Planctopirus ephydatiae, a novel Planctomycete isolated from a freshwater sponge.

The microbiome of freshwater sponges is rarely studied, and not a single novel bacterial species has been isolated and subsequently characterized from a freshwater sponge to date. A previous study showed that 14.4% of the microbiome from Ephydatia fluviatilis belong to the phylum Planctomycetes. Therefore, we sampled an Ephydatia sponge from a freshwater lake and employed enrichment techniques targeting bacteria from the phylum Planctomycetes. The obtained strain spb1T was subject to genomic and phenomic characterization and found to represent a novel planctomycetal species proposed as Planctopirus ephydatiae sp. nov. (DSM 106606 = CECT 9866). In the process of differentiating spb1T from its next relative Planctopirus limnophila DSM 3776T, we identified and characterized the first phage - Planctopirus phage vB_PlimS_J1 - infecting planctomycetes that was only mentioned anecdotally before. Interestingly, classical chemotaxonomic methods would have failed to distinguish Planctopirus ephydatiae strain spb1T from Planctopirus limnophila DSM 3776T. Our findings demonstrate and underpin the need for whole genome-based taxonomy to detect and differentiate planctomycetal species.

A new fistulose demosponge species from the Persian Gulf.

During a scientific expedition to the Palinurus Rock, Persian Gulf, Iraq, a reef, which was discovered first in 2012, we found a new species which we tentatively assigned to Ciocalypta (Porifera, Demospongiae, Suberitida, Halichondriidae). Genetic results from different authors (Morrow Cardenas, 2015, Redmond et al., 2013, Erpenbeck et al., 2012) suggest that several species of Ciocalypta and other species from Suberitida (e.g. several Axinyssa, Petromica, Topsentia, Cymbastela, Halichondria (Eumastia)) are indeed no Suberitida but belong to taxa yet unnamed. The species described here genetically clearly belongs to this new taxon outside Suberitida which awaits definition. Morphologically the new species clearly would be classified as Ciocalypta. Therefore the new species is described and compared to similar morphological species, some of them, as the type species, true Suberitida and true Ciocalypta, others belong to taxa still in need of a name.

Physics at the [Formula: see text] linear collider.

A comprehensive review of physics at an [Formula: see text] linear collider in the energy range of [Formula: see text] GeV-3 TeV is presented in view of recent and expected LHC results, experiments from low-energy as well as astroparticle physics. The report focusses in particular on Higgs-boson, top-quark and electroweak precision physics, but also discusses several models of beyond the standard model physics such as supersymmetry, little Higgs models and extra gauge bosons. The connection to cosmology has been analysed as well.

Some possibilities to reduce the biofilm formation on transparent siloxane coatings.

Presence of biofilms is a significant problem to a variety of industrial areas, underwater sensors, shipping, etc. Therefore solutions are sought to inhibit biofilm formation and to permit biofilm removal. Surface modification by suitable coating could be one of them. The present study reports the potential of new transparent biocides-free siloxane antifouling coatings, containing low toxic additives, such as TiO(2) nanoparticles, surfactants and antioxidants, to reduce biofilm formation in mimicking marine environment, laboratory conditions. As evaluated with several parameters: chlorophyll content, carotenoids content, total protein and total dry mass, the biofilm formation was most sharply reduced by the composition coatings containing non-ionic surfactant, super spreader Y17112, followed by those containing antioxidant, α-tocopherol. Depending on the amount of the super spreader (0.1-1.0 wt.%) and the tested parameter, approximately 3-8-fold reduction was observed in the biofilm formation. It is supposed, that the effect of the studied additives, both surfactant and antioxidant, is due to some inhibition of the adhesive extra cellular substances cross-linking with impact onto the biofilm cohesion strength and its adhesion.

Trehalose and vitreous states: desiccation tolerance of dormant stages of the crustaceans Triops and Daphnia.

