Contribution to drug discovery and development for tauopathies using yeast as a model

This work aimed to contribute to drug discovery and development (DDD) for tauopathies, while expanding our knowledge on this group of neurodegenerative disorders, including Alzheimer’s disease (AD). Using yeast, a recognized model for neurodegeneration studies, useful models were produced for the study of tau interaction with beta-amyloid (Aβ), both AD hallmark proteins. The characterization of these models suggests that these proteins co-localize and that Aβ1-42, which is toxic to yeast, is involved in tau40 phosphorylation (Ser396/404) via the GSK-3β yeast orthologue, whereas tau seems to facilitate Aβ1-42 oligomerization. The mapping of tau’s interactome in yeast, achieved with a tau toxicity enhancer screen using the yeast deletion collection, provided a novel framework, composed of 31 genes, to identify new mechanisms associated with tau pathology, as well as to identify new drug targets or biomarkers. This genomic screen also allowed to select the yeast strain mir1Δ-tau40 for development of a new GPSD2TM drug discovery screening system. A library of unique 138 marine bacteria extracts, obtained from the Mid-Atlantic Ridge hydrothermal vents, was screened with mir1Δ-tau40. Three extracts were identified as suppressors of tau toxicity and constitute good starting points for DDD programs. mir1Δ strain was sensitive to tau toxicity, relating tau pathology with mitochondrial function. SLC25A3, the human homologue of MIR1, codes for the mitochondrial phosphate carrier protein (PiC). Resorting to iRNA, SLC25A3 expression was silenced in human neuroglioma cells, as a first step towards the engineering of a neural model for replicating the results obtained in yeast. This model is essential to understand the mechanisms of tau toxicity at the mitochondrial level and to validate PiC as a relevant drug target. The set of DDD tools here presented will foster the development of innovative and efficacious therapies, urgently needed to cope with tau-related disorders of high human and social-economic impact.

Morphology of terebelliform polychaetes (Annelida: Polychaeta: Terebelliformia), with a focus on Terebellidae

The morphology of terebelliform polychaetes was investigated for a phylogenetic study focused on Terebellidae. For this study, specimens belonging to 147 taxa, preferably type material or specimens from type localities or areas close to them, were examined under stereo, light and scanning electron microscopes. The taxa examined were 1 Pectinariidae, 2 Ampharetidae, 2 Alvinellidae, 8 Trichobranchidae, and 134 Terebellidae, which included 8 Polycirrinae, 15 Thelepodinae, and 111 Terebellinae. A comparison of the morphology, including prostomium, peristomium, anterior segments and lobes, branchiae, glandular venter, nephridial and genital papillae, notopodia and notochaetae, neuropodia and neurochaetae, and posterior end, was made of all the currently recognized families of terebelliform polychaetes, with special emphasis on Terebellidae. A discussion of the characters useful to distinguish between genera is given. This character set will be used in a subsequent phylogenetic study (Nogueira & Hutchings in prep.)

Biogeochemistry of a deep-sea whale fall: sulfate reduction, sulfide efflux and methanogenesis

Deep-sea whale falls create sulfidic habits Supporting chemoautotrophic communities, but microbial processes underlying the formation Of Such habitats remain poorly evaluated. Microbial degradation processes (sulfate reduction, methanogenesis) and biogeochemical gradients were studied in a whale-fall habitat created by a 30 t whale carcass deployed at 1675 m depth for 6 to 7 yr on the California margin. A variety of measurements were conducted including photomosaicking, microsensor measurements, radio-tracer incubations and geochemical analyses. Sediments were Studied at different distances (0 to 9 in) from the whale fall. Highest microbial activities and steepest vertical geochemical gradients were found within 0.5 m of the whale fall, revealing ex situ sulfate reduction and in vitro methanogenesis rates of up to 717 and 99 mmol m(-2) d(-1), respectively. In sediments containing whale biomass, methanogenesis was equivalent to 20 to 30%, of sulfate reduction. During in vitro sediment studies, sulfide and methane were produced within days to weeks after addition of whale biomass, indicating that chemosynthesis is promoted at early stages of the whale fall. Total sulfide production from sediments within 0.5 m of the whale fall was 2.1 +/- 3 and 1.5 +/- 2.1 mol d(-1) in Years 6 and 7, respectively, of which similar to 200 mmol d(-1) were available as free sulfide. Sulfate reduction in bones was much lower, accounting for a total availability of similar to 10 mmol sulfide d(-1). Over periods of at least 7 yr, whale falls can create sulfidic conditions similar to other chemosynthetic habitats Such as cold seeps and hydrothermal vents.

