The Genetic Basis of Escherichia coli Pathoadaptation to Macrophages

Antagonistic interactions are likely important driving forces of the evolutionary process underlying bacterial genome complexity and diversity. We hypothesized that the ability of evolved bacteria to escape specific components of host innate immunity, such as phagocytosis and killing by macrophages (MΦ), is a critical trait relevant in the acquisition of bacterial virulence. Here, we used a combination of experimental evolution, phenotypic characterization, genome sequencing and mathematical modeling to address how fast, and through how many adaptive steps, a commensal Escherichia coli (E. coli) acquire this virulence trait. We show that when maintained in vitro under the selective pressure of host MΦ commensal E. coli can evolve, in less than 500 generations, virulent clones that escape phagocytosis and MΦ killing in vitro, while increasing their pathogenicity in vivo, as assessed in mice. This pathoadaptive process is driven by a mechanism involving the insertion of a single transposable element into the promoter region of the E. coli yrfF gene. Moreover, transposition of the IS186 element into the promoter of Lon gene, encoding an ATP-dependent serine protease, is likely to accelerate this pathoadaptive process. Competition between clones carrying distinct beneficial mutations dominates the dynamics of the pathoadaptive process, as suggested from a mathematical model, which reproduces the observed experimental dynamics of E. coli evolution towards virulence. In conclusion, we reveal a molecular mechanism explaining how a specific component of host innate immunity can modulate microbial evolution towards pathogenicity.     

Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry

Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.     

Chemodiversity of Nonacosan‐10‐ol and n‐Alkanes in the Needle Wax of Pinus heldreichii

This is the first report of individual variability and population diversity of the contents of nonacosan‐10‐ol and n‐alkanes in the needle cuticular waxes of Bosnian pines originated from Montenegro, regarded as Pinus heldreichii var. leucodermis, and from Serbia, regarded as P. heldreichii var. pan?i?i. The amount of nonacosan‐10‐ol varied individually from 27.4 to 73.2% (55.5% in average), but differences between the four investigated populations were not statistically confirmed. The size of the n‐alkanes ranged from C₁₈ to C₃₃. The most abundant n‐alkanes were C₂₃, C₂₇, and C₂₅ (12.2, 11.2, and 10.8% in average, resp.). The carbon preference index (CPI) of the n‐alkanes ranged from 0.8 to 3.1 (1.6 in average), while the average chain length (ACL) ranged from 20.9 to 26.5 (24.4 in average). Long‐chain and mid‐chain n‐alkanes prevailed (49.6 and 37.9% in average, resp.). It was also found that the populations of P. heldreichii var. leucodermis had predominantly a narrower range of n‐alkanes (C₁₈? C₃₁) than the trees of the variety pan?i?i (C₁₈? C₃₃). Differences between the varieties were also significant for most of the other characteristics of the n‐alkane pattern (e.g., most abundant n‐alkanes, CPI, ACL, and relative proportion of short‐, mid‐, and long‐chain n‐alkanes). The principle component and cluster analyses of eleven n‐alkanes confirmed the significant diversity of these two varieties.     

No Concordant Phylogeographies of the Rose Gall Wasp Diplolepis rosae (Hymenoptera,Cynipidae) and Two Associated Parasitoids across Europe

According to the Host-tracking Hypothesis, species of higher trophic levels with a close relationship to their hosts, such as parasites or parasitoids, are expected to show spatio-temporal phylogeographic patterns similar to those of their host. Alternatively, with ecological sorting, a subset of the local species pools might shift to a related host species, thereby disengaging common phylogeographic patterns. Here, we compare the phylogeographic structures of the cynipid rose gall wasp Diplolepis rosae across Europe and of two of its most common parasitoids, the wasps Orthopelma mediator and Glyphomerus stigma, by analysing the sequences of two gene fragments (COI and ITS 2). The phylogeographic structures of the three species associated with roses were incongruent. D. rosae had the lowest genetic diversity with one major clade, O. mediator showed the classical phylogeographic structure for Europe with one eastern and one western clade, and G. stigma had the highest diversity but no geographical structuring. This discordance of geographical patterns may be explained by 1) the dispersal propensity of adult parasitoids or 2) the parasitoids having the ability to switch to another host, while the primary host becomes rare or is even not available. Furthermore there was no indication that phylogenetic patterns were affected by Wolbachia infections. Our results document that communities of closely interacting species may be the result of idiosyncratic biogeographic histories.     

