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Additional file 1 Electronic Supplementary Materials. Supplementary materials (pdf format), contain Supplementary Information 1–4, Supplementary Figures. S1-S12, and Supplementary References (references that were used in Supplementary Information 1–4). Supplementary Information 1–4: additional information describing the results of this study but not the necessary information to be provided in the main text. Supplementary Figures. S1-S13: additional data with legends to support the manuscript.

Polyextremophilic algae, such as unicellular red algae known as Cyanidiophyceae, have the intrinsic capacity to selectively mobilize and adsorb metals, since they are adapted to live in geothermal and volcanic sites characterized by elevated concentration of heavy and rare metals. In this work we evaluated the ability of 3 strains of the genus Galdieria (G. maxima, G. sulphuraria, G. phlegrea) along with one strain of Cyanidium caldarium to tolerate different concentrations of rare metal as Cl4K2Pd and AuCl4K by monitoring changes in algal growth in culture exposed to different concentration of each metal and investigating algae metabolic response and possible oxidative stress induced by these metals.

Introduction: In CNS, aberrant proliferation causes cancer and im- paired survival of differentiated neurons induces neurodegenerative disorders. In order to find novel ther- apeutic targets able to inhibit aber- rant cell proliferation and/or enhance differentiated cells survival, we ana- lyzed properties of aqueous extract of Ruta graveolens on differentiated and proliferating neural cells. Ruta g. is currently used for its diuretic, sedative, and analgesic effects and recent studies described antiprolifer- ative effects on different cancer cells. Materials and methods: We used a mouse mesencephalic embryonic cell line, A1 mes-c-myc cells (A1) that are proliferating/undifferenti- ated in the presence of serum. They cease to proliferate and differentiate when serum is withdrawn and cAMP is added. Aqueous extracts (Ruta g. a.e.) were obtained from young leaves chopped, infused in boil- ing water and lyophilized. Extract concentrations of 10 mg/ml, 1 mg/ ml and 0.1 mg/ml were tested. Cell counting was performed by MTT assay and Trypan blue method. Cell cycle was analyzed by cytometry af- ter PI incorporation. Cell signalling was analyzed by western blotting. Results: Ruta g. a.e. inhibits A1 cells proliferation and induces increase in ERK phosphorylation. In presence of the ERK pathway inhibitor, PD, Ruta g. a.e. is unable to induce cell death indicating that ERK is involved in the Ruta g. effect on A1 proliferating cells. On the other hand, when Ruta g. a.e. is added, the number of dif- ferentiated A1 cells appears signifi- cantly higher as compared to control conditions and the analysis of the cell cycle showed an increased number of cells in G2/M phase in differentiated cells treated with Ruta g. a.e. Conclu- sions: Ruta g. a.e. could represent an interesting therapeutic tool since it is able at the same time to inhibit un- differentiated cell proliferation and to induce re-entry in the cell cycle of differentiated neurons.

The high temperature, acidity, and heavy metal-rich environments associated with hot springs have a major impact on biological processes in resident cells. One group of photosynthetic eukaryotes, the Cyanidiophyceae (Rhodophyta), has successfully thrived in hot springs and associated sites worldwide for more than 1 billion years. Here, we analyze chromosome-level assemblies from three representative Cyanidiophyceae species to study environmental adaptation at the genomic level. We find that subtelomeric gene duplication of functional genes and loss of canonical eukaryotic traits played a major role in environmental adaptation, in addition to horizontal gene transfer events. Shared responses to environmental stress exist in Cyanidiales and Galdieriales, however, most of the adaptive genes (e.g., for arsenic detoxification) evolved independently in these lineages. Our results underline the power of local selection to shape eukaryotic genomes that may face vastly different stresses in adjacent, extreme microhabitats.