Brazilian research on extremophiles in the context of astrobiology

Extremophiles are organisms adapted to grow at extreme ranges of environmental variables, such as high or low temperatures, acid or alkaline medium, high salt concentration, high pressures and so forth. Most extremophiles are micro-organisms that belong to the Archaea and Bacteria domains, and are widely spread across the world, which include the polar regions, volcanoes, deserts, deep oceanic sediments, hydrothermal vents, hypersaline lakes, acid and alkaline water bodies, and other extreme environments considered hostile to human life. Despite the tropical climate, Brazil has a wide range of ecosystems which include some permanent or seasonally extreme environments. For example, the Cerrado is a biome with very low soil pH with high Al+3 concentration, the mangroves in the Brazilian coast are anaerobic and saline, Pantanal has thousands of alkaline-saline lakes, the Caatinga arid and hot soils and the deep sea sediments in the Brazilian ocean shelf. These environments harbour extremophilic organisms that, coupled with the high natural biodiversity in Brazil, could be explored for different purposes. However, only a few projects in Brazil intended to study the extremophiles. In the frame of astrobiology, for example, these organisms could provide important models for defining the limits of life and hypothesize about life outside Earth. Brazilian microbiologists have, however, studied the extremophilic micro-organisms inhabiting non-Brazilian environments, such as the Antarctic continent. The experience and previous results obtained from the Brazilian Antarctic Program (PROANTAR) provide important results that are directly related to astrobiology. This article is a brief synopsis of the Brazilian experience in researching extremophiles, indicating the most important results related to astrobiology and some future perspectives in this area. Received 29 February 2012, accepted 25 May 2012, first published online 11 July 2012

Image analysis from TEM and AFM micrograph, Table S1, S2, S3, Figure S1, Figure S2

We report on metal enrichment along a natural pH gradient owing to increased CO2 degassing at cold, shal- low seeps of Vulcano Island in the Mediterranean Sea, off Sicily. We assessed composition of unfiltered and filtered seawater (b100 nm) along acidic zones ranging between ambient and pH 5, and showed that most seep derived elements are present as nanoclusters which then aggregate into larger colloids while mixing with ambient seawater along a pH gradient. Size and elemental composition of such naturally occurring nanoparticles assessed by modern characterisation methods were in good agreement with the results from conventional analytical methods. We provide analytical evidence for the presence in the water column of a large fraction of seep derived ele- ments (e.g. approximately 50% of iron, over 80% of Mn, 100% of Cr, S and Zn) in the form of nano sized par- ticles (e.g. b100 nm) even at typical open ocean pHs. We launch in situ sampling protocols and sample preparation procedures for multi-method suitable to obtain accurate measurements on nanoparticles from environmental samples. Based on our results a first insight to the formation of natural nanoparticles at cold CO2 seeps is presented and the persistence of such nano-clusters in the surrounding seawater is stipulated.

