and function in Arctic snow. Snowpack chemistry (inorganic and organic ions) including mercury (Hg) speciation was studied in samples collected during a two-month field study in a high Arctic site, Svalbard, Norway (79 °N). Shifts in microbial community structure were determined by using a 16S rRNA gene phylogenetic microarray. We linked snowpack and meltwater chemistry to changes in microbial community structure by using co-inertia analyses (CIA) and explored changes in community function due to Hg contamination by q-PCR quantification of Hg-resistance genes in metagenomic samples. Based on the CIA, chemical and microbial data were linked (p = 0.006) with bioavailable Hg (BioHg) and methylmercury (MeHg) contributing significantly to the ordination of samples. Mercury was shown to influence community function with increases in merA gene copy numbers at low BioHg levels. Our results show that snowpacks can be considered as dynamic habitats with microbial and chemical components responding rapidly to environmental changes.

Insertion Sequence (IS) elements are mobile genetic elements widely distributed among bacteria. Their activities cause mutations, promoting genetic diversity and sometimes adaptation. Previous studies have examined their copy number and distribution in Escherichia coli K-12 and natural isolates. Here, we map most of the IS elements in E. coli B and compare their locations with the published genomes of K-12 and O157:H7.The genomic locations of IS elements reveal numerous differences between B, K-12, and O157:H7. IS elements occur in hok-sok loci (homologous to plasmid stabilization systems) in both B and K-12, whereas these same loci lack IS elements in O157:H7. IS elements in B and K-12 are often found in locations corresponding to O157:H7-specific sequences, which suggests IS involvement in chromosomal rearrangements including the incorporation of foreign DNA. Some sequences specific to B are identified, as reported previously for O157:H7. The extent of nucleotide sequence divergence between B and K-12 is < 2% for most sequences adjacent to IS elements. By contrast, B and K-12 share only a few IS locations besides those in hok-sok loci. Several phenotypic features of B are explained by IS elements, including differential porin expression from K-12.These data reveal a high level of IS activity since E. coli B, K-12, and O157:H7 diverged from a common ancestor, including IS association with deletions and incorporation of horizontally acquired genes as well as transpositions. These findings indicate the important role of IS elements in genome plasticity and divergence.

Abstract The loss of preexisting genes or gene activities during evolution is a major mechanism of ecological specialization. Evolutionary processes that can account for gene loss or inactivation have so far been restricted to one of two mechanisms: direct selection for the loss of gene activities that are disadvantageous under the conditions of selection (i.e., antagonistic pleiotropy) and selection-independent genetic drift of neutral (or nearly neutral) mutations (i.e., mutation accumulation). In this study we demonstrate with an evolved strain of Escherichia coli that a third, distinct mechanism exists by which gene activities can be lost. This selection-dependent mechanism involves the expropriation of one gene’s upstream regulatory element by a second gene via a homologous recombination event. Resulting from this genetic exchange is the activation of the second gene and a concomitant inactivation of the first gene. This gene-for-gene expression tradeoff provides a net fitness gain, even if the forfeited activity of the first gene can play a positive role in fitness under the conditions of selection.

Abstract BACKGROUND. Mutations in the Wilms tumor (WT) suppressor 1 gene ( WT1 ) and the cadherin‐associated protein β1 gene ( CTNNB1 ) are found predominantly in stromal type WT, defining a genetic subgroup. The clinical relevance of these mutations remains to be determined. METHODS. A long‐term follow‐up study was performed for 71 patients (International Society of Pediatric Oncology Study 9/Society for Pediatric Oncology; n = 77 tumors) with known molecular genetic status. Eight patients had bilateral disease, including 2 patients with a WT in both kidneys and 5 patients with a WT in 1 kidney and nephrogenic rests (NRs) in the other kidney. The response to preoperative chemotherapy, relapses, metastases, metachronous tumor development, and deaths were evaluated with a median follow‐up of 12 years and 4 months. RESULTS. Nineteen patients (n = 24 tumors) had WT1 mutations, and 16 were constitutional mutations. Three patients with germline mutations had second tumor events: Two patients developed a WT in the kidney with NRs 3 years and 11 years after the first tumor; and 1 patient developed second tumors after 2 years, 1 in the kidney with a previous WT and 1 in the kidney with a previous NR. Eighteen of the WT1 mutant tumors were analyzed for CTNNB1 mutations, and all had mutations. A poor volumetric response (progression and &lt;50% reduction) was observed in all patients who had tumors with a WT1 mutation and in 23 of 52 nonmutant tumors. CONCLUSIONS. Patients with WT1 germline mutations had an increased risk for bilateral disease and second tumor events. Therefore, the authors concluded that tumor surveillance until adulthood should be considered. Although tumors with both WT1 and CTNNB1 mutations had a poor volumetric response, there was no significant difference in overall survival in this cohort of patients with and without WT1 mutations. Cancer 2008. © 2008 American Cancer Society.

We evaluated the prevalence of Chagas disease using a rapid screening test (Chagas Stat-Pak), confirmed by ELISA, in Caraparí, a village of 9000 inhabitants in southern Bolivian Chaco. The prevalence of Trypanosoma cruzi was estimated in a sample of 995 people. The prevalence adjusted on age was 51.2% and was proportionally related to age. We also observed a very significant cline from the south to the north of the locality, where the prevalence ranged from 40 to 80%. In children younger than 11 years, the prevalence was 21.5%, which confirmed the importance of residual vector transmission despite several years of vector control. Among women of procreation age, the prevalence was 63.9%, resulting in a high risk of congenital transmission. The control of the disease requires an increase in vector control and improvement of dwellings before considering children's treatment with trypanocide.