Exploration of life in extreme environments allows the discovery of intriguing organisms with extraordinary biotechnological potential. An example of extreme environments is represented by hot springs, where harsh conditions (pH < 1; Temperature > 50°C; high concentrations of metals) are prohibitive for most living organisms, except for archaea, bacteria and a few eukaryotes like the unicellular red alga Galdieria. Phylogenetic analysis based on a few plastid and nuclear genes highlighted the intricate genetic structure of Galdieria and the hypothesis of diverging clades within the G. sulphuraria species. To resolve enigmatic relationships between lineages, we used plastid, mitochondrial and nuclear genome-scale data obtained from numerous strains from around the world. The resulting phylogenomic analysis identified: i) the divergence of each of the mitochondrial, plastid, and nuclear genomes into the same six clear lineages; ii) the independent evolution of the lineages; iii) the incongruent interlineages relationships between the three genomes. Differential evolutionary pressure between the strains and the genomes were also highlighted by synonymous and non-synonymous substitutions.

Extremophiles, while typically bacteria and archaea, are also found in the eukaryotic domain of life. The eukaryote Galdieria sulphuraria is a thermoacidophilic red alga belonging to the class Cyanidiophyceae, an especially unique class as it comprises the basal clade of eukaryotic extremophiles. Galdieria species can grow both photosynthetically and heterotrophically on a variety of carbon sources, thriving down to pH 0 and temperatures up to 56 °C, while tolerating high levels of reactive oxygen species and high levels of heavy metals. Here we report whole-genome sequencing of three G. sulphuraria strains, uncovering a compact (13.1 Mb – 16.0 Mb) nuclear genome with 72-73 chromosomes, dependent on the strain.Comparative analyses of the macro synteny revealed significant structural rearrangement between G. sulphuraria isolates and the genome shows signs of sexual recombination. This, along with the large number of nuclear chromosomes compared to the genome size, reveals a mechanism of intrinsic adaptability in this eukaryotic extremophile, uncovering how G. sulphuraria can thrive in a rapidly changing extreme environment.

The lanthanides are among the rare earth elements (REEs), which are indispensable constituents of modern technologies and are often challenging to acquire from natural resources. The demand for REEs is so high that there is a clear need to develop efficient and environmentally-friendly recycling methods. In the present study, living cells of the extremophile Galdieria sulphuraria were used to remove four REEs, Yttrium, Cerium, Europium, and Terbium, from single- and quaternary-metal aqueous solutions. Two different strains, SAG 107.79 and ACUF 427, were exposed to solutions buffered at pH 2.5, 3.5, 4.5, and 5.5. Our data demonstrated that the removal performances were strain and pH dependent for all metal ions. At lower pH, ACUF 427 outperformed SAG 107.79 considerably. By increasing the pH of the solutions, there was a significant surge in the aqueous removal performance of both strains. The same trend was highlighted using quaternary-metal solutions, even if the quantities of metal removed were significantly lower. The present study provided the first insight into the comparative removal capacity of the Galdieria sulphuraria strains. The choice of the appropriate operational conditions such as the pH of the metal solutions is an essential step in developing efficient, rapid, and straightforward biological methods for recycling REEs.

Polyextremophilic red algae, which belong to the class Cyanidiophyceae, are adapted to live in geothermal and volcanic sites. These sites often have very high concentrations of heavy and precious metals. In this study, we assessed the capacity of three strains of Galdieria (G. maxima, G. sulphuraria, and G. phlegrea) and one strain of Cyanidiumcaldarium to tolerate different concentrations of precious metals, such as palladium (Cl4K2Pd) and gold (AuCl4K) by monitoring algal growths in cultures exposed to metals, and we investigated the algae potential oxidative stress induced by the metals. This work provides further understanding of metals responses in the Cyanidiophyceae, as this taxonomic class is developed as a biological refinement tool.

