Comparative analysis of a genome fragment of an uncultivated mesopelagic crenarchaeote reveals multiple horizontal gene transfers

Marine planktonic crenarchaeota have escaped all cultivation attempts to date, all crenarchaeota growing in pure culture so far being hyperthermophiles. Here, we present a comparative genomic analysis of a 16S- plus 23S-rDNA-containing fragment of a crenarchaeote retrieved from an environmental genomic library constructed from picoplankton collected at 500 m depth in the Antarctic Polar Front. The clone DeepAnt-EC39 contained an insert of 33.3 kbp, which was completely sequenced. DeepAnt-EC39 appears to represent a lineage specific to deep-sea waters but widespread geographically, as revealed by the analysis of the 16S-23S-rDNA intergenic spacer region. A comparison with previously sequenced marine crenarchaeotal genomic clones also containing an rrn operon (74A4, 4B7 and Cenarchaeum symbiosum strains A and B) revealed a highly variable structure involving gene rearrangements and insertions/deletions. The surroundings of the rrn operon and the contiguous glutamate-1-semialdehyde aminotransferase gene appear hot spots for recombination. Phylogenetic analyses of all individual predicted proteins revealed the existence of several likely cases of horizontal gene transfer both, between the two archaeal kingdoms and between the two prokaryotic domains. The most frequent horizontal transfers appear to involve genes from mesophilic methanogenic euryarchaeota related to Methanosarcinales. We hypothesise that the acquisition of genes from mesophilic bacteria and euryarchaeota has played a major role in the adaptation of Group I crenarchaeota to life at lower temperatures.     

Study of the first hours of microvinification by the use of osmotic stress-response genes as probes

When yeast cells are inoculated into grape must for vinification they find stress conditions because of osmolarity, which is due to very high sugar concentration, and pH lower than 4. In this work an analysis of the expression of three osmotic stress induced genes (GPD1, HSP12 and HSP104) under microvinification conditions is shown as a way to probe those stress situations and the regulatory mechanisms that control them. The results indicate that during the first hours of microvinification there is an increase in the GPDI mRNA levels with a maximum about one hour after inoculation, and a decrease in the amount of HSP12 and HSP104 mRNAs, although with differences between them. The RNA steady-state levels of all the genes considered, and in some cases the microvinification progress are significantly affected by the composition of the must (pH, nature of the osmotic agent and carbon source). These results point out the importance of the control of these parameters and the yeast molecular response during the first hours of vinification for an accurate winemaking process.     

Susceptibility to T cell-mediated injury in immune complex disease is linked to local activation of renin-angiotensin system: the role of NF-AT pathway

FcR provides a critical link between ligands and effector cells in immune complex diseases. Emerging evidence reveals that angiotensin (Ang)II exerts a wide variety of cellular effects and contributes to the pathogenesis of inflammatory diseases. In anti-glomerular basement membrane Ab-induced glomerulonephritis (GN), we have previously noted that FcR-deficient mice (gamma(-/-)) surviving from lethal initial damage still developed mesangial proliferative GN, which was drastically prevented by an AngII type 1 receptor (AT1) blocker. We further examined the mechanisms by which renin-Ang system (RAS) participates in this immune disease. Using bone marrow chimeras between gamma(-/-) and AT1(-/-) mice, we found that glomerular injury in gamma(-/-) mice was associated with CD4(+) T cell infiltration depending on renal AT1-stimulation. Based on findings in cutaneous delayed-type hypersensitivity, we showed that AngII-activated renal resident cells are responsible for the recruitment of effector T cells. We next examined the chemotactic activity of AngII-stimulated mesangial cells, as potential mechanisms coupling RAS and cellular immunity. Chemotactic activity for T cells and Th1-associated chemokine (IFN-gamma-inducible protein-10 and macrophage-inflammatory protein 1alpha) expression was markedly reduced in mesangial cells from AT1(-/-) mice. Moreover, this activity was mainly through calcineurin-dependent NF-AT. Although IFN-gamma-inducible protein-10 was NF-kappaB-dependent, macrophage-inflammatory protein 1alpha was dominantly regulated by NF-AT. Furthermore, AT1-dependent NF-AT activation was observed in injured glomeruli by Southwestern histochemistry. In conclusion, our data indicate that local RAS activation, partly via the local NF-AT pathway, enhances the susceptibility to T cell-mediated injury in anti-glomerular basement membrane Ab-induced GN. This novel mechanism affords a rationale for the use of drugs interfering with RAS in immune renal diseases.     