Several aquatic organisms are able to withstand extreme desiccation in at least one of their life stages. This is commonly known as "anhydrobiosis." It was often thought that to tolerate such a desiccated state required high amounts of compatible solutes such as the nonreducing disaccharide trehalose, which protects cellular structures by water replacement and glass formation. Trehalose levels of dormant eggs and cysts of five freshwater crustaceans (Daphnia magna, Daphnia pulex, Triops longicaudatus, Triops cancriformis, and Triops australiensis) were observed in different states of hydration and dehydration. Although trehalose was detected in all species, the concentration was under 0.5% of the dry weight (0.05 µg/µg protein), and no change between the different states was observed. Differential scanning calorimetry (DSC) measurements indicated that dried cysts of all Triops species were in a glassy state, supporting the vitrification hypothesis. No indication for a vitreous state was found in dried resting eggs of Daphnia.

Ice crystallization and freeze tolerance in embryonic stages of the tardigrade Milnesium tardigradum.

In tardigrades, tolerance to low temperature is well known and allows them to cope with subzero temperatures in their environment. Although the ability to tolerate freezing body water has been demonstrated in some tardigrades, freeze tolerance of embryonic stages has been little studied, although this has ecological significance. In this study, we evaluated the subzero temperature survival of five different developmental stages of the eutardigrade species Milnesium tardigradum after freezing to -30 degrees C. Embryos were exposed to five different cooling rates between room temperature and -30 degrees C at 1 degrees C/h, 3 degrees C/h, 5 degrees C/h, 7 degrees C/h, and 9 degrees C/h followed by a warming period at 10 degrees C/h. The results showed that the developmental stage and the cooling rate have a significant effect on the hatching rate. Less developed embryonic stages were more sensitive to freezing at higher freezing rates than more developed stages. Differential Scanning Calorimetry (DSC) was used to determine the temperature of crystallization (Tc) in single embryos of the different developmental stages and revealed no differences between the stages. Based on the calorimetric data, we also conclude that the ice nucleation is homogeneous in embryonic stages in tardigrades, as also recently shown for fully developed tardigrades, and not triggered by nucleating agents.

High-temperature tolerance in anhydrobiotic tardigrades is limited by glass transition.

Survival in microhabitats that experience extreme fluctuations in water availability and temperature requires special adaptations. To withstand such environmental conditions, tardigrades, as well as some nematodes and rotifers, enter a completely desiccated state known as anhydrobiosis. We examined the effects of high temperatures on fully desiccated (anhydrobiotic) tardigrades. Nine species from the classes Heterotardigrada and Eutardigrada were exposed to temperatures of up to 110 degrees C for 1 h. Exposure to temperatures of up to 80 degrees C resulted in a moderate decrease in survival. Exposure to temperatures above this resulted in a sharp decrease in survival, with no animals of the families Macrobiotidae and Echiniscidae surviving 100 degrees C. However, Milnesium tardigradum (Milnesidae) showed survival of >90% after exposure to 100 degrees C; temperatures above this resulted in a steep decrease in survival. Vitrification is assumed to play a major role in the survival of anhydrobiotic organisms during exposure to extreme temperatures, and consequently, the glass-transition temperature (T(g)) is critical to high-temperature tolerance. In this study, we provide the first evidence of the presence of a glass transition during heating in an anhydrobiotic tardigrade through the use of differential scanning calorimetry.

Freeze tolerance, supercooling points and ice formation: comparative studies on the subzero temperature survival of limno-terrestrial tardigrades.

Many limno-terrestrial tardigrades live in unstable habitats where they experience extreme environmental conditions such as drought, heat and subzero temperatures. Although their stress tolerance is often related only to the anhydrobiotic state, tardigrades can also be exposed to great daily temperature fluctuations without dehydration. Survival of subzero temperatures in an active state requires either the ability to tolerate the freezing of body water or mechanisms to decrease the freezing point. Considering freeze tolerance in tardigrades as a general feature, we studied the survival rate of nine tardigrade species originating from polar, temperate and tropical regions by cooling them at rates of 9, 7, 5, 3 and 1 degrees C h(-1) down to -30 degrees C then returning them to room temperature at 10 degrees C h(-1). The resulting moderate survival after fast and slow cooling rates and low survival after intermediate cooling rates may indicate the influence of a physical effect during fast cooling and the possibility that they are able to synthesize cryoprotectants during slow cooling. Differential scanning calorimetry of starved, fed and cold acclimatized individuals showed no intraspecific significant differences in supercooling points and ice formation. Although this might suggest that metabolic and biochemical preparation are non-essential prior to subzero temperature exposure, the increased survival rate with slower cooling rates gives evidence that tardigrades still use some kind of mechanism to protect their cellular structure from freezing injury without influencing the freezing temperature. These results expand our current understanding of freeze tolerance in tardigrades and will lead to a better understanding of their ability to survive subzero temperature conditions.