Fluoroquinolone resistance has been poorly studied in Legionella pneumophila, an intracellular pathogen responsible for legionellosis. Our goal was to further characterize molecular mechanisms involved in fluoroquinolone resistance in this species. Eight independent lineages were founded from a common fluoroquinolone-susceptible L. pneumophila ancestor and propagated by serial passages in moxifloxacin-containing culture medium. We identified the substituted mutations that affected the DNA topoisomerase II-encoding genes, determined the order of substitution of the mutations leading to the stepwise MIC increases of moxifloxacin over evolutionary time and demonstrated their direct involvement in the resistance process. Adaptation occurred through parallel stepwise increases in the moxifloxacin MICs up to 512-fold the MIC for the parental strain. Mutations affected the topoisomerase II-encoding genes gyrA, parC and gyrB, reflecting a high degree of genetic parallelism across the independent lineages. During evolution, the T83I change in GyrA occurred first, followed by G78D or S80R in ParC and D87N in GyrA, or S464Y or D426N in GyrB. By constructing isogenic strains, we showed that the progressive increase in resistance was linked to a precise order of mutation substitution, but also to the co-existence of several subpopulations of bacteria bearing different mutations. Specific mutational trajectories were identified, strongly suggesting that intermolecular epistatic interactions between DNA topoisomerases underlie the mechanism of fluoroquinolone resistance in L. pneumophila. Our results suggest that L. pneumophila has strong potential to become resistant to fluoroquinolone compounds and warrant further investigation of resistance in clinical and environmental strains of this pathogen.

Abstract The synthesis of 37 1‐(1 H ‐indol‐3‐yl)ethanamine derivatives, including 12 new compounds, was achieved through a series of simple and efficient chemical modifications. These indole derivatives displayed modest or no intrinsic anti‐staphylococcal activity. By contrast, several of the compounds restored, in a concentration‐dependent manner, the antibacterial activity of ciprofloxacin against Staphylococcus aureus strains that were resistant to fluoroquinolones due to overexpression of the NorA efflux pump. Structure–activity relationships studies revealed that the indolic aldonitrones halogenated at position 5 of the indole core were the most efficient inhibitors of the S. aureus NorA efflux pump. Among the compounds, ( Z )‐ N ‐benzylidene‐2‐( tert ‐butoxycarbonylamino)‐1‐(5‐iodo‐1 H ‐indol‐3‐yl)ethanamine oxide led to a fourfold decrease of the ciprofloxacin minimum inhibitory concentration against the SA‐1199B strain when used at a concentration of 0.5 mg L −1 . To the best of our knowledge, this activity is the highest reported to date for an indolic NorA inhibitor. In addition, a new antibacterial compound, tert ‐butyl (2‐(3‐hydroxyureido)‐2‐(1 H ‐indol‐3‐yl)ethyl)carbamate, which is not toxic for human cells, was also found.

Resistance to antibiotics often evolves when bacteria encounter antibiotics. However, bacterial strains and species without any known exposure to these drugs also vary in their intrinsic susceptibility. In many cases, evolved resistance has been shown to be costly to the bacteria, such that resistant types have reduced competitiveness relative to their sensitive progenitors in the absence of antibiotics. In this study, we examined changes in the susceptibilities of 12 populations of Escherichia coli to 15 antibiotics after 2,000 and 50,000 generations without exposure to any drug. The evolved bacteria tended to become more susceptible to most antibiotics, with most of the change occurring during the first 2,000 generations, when the bacteria were undergoing rapid adaptation to their experimental conditions. On balance, our findings indicate that bacteria with low levels of intrinsic resistance can, in the absence of relevant selection, become even more susceptible to antibiotics.

Microbial evolution experiments offer a powerful approach for coupling changes in complex phenotypes, including fitness and its components, with specific mutations. Here we investigate mutations substituted in 15 lines of Escherichia coli that evolved for 1000 generations under freeze-thaw-growth (FTG) conditions. To investigate the genetic basis of their improvements, we screened many of the lines for mutations involving insertion sequence (IS) elements and identified two genes where multiple lines had similar mutations. Three lines had IS150 insertions in cls, which encodes cardiolipin synthase, and 8 lines had IS150 insertions in the uspA-uspB intergenic region, encoding two universal stress proteins. Another line had an 11-bp deletion mutation in the cls gene. Strain reconstructions and competitions demonstrated that this deletion is beneficial under the FTG regime in its evolved genetic background. Further experiments showed that this cls mutation helps maintain membrane fluidity after freezing and thawing and improves freeze-thaw (FT) survival. Reconstruction of isogenic strains also showed that the IS150 insertions in uspA/B are beneficial under the FTG regime. The evolved insertions reduce uspB transcription and increase both FT survival and recovery, but the physiological mechanism for this fitness improvement remains unknown.

Francisella tularensis subsp. holarctica strains are classified as biovars I and II, which are susceptible and naturally resistant to the macrolide erythromycin, respectively. The present study was aimed at both selecting biovar I strains with increased levels of erythromycin resistance and characterizing the underlying genetic mechanisms. Serial cultures in the presence of increasingly high erythromycin concentrations were performed to select independent high- and intermediate-level erythromycin-resistant mutants from each of three different biovar I strains. The mutants were characterized for cross-resistance to several antibiotics, presence of mutations in the genes encoding the 23S rRNA and the L4 and L22 ribosomal proteins, and overexpression of efflux pumps. Mutants displayed cross-resistance to all macrolide compounds tested but not to other classes of antibiotics. We found mutations in domain V of the 23S rRNA gene (G2057A, A2058G, A2058T and C2611T) and in the gene encoding L22, leading to either the G91D substitution or the M82K83R84 deletion. Analysis of mutants with intermediate resistance levels obtained over the course of the selection process revealed both a positive correlation between the number of mutated ribosomal operons and the resistance level, and an additional resistance mechanism in the early steps of selection. We showed that high-level resistance to macrolides can be easily obtained in vitro in F. tularensis subsp. holarctica biovar I strains, thereby suggesting that in vivo selection for resistance may explain reported failures of antibiotic treatment. Ketolides were the most effective macrolides tested, which may limit the risk of selection for resistance.

Plasmodium vivax is a major cause of illness in areas with low transmission of malaria in Latin America, Asia, and the Horn of Africa. However, pregnancy-associated malaria remains poorly characterized in such areas. Using a hospital-based survey of women giving birth and an antenatal survey, we assessed the prevalence rates of Plasmodium spp. infections in pregnant women in Bolivia, and evaluated the consequences of malaria during pregnancy on the health of mothers and newborns. P. vivax infection was detected in 7.9% of pregnant women attending antenatal visits, and placental infection occurred in 2.8% of deliveries; these rates did not vary with parity. Forty-two percent of all P. vivax malaria episodes were symptomatic. P. vivax-infected pregnant women were frequently anemic (6.5%) and delivered babies of reduced birthweight. P. vivax infections during pregnancy are clearly associated with serious adverse outcomes and should be considered in prevention strategies of pregnancy-associated malaria.

