Automated flow cytometry as a tool to obtain a fine-grain picture of marine prokaryote community structure along an entire oceanographic cruise

On a standard oceanographic cruise, flow cytometry data are usually collected sparsely through a bottle-based sampling and with stations separated by kilometers leading to a fragmented view of the ecosystem; to improve the resolution of the datasets produced by this technique here it is proposed the application of an automatic method of sampling and staining. The system used consists of a flow-cytometer (Accuri-C6) connected to an automated continuous sampler (OC-300) that collects samples of marine surface waters every 15 min. We tested this system for five days during a brief Mediterranean cruise with the aim of estimating the abundance, relative size and phenotypic diversity of prokaryotes. Seawater was taken by a faucet linked to an inlet pump (ca. 5 m depth). Once the sample was taken, the Oncyt-300 stained it and sent it to the flow cytometer. A total of 366 samples were collected, effectively achieving a fine-grained scale view of microbial community composition both through space and time. A significative positive relationship was found comparing data obtained with the automatic method and 10 samples collected from the faucet but processed with the standard protocol. Abundance values retrieved varied from 3.56·105 cell mL−1 in the coastal area till 6.87 105 cell mL−1 in open waters, exceptional values were reached in the harbor area where abundances peaked to 1.28 106 cell mL−1. The measured features (abundance and size) were associated with metadata (temperature, salinity, conductivity) also taken in continuous, of which conductivity was the one that better explained the variability of abundance. A full 24 h measurement cycle was performed resulting in slightly higher median bacterial abundances values during daylight hours compared to night. Alpha diversity, calculated using computational cytometry techniques, showed a higher value in the coastal area above 41° of latitude and had a strong inverse relationship with both salinity and conductivity. This is the first time to our knowledge that the OC-300 is directly applied to the marine environment during an oceanographic cruise; due to its high-resolution, this set-up shows great potential both to cover large sampling areas, and to monitor day-night cycles in situ

Marked changes in diversity and relative activity of picoeukaryotes with depth in the world ocean

Microbial eukaryotes are key components of the ocean plankton. Yet, our understanding of their community composition and activity in different water layers of the ocean is limited, particularly for picoeukaryotes (0.2–3 µm cell size). Here, we examined the picoeukaryotic communities inhabiting different vertical zones of the tropical and subtropical global ocean: surface, deep chlorophyll maximum, mesopelagic (including the deep scattering layer and oxygen minimum zones), and bathypelagic. Communities were analysed by high-tthroughput sequencing of the 18S rRNA gene (V4 region) as represented by DNA (community structure) and RNA (metabolism), followed by delineation of Operational Taxonomic Units (OTUs) at 99% similarity. We found a stratification of the picoeukaryotic communities along the water column, with assemblages corresponding to the sunlit and dark ocean. Specific taxonomic groups either increased (e.g., Chrysophyceae or Bicosoecida) or decreased (e.g., Dinoflagellata or MAST-3) in abundance with depth. We used the rRNA:rDNA ratio of each OTU as a proxy of metabolic activity. The highest relative activity was found in the mesopelagic layer for most taxonomic groups, and the lowest in the bathypelagic. Altogether, we characterize the change in community structure and metabolic activity of picoeukaryotes with depth in the global ocean, suggesting a hotspot of activity in the mesopelagic

Immunotherapy for recurrent ovarian cancer: a further piece of the puzzle or a striking strategy?

Introduction: Treatment of ovarian cancer has been long standardized with the inclusion of surgery and chemotherapy based on platinum and taxanes, this strategy reaching high remission rates. However, when this treatment fails, further options are available with little benefit. Since ovarian cancer has specific immunologic features, actually immunotherapy is under evalua- 15 tion to overcome treatment failure in patients experiencing recurrence. Areas covered: Immunogenicity of ovarian cancer and its relationship with clinical outcomes is briefly reviewed. The kinds of immunotherapeutic strategies are summarized. The clinical trials investigating immunotherapy in recurrent ovarian cancer patients are reported. 20 Expert opinion: The results of these clinical trials about immunotherapy are interesting, but little clinical benefit has been achieved until now. For this reason, we could conclude that immunotherapy is quite different from other treatment options and it could change the global approach for recurrent ovarian cancer treatment. However, to date only fragmentary findings are 25 available to define the real role of immunotherapy in this setting

Marine protist diversity in European coastal waters and sediments as revealed by high-throughput sequencing