Synthesis and biological activity of naphthalene analogues of phenstatins: naphthylphenstatins

Novel phenstatin analogues with a 2-naphthyl moiety combined with either a 2,3,4- or a 3,4,5-trimethoxyphenyl ring have been synthesized, and their tubulin polymerization inhibiting and cytotoxic activities have been evaluated. The 2-naphthyl ring is a better replacement for the 3-hydroxy-4-methoxyphenyl ring in the phenstatin series than in the combretastatin series. For the naphthylphenstatins, the carbonyl is required, and the preferred orientation of the trimethoxyphenyl ring is the one found in combretastatins.     

Life cycle and molecular phylogeny of the dinoflagellates Chytriodinium and Dissodinium,ectoparasites of copepod eggs

The dinoflagellates Chytriodinium affine, C. roseum and Dissodinium pseudolunula are ectoparasites of crustacean eggs. Here, we present new observations regarding their life cycle based on coastal plankton samples and incubations and analyze their molecular phylogeny using the small subunit ribosomal RNA gene (SSU rDNA) as a marker. In contrast to the typical stages already documented for its life cycle, we observed that D. pseudolunula dinospores may exceptionally differentiate inside a globular cyst. Despite its parasitic life style, the cysts and dinospores of D. pseudolunula contain chlorophyll a. We obtained the first SSU rDNA sequences for the genera Chytriodinium (the type C. roseum and C. affine) and Dissodinium (D. pseudolunula). Classical taxonomical schemes have ascribed these genera to the order Blastodiniales. However, our SSU rDNA-based phylogenetic analysis shows that these ectoparasites form a clade in the Gymnodinium sensu stricto group, unarmored dinokaryotic dinoflagellates of the order Gymnodiniales. They branch in a subgroup composed of warnowiids, polykrikoids, the type of Gymnodinium, G. fuscum and G. aureolum. Although Chytriodinium and Dissodinium appear to be relatives based on SSU rDNA phylogeny, feeding and host specificity, their life cycles are substantially different. Based on these data we consider that the type of life cycle is a poor criterion for classification at the family level. We suggest that the morphology of the infective cell is probably the most reliable phenotypic characteristic to determine the systematic position of parasitic dinoflagellates.     

Marked seasonality and high spatial variability of protist communities in shallow freshwater systems

Small eukaryotes have key roles in aquatic ecosystems, influencing their local environment, global biogeochemical cycles and climate. Their impact depends on community structure, which varies along time. However, very few studies take into account temporal variation. This is especially true for small, shallow freshwater systems, which remain largely understudied despite their wide variety, global surface and intense microbial activity. We have monthly followed changes in the community structure of small microbial eukaryotes (0.2–5 μm cell diameter) for 2 years in four ponds and one brook located in North-Western France based on massive 18S rDNA amplicon 454 pyrosequencing. We detected a total of 3742 stringently defined operational taxonomic units (OTUs) encompassing all recognized eukaryotic supergroups and lineages of uncertain affiliation. Although geographically close, protist communities in the five ecosystems were contrasting, with very few shared OTUs, suggesting that environmental selection mainly drives community structure. The temporal dynamics of different high-rank taxa appeared complex and rapid at monthly scales. Despite this, a clear and reproducible seasonality was observed. As expected, low-abundance OTUs dominated the community. Although some of them appeared sporadically or remained at low frequencies during the survey, others occasionally reached relatively high abundances, sometimes recurrently. This shows that at least a fraction of low-abundance eukaryotes constitutes a seed bank. The annual proportion of primary producers, free-living heterotrophs and parasites appeared remarkably constant among the different ecosystems, suggesting underlying trends of ecosystem carrying capacity for these functional groups.     

The extent of protist diversity: insights from molecular ecology of freshwater eukaryotes

Classical studies on protist diversity of freshwater environments worldwide have led to the idea that most species of microbial eukaryotes are known. One exemplary case would be constituted by the ciliates, which have been claimed to encompass a few thousands of ubiquitous species, most of them already described. Recently, molecular methods have revealed an unsuspected protist diversity, especially in oceanic as well as some extreme environments, suggesting the occurrence of a hidden diversity of eukaryotic lineages. In order to test if this holds also for freshwater environments, we have carried out a molecular survey of small subunit ribosomal RNA genes in water and sediment samples of two ponds, one oxic and another suboxic, from the same geographic area. Our results show that protist diversity is very high. The majority of phylotypes affiliated within a few well established eukaryotic kingdoms or phyla, including alveolates, cryptophytes, heterokonts, Cercozoa, Centroheliozoa and haptophytes, although a few sequences did not display a clear taxonomic affiliation. The diversity of sequences within groups was very large, particularly that of ciliates, and a number of them were very divergent from known species, which could define new intra-phylum groups. This suggests that, contrary to current ideas, the diversity of freshwater protists is far from being completely described.     