Galdieria maxima is a polyextremophilic alga capable of diverse metabolic processes. Ammonia is widely used in culture media typical of laboratory growth. Recent reports that this species can grow on wastes promote the concept that G. maxima might have biotechnological utility. Accordingly, there is a need to know the range of pH levels that can support G. maxima growth in a given nitrogen source. Here, we examined the combined effect of pH and nitrate/ammonium source on the growth and long-term response of the photochemical process to a pH gradient in different G. maxima strains. All were able to use differing nitrogen sources, despite both the growth rate and photochemical activity were significantly affected by the combination with the pH. All strains acidified the NH4+-medium (pH &lt, 3) except G. maxima IPPAS P507. Under nitrate at pH &ge, 6.5, no strain was able to acidify the medium, noteworthy, G. maxima ACUF551 showed a good growth performance under nitrate at pH 5, despite the alkalization of the medium.

Galdieria is a photosynthetic unicellular protist, inhabiting thermoacidic environments around the world. The synchronicity of these thermoacidophilic algae with their extreme habitats makes them unable to thrive in different ecological conditions. The genetic structure of Galdieria populations has not yet been studied. In this report, the level of genetic diversity and struc- ture of five Galdieria populations from Iceland were assessed through both random amplified polymorphic DNA (RAPD) markers, and a partial calmodulin gene fragment (previously used to assess the population structure of these extremophilic algae). The level of population differentiation from both the RAPD and CaM markers was estimated using PLP, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon’s information index, Ks, Kst. The migration ability of the Galdieria populations was suggested by the high level of genetic variations scored within each Galdieria population and by the small number of polymorphisms detected between the different Icelandic populations. The low genetic diversity between the two species was highlighted by RAPD and calmodulin markers, suggesting for the first time an interspecific genetic flow between species as strategy to evolve in stressful environments. Galdieria is a photosynthetic unicellular protist, inhabiting thermoacidic environments around the world. The synchronicity of these thermoacidophilic algae with their extreme habitats makes them unable to thrive in different ecological conditions. The genetic structure of Galdieria populations has not yet been studied. In this report, the level of genetic diversity and structure of five Galdieria populations from Iceland were assessed through both random amplified polymorphic DNA (RAPD) markers, and a partial calmodulin gene fragment (previously used to assess the population structure of these extremophilic algae). The level of population differentiation from both the RAPD and CaM markers was estimated using PLP, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon’s information index, Ks, Kst. The migration ability of the Galdieria populations was suggested by the high level of genetic variations scored within each Galdieria population and by the small number of polymorphisms detected between the different Icelandic populations. The low genetic diversity between the two species was highlighted by RAPD and calmodulin markers, suggesting for the first time an interspecific genetic flow between species as strategy to evolve in stressful environments.

Heterotrophic components of biofilms on wood artefacts were studied at the Conservation Laboratory for Wood Artefacts of the University Suor Orsola Benincasa of Naples, Italy. The aim of the study was to add new information on the micro-habitats represented by biofilms formed by wood-dwelling organisms. Light and electron microscopy of histological features of woods used to make the artefacts showed that the woods belonged to species of lime (Tilia sp.), poplar (Populus sp.) and pear (Pyrus sp.). A Denaturing Gradient Gel Electrophoresis analysis performed on heterotrophic microorganisms colonizing the artefacts led to identify four species of bacteria, namely Bacillus cereus, B. mycoides, B. subtilis and Microbacterium oleivorans, and seven species of fungi, namely Alternaria alternata, Aspergillus fumigans, A. versicolor, Cladosporium cladosporioides, C. oxysporum, Fusarium oxysporum and Penicillium chrysogenum. Based on its morphological features, an insect found on some artefacts was identified as the xylophagous beetle Nicobium castaneum (Anobiidae). The influence of wood type and environmental conditions on the diversity of microorganisms was discussed.