Congenital transmission of Chagas disease stand out as a major public health problem since the vector control was performed in all endemic areas and has shown its effectiveness. An epidemiological study was performed in three maternity hospitals of the city of Santa Cruz de la Sierra, Bolivia from 2006 to 2008. The serological screening for Trypanosoma cruzi infection was carried out in 15,767 pregnant women. Chagas infection was detected in 3725 women (23.6%), who gave birth to 125 newborns infected by T. cruzi at birth, representing an incidence of 790 per 100,000 births during a period of 16 months and a vertical transmission rate by 3.4%. There was a significant difference between hospitals that might be explained by socio-economic origins of mothers and diagnostic constraints.

Francisella tularensis is a Gram-negative bacterium causing tularaemia. Classified as possible bioterrorism agent, it may be transmitted to humans via animal infection or inhalation leading to severe pneumonia. Its virulence is related to iron homeostasis involving siderophore biosynthesis directly controlled at the transcription level by the ferric uptake regulator Fur, as presented here together with the first crystal structure of the tetrameric F. tularensis Fur in the presence of its physiological cofactor, Fe2+. Through structural, biophysical, biochemical and modelling studies, we show that promoter sequences of F. tularensis containing Fur boxes enable this tetrameric protein to bind them by splitting it into two dimers. Furthermore, the critical role of F. tularensis Fur in virulence and pathogenesis is demonstrated with a fur-deleted mutant showing an attenuated virulence in macrophage-like cells and mice. Together, our study suggests that Fur is an attractive target of new antibiotics that attenuate the virulence of F. tularensis.

This study aims to evaluate tumor motion with controlled changes of the bladder volume, and to assess the reproducibility of bladder (and tumor) position using a urinary catheter balloon as an immobilization device.First, three patients with tumor growths in three different bladder regions (trigone, left lateral wall, anterior wall) were evaluated. Three-dimensional CT-based reconstructed images were used to measure the displacement of the tumors when 100 cc were removed from a bladder originally filled with 170 cc of contrast. The 3D calculated boost beam arrangements and field sizes for the three tumors in the partially emptied bladders were used to simulate treatment of the same tumors in the maximally filled bladders. Dose-volume histograms were obtained. Second, verification of an ellipsoid model for bladder volume changes was undertaken in 41 patients. Third, in eight additional patients a urinary catheter balloon filled with 80-cc sterile saline solution was used in an attempt to reproduce the shape and spatial coordinates of the bladder during the boost treatment. A pair of orthogonal films with the 80-cc balloon filled with contrast material were taken at simulation and repeated twice at weekly intervals during radiotherapy. The reproducibility was quantified by sequentially calculating the common surface of the bladder images in each orthogonal view.Target motion, especially in the craniocaudal axis, appeared to be more relevant for tumors arising in the bladder walls (15 mm) than in the trigone (5 mm). Underdosage (<95% of the prescribed dose to the target volume) was observed in 20, 20, and 50% (with 1 cm margins around the tumor) and in 10, 10, and 15% (with 1.5 cm margins around the tumor) of the tumors arising in the trigone, left lateral wall, and anterior wall, respectively. The ellipsoidal model was validated with a strong correlation coefficient allowing to establish a predictive model for bladder wall displacements as a function of bladder volume. In the balloon reproducibility study, mean reproducibility factors of 0.84 (+/-0.06) and 0.82 (+/-0.07) were obtained for both anteroposterior and lateral views, respectively.Changes in bladder volume and shape related to bladder filling can result in clinically significant displacements of the target volume. A minimum of 2-cm margins around the target may compensate for extreme bladder volume changes during boost treatment. An ellipsoidal model for the bladder is consistent with these observations. Although an 80-cc urinary catheter balloon helped to immobilize the bladder, reproducibility was less than perfect.

2-(2(')-Hydroxyphenyl)benzenesulfinate desulfinase (HPBS desulfinase) catalyzes the cleavage of the carbon-sulfur bond of 2-(2(')-hydroxyphenyl)benzenesulfinate (HPBS) to form hydroxybiphenyl and sulfite. This is the final step in the desulfurization of dibenzothiophene, the organosulfur compound used to study biodesulfurization of petroleum middle distillate. HPBS desulfinase was purified 1600-fold from Rhodococcus IGTS8. The purification was monitored using a spectrofluorimetric assay and SDS-PAGE. The pI of HPBS desulfinase is 5.6, the temperature optimum is 35 degrees C, and the pH optimum is 7.0. HPBS desulfinase has a K(m) of 0.90+/-0.15 microM and a k(cat) of 1.3+/-0.07 min(-1). Several analogs were tested for their ability to act as substrates or inhibitors of HPBS desulfinase. No alternative substrates and very few inhibitors were identified. HPBS desulfinase activity decreases in the presence of Cu(2+) and Zn(2+), while no metals significantly enhance enzyme activity. HPBS desulfinase is susceptible to tyrosine, tryptophan, and cysteine specific modification agents.

The lvh region of the Legionella pneumophila genome, which encodes a type IV secretion system, is located on a plasmid-like element in strains Paris (pP36) and Philadelphia (pLP45). The pP36 element has been described either integrated in the chromosome or excised as a multi-copy plasmid, in a similar manner to pLP45. In this paper, the chromosomal integration of pP36 in the Paris strain genome was described, occurring through site-specific recombination at the 3′ end of a transfer-messenger RNA gene by recombination between attachment sites, in a similar manner to pathogenicity islands. This integration was growth-phase dependent, occurring during the exponential phase. Several pP36-borne genes were expressed during the lag phase of bacterial growth, coinciding with the peak amount of the episomal form of pP36. Expression of the same genes decreased during the exponential and stationary phases, owing to the integration phenomenon and a loss of episomal copies of pP36. A similar plasmid-like element was described in the Lens strain genome, suggesting that the mobility of the lvh region is a phenomenon widespread among Legionella sp.

