e13546 Background: High sensitivity detection of cancer biomarkers facilitates early diagnosis and provides insight for intervention including stratification for drug treatment and monitoring changes in tumor mutation profiles by analyzing DNA isolated from serum/plasma or circulating tumor cells. PIK3CA, encoding the PI3K p110α catalytic subunit, is mutated frequently in common cancers, including breast and colon carcinomas. 80% of these mutations are represented by three amino acid substitutions in the enzyme’s helical (E542K and E545K) or kinase (H1047R) domains. Methods: In this study, we have applied improved and complete enrichment CO-amplification at lower denaturation temperature PCR (Ice COLD-PCR) technology for high sensitivity detection and robust enrichment of any mutations present in a DNA sample. Ice-COLD-PCR preferentially amplifies low levels of any mutant DNA in a sample containing a vast excess of wild-type DNA. The use of a reference sequence oligonucleotide complimentary to one of the wild-type DNA strands results in linear amplification of one of the wild type strands while both mutant strands amplify exponentially during ice COLD- PCR. After Ice COLD-PCR, there is sufficient mutant allele enrichment to allow Sanger sequencing confirmation of the mutation. Results: The use of Ice COLD-PCR allows for the enrichment of mutations any of the key mutations in the PIK3CA gene that cannot be detected by standard PCR followed by Sanger sequencing methods. Initial limit of detection experiments indicate that the detection and sequence confirmation of mutations in PIK3CA more sensitive than standard PCR amplification or allele specific probe methodologies. Conclusions: After Ice COLD-PCR, low level PIK3CA mutant alleles are enriched over the wild-type allele such that simple sequence confirmation on multiple downstream systems such as Sanger Sequencing or allele-specific probe platforms is possible. Ice COLD-PCR amplification of PIK3CA and other genes requires only a standard thermocycler coupled to any downstream sequence analysis platform and therefore can be developed as a powerful, highly sensitive diagnostic assay.
Abstract. The Earth's oceans are one of the largest sinks in the Earth system for anthropogenic CO2 emissions, acting as a negative feedback on climate change. Earth system models project that climate change will lead to a weakening ocean carbon uptake rate as warm water holds less dissolved CO2 and as biological productivity declines. However, most Earth system models do not incorporate the impact of warming on bacterial remineralisation and rely on simplified representations of plankton ecology that do not resolve the potential impact of climate change on ecosystem structure or elemental stoichiometry. Here, we use a recently developed extension of the cGEnIE (carbon-centric Grid Enabled Integrated Earth system model), ecoGEnIE, featuring a trait-based scheme for plankton ecology (ECOGEM), and also incorporate cGEnIE's temperature-dependent remineralisation (TDR) scheme. This enables evaluation of the impact of both ecological dynamics and temperature-dependent remineralisation on particulate organic carbon (POC) export in response to climate change. We find that including TDR increases cumulative POC export relative to default runs due to increased nutrient recycling (+∼1.3 %), whereas ECOGEM decreases cumulative POC export by enabling a shift to smaller plankton classes (-∼0.9 %). However, interactions with carbonate chemistry cause opposite sign responses for the carbon sink in both cases: TDR leads to a smaller sink relative to default runs (-∼1.0 %), whereas ECOGEM leads to a larger sink (+∼0.2 %). Combining TDR and ECOGEM results in a net strengthening of POC export (+∼0.1 %) and a net reduction in carbon sink (-∼0.7 %) relative to default. These results illustrate the degree to which ecological dynamics and biodiversity modulate the strength of the biological pump, and demonstrate that Earth system models need to incorporate ecological complexity in order to resolve non-linear climate–biosphere feedbacks.