Phlegrean Fields is a large volcanic area situated southwest of Naples (Italy), including both cave and thermoacidic habitats. These extreme environments host the genus Cyanidium; the species C. chilense represents a common phototrophic micro- organism living in anthropogenic caves. With a view to provide a comprehensive characterization for a correct taxonomic classification, morpho-ultrastructural investigations ofC. chilense from Syb’s cave (Phlegren Fields) was herein car-ried out and compared with the thermoacidophilic C. caldarium. The biofilm was also analyzed to define the roleofC. chilensein the establishment of a bio film within caveenvironments. Despite the peculiar ecological and molecular divergences,C. chilenseandC. caldariumshared all the maindiacritic features, suggesting morphological convergence within the genus; cytological identity was found amongC. chilensestrains geographically distant and adapted to different substrates, such as the porous yellow tuff of Sybil cave and calcyte, magnesite and basaltic rocks from other caves. C. chilense is generally dominant in all biofilms, developing monospecific islets, developing both super ficially or betweenfungal hyphae and coccoid cyanobacteria. Extracellular polymeric substances (EPS) were recorded in C. chilense bio filmsfrom Sybil cave, confirming the role of EPS in facilitating cellsadhesion to the surface, creating a cohesive network of inter-connecting biofilm cells

Algal based wastewater treatment offers the opportunity to recover, in the form of biomass, the nutrients and internal chemical energy of wastewater. Recently, there has been a growing interest in the use of extremophilic microalgae, as they can easily adapt to difficult and often pollutant-rich environments. The thermo-acidophilic microalga Galdieria phlegrea is a species of recent discovery and great metabolic versatility, but it has still been poorly studied. Here, G. phlegrea was cultivated using raw municipal wastewater in 1 L Erlenmeyer flasks with 700 mL working volume at 37 °C for up to nine days. During the cultivation phase, biomass growth, phycocyanin content, ammonium and phosphate removal from the wastewater, lipid fraction, total carbon and nitrogen in the biomass, and variation in δ13C and δ15N isotopic ratios (a novel analytical contribution in these experiments) were monitored. Results indicated that G. phlegrea was able to grow in raw effluent, where it removed more than 50% ammonium and 20% phosphate in 24 h, total lipid content was in the range of 11–22%, while average C-N content was of 45% and 6%, respectively, isotopic analyses proved to be a useful support in identifying C and N metabolic pathways from effluent to biomass. Overall, G. phlegrea showed consistent performance with similar Cyanidiophyceae and is a potentially viable candidate for municipal wastewater valorization from a circular economy perspective.

In plants and algae, sulfate assimilation and cysteine synthesis are regulated by sulfur accessibility from the environment. This study reports the effects of sulfur (S)-deprivation in autotrophic and heterotrophic cultures of Galdieria phlegrea (Cyanidiophyceae), a unicellular red alga isolated in the Solfatara crater located in Campi Flegrei (Naples, Italy), where H2S is the prevalent form of gaseous sulfur in the fumarolic fluids and sulfur is widespread in the soils near the fumaroles. This is the first report on the effects of S-deprivation on a sulfurous microalga that is also able to grow heterotrophically in the dark. The removal of sulfur from the culture medium of illuminated cells caused a decrease in the soluble protein content and a significant decrease in the intracellular levels of glutathione. Cells from heterotrophic cultures of Galdieria phlegrea exhibited high levels of internal proteins and glutathione content, which did not diminish during S-starvation, but rather glutathione significantly increased.The activity of O-acetylserine(thiol)lyase (OASTL), the enzyme synthesizing cysteine, was enhanced under S-deprivation in a time-dependent manner in autotrophic but not in heterotrophic cells. Analysis of the transcript abundance of the OASTL gene supports the OASTL activity increase in autotrophic cultures under S-deprivation.