Genome-wide profiling has allowed the regulatory interaction networks of many organisms to be visualised and the pattern of connections between genes to be studied. These networks are non-random, following a power-law distribution with a small number of well-connected 'hubs' and many genes with only one or a few connections. Theoretical work predicts that power-law networks display several unique properties. One of the most biologically interesting of these is an intrinsic robustness to disturbance such that removal of a random gene will have little effect on network function. Conversely, targeted removal of a hub gene is expected to have a large effect.We compared the response of Escherichia coli to environmental and mutational stress following disruption of random or hub genes. We found that disruption of random genes had less effect on robustness to environmental stress than did the targeted disruption of hub genes. In contrast, random disruption strains were slightly less robust to the effect of mutational stress than were hub disruption strains. When we compared the effect of each disruption on environmental and mutational stress, we found a negative relationship, such that strains that were more environmentally robust tended to be less robust to mutational stress.Our results demonstrate that mutant strains of E. coli respond differently to stress, depending on whether random or hub genes are disrupted. This difference indicates that the power-law distribution of regulatory interactions has biological significance, making random disruptions less deleterious to organisms facing environmental stress. That E. coli can reduce the effect of environmental stress without reducing the phenotypic effect of additional mutations, indicates that robustness and evolvability need not be antagonistic.

Summary Objective To compare the results of an immunochromatographic test performed on whole blood, Chagas Stat‐Pak ® , with those of an ELISA test using recombinant antigens. Method We tested 995 subjects of a rural population of all ages in the south of Bolivia, 459 pregnant women of the same population and 1030 urban women giving birth from the east of Bolivia. Results The sensitivity of the CSP test for the entire studied population ( n = 2484) was 94.73% [93.35–96.10]; the specificity was 97.33% [96.50–98.15]. However, the specificity differed significantly between rural pregnant and urban birthing women, which could be attributed either to differences of parasite strain or Chagas prevalence. Conclusion The test is simple of use, reliable, relatively inexpensive (&lt;2 US$ each test) and its performances are compatible with a field use for large‐scale screenings.

Patients with early-stage cervical cancer may be treated appropriately with either radical surgery or radiation therapy. As most patients will be cured of their disease, side-effects of therapy and quality of life become of great importance. Individualization of treatment to reduce therapy-associated morbidity should be the main goal in cervical cancer management. Recent developments in surgical techniques, such as laparoscopy, nerve-sparing radical hysterectomy, sentinel lymph node biopsy, trachelectomy and ‘less radical’ hysterectomy, have contributed to reduce the morbidity of the surgical treatment. The use of postoperative radiotherapy or chemoradiation leads to more pronounced side effects than after either surgery or irradiation alone. Therefore, prognostic factors should be used to select patients for either surgery or radiotherapy alone to minimize the increased toxicities associated with the combination. The objectives of this review are to discuss the evidence supporting radical surgery, ‘less radical’ surgery and radiotherapy with regard to complication rate and quality of life.

Summary Objective To demonstrate the feasibility of a house‐to‐house screening system used for congenital Chagas disease in rural areas based on an active search for pregnant women and newborns in their homes in addition to passive case detection in health facilities. Methods Exploratory phase conducted by the research team followed by an operational period coordinated by municipal health service. A blood sample was taken for serological and parasitological tests of Trypanosoma cruzi from pregnant women who were searching antenatal care or visited at home by field investigators. Infants born to T. cruzi ‐infected women were examined for infection at birth and again at 1 and 7 months of age. Results 64.5% of the pregnant women were infected. Congenital infection was diagnosed at birth in 4.0% (12/299) of the children born to seroreactive mothers. Twelve additional cases of infection (4%) were diagnosed in children between 1 and 7 months of age. Finally, 37% of the children were lost to follow‐up in the exploratory phase and 53% during the operational phase ( P = 0.002), significantly fewer than in most passive case detection studies. Conclusion Despite poorer outcomes after door‐to‐door screening activities have been transferred to the health system, a combined strategy based on active and passive case detection appeared to be efficient for identifying rural cases of congenital Chagas disease.

DNA duplications constitute important precursors for genome variation. Here we analyzed an unequal duplication harboring a beneficial mutation that may provide alternative evolutionary outcomes.We characterized this evolutionary event during experimental evolution for only 100 generations of an Escherichia coli strain under glucose limitation within chemostats. By combining Insertion Sequence based Restriction Length Polymorphism experiments, pulsed field gel electrophoresis and two independent genome re-sequencing experiments, we identified an evolved lineage carrying a 180 kb duplication of the 46' region of the E. coli chromosome. This evolved duplication revealed a heterozygous state, with one copy harboring a 2668 bp deletion that included part of the ogrK gene and both the yegR and yegS genes. By genetically manipulating ancestral and evolved strains, we showed that the single yegS inactivation was sufficient to confer a frequency dependent fitness increase under the chemostat selective conditions in both the ancestor and evolved genetic contexts, implying that the duplication itself was not a direct fitness contributor. Nonetheless, the heterozygous duplicated state was relatively stable in the conditions prevailing during evolution in chemostats, in striking contrast to non selective conditions in which the duplication resolved at high frequency into either its ancestral or deleted copy.Our results suggest that the duplication state may constitute a second order selection process providing higher evolutionary potential. Moreover, its heterozygous nature may provide differential evolutionary opportunities in alternating environments. Our results also highlighted how careful analyses of whole genome data are needed to identify such complex rearrangements.

Metabolic cross-feeding interactions between microbial strains are common in nature, and emerge during evolution experiments in the laboratory, even in homogeneous environments providing a single carbon source. In sympatry, when the environment is well-mixed, the reasons why emerging cross-feeding interactions may sometimes become stable and lead to monophyletic genotypic clusters occupying specific niches, named ecotypes, remain unclear. As an alternative to evolution experiments in the laboratory, we developed Evo2Sim, a multi-scale model of in silico experimental evolution, equipped with the whole tool case of experimental setups, competition assays, phylogenetic analysis, and, most importantly, allowing for evolvable ecological interactions. Digital organisms with an evolvable genome structure encoding an evolvable metabolic network evolved for tens of thousands of generations in environments mimicking the dynamics of real controlled environments, including chemostat or batch culture providing a single limiting resource. We show here that the evolution of stable cross-feeding interactions requires seasonal batch conditions. In this case, adaptive diversification events result in two stably co-existing ecotypes, with one feeding on the primary resource and the other on by-products. We show that the regularity of serial transfers is essential for the maintenance of the polymorphism, as it allows for at least two stable seasons and thus two temporal niches. A first season is externally generated by the transfer into fresh medium, while a second one is internally generated by niche construction as the provided nutrient is replaced by secreted by-products derived from bacterial growth. In chemostat conditions, even if cross-feeding interactions emerge, they are not stable on the long-term because fitter mutants eventually invade the whole population. We also show that the long-term evolution of the two stable ecotypes leads to character displacement, at the level of the metabolic network but also of the genome structure. This difference of genome structure between both ecotypes impacts the stability of the cross-feeding interaction, when the population is propagated in chemostat conditions. This study shows the crucial role played by seasonality in temporal niche partitioning and in promoting cross-feeding subgroups into stable ecotypes, a premise to sympatric speciation.