Reliability and accuracy of handheld stopwatches compared with electronic timing in measuring sprint performance

This study assessed reliability of split times obtained by handheld stopwatches (HHSs) compared with electronic timing (ET) during a 200-m sprint. Two HHS timing methods were compared with ET: single-split timers (SST) and multiple-split timers (MST). Twenty-six timers without previous experience were given instruction and completed practice trials until good agreement was achieved between ET and HHS. Trained runners (8 males, 10 females) were timed for each 25-m interval on a standard 200-m course. Repeated-measures analysis of variance and intraclass correlation models were used to determine reliability. A total of 248 split times were analyzed. No significant differences were found between the three timing methods (p > 0.99), and calculated intraclass correlation values were high (0.988). Mean error between SST, MST, and ET (-0.04 +/- 0.24 and -0.05 +/- 0.24 seconds, respectively) indicated faster HHS times, though not significantly. However, absolute errors were considerably larger (0.15 +/- 0.20 and 0.16 +/- 0.19 between SST, MST, and ET, respectively). The HHS-recorded splits were faster than ET in 67.3% of splits and slower in 29.4%. The distribution of errors made the development of a reliable correction factor to convert HHS to ET impossible. It was concluded that on the basis of the small mean error and high intraclass correlations, the use of HHSs may be a viable alternative to ET in collecting group data. However, on the basis of the absolute error between HHS and ET, when high degrees of precision are required, ET should be used, and reliable correction of HHS to ET values is not possible. It was further concluded that HHS times should be reported without attempting correction and interpreted in light of the shortcomings of the HHS method.     

Gamma-tubulin37C and gamma-tubulin ring complex protein 75 are essential for bicoid RNA localization during drosophila oogenesis

bicoid (bcd) RNA localization requires the activity of exuperantia and swallow at sequential steps of oogenesis and is microtubule dependent. In a genetic screen, we identified two novel genes essential for bcd RNA localization. They encode maternal gamma-Tubulin37C (gammaTub37C) and gamma-tubulin ring complex protein 75 (Dgrip75), both of which are gamma-tubulin ring complex components. Mutations in these genes specifically affect bcd RNA localization, whereas other microtubule-dependent processes during oogenesis are not impaired. This provides direct evidence that a subset of microtubules organized by the gamma-tubulin ring complex is essential for localization of bcd RNA. At stage 10b, we find gammaTub37C and Dgrip75 anteriorly concentrated and propose the formation of a microtubule-organizing center at the anterior pole of the oocyte.     

Chemical defense of brown algae (Dictyopteris spp.) against the herbivorous amphipod Ampithoe longimana

Terpenoids, polyphenols, and C₁₁ metabolites are broadly distributed among brown seaweeds. Terpenoids and polyphenols have often been investigated as chemical defenses against herbivores, while there are only few investigations of the fatty-acid-derived C₁₁ hydrocarbons and C₁₁ sulfur compounds as potential defenses. We investigated effects of C₁₁ sulfur metabolites from the cosmopolitan brown alga Dictyopteris membranacea on feeding and fitness of the herbivorous amphipod Ampithoe longimana. In choice tests between freshly collected thalli of D. hoytii (which lacks C₁₁ sulfur compounds) and D. membranacea (which contains C₁₁ sulfur compounds) amphipods consumed about 4 times more of the species lacking the C₁₁ sulfur compounds. The same feeding preference was observed when these plants were finely ground and embedded in an agar matrix to destroy morphological differences. When a diet made from field-collected thalli of D. membranacea containing C₁₁ sulfur compounds was tested against a diet made from a laboratory culture of D. membranacea that had lost the ability to produce C₁₁ sulfur compounds, the same magnitude of preference was observed for the population lacking the sulfur compounds. In addition to the C₁₁ sulfur compounds, a water-soluble C9-oxo acid that appears to be a by-product in the biosynthesis of the C₁₁ metabolites also suppressed amphipod feeding to a comparable extent. Both classes of compound may contribute to the effective chemical protection of D. membranacea. When juvenile amphipods were reared for 28 days on artificial diets containing the above compounds, their survivorship (⢪%) closely resembled that of a starved treatment, but differed dramatically from a control treatment (60%) consisting of the same food, but without the metabolites. Most other classes of brown algal secondary metabolites are defensive against a broad spectrum of larger herbivores, but relatively ineffective against the amphipod studied here. In contrast, the fatty-acid-derived sulfur compounds and the C9-oxo acid strongly deter Ampithoe-like mesograzers but appear less effective against other herbivores, suggesting that these metabolites could be ecologically important in defending zygotes and germlings against these small consumers.     

