Acquisition and maintenance of resistance to viruses in eukaryotic phytoplankton populations

Viruses are known to play a key role in the regulation of eukaryotic phytoplankton population densities; however, little is known about the mechanisms of how they interact with their hosts and how phytoplankton populations mediate their regulations. Viruses are obligate parasites that depend on host cell machinery for their dissemination in the environment (most of the time through host cell lysis that liberates many new particles). But viruses also depend on a reliable host population to carry on their replication before losing their viability. How do hosts cells survive when they coexist with their viruses? We show that clonal lines of three picoeukaryotic green algae (i.e. Bathycoccus sp., Micromonas sp., Ostreococcus tauri) reproducibly acquire resistance to their specific viruses following a round of infection. Our observations show that two mechanisms of resistance may operate in O. tauri. In the first resistant type, viruses can attach to their host cells but no new particles develop. In the second one, O. tauri acquires tolerance to its virus and releases these viruses consistently. These lines maintained their resistance over a 3‐year period, irrespective of whether or not they were re‐challenged with new viral inoculations. Co‐culturing resistant and susceptible lines revealed resistance to be associated with reduced host fitness in terms of growth rate.     

Identification of bilirubin reduction products formed by Clostridium perfringens isolated from human neonatal fecal flora

Urobilinoids belong to the heterogenous group of degradation products of bilirubin formed in the gastrointestinal tract by intestinal microflora. Among them urobilinogen and stercobilinogen with their respective oxidation products, urobilin and stercobilin, are the most important compounds. The aim of present study was to analyze the products of bacterial reduction of bilirubin in more detail. The strain of Clostridium perfringens isolated from neonatal stools, capable of reducing bilirubin, was used in the study. Bacteria were incubated under anaerobic conditions with various native as well as synthetic bile pigments, including radiolabeled unconjugated bilirubin (UCB). Their reduction products were extracted from media and separated following thin layer chromatography. Pigments isolated were analyzed by spectrophotometry, spectrofluorometry and mass spectrometry. In a special set of experiments, bilirubin diglucuronide was incubated with either bacterial lysate or partially purified bilirubin reductase and beta-glucuronidase to reveal whether bilirubin glucuronides may be directly reduced onto conjugated urobilinoids. A broad substrate activity was detected in the investigated strain of C. perfringens and a series of bilirubin reduction products was identified. These products were separated in the form of their respective chromogens and further oxidized. Based on their physical-chemical properties, as well as mass spectra, end-catabolic bilirubin products were identified to belong to urobilinogen species. The reduction process, catalyzed enzymatically by the studied bacterial strain, does not proceed to stercobilinogen. Bilirubin diglucuronide is not reduced onto urobilinoid conjugates, glucuronide hydrolysis must precede double bond reduction and thus UCB is reduced much faster.     

The effects of plant traits on species' responses to present and historical patch configurations and patch age

Patch configuration is viewed as an important factor affecting the distributions of plant species. Although a number of studies have explored the relationship between plant life‐history traits and species’ distributions in fragmented landscapes, the effect of individual traits on the dependence of species on historical versus current landscape configurations remains unclear. We identified the extent to which present (2000s) and historical (1843, 1954, 1980s) patch configurations (area and connectivity) and patch age affected the distributions of 99 species inhabiting dry calcareous grasslands. We used traits related to dispersal, survival, growth and habitat preferences to explain the dependence of 60 of these species on present and historical configurations, and the age of grassland patches. We found that most of the species had an affinity to currently or historically large, older and more isolated patches. This suggests that many dry grassland species are not in equilibrium with the current landscape, as their distributions still reflect past landscape structures. Rapidly growing species with higher seed bank longevity and nutrient requirements primarily occur in young, large grassland patches whereas species with the opposite traits occur in older, historically large and currently more isolated smaller patches. We hypothesise that patch quality is the reason why different species occupy patches of different age. Species occupying young, large patches commonly disperse by endozoochory. By contrast, no dispersal trait is associated with species occupying old, usually isolated patches. Our results suggest that species occupying old patches are exposed to higher potential risk of extinction, as their distributions are probably limited by the low number and connectivity of available suitable patches and poor dispersal ability. We thus suggest that the dynamics of these species can effectively be supported by improving the quality of young grassland patches.     