(Table S1) Molecular analyses and morphological comparisons of deep-sea clams

Large vesicomyid clams are common inhabitants of sulphidic deep-sea habitats such as hydrothermal vents, hydrocarbon seeps and whale-falls. Yet, the species- and genus-level taxonomy of these diverse clams has been unstable due to insufficiencies in sampling and absence of detailed taxonomic studies that would consistently compare molecular and morphological characters. To clarify uncertainties about species-level assignments, we examined DNA sequences from mitochondrial cytochrome-c-oxidase subunit I (COI) in conjunction with morphological characters. New and published COI sequences were used to create a molecular database for 44 unique evolutionary lineages corresponding to species. Overall, the congruence between molecular and morphological characters was good. Several discrepancies due to synonymous species designations were recognized, and acceptable species names were rectified with published COI sequences in cases where morphological specimens were available. We identified seven species with trans-Pacific distributions, and two species with Indo-Pacific distributions. Presently, 27 species have only been documented from one region, which might reflect limited ranges, or insufficient geographical sampling. Vesicomyids exhibit the greatest species diversity along the northwest Pacific ridge systems and in the eastern Pacific, along the western America margin, where depth zonation typically results in segregation of closely related species. The broad distributions of several vesicomyid species suggest that their required chemosynthetic habitats might be more common than previously recognized and occur along most continental margins.

Sediment properties, contents and accumulation rates of Hg in cores of Upper Quaternary sediments from the Deryugin Basin, Okhotsk Sea

The Hg distribution and some mineralogical-geochemical features of bottom sediments up to a depth of 10 m in the Deryugin Basin showed that the high and anomalous Hg contents in the Holocene deposits are confined to a spreading riftogenic structure and separate fluid vents within it. The accumulations of Hg in the the sediments were caused by its fluxes from gas and low-temperature hydrothermal vents under favorable oceanological conditions in the Holocene. The two mainly responsible for the high and anomalous Hg contents are infiltration (fluxes of hydrothermal or gas fluids from the sedimentary cover) and plume (Hg precipitation from water plumes with certain hydrochemical conditions forming above endogenous sources). The infiltration anomalies of Hg were revealed in the following environments: (1) near gas vents on the northeastern Sakhalin slope, where high Hg contents are associated only with Se and were caused by the accumulation of gases ascending from beneath the gas hydrate layer; (2) in the area of inferred occasionally operating low-temperature hydrothermal seeps in the central part of the Deryugin Basin, in which massive barite chimneys, hydrothermal Fe-Mn crusts, and anomalous contents of Mn, Ba, Zn, and Ni in sediments develop.

(Table 2) Concentration of suspended matter and dissolved Mn in water samples above the Mid-Atlantic Ridge rift zone near 29°N

Data on nephelometry, CTD probing, and dissolved manganese obtained during Cruise 34 of R/V Akademik Mstislav Keldysh showed that a multiplume was found over the Mid-Atlantic Ridge rift zone near 29°N. This multiplume comprises plumes of several hydrothermal vents and is separate from the plume of the Broken Spur hydrothermal field. Approximate coordinates were estimated for three vents.

Results of H2-consumption experiments during METEOR cruise M64/2

The discovery of deep-sea hydrothermal vents in 1977 revolutionized our understanding of the energy sources that fuel primary productivity on Earth. Hydrothermal vent ecosystems are dominated by animals that live in symbiosis with chemosynthetic bacteria. So far, only two energy sources have been shown to power chemosynthetic symbioses: reduced sulphur compounds and methane. Using metagenome sequencing, single-gene fluorescence in situ hybridization, immunohistochemistry, shipboard incubations and in situ mass spectrometry, we show here that the symbionts of the hydrothermal vent mussel Bathymodiolus from the Mid-Atlantic Ridge use hydrogen to power primary production. In addition, we show that the symbionts of Bathymodiolus mussels from Pacific vents have hupL, the key gene for hydrogen oxidation. Furthermore, the symbionts of other vent animals such as the tubeworm Riftia pachyptila and the shrimp Rimicaris exoculata also have hupL. We propose that the ability to use hydrogen as an energy source is widespread in hydrothermal vent symbioses, particularly at sites where hydrogen is abundant.

