Mapping the Binding Interface between an HIV-1 Inhibiting Intrabody and the Viral Protein Rev

HIV-1 Rev is the key protein in the nucleocytoplasmic export and expression of the late viral mRNAs. An important aspect for its function is its ability to multimerize on these mRNAs. We have recently identified a llama single-domain antibody (Nb₁₉₀) as the first inhibitor targeting the Rev multimerization function in cells. This nanobody is a potent intracellular antibody that efficiently inhibits HIV-1 viral production. In order to gain insight into the Nb₁₉₀-Rev interaction interface, we performed mutational and docking studies to map the interface between the nanobody paratope and the Rev epitope. Alanine mutants of the hyper-variable domains of Nb₁₉₀ and the Rev multimerization domains were evaluated in different assays measuring Nb₁₉₀-Rev interaction or viral production. Seven residues within Nb₁₉₀ and five Rev residues are demonstrated to be crucial for epitope recognition. These experimental data were used to perform docking experiments and map the Nb₁₉₀-Rev structural interface. This Nb₁₉₀-Rev interaction model can guide further studies of the Nb₁₉₀ effect on HIV-1 Rev function and could serve as starting point for the rational development of smaller entities binding to the Nb₁₉₀ epitope, aimed at interfering with protein-protein interactions of the Rev N-terminal domain.     

Ecological indicators based on trophic spectrum as a tool to assess ecosystems fishing impacts

Trophic indicators were used to compare two Malian freshwater reservoirs whose main differences are based on their different fishing pressures. Data were collected from a scientific survey of small-scale fishery landings conducted in 2002/2003. The trophic levels of fish species caught by artisanal fisheries are estimated from observations of scientific fishing or from the metabase Fishbase. Important differences exist in the trophic structure of both reservoirs. In Selingue (with high fishing pressure), very few top predators are found in the catches while the low trophic level fishes increase in total catches. In Manantali (with low fishing pressure), the top predators contribute twice as much to catches compared to Selingue. Hence, the mean trophic level of catches in Selingue (2.80) is lower than in Manantali (2.97). When comparing these results with those of study made in 1994/1995, it clearly appears that the effects of the fishing pressure in Selingue are obvious through a decrease of 0.12 in the mean trophic level while in Manantali this mean level has increased by 0.33 due to a recent strategic targeting of top predators. Trophic spectra seem to be relevant tools to characterize exploited fish communities from multi-specific and multi-gear small-scale fisheries catch data.     

A unifying quantitative framework for exploring the multiple facets of microbial biodiversity across diverse scales

Recent developments of molecular tools have revolutionized our knowledge of microbial biodiversity by allowing detailed exploration of its different facets and generating unprecedented amount of data. One key issue with such large datasets is the development of diversity measures that cope with different data outputs and allow comparison of biodiversity across different scales. Diversity has indeed three components: local (α), regional (γ) and the overall difference between local communities (β). Current measures of microbial diversity, derived from several approaches, provide complementary but different views. They only capture the β component of diversity, compare communities in a pairwise way, consider all species as equivalent or lack a mathematically explicit relationship among the α, β and γ components. We propose a unified quantitative framework based on the Rao quadratic entropy, to obtain an additive decomposition of diversity (γ = α + β), so the three components can be compared, and that integrate the relationship (phylogenetic or functional) among Microbial Diversity Units that compose a microbial community. We show how this framework is adapted to all types of molecular data, and we highlight crucial issues in microbial ecology that would benefit from this framework and propose ready‐to‐use R‐functions to easily set up our approach.     

An alternate sucrose binding mode in the E203Q Arabidopsis invertase mutant: an X-ray crystallography and docking study

In the present study, we report on the X-ray crystallographic structure of a GH32 invertase mutant, (i.e., the Arabidopsis thaliana cell-wall invertase 1-E203Q, AtcwINV1-mutant) in complex with sucrose. This structure was solved to reveal the features of sugar binding in the catalytic pocket. However, as demonstrated by the X-ray structure the sugar binding and the catalytic pocket arrangement is significantly altered as compared with what was expected based on previous X-ray structures on GH-J clan enzymes. We performed a series of docking and molecular dynamics simulations on various derivatives of AtcwINV1 to reveal the reasons behind this modified sugar binding. Our results demonstrate that the E203Q mutation introduced into the catalytic pocket triggers conformational changes that alter the wild type substrate binding. In addition, this study also reveals the putative productive sucrose binding modus in the wild type enzyme.     