In the last ten years ivermectin appeared an efficient and safe alternative to diethylcarbamazine which is known to induce severe adverse reactions in loiasis, including encephalitis. After these results, large scale ivermectin treatments against onchocerciasis were carried out in Central Africa where loiasis is also endemic; and seven cases of severe reaction were reported in Cameroon since 1991, during these mass ivermectin treatments. In order to study adverse reactions in patients harbouring high load of Loa loa microfilariae (mf), we realized careful hospital based treatment in 112 patients with more than 3,000 mf/ml (ml) blood. Patients received once 200 micrograms ivermectin per kilogram at day 0 (D0). Clinical examination was made daily during the four following days (D1 to D4). Blood and urine samples were analysed before treatment and at D1 and D3. Lumbar puncture was made at D1 for 39 patients with more than 10,000 mf/ml; at D3 for the 49 following patients without consideration for the level of parasitaemia, and at D0 and D3 for ten voluntary patients. For analysis the patients were distributed in 3 groups according to initial parasitaemia: the first group included 50% out of the patients, those whose parasitaemia was fewer than 15,000 mf/ml blood; the second group included 25% patients whose parasitaemia was between 15,000 and 30,000 mf/ml; the third group included the last 25% patients whose parasitaemia was higher than 30,000 mf per ml blood. Adverse reactions were observed in 71% out of the patients. Symptoms described were fever, pruritus, headache, arthralgia. Most symptoms appeared 24 to 36 hours after treatment. Temperature increased significantly in group 3. Microfilaraemia decreased by 85% in the 3 groups during the 4 days following treatment. C-reactive-protein increased dramatically after treatment in all patients (p < 10(-4)). Some patients presented blood in urine in three groups but haematuria reached 35% of patients in group 3. Proteinuria is noted among 33% of all patients but 20% in group 1 and 2 versus 70% in group 3. Loa loa mf were observed in urine of half the patients, but in low amounts (< 10 mf per 50 ml urine). In cerebro-spinal fluid (CSF), some mf appeared at D1 or D3 in people heavily infected with Loa loa, reaching 80% of the patients of group 3. LP made at D0 in ten patients with parasitaemia higher than 30,000 mf/ml blood confirmed that CSF was naturally microfilaria free before treatment. One patient presented severe troubles with fever, asthenia and conscience troubles beginning at D3, reactive coma at D4, renal impairment with transitory anuria; progressive improvement in 2 weeks and complete recovery at D22; he presented 102 mf/ml CSF at D6. The study confirmed that ivermectin treatment is generally well tolerated. Among people with high Loa loa parasitaemia the symptoms after treatment are frequent but mild. However severe cases with conscience troubles are possible, and may occur in about 1% of subjects with more than 3,000 mf/ml blood. Severity of adverse reactions was linked to level of parasitaemia before treatment. The critical parasitaemia level which could lead to expect serious adverse effects seems to be 30,000 ml/ml blood. These informations should induce carefulness to carry out large scale treatments against filariosis in endemic areas of Loa loa.

Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets.

A collection of 3-substituted indole derivatives was prepared using nucleophilic addition of indoles to nitrones. The compounds were then tested for their antibacterial activity against almost thirty bacterial strains representative of common human pathogens. Two types of indolic molecules inhibit the growth of Staphylococcus aureus, including MRSA and VISA strains, with MIC values ranging from 8 to 16 mg/L.

A randomised, unblinded, clinical trial comparing two benznidazole regimens for congenital Chagas disease was carried out to determine whether simplification and reduction in the length of treatment could lead to better treatment compliance.This study was conducted in Santa Cruz, Bolivia. Serological screening was carried out in pregnant women, and parasites were sought in the blood of newborns from seropositive mothers. Infected infants were randomly assigned to two treatment groups. Recovery was assessed by parasite seeking at 1 month and 2 months as well as serological tests at 9 months. Assessment of treatment adherence was based on weekly home visits and use of electronic monitors.Benznidazole was given to 63 newborns in group A (5 mg/kg in two daily doses for 60 days) and 61 newborns in group B (7.5 mg/kg in a single daily dose for 30 days). There was no difference in compliance between the two groups. The study confirmed the efficacy and good tolerance of both benznidazole regimens in the treatment of congenital Chagas disease.The short treatment should be preferred as it allows reducing the dose of benznidazole as well as the cost of treatment.

ABSTRACT The phenotypic plasticity of global regulatory networks provides bacteria with rapid acclimation to a wide range of environmental conditions, while genetic changes in those networks provide additional flexibility as bacteria evolve across long time scales. We previously identified mutations in the global regulator-encoding gene fis that enhanced organismal fitness during a long-term evolution experiment with Escherichia coli . To gain insight into the effects of these mutations, we produced two-dimensional protein gels with strains carrying different fis alleles, including a beneficial evolved allele and one with an in-frame deletion. We found that Fis controls the expression of the major porin-encoding gene ompF in the E. coli B-derived ancestral strain used in the evolution experiment, a relationship that has not been described before. We further showed that this regulatory connection evolved over two different time scales, perhaps explaining why it was not observed before. On the longer time scale, we showed that this regulation of ompF by Fis is absent from the more widely studied K-12 strain and thus is specific to the B strain. On a shorter time scale, this regulatory linkage was lost during 20,000 generations of experimental evolution of the B strain. Finally, we mapped the Fis binding sites in the ompF regulatory region, and we present a hypothetical model of ompF expression that includes its other known regulators.

Abstract We investigated the relationship between genomic and phenotypic evolution among replicate populations of E scherichia coli evolved for 1000 generations in four different environments. By resequencing evolved genomes, we identified parallel changes in genes encoding transcription regulators within and between environments. Depending on both the environment and the altered gene, genetic parallelism at the gene level involved mutations that affected identical codons, protein domains or were widely distributed across the gene. Evolved clones were characterized by parallel phenotypic changes in their respective evolution environments but also in the three alternative environments. Phenotypic parallelism was high for clones that evolved in the same environment, even in the absence of genetic parallelism. By contrast, clones that evolved in different environments revealed a higher parallelism in correlated responses when they shared mutated genes. Altogether, this work shows that after an environmental change or the colonization of a new habitat, similar ecological performance might be expected from individuals that share mutated genes or that experienced similar past selective pressures.