Spirostomum spp. (Ciliophora, Protista), a suitable system for endocytobiosis research.

Among ciliate genera, only Paramecium and Euplotes species have been studied extensively as host organisms of bacterial endocytobionts. In this article, we show that members of the genus Spirostomum may also serve as a suitable system for endocytobiosis research. Two strains of Spirostomum minus (Heterotrichea, Ciliophora) collected in Germany and Italy, respectively, were found to harbor different types of bacterial infections. Bacteria of various sizes and shapes were observed in the cytoplasm or in the nuclei of the ciliates. The bacteria in the cytoplasm were either surrounded by a peribacterial membrane or lay naked. One of the bacterial species was found in the vicinity of the contractile fibrillar system (myonemes) of the ciliates. In rare cases, another type of bacteria was observed associated with mitochondria. The macronuclei of both the Italian and the German strains were crowded with endocytobionts. The endonuclear bacteria in the two S. minus strains differed with respect to their cytoplasmic structures but they were of similar size and both were rod shaped. According to the results of in situ hybridization, the endonuclear bacteria of the Italian strain belong to the subgroup of alphaproteobacteria, whereas the bacteria associated with the fibrillar system appeared to be gram-positive bacteria with high G+C content. While both the German and the Italian strains were found to permanently maintain their endocytobionts, they were at least partly colonized by different bacteria. This is taken as an indication that geographically separated populations of ciliates may be stably infected by different endocytobionts, possibly due to different ecological conditions. For S. minus and S. ambiguum a total of 7 different bacterial endocytobionts have now been recorded. We recommend the members of the genus Spirostomum as a suitable system for endocytobiosis research.

Cloning, recombinant expression and biochemical characterisation of novel esterases from Bacillus sp. associated with the marine sponge Aplysina aerophoba.

Two novel esterases (EstB1 and EstB2) were isolated from a genomic library of Bacillus sp. associated with the marine sponge Aplysina aerophoba. EstB1 shows low identity (26-44%) with the published hydrolases of the genus Bacillus, whereas EstB2 shows high identity (73-74%) with the carboxylesterases from B. cereus and B. anthracis. Both esterases were efficiently expressed in Escherichia coli under the control of T7 promoter using the vector pET-22b(+). Recombinant EstB1 was purified in a single step to electrophoretic homogeneity by IMAC. A method for the refolding of inclusion bodies formed by the recombinant EstB2 was established to obtain active enzyme. Substrate specificity of the two enzymes towards p-nitrophenyl and methyl esters and the respective kinetic parameters K(m) and V(max) were determined. The temperature optima of EstB1 and EstB2 were determined to be in the range of 30-50 degrees C and 20-35 degrees C, respectively. The pH optima were found to be in the range of 6.5-7.5 and 6.5-8.0, respectively. Both enzymes showed the highest stability in up to 50% (v/v) DMSO followed by methanol, ethanol and 2-propanol. The influence of high NaCl and KCl concentrations was tested. The inhibition effect of 10-50 mM Zn(2+) and 50 mM Mg(2+) and Ca(2+) ions was observed for both esterases. One to five millimolar PMSF deactivated the enzymes, whereas beta-mercaptoethanol, DTT and EDTA had no effect on the enzymes activity.

Sustainable use of marine resources: cultivation of sponges.