To date, the main source for phycocyanin production is the thermophilic cyanobacterium Spirulina platensis, although latest researches are exploring the possibility to exploit the thermoacidophilic Cyanidiophyceae (Rhodophyta) to this purpose. Galdieria phlegrea is a polyextremophilic red alga belonging to Cyanidiophyceae, colonizing, along with G. sulphuraria, acidic and thermal environments and distinguishable from this latter on the base of molecular and ecophysiological traits. In the present paper, a characterization of C-phycocyanin from two strains of G. phlegrea with different geographic provenance (Phlegrean Fields Naples, Italy; Diyadin, Turkey) was provided under autotrophic and heterotrophic conditions. The results showed different optimal pHs and thermostability between the strains and between autotrophic and heterotrophic cells. What is intriguing, a pre-heating at 70 °C of heterotrophic cells from the Italian strain resulted in a highly heat-resistant of C-phycocyanin. We hypothesized that the different features of this pigment, currently widely used for various applicative purposes, are related to the habitat from which the microalga comes from.

The nomenclatural history of the genus Cyanidium (Tilden) Geitler is long and complex, due to uncertainties regarding its taxonomic position. In the course of the last two centuries, Cyanidium has been assigned to Cyanophyta, Chlorophyta, and finally to Rhodophyta (Ott & Sechback 1994, Albertano et al. 2000). On the basis of morphological, ultrastructural and reproductive characters, Merola et al. (1981) instituted two new genera, Galdieria and Cyanidioschyzon, strictly related to Cyanidium, and assigned all three genera to the new class Cyanidiophyceae. Later, in the treatment proposed by Ott & Sechback (1994), all species belonging to Galdieria or Cyanidioschyzon were transferred to Cyanidium. In the most recent taxonomic proposal by Yoon et al. (2014), the tripartite generic architecture has been confirmed, even though a different taxonomic hierarchy was applied at the higher ranks.

Walnut husk washing waters (WHWW), a by-product of walnut production, are indiscriminately used for irrigation without preliminary risk assessment. Basing on previous in vitro results on the toxicity of this by-product, we have followed the morphophysiological development of Zea mays, Lactuca sativa cv. Gentilina and L. sativa cv. Canasta under diluted and undiluted WHWW irrigation. Significant development alterations have been observed in root and shoot elongations for all crops as well as in total biomass and chlorophyll content. The genotoxic potential of WHWW has been concurrently verified; acridine orange/ethidium bromide staining evidenced chromatin modifications and DNA degradation and also was confirmed by DNA laddering. The DNA instability was also assessed through RAPD, thus suggesting the danger of the by-product of walnut processing and focusing the attention on the necessity of an efficient treatment of WHWWs. The findings obtained by PCA of agronomic and physiological traits suggested that establishing guidelines for the administration of WHWW for irrigation is of great importance, and it is necessary to supervise their use in agricultural soils. © 2013 Springer-Verlag Berlin Heidelberg.

We report here the de novo assembly of a eukaryotic genome using only MinION nanopore DNA sequence data by examining a novel Galdieria sulphuraria genome: strain SAG 107.79. This extremophilic red alga was targeted for full genome sequencing as we found that it could grow on a wide variety of carbon sources and could uptake several precious and rare-earth metals, which places it as an interesting biological target for disparate industrial biotechnological uses. Phylogenetic analysis clearly places this as a species of G. sulphuraria. Here we additionally show that the genome assembly generated via nanopore long read data was of a high quality with regards to low total number of contiguous DNA sequences and long length of assemblies. Collectively, the MinION platform looks to rival other competing approaches for de novo genome acquisition with available informatics tools for assembly. The genome assembly is publically released as NCBI BioProject PRJNA330791. Further work is needed to reduce small insertion-deletion errors, relative to short-read assemblies.

Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens' effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue's noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.

The aim of this study was to confirm the utility of the Comet assay as a genotoxicity screening test for evaluating the impact of walnut husk aqueous extract. Phytotoxicity assays using diluted and undiluted walnut husk aqueous extracts were performed on young roots of Raphanus sativus (radish), and the Comet assay was used to evaluate DNA integrity in isolated radish radicle nuclei. The results reveal a dose-dependent accumulation of DNA damage in radish radicles treated with walnut husks water extract and that the Kolmogorov-Smirnov test combined with Johnson SB distribution was the best approach for describing Comet assay data. © Springer Science+Business Media, LLC 2012.