Both proteins and RNAs can misfold into non-functional conformations. Protein chaperones promote native folding of nascent polypeptides and refolding of misfolded species, thereby buffering mutations that compromise protein structure and function. Here, we show that RNA chaperones can also act as mutation buffers that enhance organismal fitness. Using competition assays, we demonstrate that overexpression of select RNA chaperones, including three DEAD box RNA helicases (DBRHs) (CsdA, SrmB, RhlB) and the cold shock protein CspA, improves fitness of two independently evolved Escherichia coli mutator strains that have accumulated deleterious mutations during short- and long-term laboratory evolution. We identify strain-specific mutations that are deleterious and subject to buffering when introduced individually into the ancestral genotype. For DBRHs, we show that buffering requires helicase activity, implicating RNA structural remodelling in the buffering process. Our results suggest that RNA chaperones might play a fundamental role in RNA evolution and evolvability.

The impact of historical contingency, i.e. the past evolutionary history of a population, on further adaptation is mostly unknown at both the phenotypic and genomic levels. We addressed this question using a two-step evolution experiment. First, replicate populations of Escherichia coli were propagated in four different environmental conditions for 1000 generations. Then, all replicate populations were transferred and propagated for further 1000 generations to a single new environment.Using this two-step experimental evolution strategy, we investigated, at both the phenotypic and genomic levels, whether and how adaptation in the initial historical environments impacted evolutionary trajectories in a new environment. We showed that both the growth rate and fitness of the evolved populations obtained after the second step of evolution were contingent upon past evolutionary history. In contrast however, the genes that were modified during the second step of evolution were independent from the previous history of the populations.Our work suggests that historical contingency affects phenotypic adaptation to a new environment. This was however not reflected at the genomic level implying complex relationships between environmental factors and the genotype-to-phenotype map.

Abstract Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures. We confirmed that crp deletion had a more deleterious effect on growth rate in the LTEE-adapted genotypes; and we showed that the ptsG gene, which encodes the major glucose-PTS transporter, gained CRP (cyclic AMP receptor protein) dependence over time in the LTEE. We then further evolved the four crp-deleted genotypes in glucose minimal medium, and we found that they all quickly recovered from their growth defects by increasing glucose uptake. We showed that this recovery was specific to the selective environment and consistently relied on mutations in the cis-regulatory region of ptsG, regardless of the initial genotype. These mutations affected the interplay of transcription factors acting at the promoters, changed the intrinsic properties of the existing promoters, or produced new transcription initiation sites. Therefore, the plasticity of even a single promoter region can compensate by three different mechanisms for the loss of a key regulatory hub in the E. coli TRN.

By using a PCR approach based on conserved regions of ADP-glucose pyrophosphorylases, a glgC gene was cloned from Streptomyces coelicolor A3(2). The deduced glgC gene product showed end-to-end relatedness to other bacterial ADP-glucose pyrophosphorylases. The glgC gene is about 1,000 kb from the leftmost chromosome end and is not closely linked to either of the two glgB genes of S. coelicolor, which encode glycogen branching enzymes active in different locations in differentiated colonies. Disruption of glgC eliminated only the first of two temporal peaks of ADP-glucose pyrophosphorylase activity and glycogen accumulation and prevented cytologically observable glycogen accumulation in the substrate mycelium of colonies (phase I), while glycogen deposition in young spore chains (phase II) remained readily detectable. The cloned glgC gene therefore encodes an ADP-glucose pyrophosphorylase essential only for phase I (and it is therefore named glgCI). A second, phase II-specific, glgC gene should also exist in S. coelicolor, though it was not detected by hybridization analysis.

Journal Article Evidence of congenital transmission of Trypanosoma cruzi in a vector-free area of Bolivia Get access L. Brutus, L. Brutus aResearch Unit ‘Mother and Child Health in the Tropics’, Institut de Recherche pour le Développement, CP 9214, La Paz, Bolivia Search for other works by this author on: Oxford Academic PubMed Google Scholar D. Schneider, D. Schneider aResearch Unit ‘Mother and Child Health in the Tropics’, Institut de Recherche pour le Développement, CP 9214, La Paz, Bolivia Search for other works by this author on: Oxford Academic PubMed Google Scholar J. Postigo, J. Postigo aResearch Unit ‘Mother and Child Health in the Tropics’, Institut de Recherche pour le Développement, CP 9214, La Paz, Bolivia Search for other works by this author on: Oxford Academic PubMed Google Scholar W. Delgado, W. Delgado bInstituto Nacional de Laboratorios de Salud, Rafael Zubieta 1889 Miraflores, La Paz, Bolivia Search for other works by this author on: Oxford Academic PubMed Google Scholar S. Mollinedo, S. Mollinedo bInstituto Nacional de Laboratorios de Salud, Rafael Zubieta 1889 Miraflores, La Paz, Bolivia Search for other works by this author on: Oxford Academic PubMed Google Scholar J.-P. Chippaux J.-P. Chippaux ⁎ aResearch Unit ‘Mother and Child Health in the Tropics’, Institut de Recherche pour le Développement, CP 9214, La Paz, Bolivia ⁎Corresponding author. Tel.: +591 222 1901; fax: +591 278 2944. E-mail address: chippaux@ird.fr (J.-P. Chippaux). Search for other works by this author on: Oxford Academic PubMed Google Scholar Transactions of The Royal Society of Tropical Medicine and Hygiene, Volume 101, Issue 11, November 2007, Pages 1159–1160, https://doi.org/10.1016/j.trstmh.2007.03.015 Published: 01 November 2007 Article history Received: 24 December 2006 Revision received: 22 March 2007 Accepted: 22 March 2007 Published: 01 November 2007