Lack of Bdnf and TrkB signalling in the postnatal cochlea leads to a spatial reshaping of innervation along the tonotopic axis and hearing loss

Members of the neurotrophin gene family and their high-affinity Trk receptors control innervation of the cochlea during embryonic development. Lack of neurotrophin signalling in the cochlea has been well documented for early postnatal animals, resulting in a loss of cochlear sensory neurones and a region-specific reduction of target innervation along the tonotopic axis. However, how reduced neurotrophin signalling affects the innervation of the mature cochlea is currently unknown. Here, we have analysed the consequences of a lack of the TrkB receptor and its ligand, the neurotrophin brain-derived neurotrophic factor (Bdnf), in the late postnatal or adult cochlea using mouse mutants. During early postnatal development, mutant animals show a lack of afferent innervation of outer hair cells in the apical part of the cochlea, whereas nerve fibres in the basal part are maintained. Strikingly, this phenotype is reversed during subsequent maturation of the cochlea, which results in a normal pattern of outer hair cell innervation in the apex and loss of nerve fibres at the base in adult mutants. Measurements of auditory brain stem responses of these mice revealed a significant hearing loss. The observed innervation patterns correlate with opposing gradients of Bdnf and Nt3 expression in cochlear neurones along the tonotopic axis. Thus, the reshaping of innervation may be controlled by autocrine signalling between neurotrophins and their receptors in cochlear neurones. Our results indicate a substantial potential for re-innervation processes in the mature cochlea, which may also be of relevance for treatment of hearing loss in humans.     

Heme oxygenase microsatellite polymorphism, oxidative stress, glycemic control, and complication development in type 2 diabetes patients

Heme oxygenase-1 (HMOX-1) is activated by oxidative stress, and gene responsiveness is reportedly determined by the number of dinucleotide (GT(n)) repeats in its highly polymorphic promoter region. "Short" (S; GT(n)<25) alleles reportedly associate with higher response, lower oxidative stress, lower risk of type 2 diabetes mellitus (type 2DM), and better glycemic control and outcome, but data are conflicting. We investigated GT(n) in type 2DM subjects (all ethnic Chinese) in relation to basal glycemic control, oxidative stress, and outcome during up to 9 years' follow-up. Fasting blood from 418 type 2 DM subjects was collected at entry for GT(n) genotyping, glycated hemoglobin, glucose, lipids, and biomarkers of oxidative stress and antioxidants. A subset (n=368) was followed for up to 9 years for incident complications or death. GT(n) genotype distribution was 128, 182, and 108 for, respectively, S/S, S/L, and L/L. No significant differences in glycemic control, lipids, or oxidative stress were seen across genotypes. During follow-up, 168/368 subjects developed complications. No association was seen with GT(n). No difference in plasma HO-1 was seen between genotypes in a small substudy (S/S n=21 vs L/L n=23). Glycated hemoglobin and lymphocytic DNA damage was higher (p<0.05) at entry in the incident complications group. No other significant differences were seen in oxidative stress or antioxidants. Data do not support the postulated link between HMOX-1 microsatellite polymorphism and type 2 DM or the putative beneficial effect of the S allele on glycemic control, oxidative stress, or outcome in type 2 DM patients, at least in this particular population.