A mouse model for Li-Fraumeni-Like Syndrome with cardiac angiosarcomas associated to POT1 mutations

The shelterin protein POT1 has been found mutated in many different familial and sporadic cancers, however, no mouse models to understand the pathobiology of these mutations have been developed so far. To address the molecular mechanisms underlying the tumorigenic effects of POT1 mutant proteins in humans, we have generated a mouse model for the human POT1^R117C mutation found in Li-Fraumeni-Like families with cases of cardiac angiosarcoma by introducing this mutation in the Pot1a endogenous locus, knock-in for Pot1a^R117C. We find here that both mouse embryonic fibroblasts (MEFs) and tissues from Pot1a^+/ki mice show longer telomeres than wild-type controls. Longer telomeres in Pot1a^+/ki MEFs are dependent on telomerase activity as they are not found in double mutant Pot1a^+/ki Tert^-/- telomerase-deficient MEFs. By using complementation assays we further show that POT1a pR117C exerts dominant-negative effects at telomeres. As in human Li-Fraumeni patients, heterozygous Pot1a^+/ki mice spontaneously develop a high incidence of angiosarcomas, including cardiac angiosarcomas, and this is associated to the presence of abnormally long telomeres in endothelial cells as well as in the tumors. The Pot1a^+/R117C mouse model constitutes a useful tool to understand human cancers initiated by POT1 mutations.     

A physiological and molecular study of the effects of nickel deficiency and phenylphosphorodiamidate (PPD) application on urea metabolism in oilseed rape (Brassica napus L.)

Background and aims

Urea is the major nitrogen (N) form supplied as fertilizer in agriculture. However, urease, a nickel-dependent enzyme, allows plants to use external or internally generated urea as a nitrogen source. Since a urease inhibitor is frequently applied in conjunction with urea fertilizer, the N-metabolism of plants may be affected. The aim of this study was to determine physiological and molecular effects of nickel deficiency and a urease inhibitor on urea uptake and assimilation in oilseed rape.

Methods

Plants were grown on hydroponic solution with urea as the sole N source under three treatments: plants treated with nickel (+Ni) as a control, without nickel (?Ni) and with nickel and phenylphosphorodiamidate (+Ni+PPD). Urea transport and assimilation were investigated.

Results

The results show that Ni-deficiency or PPD supply led to reduced growth and reduced ¹⁵N-uptake from urea. This effect was more pronounced in PPD-treated plants, which accumulated high amounts of urea and ammonium. Thus, Ni-deficiency or addition of PPD, limit the availability of N and decreased shoot and root amino acid content. The up-regulation of BnDUR3 in roots indicated that this gene is a component of the stress response to nitrogen-deficiency. A general decline of glutamine synthetase (GS) activity and activation of glutamate dehydrogenase (GDH) and increases in its expression level were observed in control plants. At the same time, in (?N) or (+Ni+PPD) treated plants, no increases in GS or GDH activities and expression level were found.

Conclusions

Overall results showed that plants require Ni as a nutrient (while most widely used nutrient solutions are devoid of Ni), whether they are grown with or without a urea supply, and that urease inhibitors may have deleterious effects at least in hydroponic grown oilseed rape.     

Outer pore topology of the ECaC-TRPV5 channel by cysteine scan mutagenesis

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.     

M234Glu is a component of the proton sponge in the reaction center from photosynthetic bacteria

Bacterial reaction centers use light energy to couple the uptake of protons to the successive semi-reduction of two quinones, namely Q_A and Q_B. These molecules are situated symmetrically in regard to a non-heme iron atom. Four histidines and one glutamic acid, M234Glu, constitute the five ligands of this atom. By flash-induced absorption spectroscopy and delayed fluorescence we have studied in the M234EH and M234EL variants the role played by this acidic residue on the energetic balance between the two quinones as well as in proton uptake. Delayed fluorescence from the P⁺Q_A^? state (P is the primary electron donor) and temperature dependence of the rate of P⁺Q_A^? charge recombination that are in good agreement show that in the two RC variants, both Q_A^? and Q_B^? are destabilized by about the same free energy amount: respectively ～ 100 ± 5 meV and 90 ± 5 meV for the M234EH and M234EL variants, as compared to the WT. Importantly, in the M234EH and M234EL variants we observe a collapse of the high pH band (present in the wild-type reaction center) of the proton uptake amplitudes associated with formation of Q_A^? and Q_B^?. This band has recently been shown to be a signature of a collective behaviour of an extended, multi-entry, proton uptake network. M234Glu seems to play a central role in the proton sponge-like system formed by the RC protein.     