Archaeal overdominance close to life-limiting conditions in geothermally influenced hypersaline lakes at the Danakil Depression,Ethiopia

The Dallol protovolcanic area on the Danakil Depression (Afar region, Ethiopia) exhibits unique hydrothermal manifestations in hypersaline context, yielding varied polyextreme physicochemical conditions. Previous studies identified a wide archaeal diversity in less extreme brines but failed to identify microorganisms thriving in either high-chaotropicity, low-water-activity brines or hyperacidic-hypersaline Na-Fe-rich brines. Recently, we accessed several small lakes under intense degassing activity adjacent to the Round Mountain, west to the Dallol dome [Western Canyon Lakes (WCL); WCL1-5]. They exhibited intermediate parameter combinations (pH ~ 5, 34%–41% (weight/volume) NaCl-dominated salts with relatively high levels of chaotropic Mg-Ca salts) that should allow to better constrain life limits. These lakes were overwhelmingly dominated by Archaea, encompassing up to 99% of prokaryotic 16S rRNA gene amplicon sequences in metabarcoding studies. The majority belonged to Halobacteriota and Nanohaloarchaeota, the latter representing up to half of prokaryotic sequences. Optical and epifluorescence microscopy showed active cells in natural samples and diverse morphotypes in enrichment cultures. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy revealed tiny cells (200–300 nm diameter) epibiotically associated with somewhat larger cells (0.6–1 μm) but also the presence of silica-dominated precipitates of similar size and shape, highlighting the difficulty of distinguishing microbes from mineral biomorphs in this kind of low-biomass systems.     

Global eukaryote phylogeny: Combined small- and large-subunit ribosomal DNA trees support monophyly of Rhizaria, Retaria and Excavata

Resolution of the phylogenetic relationships among the major eukaryotic groups is one of the most important problems in evolutionary biology that is still only partially solved. This task was initially addressed using a single marker, the small-subunit ribosomal DNA (SSU rDNA), although in recent years it has been shown that it does not contain enough phylogenetic information to robustly resolve global eukaryotic phylogeny. This has prompted the use of multi-gene analyses, especially in the form of long concatenations of numerous conserved protein sequences. However, this approach is severely limited by the small number of taxa for which such a large number of protein sequences is available today. We have explored the alternative approach of using only two markers but a large taxonomic sampling, by analysing a combination of SSU and large-subunit (LSU) rDNA sequences. This strategy allows also the incorporation of sequences from non-cultivated protists, e.g., Radiozoa (=radiolaria minus Phaeodarea). We provide the first LSU rRNA sequences for Heliozoa, Apusozoa (both Apusomonadida and Ancyromonadida), Cercozoa and Radiozoa. Our Bayesian and maximum likelihood analyses for 91 eukaryotic combined SSU+LSU sequences yielded much stronger support than hitherto for the supergroup Rhizaria (Cercozoa plus Radiozoa plus Foraminifera) and several well-recognised groups and also for other problematic clades, such as the Retaria (Radiozoa plus Foraminifera) and, with more moderate support, the Excavata. Within opisthokonts, the combined tree strongly confirms that the filose amoebae Nuclearia are sisters to Fungi whereas other Choanozoa are sisters to animals. The position of some bikont taxa, notably Heliozoa and Apusozoa, remains unresolved. However, our combined trees suggest a more deeply diverging position for Ancyromonas, and perhaps also Apusomonas, than for other bikonts, suggesting that apusozoan zooflagellates may be central for understanding the early evolution of this huge eukaryotic group. Multiple protein sequences will be needed fully to resolve basal bikont phylogeny. Nonetheless, our results suggest that combined SSU+LSU rDNA phylogenies can help to resolve several ambiguous regions of the eukaryotic tree and identify key taxa for subsequent multi-gene analyses.     

Conventional and molecular cytogenetic diagnostic methods in stem cell research: a concise review

Regenerative medicine and cell therapy are emerging clinical disciplines in the field of stem cell biology. The most important sources for cell transplantation are human embryonic and adult stem cells. The future use of these human stem cell lines in humans requires a guarantee of exhaustive control with respect to quality control, safety and traceability. Genetic instability and chromosomal abnormalities represent a potential weakness in basic studies and future therapeutic applications based on these stem cell lines, and may explain, at least in part, their usual tumourigenic properties. So, the introduction of the cytogenetic programme in the determination of the chromosomal stability is a key point in the establishment of the stem cell lines. The aim of this review is to provide readers with an up-to-date overview of all the cytogenetic techniques, both conventional methods and molecular fluorescence methods, to be used in a stem cell bank or other stem cell research centres. Thus, it is crucial to optimize and validate their use in the determination of the chromosomal stability of these stem cell lines, and assess the advantages and limitations of these cutting-edge cytogenetic technologies.