Among all metazoan phyla, sponges are known to produce the largest number of bioactive compounds, some of them metabolites with human therapeutic value. Therefore, an increasing interest in basic cell biology research up to biochemical engineering can be observed aiming at the production of sponge metabolites under completely controlled conditions. One major obstacle is the limited availability of larger quantities of defined sponge material--the so-called supply problem. In this chapter, different approaches used so far for producing sponge biomass by in situ aquaculture as well as some significant progress in the maintenance of sponges in aquaria are reviewed. These approaches are mainly based on old methods for producing commercial bath sponges as well as on experience in maintaining sponges in public aquaria and on the usage of artificial substrates for a natural-like colonization structure. In recent years, great efforts have been made to set up in vitro culture systems for the cultivation of sponge cells. One of the major advantages of cell cultures is the possibility to control and manipulate the cultivation conditions depending on the sponge species and the target metabolite. Up to now, monolayer cultures of dissociated sponge cells have been shown in a few cases to produce the desired product. However, to date, no continuously growing sponge cell line has been established. Organotypic culture systems, which maintain or mimic the natural tissue structure, have been developed in recent years and demonstrate a promising way towards the biotechnology of sponges. Successful attempts to produce sponge metabolites using the three-dimensional growing primmorphs are given. The use of sponge fragments, another three-dimensional approach, has reappeared and has also been successfully used as an in vitro approach as well as for the biotechnological production of boreal sponge tissue.

Sustainable production of bioactive compounds from sponges: primmorphs as bioreactors.

Sponges [phylum Porifera] are a rich source for the isolation of biologically active and pharmacologically valuable compounds with a high potential to become effective drugs for therapeutic use. However, until now, only one compound has been introduced into clinics because of the limited amounts of starting material available for extraction. To overcome this serious problem in line with the rules for a sustainable use of marine resources, the following routes can be pursued; first, chemical synthesis, second, cultivation of sponges in the sea (mariculture), third, growth of sponge specimens in a bioreactor, and fourth, cultivation of sponge cells in vitro in a bioreactor. The main efforts to follow the latter strategy have been undertaken with the marine sponge Suberites domuncula. This species produces compounds that affect neuronal cells, such as quinolinic acid, a well-known neurotoxin, and phospholipids. A sponge cell culture was established after finding that single sponge cells require cell-cell contact in order to retain their telomerase activity, one prerequisite for continuous cell proliferation. The sponge cell culture system, the primmorphs, comprises proliferating cells that have the potency to differentiate. While improving the medium it was found that, besides growth factors, certain ions (e.g. silicate and iron) are essential. In the presence of silicate several genes required for the formation of the extracellular matrix are expressed (silicatein, collagen and myotrophin). Fe3+ is essential for the synthesis of the spicules, and causes an increased expression of the ferritin-, septin- and scavenger receptor genes. Furthermore, high water current is required for growth and canal formation in the primmorphs. The primmorph system has already been successfully used for the production of pharmacologically useful, bioactive compounds, such as avarol or (2'-5')oligoadenylates. Future strategies to improve the sponge cell culture are discussed; these include the elucidation of those genes which control the proliferation phase and the morphogenesis phase, two developmental phases which the cells in primmorphs undergo. In addition, immortalization of sponge cells by transfection with genomic DNA appears to be a promising way, since recent studies underscore the applicability of this technique for sponges.

Comparative studies on two potential methods for the biotechnological production of sponge biomass.

The production of marine sponge biomass is one of the main outstanding goals of marine biotechnology. Due to the increased number of sponge secondary metabolites of economical value the interest in sponge cultivation increased over the last years, too. Therefore, we examined cultivation properties of 11 Mediterranean sponge species. Two methodologies were tested: functional fragment culture and multicell reaggregate culture. The in vitro cultivation of sponge fragments without further dissociation and reaggregation is a method formerly not reported. Reaggregates and functional fragments are promising attempts for culture system development. A broad spectrum of reaggregation properties was found among the species tested. In three species multicell aggregate cultures could be maintained for several months: Petrosia ficiformis, Suberites domuncula and Acanthella acuta. Our results indicate that cellular aggregates or fragments of sponges can be valuable tools in the development of methods for biotechnological production of sponge biomass. Further focus on nutritional demands and the biochemical status of the cells in these kind of cellular associations are needed in order to obtain functional aggregates and fragments.