International audienceAlthough protists are critical components of marine ecosystems, they are still poorly characterized. Here we analysed the taxonomic diversity of planktonic and benthic protist communities collected in six distant European coastal sites. Environmental deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) from three size fractions (pico-, nano- and micro/mesoplankton), as well as from dissolved DNA and surface sediments were used as templates for tag pyrosequencing of the V4 region of the 18S ribosomal DNA. Beta-diversity analyses split the protist community structure into three main clusters: picoplankton-nanoplankton-dissolved DNA, micro/mesoplankton and sediments. Within each cluster, protist communities from the same site and time clustered together, while communities from the same site but different seasons were unrelated. Both DNA and RNA-based surveys provided similar relative abundances for most class-level taxonomic groups. Yet, particular groups were overrepresented in one of the two templates, such as marine alveolates (MALV)-I and MALV-II that were much more abundant in DNA surveys. Overall, the groups displaying the highest relative contribution were Dinophyceae, Diatomea, Ciliophora and Acantharia. Also, well represented were Mamiellophyceae, Cryptomonadales, marine alveolates and marine stramenopiles in the picoplankton, and Monadofilosa and basal Fungi in sediments. Our extensive and systematic sequencing of geographically separated sites provides the most comprehensive molecular description of coastal marine protist diversity to date

Experimental study of differentially rotating supersonic plasma flows produced by aluminium wire array Z-pinches

A novel approach to cylindrical wire array z-pinches has been developed in order to create a rotating plasma flow analogous to astrophysical accretion discs. The method involves subjecting the wire array to a cusp magnetic field (B_r) to create converging off axis ablation streams to form a rotating flow. The rotation is sustained by the ram pressure of the ablation streams in a quasi-equilibrium state for approximately 150 ns. This corresponds to one full rotation of the plasma about the axis. The rotating plasma is supersonic with Mach number ~2 and a radially constant rotation velocity between 60 and 75 km/s; the angular velocity therefore has an r^-1 dependence and the flow is differential. A Thomson scattering diagnostic is used to measure the electron and ion temperatures as Te ~30 eV and Ti >55 eV and the ionisation of the plasma (Z) between 6 and 8. These parameters are used to calculate the Reynolds number (10^5 to 10^6) and magnetic Reynolds numbers (20 to 100) which are large enough for viscous and resistive effects to be negligible on the large scale of the flow. These are of sufficient magnitude for the experiment to be scalable to astrophysical accretion discs. Further more the Reynolds number for the experiment is large enough for shear instabilities to manifest in the plasma. Some evidence for this can be seen in XUV images and Thomson spectra which indicate the development of perturbations and vorticity within the flow. Predictions for the growth rate of the Kelvin Helmholtz instability, 12 to 40 ns, agree reasonably well with the observed perturbation growth of ~30 ns. It is also possible that shear instabilities are driving hydrodynamic turbulence. Turbulent heating of the plasma could explain the approximately 500 eV increase in the ion temperature observed from some Thomson spectra. Further work is required however to prove the existence of shear flows and turbulence within the experiments.Open Acces

A Short Comparison of Two Marine Planktonic Diazotrophic Symbioses Highlights an Un-quantified Disparity

Some N2-fixing cyanobacteria form symbiosis with diverse protists. In the plankton two groups of diazotrophic symbioses are described: (1) a collective group of diatoms which associate with heterocystous cyanobacteria (Diatom Diazotroph Associations, DDA), and (2) the microalgal prymnesiophyte Braarudosphaera bigelowii and its relatives which associate with the unicellular cyanobacterium Candidatus Atelocyanobacterium thalassa (hereafter as UCYN-A). Both symbiotic systems co-occur, and in both partnerships the symbionts function as a nitrogen (N) source. In this perspective, we provide a brief comparison between the DDAs and the prymnesiophyte-UCYN-A symbioses highlighting similarities and differences in both systems, and present a bias in the attention and current methodology that has led to an under-detection and under-estimation of the DDAs

Maximum Likelihood phylogenetic trees for eukaryotic supergroups.

<p>Trees include several taxonomic groups within Alveolata (A), Stramenopiles (B), and are done with sequences representative of each OTU obtained clustering at 0.05 distance (A) and 0.01 distance (B). The number of sequences (about 550 bp in length) per tree is 798 and 523 respectively. Red dots represent bootstrap values above 75 and orange dots values above 50.</p

Comparison of partial and full-length 18S rDNA sequences to infer genetic distances.

<p>The three panels show pair-wise genetic distances (Jukes Cantor corrected) of the complete gene against partial regions (V4–V5 in dark grey or V9 in light grey) for sequences within Stramenopiles (A), Alveolata (B), and Rhizaria (C). Slopes (m) and coefficients (R) of the correlations are shown at the top of the graphs.</p

Intragroup phylogenetic distance and trunk length of Stramenopiles groups.

<p>A complementary view of phylogenetic structure of Stramenopiles is shown by displaying the trunk length (vertical lines) and the Mean Phylogenetic Distance (vertical boxes) of each group (based on tree in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057170#pone-0057170-g002" target="_blank">Figure 2B</a>).</p

Maximum Likelihood phylogenetic trees for eukaryotic supergroups.

<p>Trees include several taxonomic groups within CCTH (A), and Rhizaria (B) and are done with sequences representative of each OTU obtained clustering at 0.05 distance. The number of sequences (about 550 bp in length) per tree 218 and 303 respectively. Red dots represent bootstrap values above 75.</p