Abstract Objectives We report the synthesis, antibacterial activity and toxicity of 24 bis-indolic derivatives obtained during the development of new ways of synthesis of marine bis-indole alkaloids from the spongotine, topsentin and hamacanthin classes. Methods Innovative ways of synthesis and further structural optimizations led to bis-indoles presenting either the 1-(1H-indol-3′-yl)-1,2-diaminoethane unit or the 1-(1H-indol-3-yl)ethanamine unit. MIC determination was performed for reference and clinical strains of Staphylococcus aureus and CoNS species. MBC, time–kill kinetics, solubility, hydrophobicity index, plasma protein-binding and cytotoxicity assays were performed for lead compounds. Inhibition of the S. aureus NorA efflux pump was also tested for bis-indoles with no antistaphylococcal activity. Results Lead compounds were active against both S. aureus and CoNS species, with MICs between 1 and 4 mg/L. Importantly, the same MICs were found for MRSA and vancomycin-intermediate S. aureus strains. Early concentration-dependent bactericidal activity was observed for lead derivatives. Compounds with no intrinsic antibacterial activity could inhibit the S. aureus NorA efflux pump, which is involved in resistance to fluoroquinolones. At 0.5 mg/L, the most effective compound led to an 8-fold reduction of the ciprofloxacin MIC for the SA-1199B S. aureus strain, which overexpresses NorA. However, the bis-indole compounds displayed a high hydrophobicity index and high plasma protein binding, which significantly reduced antibacterial activity. Conclusions We have synthesized and characterized novel bis-indole derivatives as promising candidates for the development of new antistaphylococcal treatments, with preserved activity against MDR S. aureus strains.

Relationships between iron deficiency, cell-mediated immunity (CMI) and morbidity were studied in 220 children in rural south Togo. Iron deficiency was defined by abnormal values of at least two biochemical indicators of iron status viz. (i) plasmat ferritin, (ii) transferrin saturation, (iii) erythrocyte protophorphyrin. In children without any sign of inflammation, the percentage of B lymphocytes was higher in iron-deficient than in iron-sufficient children (26.7±2.9 vs 18.1±1.5). Mature T lymphocyte and helper-inducer T lymphocyte percentages were lower (51.6±3.7 vs 62.2±1.6 and 32.5±2.4 vs 38.7±1.4, p<0.05 respectively). The number of mature T and helper-inducer T lymphocytes was inversely related to iron status. No alteration of the CMI function assessed by delayed skin hypersensitivity was observed. Prevalence of diarrhea, upper respiratory tract infections and febrile episodes was increased in iron-deficient children.

The need to control processes with time delays can be found in many aspects of the metallurgical industry from refining to rolling mills. When the time delay becomes long compared with the system time constant, or when the time delay varies, then control of the process becomes difficult using classical control methods. Three controllers are evaluated for their applicability to this problem: the classic proportional-integral (P-I) controller; the Smith linear predictor; and the Clarke-Gawthrop self-tuning controller. Each of the controllers is implemented on an example process which is first-order with a long time delay. All three control strategies are evaluated for their response to step reference changes, step load disturbances, and variations in the process time delay. No one control strategy has the perfect solution, so the advantages and limitations of each strategy are presented and discussed.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

To compare the drop of Chagas antibody titres between non-infected and congenitally infected newborns treated by two doses of benznidazole, aiming at evaluating the recovery time and giving recommendations regarding serological criteria of recovery.During a clinical trial, the drop of Trypanosoma cruzi antibody titres measured by ELISA tests was followed during the first year of life in congenitally infected newborns treated with different doses of benznidazole and compared to T. cruzi antibody titres in non-parasitaemic newborns. Confirmation of recovery was given by two negative serological tests: Chagas Stat-Pak (CSP) (immunochromatography) and Chagatest v3.0 (ELISA).In non-parasitaemic infants of infected mothers, antibodies of maternal origin disappeared in <8 months while in infected infants, T. cruzi antibodies decreased more slowly and disappeared in 9-16 months allowing to confirm the recovery. All CSP tests were negative before the ninth month while about 10% of ELISA tests remained positive at the 12th month.Recovery may be confirmed in most cases at 10 months. The CSP test was compared to Chagatest v3.0 ELISA and appeared to give a reliable response. The decrease rate of antibodies does not depend on treatment modes.

A better control of major neglected tropical diseases such as schistosomiasis is urgently needed to reduce their impact on public health in developing countries. To optimize the efficiency of intervention campaigns, we assessed the influence of individual human factors on the level of Schistosoma haematobium infection and morbidity in a typical Sahelian country (Niger). Random samples of 246 and 257 individuals were selected from general census in two villages with distinct patterns of schistosome transmission. One village (Lossa) is located in an area of perennial transmission whereas transmission is seasonal in the other village (Tara). Despite comparable levels of both egg excretion and lower tract pathology in the two villages, the inhabitants of Lossa had a higher risk (OR: 2.1, 95% CI: 1.1-3.9) of developing upper tract lesions compared to those living in Tara. In both villages, bladder lesions were more serious in males than in females. Children between 7 and 15 years old were the most at risk to experience heavy infections (OR: 3.4, 95% CI: 2.1-5.7), bladder (OR: 4.5, 95% CI: 2.6-7.8) and upper tract (OR: 10.4, 95% CI: 2.4-45.0) lesions, independently of gender and village. These results confirm that targeted intervention campaigns should include foci regardless of their schistosome transmission pattern and focus on the school-aged population.

Plasmid pGBG1 was constructed to isolate mobile genetic elements in a wide variety of gram-negative bacteria. The mutation target, carried on a broad-host-range vector, allows positive selection for tetracycline resistance. In tests using several gram-negative bacteria we could detect transposition events of either insertion sequences or transposons. A new insertion sequence (IS) element was identified in Ralstonia eutropha.

We consider a large class of fast growing sequences of numbers U n like the nth superfactorial [Formula: see text], the nth hyperfactorial [Formula: see text] and similar ones. We show that their mantissas are distributed following Benford's law in the sense of the natural density. We prove that this is also verified by [Formula: see text], by [Formula: see text] and is passed down to all the sequences obtained by iterating this design process. We also consider the superprimorial numbers and the products of logarithms of integers.

The economic importance of the trematode Schistosoma bovis in African livestock has justified the development of a specific vaccine. Administered preventively to sheep, rSb28GST—the only molecule cloned from S. bovis which has demonstrated vaccine potentialities in goats and cattle—reduced the mean worm burden in vaccinated animals and improved their health status compared with that of non-vaccinated controls. As in goats, but not in bovines, the fecundity of the settled worm pairs was not modified. Therefore, rSb28GST can be proposed as a universal tool for the prevention of clinical disorders engendered by the main schistosome species affecting domestic ruminants in the African continent.