Variable fluorescence normalized to maximal fluorescence, F v /F m , determined by Fast Repetition Rate Fluorometry (FRRF) is being increasingly used to compare photosynthetic electron transport capacity in natural phytoplankton communities. Interpreting results of such studies is, however, complicated by the fact that both nutrient status and light history (photoinhibition under in situ conditions) are known to influence F v /F m . Thus, the value of F v /F m measurements in the field would be greatly enhanced if the light history signal could be separated from other influences. Here, both field and laboratory studies demonstrate that dark treatment (30 min‐4 h) before FRRF measurement is not sufficient to remove a light history signal in F v /F m . The signal could, however, be essentially eliminated by incubation of samples in low light prior to F v /F m determination. For the study conditions tested, the most effective treatment for removal of the signal was 4 h at 50 µmol m ‐2 s ‐1 . However, the effectiveness of the light treatment in removing the light history was influenced by temperature. Therefore, no universal protocol for eliminating the light history signal can be developed, but recommendations are given for developing site‐specific approaches for separating the light history signal from other factors influencing F v /F m . Carrying out light incubations before determining F v /F m not only provides the possibility for eliminating a light history signal in the measurements but the difference between F v /F m measured after light and dark incubations appears also to be a potentially useful indicator of the degree of photoinhibition experienced by phytoplankton under natural conditions.
SUMMARY Phalacroma currently comprises 69 species of mostly marine heterotrophic dinophysoids. With a round cell body and short sulcal and cingular lists, Phalacroma spp. have a simple morphology compared to other dinophysoid genera. Therefore, species identification is not always a trivial matter. A few Arctic species have been described and, with the exception of Ph. rotundatum , they are not only infrequently recorded but also occur in low cell abundances. Here, we studied a Lugol fixed sample from Denmark Strait. From this sample 13 cells with a Phalacroma ‐like morphology were isolated under a stereo microscope, photo documented, and used for single‐cell PCR determination of nuclear‐encoded LSU rDNA sequences. The sequences fell in three groups defined by their ribotype. A single ribotype sequence representing each group was added to an alignment containing a diverse assemblage of dinophysoids and analyzed using Bayesian analysis and maximum likelihood. Based on comparative light microscopy one of the ribotypes (ribotype 1) was similar but not identical to Ph. ruudi and ribotype 2 could not be matched to a known species of Phalacroma . In the phylogeny both clustered with other species of Phalacroma including the type species. The morphology of ribotype 3 was similar but not identical to Ph. braarudii . In the phylogeny ribotype 3 clustered outside the core group of Phalacroma species. Hence, ribotype 3 was designated ‘ Phalacroma’ sp. 3 and has to be described as a new genus pending additional data. This study has revealed the existence of cryptic species diversity within the Phalacroma morphotype boundary. Two additional Phalacroma species ( Ph. apicatum and Ph . cf. argus ) also clustered outside Phalacroma sensu stricto further emphasizing the presence of cryptic species within the genus. Future studies with a polyphasic approach are needed to better address the taxonomy of Phalacroma and Phalacroma ‐like dinophysoids.
Summary Phytoplankton assemblages in the open ocean are usually assumed to be mixed on local scales unless large semi‐permanent density discontinuities separating water masses are present. Recent modelling studies have, however, suggested that ephemeral submesoscale oceanographic features leading to only subtle density discontinuities may be important for controlling phytoplankton alpha‐ and beta‐diversity patterns. Until now, no empirical evidence has been presented to support this hypothesis. Using hydrographic and taxonomic composition data collected near Iceland during the period of the 2008 spring bloom, we show that the distribution of phytoplankton alpha‐ and beta‐diversity was related to submesoscale heterogeneity in oceanographic conditions. Distinct phytoplankton communities as well as differences in richness were identified on either side of a front delimiting surface waters of slightly different (˜0.03) salinities. Alpha‐diversity was significantly higher on the high salinity side of the front compared to the low salinity side. This difference was primarily driven by the presence of several large diatom species in the high salinity region, especially of the genus Chaetoceros which dominated the biomass here. By investigating beta‐diversity in relation to environmental and spatiotemporal variables, we show that the regional distribution of phytoplankton taxa was influenced by both different environmental conditions on either side of the front and dispersal limitation across the front. Changes in beta‐diversity were primarily driven by turnover rather than nestedness and were apparently controlled by different processes in each region. Synthesis . This study shows that small‐scale and ephemeral density discontinuities created by submesoscale frontal dynamics can play a major role in structuring patterns of phytoplankton diversity. Evidence is presented that they can generate changes in environmental conditions (leading to environmental filtering) and act as physical (dispersal) barriers for phytoplankton transport. The study suggests that dispersal barriers are potentially of much greater importance for phytoplankton diversity at local scales than currently recognized and indicates that drivers of marine phytoplankton diversity are similar to those structuring diversity of land plants.