Dinoflagellates from marine algal blooms produce neurotoxic compounds: effects on free calcium levels in neuronal cells and synaptosomes.

In this report, evidence is presented that the marine unicellular eukaryotic dinoflagellates can cause neurotoxicity very likely by an increase in intracellular free calcium ions ([Ca(2+)](i)). Determinations of the effects of culture supernatants from different clones of the dinoflagellate Alexandrium sp. isolated from algal blooms on the viability of rat primary neuronal cells revealed that all clones tested were toxic for these cells. In addition, all Alexandrium clones tested, except for A. ostenfeldii BAH ME-141, were found to be toxic for rat pheochromocytoma PC12 cells. No toxicity was observed for culture supernatants from Gonyaulax and Coolia monotis. Calcium ions are important in the process of apoptotic cell death; our studies revealed that the dinoflagellate supernatants from A. lusitanicum K2, A. lusitanicum BAH ME-091, and A. tamarense 1M caused an increase in [Ca(2+)](i) levels in both PC12 cells and primary neuronal cells. These dinoflagellate supernatants, as well as the A. tamarense ccmp 115 supernatant, were found to cause also an increase in free calcium concentration in isolated synaptosomes. Our results suggest that the neurotoxic effects of certain dinoflagellate supernatants may be associated with disturbances in [Ca(2+)](i) levels.

Primmorphs generated from dissociated cells of the sponge Suberites domuncula: a model system for studies of cell proliferation and cell death.

Sponges (Porifera) represent the lowest metazoan phylum; they have been shown to be provided with the characteristic metazoan structural and functional molecules. One autapomorphic character of sponges is the presence of high levels of telomerase activity in all cells (or almost all cells, including somatic cells). In spite of this fact previous attempts to cultivate sponge cells remained unsuccessful. It was found that dissociated sponge cells do not replicate DNA and lose their telomerase activity. In addition, no nutrients or metabolites have been detected that would stimulate sponge cells to divide. In the present study we report the culture conditions required for the formation of multicellular aggregates from dissociated single cells of Suberites domuncula, termed primmorphs. These primmorphs are formed in seawater without addition of further supplements, and have an organised tissue-like structure; they have been cultured for more than 5 months. Cross-sections revealed a distinct external layer covered by a continuous pinacoderm, and a central zone composed primarily of spherulous cells. After their association into primmorphs, the cells turn from the telomerase-negative state into the telomerase-positive state; a telomerase level of 4.7 total product generated (TPG) units/5 x 10(3) cell equivalents has been determined. Moreover, a major fraction of the cells in the primmorphs undergoes DNA synthesis and hence has the capacity to grow. Applying the BrdU-labelling and detection assay it is demonstrated that up to 33.8% of the cells in the primmorphs are labelled with BrdU after an incubation period of 12 h. It is proposed that the primmorph system described here is a powerful novel model system to study basic mechanisms of cell proliferation and cell interaction, as well as of morphogenesis, ageing and apoptosis.

Bacteria of the Genus Roseobacter Associated with the Toxic Dinoflagellate Prorocentrum lima.

The dinoflagellate Prorocentrum lima is known to produce diarrhetic shellfish poisons. However, it is yet unclear if the dinoflagellates themselves or the bacteria associated with them produce the toxins. Here we analyze the toxicity as well as the spectrum of bacteria in two cultures of P. lima, namely P. lima-SY and P. lima-ST, which initially derived from the same P. lima strain PL2V. Toxicity tests, applying the Artemia bioassay revealed in both cultures high levels of toxins. The bacteria, associated with the two cultures, were identified by PCR/nucleotide sequence analysis of the 16S rRNA gene. From cultures of P. lima-SY the dominant sequence was found to share a 93.7% similarity with the sequence of Roseobacter algocolus [R. algicola]; the relative abundance was determined to be 83%. In addition three further sequences of bacteria, grouped to the α-Protobacteria have been identified: Paracoccus denitrificans [90.8%], R. algocolus [94.4%] and Rhizobium huakuii [92.6%]. The identification of bacteria in P. lima-ST revealed that most share highest similarity with Bartonella taylorii but with a relatively low score of 87%. In addition to this sequence, two sequences with high similarity to the genus Roseobacter were obtained. The other sequences identified have not been detected in P. lima-SY. Studies with pure bacterial strains, previously isolated from a culture of P. lima-ST and subsequently cultured on agar plates, revealed that none of them was identical to those identified in the dinoflagellate culture itself. An explanation for the change of the spectrum of bacteria in the different cultures can only be expected when axenic cultures from P. lima are available.

