Bioguided Fractionation and Isolation of an Antiplasmodial Saponin from the Roots of Nauclea xanthoxylon (A.Chev.) Aubrév. (Rubiaceae)

The root extract of Nauclea xanthoxylon (A.Chev.) Aubrév. displayed significant 50 % inhibition concentration (IC₅₀s) of 0.57 and 1.26 μg/mL against chloroquine resistant and sensitive Plasmodium falciparum (Pf) Dd2 and 3D7 strains, respectively. Bio-guided fractionation led to an ethyl acetate fraction with IC₅₀s of 2.68 and 1.85 μg/mL and subsequently, to the new quinovic acid saponin named xanthoxyloside ( 1 ) with IC₅₀s of 0.33 and 1.30 μM, respectively against the tested strains. Further compounds obtained from ethyl acetate and hexane fractions were the known clethric acid ( 2 ), ursolic acid ( 3 ), quafrinoic acid ( 4 ), quinovic acid ( 5 ), quinovic acid 3-O-β-D-fucopyranoside ( 6 ), oleanolic acid ( 7 ), oleanolic acid 3-acetate ( 8 ), friedelin ( 9 ), β-sitosterol ( 10a ), stigmasterol ( 10b ) and stigmasterol 3-O-β-D-glucopyranoside ( 11 ). Their structures were characterised with the aid of comprehensive spectroscopic methods (1 and 2D NMR, Mass). Bio-assays were performed using nucleic acid gel stain (SYBR green I)-based fluorescence assay with chloroquine as reference. Extracts and compounds exhibited good selectivity indices (SIs) of >10. Significant antiplasmodial activities measured for the crude extract, the ethyl acetate fraction and xanthoxyloside ( 1 ) from that fraction can justify the use of the root of N. xanthoxylon in ethnomedicine to treat malaria.     

Stress survival islet 1 (SSI-1) survey in Listeria monocytogenes reveals an insert common to listeria innocua in sequence type 121 L. monocytogenes strains

Listeria monocytogenes strains (n = 117) were screened for the presence of stress survival islet 1 (SSI-1). SSI-1(+) strains (32.5%) belonged mainly to serotypes 1/2c, 3b, and 3c. All sequence type 121 (ST-121) strains included (n = 7) possessed homologues to Listeria innocua genes lin0464 and lin0465 instead of SSI-1.     

Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.     

Chemical soil factors influencing plant assemblages along copper-cobalt gradients: implications for conservation and restoration

Aims

Define the chemical factors structuring plant communities of three copper-cobalt outcrops (Tenke-Fungurume, Katangan Copperbelt, D. R. Congo) presenting extreme gradients.

Methods

To discriminate plant communities, 172 vegetation records of all species percentage cover were classified based on NMDS and the Calinski criterion. Soil samples were analyzed for 13 chemical factors and means compared among communities with ANOVA. Partial canonical correspondence analysis (pCCA) was used to determine amount of variation explained individually by each factor and site effect.

Results

Seven communities were identified. Six of the studied communities were related to distinct sites. Site effect (6.0 % of global inertia) was identified as the most important factor related to plant communities’ variation followed by Cu (5.5 %), pH (3.6 %) and Co (3.5 %). Unique contribution of site effect (3.8 %) was higher than that of Cu (1.1 %) and Co (1.0 %).

Conclusions

In restoration, not only Cu and Co contents will be important to maintain vegetation diversity, attention should also be given to co-varying factors potentially limiting toxicity of metals: pH, organic matter, Ca and Mn. Physical parameters were also identified as important in the creation of adequate conditions for diverse communities. Further studies should focus on the effect of physical parameters and geology.     

Multiple origin of metallicolous populations of the pseudometallophyte Arabidopsis halleri (Brassicaceae) in central Europe: the cpDNA testimony

The population structure of the pseudo-metallophyte herb, Arabidopsis halleri, was studied using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) on chloroplast DNA (cpDNA). The history of metallicolous (M) populations showing increased zinc tolerance was investigated. Eight primer-enzyme combinations out of 72 tested were applied to a total of 625 individuals from 28 widespread populations, 14 of them being M. Eleven distinct chlorotypes were found: five were common to nonmetallicolous (NM) and M populations, whereas six were only observed in one edaphic type (five in NM and one in M). No difference in chlorotype diversity between edaphic types was detected. Computed on the basis of chlorotype frequencies, the level of population differentiation was high but remained the same when taking into account levels of molecular divergence between chlorotypes. Isolation by distance was largely responsible for population differentiation. Geographically isolated groups of M populations were more genetically related to their closest NM populations than to each other. Our results suggest that M populations have been founded separately from distinct NM populations without suffering founding events and that the evolution towards increased tolerance observed in the distinct M population groups occurred independently.     

Roles for alpha(2)p24 and COPI in endoplasmic reticulum cargo exit site formation.

A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg(2+)ATP and Mg(2+)GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins alpha(2)p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of alpha(2)p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of alpha(2)p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPgammaS, inhibition by Brefeldin A (BFA), or depletion of beta-COP from cytosol. Therefore, the p24 family member, alpha(2)p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the generation of ER cargo exit sites.     

Refuge from predation, the benefit of living in an extreme acidic environment?

Organisms living in extreme habitats require costly adaptations to cope with these conditions. Among the suggested potential benefits that trade off these costs is refuge from predation. To study these interactions in extreme environments, samples were taken in the cave Cueva de Villa Luz, Tabasco, Mexico, where more than 32 subterranean springs, some H(2)S rich, rise from the floor. Hydrogen sulfide gas plus oxygen is absorbed by freshwater, and oxidation forms concentrated sulfuric acid. Snottites, whitish hollow mucous tubes, hang from the ceiling of the cave. Fluid drops from these snottites were recorded as having pH values of 0-3. We report the discovery of a new species of nematode that thrives in the highly acidic environment of the snottite. Micro CT scan of snottites reveals a complex interaction between the acidic snottite, nematodes, and abundant nematode-eating mites. The nematode adaptation to low pH probably protects them against mite predation, for which nematodes are most likely the most important source of carbon in this sulfur-driven ecosystem.     

In silico and empirical evaluation of twelve metabarcoding primer sets for insectivorous diet analyses

During the most recent decade, environmental DNA metabarcoding approaches have been both developed and improved to minimize the biological and technical biases in these protocols. However, challenges remain, notably those relating to primer design. In the current study, we comprehensively assessed the performance of ten COI and two 16S primer pairs for eDNA metabarcoding, including novel and previously published primers. We used a combined approach of in silico, in vivo‐mock community (33 arthropod taxa from 16 orders), and guano‐based analyses to identify primer sets that would maximize arthropod detection and taxonomic identification, successfully identify the predator (bat) species, and minimize the time and financial costs of the experiment. We focused on two insectivorous bat species that live together in mixed colonies: the greater horseshoe bat (Rhinolophus ferrumequinum) and Geoffroy's bat (Myotis emarginatus). We found that primer degeneracy is the main factor that influences arthropod detection in silico and mock community analyses, while amplicon length is critical for the detection of arthropods from degraded DNA samples. Our guano‐based results highlight the importance of detecting and identifying both predator and prey, as guano samples can be contaminated by other insectivorous species. Moreover, we demonstrate that amplifying bat DNA does not reduce the primers' capacity to detect arthropods. We therefore recommend the simultaneous identification of predator and prey. Finally, our results suggest that up to one‐third of prey occurrences may be unreliable and are probably not of primary interest in diet studies, which may decrease the relevance of combining several primer sets instead of using a single efficient one. In conclusion, this study provides a pragmatic framework for eDNA primer selection with respect to scientific and methodological constraints.