Synthesis and cytotoxic activity of various 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils in their racemic form

The preparation of various 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils with alkyl chain lengths C(1)-C(12) is described. The synthesis is based on the preparation of 5-[chloro-(4-nitro-phenyl)-methyl]-uracil and subsequent substitution of chlorine with appropriate alcohols. The resulting ethers were tested for their cytotoxic activity in vitro against five cancer cell lines. The compounds were less active in lung resistance protein expressing cell lines, suggesting the involvement of this multidrug resistant protein in control of the biological activity. Cytotoxic substances induced rapid inhibition of DNA and modulation of RNA synthesis followed by induction of apoptosis. The data indicate that the biological activity of 5-[alkoxy-(4-nitro-phenyl)-methyl]-uracils depends on the alkyl chain length.     

Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans

Leptosphaeria maculans, a dothideomycete fungus causing stem canker on oilseed rape, develops gene-for-gene interactions with its host plants. It has the ability to rapidly adapt to selection pressure exerted by cultivars harbouring novel resistance genes as exemplified recently by the 3-year evolution towards virulence at the AvrLm1 locus in French populations. The AvrLm1 avirulence gene was recently cloned and shown to be a solo gene within a 269 kb non-coding, heterochromatin-like region. Here we describe the sequencing of the AvrLm1 genomic region in one avirulent and two virulent isolates to investigate the molecular basis of evolution towards virulence at the AvrLm1 locus. For these virulent isolates, the gain of virulence was linked to a 260 kb deletion of a chromosomal segment spanning AvrLm1 and deletion breakpoints were identical or similar. Among the 460 isolates analysed from France, Australia and Mexico, a similar large deletion was apparent in > 90% of the virulent isolates. Deletion breakpoints were also strongly conserved in most of the virulent isolates, which led to the hypothesis that a unique deletion event leading to the avrLm1 virulence has diffused in pathogen populations. These data finally suggest that retrotransposons are key drivers in genome evolution and adaptation to novel selection pressure in L. maculans.     

Synthesis and biological activity of desmethoxy analogues of coruscanone A

A series of simple desmethoxy analogues of coruscanone A was prepared via a novel version of Ti(iPrO)(4)-mediated Knoevenagel condensation of cyclopentenedione with substituted benzaldehydes and cinnamic aldehydes, and the compounds were evaluated for antifungal activity and cytotoxicity. The most potent 2-benzylidenecyclopent-4-ene-1,3-dione possessed antifungal effect comparable to coruscanone A and a somewhat broader spectrum of activity against Candida species. The compound was also superior to fluconazole against several non-albicans Candida sp. Evaluation of the ability of the compound to influence cell proliferation using two different assays showed that 2-benzylidenecyclopent-4-ene-1,3-dione has lower cytotoxicity compared to the natural product.     

Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization

The interactions of the AMPA receptor (AMPAR) auxiliary subunit Stargazin with PDZ domain-containing scaffold proteins such as PSD-95 are critical for the synaptic stabilization of AMPARs. To investigate these interactions, we have developed biomimetic competing ligands that are assembled from two Stargazin-derived PSD-95/DLG/ZO-1 (PDZ) domain-binding motifs using 'click' chemistry. Characterization of the ligands in vitro and in a cellular FRET-based model revealed an enhanced affinity for the multiple PDZ domains of PSD-95 compared to monovalent peptides. In cultured neurons, the divalent ligands competed with transmembrane AMPAR regulatory protein (TARP) for the intracellular membrane-associated guanylate kinase resulting in increased lateral diffusion and endocytosis of surface AMPARs, while showing strong inhibition of synaptic AMPAR currents. This provides evidence for a model in which the TARP-containing AMPARs are stabilized at the synapse by engaging in multivalent interactions. In light of the prevalence of PDZ domain clusters, these new biomimetic chemical tools could find broad application for acutely perturbing multivalent complexes.