Bioeffects of diagnostic ultrasound in vitro.

Biological effects induced by ultrasound were frequently reported for continuous wave (cw) mode. Thresholds for the onset of bioeffects of pulsed ultrasound, starting from diagnostic conditions, have not yet been defined by standardized in vitro models. We therefore investigated the effects of pulsed ultrasound on cultured cells using diagnostic ultrasound devices, a selfmade transducer and a sonochemical laboratory reactor tunable from pulsed diagnostic conditions to cw ultrasound. Additionally, we determined physical parameters of the ultrasonic field by different types of hydrophones. Sonochemical reactions and the effects induced by the ultrasonic fields in cultured cells indicated a threshold for bioeffects.

Disturbance of cellular calcium homeostasis by in vitro application of shock waves.

Extracorporeally generated shock waves used in lithotripsy of urinary and biliary stones exhibit tissue lesions in vivo and destroy or damage cells in vitro. The involvement of cavitation-generated free radicals in these harmful effects is discussed controversially. We investigated changes in cytoplasmic calcium concentration and intracellular calcium localization after shock-wave treatment of suspended cell cultures using flow cytometry and electron microscopy and present evidence for the disturbance of mitochondrial Ca2+ a sequestration and, therefore, for a chemically induced cell injury.

Reduced cavitation-induced cellular damage by the antioxidative effect of vitamin E.

Fragmentation of human urinary and biliary stones by shock waves in extracorporeal lithotripsy is accompanied by tissue damage. Both the fragmentation as well as the side effects are often attributed to cavitation. The hazardous potential of cavitation is not only of a physical nature but also of a chemical nature, because of the generation of free radicals, e.g. .OH, .H and .O2. After the application of shock waves, we have demonstrated cavitation-generated free radicals in cell-free solutions and also in the surviving and intact suspended MGH-U1 cells by hydroethidine measurements. Under electron microscopical inspection, the same cells exhibited perinuclear cisternae, damaged mitochondria and numerous intracellular vacuoles. The contribution of free radicals to cell damage was investigated by reducing the vitamin E level in rats by a tocopherol free diet and by incubating L1210 cells in a tocopherol enriched medium. After 250 shock waves, ex vivo erythrocytes revealed a 75% increase in total cell disruption over cells from non-depleted rats. The in vitro experiments with L1210 cells exhibited a moderate protection by the addition of this scavenger of free radicals.

Dye and electric coupling between osteoblast-like cells in culture.

Primary cultures of osteoblast-like cells (OB) derived from calvarial fragments of newborn rats and juvenile guinea pigs formed numerous gap junctions between neighboring cells in vitro. Intracellular injection of Lucifer yellow led to a staining of up to 30 adjacent cells. Parallel intracellular recordings showed that amplitudes of stimulated membrane potential changes (4-5 mV) were closely related between coupled cells. The coupling factor, which was derived from the ratio of these amplitudes, ranged between 0.1 and 0.8. The coupling factor (1) was not dependent on the membrane potential or the injected current strength; (2) strong acidosis (pH < 6.6) and hypercapnia (pCO2 > 80 mm Hg) did not affect electric or dye coupling; (3) elevation of intracellular cAMP level was ineffective; (4) rise of the extra- and intracellular Ca2+ concentration did not effect the electric coupling; (5) the anticonvulsant drugs carbamazepine and phenytoin impaired the coupling factor up to 59%. The findings show that cell-cell communication between OB via gap junctions proved stable under various conditions which, in other tissues, were found to reduce the coupling strength of gap junctions.