Timing and abundance as key mechanisms affecting trophic interactions in variable environments

Climatic changes are disrupting otherwise tight trophic interactions between predator and prey. Most of the earlier studies have primarily focused on the temporal dimension of the relationship in the framework of the match–mismatch hypothesis. This hypothesis predicts that predator's recruitment will be high if the peak of the prey availability temporally matches the most energy‐demanding period of the predators breeding phenology. However, the match–mismatch hypothesis ignores the level of food abundance while this can compensate small mismatches. Using a novel time‐series model explicitly quantifying both the timing and the abundance component for trophic relationships, we here show that timing and abundance of food affect recruitment differently in a marine (cod/zooplankton), a marine–terrestrial (puffin/herring) and a terrestrial (sheep/vegetation) ecosystem. The quantification of the combined effect of abundance and timing of prey on predator dynamics enables us to come closer to the mechanisms by which environment variability may affect ecological systems.     

Development of a 16S rRNA gene-based prototype microarray for the detection of selected actinomycetes genera

Actinomycetes are known for their secondary metabolites, which have been successfully used as drugs in human and veterinary medicines. However, information on the distribution of this group of Gram-positive bacteria in diverse ecosystems and a comprehension of their activities in ecosystem processes are still scarce. We have developed a 16S rRNA-based taxonomic microarray that targets key actinomycetes at the genus level. In total, 113 actinomycete 16S rRNA probes, corresponding to 55 of the 202 described genera, were designed. The microarray accuracy was evaluated by comparing signal intensities with probe/target-weighted mismatch values and the Gibbs energy of the probe/target duplex formation by hybridizing 17 non-actinomycete and 29 actinomycete strains/clones with the probe set. The validation proved that the probe set was specific, with only 1.3% of false results. The incomplete coverage of actinomycetes by a genus-specific probe was caused by the limited number of 16S rRNA gene sequences in databases or insufficient 16S rRNA gene polymorphism. The microarray enabled discrimination between actinomycete communities from three forest soil samples collected at one site. Cloning and sequencing of 16S rRNA genes from one of the soil samples confirmed the microarray results. We propose that this newly constructed microarray will be a valuable tool for genus-level comparisons of actinomycete communities in various ecological conditions.     

Comparsion ofin vitro activities of antifungal drugs and propolis against yeasts isolated from patients with superficial mycoses

Thein vitro susceptibilities of propolis and antifungal drugs were determined against some yeasts isolated from patients with superficial mycoses. The agents tested included fluconazole, itraconazole, ketoconazole, terbinafine and propolis. MICs were determined by the broth microdilution technique following National Committee for Clinical Laboratory Standards document M27-P. For allCandida albicans isolates from the patients with superficial mycoses, ketoconazole presented higher (P<0.05) efficiency than that of the other antifungal agents tested. The geometric mean MIC values of antifungal drugs and propolis against the yeasts tested ranged from 0.087 to 12.69 μg/mL and 0.4–0.6 μg/mL, respectively. Propolis also showed an important antifungal activity against the yeasts tested, MIC ranges of the propolis were between 0.01–1.65 μg/mL. Based on these results, propolis requires further investigation as a potential agent for the treatment of superficial mycoses.     

Alloparental feeding in Adélie penguins: why is it uncommon?

We investigated alloparental interactions and conditions which could facilitate or prevent the expression of alloparental behaviours in Adélie penguins (Pygoscelis adeliae), a long-lived seabird which nests in high-density colonies around Antarctica. Observation sessions were carried out during the crèche stage on 48 identified pairs and 50 identified chicks in a 217-nest subcolony. As the season progressed, young were fed less often by their own parents because these were increasingly absent from the breeding site and less responsive to their offspring’s solicitations. As a consequence, young and particularly those with a low body mass, coming from a two-chick brood, opted for gradually soliciting more from other adults to obtain food, preferentially those nesting in their direct vicinity. Unsuccessful breeders represented a low and constant part of the adult population and were not specifically solicited by unrelated young. Despite the increasing chick demand, only 4.1% (3 out of 73) of alloparental solicitations resulted in feeding, which is negligible compared to parental feeding. To investigate factors that could trigger the appearance of alloparental care, we carried out comparisons with king (Aptenodytes patagonicus) and emperor penguins (Aptenodytes forsteri) which represent the closest species for which data on alloparental behaviour were available. Our results show different trends to those observed in these species and three factors may explain the low occurrence of alloparental behaviour in Adélie penguins: (1) the low and constant proportion of unsuccessful breeders, (2) the absence of chick selectivity towards unsuccessful breeders, and (3) the late period of chick accessibility for potential alloparents.