Abstract From November 1992 to December 1997, 25 patients (inoperable or refusing cystectomy) were included in a prospective study to assess the feasibility, tolerance, and curative potential of accelerated radiotherapy (RT) and concomitant cisplatin. Median age was 74 years (range 49–86). Stage distribution was as follows: 1 T1, 10 T2, 8 T3, and 6 T4. Two patients had clinically positive pelvic nodes. The goal was to deliver a total dose of 40 Gy to the whole pelvis and bladder in 4 weeks using a concomitant boost of 20 Gy to the tumor or to the whole bladder during the third and fourth weeks (total dose 60 Gy), with daily cisplatin (6 mg/m 2 ) before RT for patients with creatinine clearance &gt; 50 ml/min. All but one patient completed the RT protocol. Daily cisplatin was sucessfully delivered in 18 patients. One patient presented with grade III ototoxicity. Diarrhea was scored grade III in two and grade IV in two patients. Acute urinary toxicity was scored grade III in one patient. Posttreatment late effects included bladder grade II and grade III in two patients and one patient, respectively; large bowel grade III in one; urethral grade III in one; and femoral head radionecrosis in one. Four‐year overall and disease‐specific survival rates were 23% and 35%, respectively. The latter was 60% for patients with T2 tumors. The 4‐year actuarial locoregional control rate for all patients was 61%. In summary, accelerated RT and concomitant cisplatin is feasible with acceptable tolerance even in relatively old patients. Although outcome was better for patients with low‐stage tumors, local control and survival rates appeared similar to those of standard RT schedules for a similar patient population. © 2001 Wiley‐Liss, Inc.

Streptomyces ambofaciens RP181110 produces the macrolide polyketide spiramycin. Like many other Streptomyces species, the RP181110 strain is prone to genetic instability involving genomic rearrangements (deletions and/or amplifications) in the large unstable region of the genome. It has previously been demonstrated that the amplification of a particular locus (AUD205) affects spiramycin biosynthesis and, conversely, the loss of this amplification is correlated with the restoration of antibiotic production. This report focuses on a 0·93 kb reiterated fragment specific for the AUD205 locus. Sequencing of 3596 bp including this reiteration revealed the presence of an ORF (orfPS) whose potential product was highly homologous to the EryA and Raps proteins, responsible for the biosynthesis of erythromycin in Saccharopolyspora erythraea and rapamycin in Streptomyces hygroscopicus, respectively. orfPS encodes a protein with at least four successive domains: ketoacyl synthase, acyltransferase, ketoreductase and acyl carrier protein. This organization is very similar to most eryA and rap modules. The reiterated sequence corresponds to the acyltransferase domain. orfPS was transcribed during rapid growth and stationary phase in RP181110 and overtranscribed in the amplified mutant. Both these results suggest that the gene encodes a type I polyketide synthase and its reorganization is responsible for the loss of spiramycin production in the amplified strains.

The combined effects of protein-energy malnutrition (PEM) and infection on thymic function evaluated by specific plasma thymulin activity were studied in Senegalese children: 29 hospitalized in Dakar for severe malnutrition and various diseases; 9 infected without sign of severe PEM, living in Dakar; 13 apparently healthy, uninfected, living in Dakar; and 7 apparently healthy, uninfected, living in Paris. Most of the free-living children in Dakar suffered from mild to moderate PEM. The specific thymulin activity (total plasma activity minus the activity recorded after adsorption of the plasma with a monoclonal antithymulin antibody) was almost undetectable in the infected children and was normal only in the children living in Paris. Such activity might be decreased by moderate and severe PEM and severe malnutrition may not be the only underlying cause of depressed level of thymulin in malnourished children from the Third World. Concurrent infections are important factors.

Adaptation depends on the rates, effects, and interactions of many mutations. We analyzed 264 genomes from 12 Escherichia coli populations to characterize their dynamics over 50,000 generations. The trajectories for genome evolution in populations that retained the ancestral mutation rate fit a model where most fixed mutations are beneficial, the fraction of beneficial mutations declines as fitness rises, and neutral mutations accumulate at a constant rate. We also compared these populations to lines evolved under a mutation-accumulation regime that minimizes selection. Nonsynonymous mutations, intergenic mutations, insertions, and deletions are overrepresented in the long-term populations, supporting the inference that most fixed mutations are favored by selection. These results illuminate the shifting balance of forces that govern genome evolution in populations adapting to a new environment.

A Streptomyces coelicolor gene, called spaA, homologous to the stationary phase regulatory gene rspA of Escherichia coli [Huisman and Kolter (1994) Science 265, 537-539], was cloned using the Streptomyces ambofaciens rspA homologue spa2 [Schneider et al. (1993) J. Gen. Microbiol. 139, 2559-2567] as a probe. Considerable differences in sequence and in genetic context were detected between spa2 of S. ambofaciens and spaA of S. coelicolor. A cloned internal fragment of spaA was used to direct integration of a phage vector into the spaA gene. The disruption caused delayed antibiotic production (undecylprodigiosin and actinorhodin) and led on further incubation to increased actinorhodin production at high, but not low, cell density. This phenotype was apparent only on the nutritionally poorest of three media tested. The attempted use of an integrating plasmid-based system for gene replacement of spaA gave rise to extensive deletions of adjacent chromosomal DNA.

Genetic instability in Streptomyces ambofaciens DSM 40697 is correlated with genomic instability characterized by multiple rearrangements (deletions and/or amplifications) occurring in a large unstable region. We have focused on one of the two amplifiable DNA loci which were mapped in this region: the amplifiable unit of DNA locus 6 (AUD6). The nucleotide sequence of one AUD6 fragment of 1·9 kb reveals the presence of two open reading frames (ORF1 and ORF2) on the basis of the typical Streptomyces base composition at each of the three positions within codons. ORF1 shows some similarity with a gene encoding a regulatory protein. The presence of potential genes in this unstable locus was unexpected because deletions occurred with high frequency within this region in the genetic instability-derived mutant strains. However, transcription analyses by S1 nuclease protection experiments on the wild-type strain showed transcription of both ORF1 and ORF2. Moreover, the amplified strain reveals increased transcription of ORF1 but no transcription of ORF2. The amplification therefore results in a switch in transcription. The unstable region of S. ambofaciens DSM 40697 therefore is not a ‘silent’ region because at least some loci are transcribed.

Genetic instability in Streptomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.