Beta- Lactam Antibiotics Stimulate Biofilm Formation in Non-Typeable Haemophilus influenzae by Up-Regulating Carbohydrate Metabolism

Non-typeable Haemophilus influenzae (NTHi) is a common acute otitis media pathogen, with an incidence that is increased by previous antibiotic treatment. NTHi is also an emerging causative agent of other chronic infections in humans, some linked to morbidity, and all of which impose substantial treatment costs. In this study we explore the possibility that antibiotic exposure may stimulate biofilm formation by NTHi bacteria. We discovered that sub-inhibitory concentrations of beta-lactam antibiotic (i.e., amounts that partially inhibit bacterial growth) stimulated the biofilm-forming ability of NTHi strains, an effect that was strain and antibiotic dependent. When exposed to sub-inhibitory concentrations of beta-lactam antibiotics NTHi strains produced tightly packed biofilms with decreased numbers of culturable bacteria but increased biomass. The ratio of protein per unit weight of biofilm decreased as a result of antibiotic exposure. Antibiotic-stimulated biofilms had altered ultrastructure, and genes involved in glycogen production and transporter function were up regulated in response to antibiotic exposure. Down-regulated genes were linked to multiple metabolic processes but not those involved in stress response. Antibiotic-stimulated biofilm bacteria were more resistant to a lethal dose (10 µg/mL) of cefuroxime. Our results suggest that beta-lactam antibiotic exposure may act as a signaling molecule that promotes transformation into the biofilm phenotype. Loss of viable bacteria, increase in biofilm biomass and decreased protein production coupled with a concomitant up-regulation of genes involved with glycogen production might result in a biofilm of sessile, metabolically inactive bacteria sustained by stored glycogen. These biofilms may protect surviving bacteria from subsequent antibiotic challenges, and act as a reservoir of viable bacteria once antibiotic exposure has ended.     

Precipitation-dependent flowering of Globularia alypum and Erica multiflora in Mediterranean shrubland under experimental drought and warming, and its inter-annual variability

Background and Aims

Relationships between autumn flowering, precipitation and temperature of plant species of Mediterranean coastal shrublands have been described, but not analysed experimentally. These relationships were analysed for two species of co-occurring, dominant, autumn-flowering shrubs, Globularia alypum and Erica multiflora, over 4 years and in experimentally generated drought and warming conditions. The aim was to improve predictions about the responses and adaptations of flowering of Mediterranean vegetation to climate change.

Methods

Beginning of anthesis and date of maximum flowering intensity (‘peak date’) were monitored over 4 years (2001–2004) on a garrigue land type in the noth-east of the Iberian Peninsula. Two experimental treatments were applied, increased temperature (+0·73°C) and reduced soil moisture (–17%) relative to untreated plots.

Key Results

Flowering of Globularia alypum and Erica multiflora differed greatly between years depending on the precipitation of the previous months and the date of the last substantial rainfall (>10 mm). Globularia alypum flowered once or twice (unimodal or bimodal) as the result of differences in the distribution and magnitude of precipitation in late-spring and summer (when floral buds develop). The drought treatment delayed and decreased flowering of Globularia alypum in 2001 and delayed flowering in 2002. Warming extended the period between the beginning of flowering and the end of the second peak for autumn flowering in 2001 and also increased peak intensity in 2002. Flowering of Erica multiflora was unaffected by either treatment.

Conclusions

Autumn flowering of Globularia alypum and Erica multiflora is more dependent on water availability than on temperature. Considerable inter-annual plasticity in the beginning of anthesis and peak date and on unimodal or bimodal flowering constitutes a ‘safe strategy’ for both species in relation to varying precipitation and temperature. However, severe changes in precipitation in spring and summer may severely affect flowering of Globularia alypum but not Erica multiflora, thus affecting development/structure of the ecosystem if such conditions persist.Key words: Globularia alypum, Erica multiflora, autumn flowering, drought, global warming, Mediterranean     

Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.