Hydrogen consumption in vent files of the equatorial Atlantic

The discovery of deep-sea hydrothermal vents in 1977 revolutionized our understanding of the energy sources that fuel primary productivity on Earth. Hydrothermal vent ecosystems are dominated by animals that live in symbiosis with chemosynthetic bacteria. So far, only two energy sources have been shown to power chemosynthetic symbioses: reduced sulphur compounds and methane. Using metagenome sequencing, single-gene fluorescence in situ hybridization, immunohistochemistry, shipboard incubations and in situ mass spectrometry, we show here that the symbionts of the hydrothermal vent mussel Bathymodiolus from the Mid-Atlantic Ridge use hydrogen to power primary production. In addition, we show that the symbionts of Bathymodiolus mussels from Pacific vents have hupL, the key gene for hydrogen oxidation. Furthermore, the symbionts of other vent animals such as the tubeworm Riftia pachyptila and the shrimp Rimicaris exoculata also have hupL. We propose that the ability to use hydrogen as an energy source is widespread in hydrothermal vent symbioses, particularly at sites where hydrogen is abundant.

Isotope compositions of oxygen and carbon from phosphates and Fe-Mn crusts and nodules sampled at Pacific seamounts

Results of a study of phosphates collected on Pacific Ocean seamounts using some physicochemical methods are described. Particular attention is paid to isotopic studies of both phosphates and their accompanying features. Oxygen isotopic compositions in phosphates of different genetic types were compared. Possible hydrothermal origin of phosphates is considered, and probable conditions of their formation are determined.

Chloroflexus spp. and Sulfurihydrogenibium spp. in microbial mats of the Nakabusa hot spring, Japan

Microbial mats develop in a wide range of aquatic habitats, such as geothermal hot springs, hypersaline ponds, marine cold seeps or hydrothermal vents. The Nakabusa hot spring is located in the Nagano Prefecture, Japan (36.3875N, 137.75E), dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65°C. The microbial community of such mats was analyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Microbial mat samples were kept in sterile plastic tubes (for molecular analysis) or glass bottles completely filled with hot spring water to avoid oxidation. Samples were transferred to the laboratory on ice and used for physiological experiments within 8h. Quantification of cell biovolumes was carried out based on images of mat sections hybridized with Sulfurihydrogenibium- and Chloroflexi-specific probes, and stained with DAPI. In situ hybridizations (CARD-FISH) of thin matsections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfurihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume).

Polymetallic ferromanganese deposits research on the Atlantic Spanish continental margin

Seamounts, submarine banks, volcanoes and undercurrent channels are prominent geomorphic features that have become an important target for minerals research and exploration with the goal of future exploitation. Polymetallic ferromanganese deposits are common types of mineralization on these settings. Co-rich ferromanganese crusts are important as potential resources of Mn and Co, but also Ti, Ni, Tl, REEs, PGEs, and other metals. Many seamounts and channels along the Atlantic Spanish continental margin are known to hold mineral deposits but are poorly studied. This work presents and briefly describes the most recent activities of the Spanish Geological Survey (IGME) on exploration and investigation of ferromanganese deposits along the Atlantic Spanish continental margin. Different submarine areas from the northwestern margin of the Iberian Peninsula to the west off Canary Islands have been surveyed by geophysical, sampling and underwater observations from 89 to 4000 m water depth. The mineral deposits cover a large diversity of submarine geological and geomorphical features: mud volcanoes and diapirs related to hydrocarbon seeps, seamounts associated with hot spot volcanism, hydrothermal vents in active magmatic volcanoes, structural basement highs and banks or contourite channels. Considering the collected dataset, we present the preliminary results of the study of these mineral deposits, including ferromanganese nodules and crusts and phosphate pavements and nodules, which can be considered as potential sources of raw materials.

Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp. 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity.

Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli. Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein. Of the five cloned DNA polymerases, the Thermococcus sp. 9 degrees N-7 DNA polymerase was chosen for biochemical characterization. Thermococcus sp. 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase. Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp. 9 degrees N-7 DNA polymerase. Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity.