Mutational analysis of ionizable residues proximal to the cytoplasmic interface of membrane spanning domain 3 of the multidrug resistance protein, MRP1 (ABCC1): glutamate 1204 is important for both the expression and catalytic activity of the transporter

The multidrug resistance protein MRP1 is an ATP-dependent transporter of organic anions and chemotherapeutic agents. A significant number of ionizable amino acids are found in or proximal to the 17 transmembrane (TM) helices of MRP1, and we have investigated 6 of these at the cytoplasmic interface of TM13-17 for their role in MRP1 expression and transport activity. Opposite charge substitutions of TM13 Arg(1046) and TM15 Arg(1131) did not alter MRP1 expression nor did they substantially affect activity. In contrast, opposite charge substitutions of TM16 Arg(1202) and Glu(1204) reduced protein expression by >80%; however, MRP1 expression was not affected when Arg(1202) and Glu(1204) were replaced with neutral or same-charge residues. In addition, organic anion transport levels of the R1202L, R1202G, and R1202K mutants were comparable with wild-type MRP1. In contrast, organic anion transport by E1204L was substantially reduced, whereas transport by E1204D was comparable with wild-type MRP1, with the notable exception of GSH. Opposite charge substitutions of TM16 Arg(1197) and TM17 Arg(1249) did not affect MRP1 expression but substantially reduced transport. Mutants containing like-charge substitutions of Arg(1197) or Arg(1249) were also transport-inactive and no longer bound leukotriene C(4). In contrast, substrate binding by the transport-compromised E1204L mutant remained intact. Furthermore, vanadate-induced trapping of azido-ADP by E1204L was dramatically increased, indicating that this mutation may cause a partial uncoupling of the catalytic and transport activities of MRP1. Thus, Glu(1204) serves a dual role in membrane expression of MRP1 and a step in its catalytic cycle subsequent to initial substrate binding.     

Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods

Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.     

Reconstructing atoll-like mounds from the Frasnian of Belgium

A succession of Frasnian mounds on the southern border of the Dinant Synclinorium (Belgium) was investigated for their facies architecture, sedimentary dynamics and palaeogeographic evolution. Seven mound facies were defined from the Arche (A) and Lion (L) members, each characterized by a specific range of textures and association of organisms (A2/L2: red or pink limestone with stromatactis, corals and crinoids; A3/L3: grey, pink or green limestone with stromatactis, corals and stromatoporoids; A4/L4: grey limestone with corals, peloids and dasycladaceens; A5/L5: grey microbial limestone; A6/L6: grey limestone with dendroid stromatoporoids; A7/L7: grey laminated limestone with fenestrae; and A8/L8: grey bioturbated limestone). Laterally equivalent sediments include substantial reworked material from the buildups and background sedimentation. Textures and fossils suggest that A2/L2 and A3/L3 facies developed close to storm wave base, in a subphotic environment. Facies A4/L4, occurring near fair weather wave base in the euphotic zone, includes lenses of A5/L5 with stromatolitic coatings and thrombolithes. A6/L6 corresponds to a slightly restricted environment and shows a progressive transition to fenestral limestone of A7/L7. This facies was deposited in a moderately restricted intertidal area. A8/L8 developed in a quiet lagoonal subtidal environment. The mounds started with A2/L2 or A3/L3 in which microbial lenses and algal facies A4/L4 became progressively more abundant upwards. Following 20 m of laterally undifferentiated facies, more restricted facies occur in the central part of the buildups. This geometry suggests the initiation of restricted sedimentation, sheltered by bindstone or floatstone facies. The facies interpretation shows that after construction of the lower part of the mounds during a transgression and a sea-level highstand, a lowstand forced reef growth to the margin of the buildups, initiating the development of atoll-like crowns during the subsequent transgressive stage. The persistence of restricted facies results from the balance between sea-level rise and reef growth.     

A broad survey of recombination in animal mitochondria

Recombination in mitochondrial DNA (mtDNA) remains a controversial topic. Here we present a survey of 279 animal mtDNA data sets, of which 12 were from asexual species. Using four separate tests, we show that there is widespread evidence of recombination; for one test as many as 14.2% of the data sets reject a model of clonal inheritance and in several data sets, including primates, the recombinants can be identified visually. We show that none of the tests give significant results for obligate clonal species (apomictic pathogens) and that the sexual species show significantly greater evidence of recombination than asexual species. For some data sets, such as Macaca nemestrina, additional data sets suggest that the recombinants are not artifacts. For others, it cannot be determined whether the recombinants are real or produced by laboratory error. Either way, the results have important implications for how mtDNA is sequenced and used.     

Predominance of phytoplankton-derived dissolved and particulate organic carbon in a highly eutrophic tropical coastal embayment (Guanabara Bay,Rio de Janeiro,Brazil)

We investigate the carbon dynamics in Guanabara Bay, an eutrophic tropical coastal embayment surrounded by the megacity of Rio de Janeiro (southeast coast of Brazil). Nine sampling campaigns were conducted for dissolved, particulate and total organic carbon (DOC, POC and TOC), dissolved inorganic carbon (DIC), partial pressure of CO₂ (pCO₂), chlorophyll a (Chl a), pheo-pigments and ancillary parameters. Highest DOC, POC and Chl a concentrations were found in confined-shallow regions of the bay during the summer period with strong pCO₂ undersaturation, and DOC reached 82 mg L^?1, POC 152 mg L^?1, and Chl a 800 μg L^?1. Spatially and temporally, POC and DOC concentrations varied positively with total pigments, and negatively with DIC. Strong linear correlations between these parameters indicate that the production of TOC translates to an equivalent uptake in DIC, with 85% of the POC and about 50% of the DOC being of phytoplanktonic origin. Despite the shallow depths of the bay, surface waters were enriched in POC and DOC relative to bottom waters in periods of high thermohaline stratification. The seasonal accumulation of phytoplankton-derived TOC in the surface waters reached about 105 g C m^?2 year^?1, representing between 8 and 40% of the net primary production. The calculated turnover time of organic carbon was 117 and 34 days during winter and summer, respectively. Our results indicate that eutrophication of coastal bays in the tropics can generate large stocks of planktonic biomass and detrital organic carbon which are permanently being produced and partially degraded and buried in sediments.     

Budget of methane emissions from soils, livestock and the river network at the regional scale of the Seine basin (France)

We used various approaches to establish a comprehensive budget of methane (CH₄) emissions from the Seine basin, including direct emissions from livestock and soils as well as emissions from the drainage network. For the direct emissions from livestock, we used official livestock census numbers and emission factors (CH₄ emitted by each animal species per head per year) available in the literature. For the emissions from soils, we based our estimates on experimental measurements in closed chambers installed on different agricultural plots, forest, and grasslands in 2008 and 2009. The results were extrapolated to the whole Seine basin, including grassland, cropland, and forest soil distributions in the Seine basin. The CH₄ emissions from the Seine drainage network were also based on measurements of sampled waters in various rivers and streams (from headwaters to estuary) during different seasons in 2007, 2008, and 2010. After chemical analysis of CH₄ concentrations in the water samples using a gas chromatographic technique and calculation of the CH₄ supersaturation by stream order in rivers of the Seine basin (from 1 to 8) and by season we could estimate the CH₄ emissions for the whole water surface area of the Seine drainage network. The livestock of the Seine basin produce CH₄ emissions amounting to 166 × 10⁶ kg C year^?1, among which cattle are responsible for 85 %. The total CH₄ emission from the Seine drainage network was estimated at 0.3 × 10⁶ kg C year^?1, large rivers being responsible for the largest proportion. Ebullition could account for an additional 0.2 × 10⁶ kg C year^?1. Soils of the Seine basin are a net sink for CH₄ (9.4 × 10⁶ kg C year^?1). The water and soils fluxes are low with regard to emissions by livestock, but domestic waste, through landfills, could contribute an additional 40 × 10⁶ kg C year^?1.     

An improved genome of the model marine alga Ostreococcus tauri unfolds by assessing Illumina de novo assemblies

Background

Cost effective next generation sequencing technologies now enable the production of genomic datasets for many novel planktonic eukaryotes, representing an understudied reservoir of genetic diversity. O. tauri is the smallest free-living photosynthetic eukaryote known to date, a coccoid green alga that was first isolated in 1995 in a lagoon by the Mediterranean sea. Its simple features, ease of culture and the sequencing of its 13 Mb haploid nuclear genome have promoted this microalga as a new model organism for cell biology. Here, we investigated the quality of genome assemblies of Illumina GAIIx 75 bp paired-end reads from Ostreococcus tauri, thereby also improving the existing assembly and showing the genome to be stably maintained in culture.

Results

The 3 assemblers used, ABySS, CLCBio and Velvet, produced 95% complete genomes in 1402 to 2080 scaffolds with a very low rate of misassembly. Reciprocally, these assemblies improved the original genome assembly by filling in 930 gaps. Combined with additional analysis of raw reads and PCR sequencing effort, 1194 gaps have been solved in total adding up to 460 kb of sequence. Mapping of RNAseq Illumina data on this updated genome led to a twofold reduction in the proportion of multi-exon protein coding genes, representing 19% of the total 7699 protein coding genes. The comparison of the DNA extracted in 2001 and 2009 revealed the fixation of 8 single nucleotide substitutions and 2 deletions during the approximately 6000 generations in the lab. The deletions either knocked out or truncated two predicted transmembrane proteins, including a glutamate-receptor like gene.

Conclusion

High coverage (>80 fold) paired-end Illumina sequencing enables a high quality 95% complete genome assembly of a compact ~13 Mb haploid eukaryote. This genome sequence has remained stable for 6000 generations of lab culture.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1103) contains supplementary material, which is available to authorized users.     

Madanidinium loirii gen. et sp. nov. (Dinophyceae), a new marine benthic dinoflagellate from Martinique Island,Eastern Caribbean

A new benthic phototrophic dinoflagellate is described from sediments of a tropical marine cove at Martinique Island and its micromorphology is studied by means of light and electron microscopy. The cell contains small golden-brown chloroplasts and the oval nucleus is posterior. It is laterally compressed, almost circular in shape when viewed laterally. It consists of a small epitheca tilted toward the right lateral side and a larger hypotheca. In the left view, the cingulum is more anterior and the epitheca is reduced. The cingulum is displaced and left-handed. This organism is peculiar in having no apical pore and its thecal plate arrangement is 2′ 1a 7′′ 5c 3s 5′′′ 1′′′′. The plates are smooth with small groups of pores scattered on their surface. An area with 60–80 densely arranged pores is found near the centre of the 2′′′ plate, on the left lateral side. Morphologically, these features are different from all other laterally compressed benthic genera. In addition, molecular genetic sequences of SSU and partial LSU form a distinct and well-supported clade among dinoflagellates and support the erection of a new genus. However, molecular phylogenies inferred from ribosomal genes failed to confirm any clear relationship with other benthic taxa and affinity with other laterally compressed dinoflagellates has not been demonstrated. Hence, the taxonomic affinity of Madanidinium loirii with a defined order and family is unclear at the moment.     

A northern rockhopper penguin unveils dispersion pathways in the Southern Ocean

We report the first record of a northern rockhopper penguin Eudyptes moseleyi on the Kerguelen Islands, Southern Indian Ocean. The penguin must have crossed the subtropical convergence to reach the island. This species was recently proved to be genetically different from the subantarctic eastern rockhopper penguin E. filholi that normally breeds on the Kerguelen Islands. The sequencing of a part of the mitochondrial control region shows that this bird may come from the population of Gough Island, 6,000 km away, in the south Atlantic Ocean. This finding confirms that the genetic isolation between these two penguin species is complete, although some individuals may sporadically disperse between the breeding sites. This first direct observation of a disperser from the Atlantic to the Indian Ocean also adds further support to a biogeographic dispersion pattern already suggested by phylogeographic patterns in other species from the Southern Ocean.     

Field surveys of capercaillie (<Emphasis Type="Italic">Tetrao urogallus</Emphasis>) in the Swiss Alps underestimated local abundance of the species as revealed by genetic analyses of non-invasive samples

An increasing number of species are becoming threatened by habitat loss and fragmentation. Therefore, solid estimates of the species’ abundance in the remaining populations are required to develop suitable conservation measures and to monitor their effectiveness. The capercaillie (Tetrao urogallus L.) has experienced a dramatic decline in central Europe and has disappeared from large areas of its former natural range. In Switzerland, the species’ distribution, habitat requirements and demographic status were studied and evaluated in an attempt to support appropriate management decisions to conserve the species. National surveys of the capercaillie in Switzerland have traditionally been obtained from male counts at leks. However, individual attendance to the lek is sex- and age-specific. Thus, male counts at leks may provide a biased estimate of local population sizes. In the present study, we compared two alternative indirect methods to estimate the sizes of local populations at eight study sites situated in the Alps and Prealps of Switzerland. We first assessed the sizes of local populations from the observed density and distribution of direct and indirect evidence of the species’ presence during field surveys. Feather and faeces samples collected during field surveys were genotyped at twelve nuclear microsatellite loci and a sex-specific nuclear gene fragment. Individual genotypes were used as genetic tags to estimate the sizes of the eight local populations using an urn model developed for small populations. The index of local population sizes assessed from field surveys was lower than the number of unique genotypes at each study site, which itself underestimated the abundances of populations in most cases. Based on our results, the genetic tagging method appeared to be less biased than the field survey method. However, an alternative faeces sampling scheme, resulting in 2–3 genotypings per individual, could further improve the accuracy of the size estimates of local populations. Our study confirms that genetic tagging methods are a valuable tool to estimate the sizes of local populations and to monitor the response of rare and elusive species to management actions.