Oxygen Productivity of Planktonic Algae under Fluctuating and Constant Light Conditions

Net oxygen productivity in cultures of Monoraphidium minutum, Cryptomonas sp. and Planktothrix agardhii exposed to fluctuating and constant light conditions was measured in a laboratory incubator. The fluctuating light climate simulated a linear up and down movement in a 2 m water column at 4 different ratios of euphotic depth to mixing depth. In addition, cultures were kept at a constant light climate simulating static incubation at 0, 0.5, 1 and 2 m depth and at the depth of the mean irradiance, respectively. Integral productivity in the simulated water column was lowest when algae were incubated at constant light in different depths, highest when the algae were incubated at constant mean photon flux density (PFD) and intermediate under fluctuating light. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The influence of photon flux density (PFD) on short term 14C incorporation into proteins,carbohydrates and lipids in freshwater algae

The effect of photon flux density (PFD) on the partitioning of photosynthetically fixed ¹⁴CO₂-C into major intracellular end products was investigated for three species of freshwater planktonic algae (Nitzschia palea, Monoraphidium minutum and Synechococcus elongatus belonging to three different classes. This study was designed to investigate the phenomenon of polysaccharide synthesis associated with the saturation of protein synthesis and to test if this process is common to all three phytoplankton species. Protein synthesis was saturated at low PFD in all three species of algae studied. However, fixed carbon was differentially stored, namely in lipids in Nitzschia palea (Bacillariophyceae), in polysaccharides in Monoraphidium minutum (Chlorophyceae), and in low molecular weight metabolites (LMW) in Synechococcus elongatus (Cyanophyceae). The results of this transient state study indicate that the metabolic pathways of algae can easily be controlled by different irradiance. Furthermore, it appears that the difference in the patterns of synthesis is taxonomy dependent.     

Plankton community and the relationship with the environment in saline lakes of Onon-Torey plain,Northeastern Mongolia

The plankton community of sixteen saline lakes located on Onon-Torey plain (Northeastern Mongolia) during the filling phase and the raising of the water level was investigated in July 2011. Thirty-five taxa of phytoplankton and thirty-one species of zooplankton were found. For phytoplankton, blue-green algae (Merismopedia elegans, Anabaenopsis elenkinii, Arthrospora fusiformis, Spirulina major, Lyngbya sp., Oscillatoria sp.) and green algae (Monoraphidium minutum, Tetrastrum komarekii, Ankyra ocellata, Oocystis sp.) were dominant. For zooplankton, Filinia longiseta, Brachionus plicatilis, B. variabilis, Hexarthra mira (Rotifera), Daphnia magna, Moina brachiata, M. mongolica (Cladocera), Arctodiaptomus bacillifer, Mixodiaptomus incrassatus, Metadiaptomus asiaticus (Copepoda) dominated. Mineralization, active hydrogen ratio, dissolved oxygen and water temperature were the main factors influencing the diversity, structure and distribution of plankton organisms in the steppe lakes during low water level. The RDA analysis for phytoplankton and zooplankton from different lakes was carried out for selected two groups which included lakes and a subset related species. The first group is of oligohaline and mesohaline lakes in which mostly green algae, rotifers and copepods inhabit. The second group is of mesohaline and polyhaline lakes with mainly blue-green algae, some crustaceans and rotifers inhabiting. High abundance and biomass of Spirulina major, Oscillatoria sp. and Brachionus variabilis were observed in lakes with high mineralization, pH and temperature.     

Influence of the filamentous cyanobacterium Planktothrix agardhii on population growth and reproductive pattern of the rotifer Brachionus calyciflorus

The population dynamics of B. calyciflorus was investigated using a green alga, Monoraphidium minutum, and a blue-green alga Planktothrix agardhii as food sources, separately and as mixtures. Growth rate (r), egg ratio (ER), and juvenile development time (D _j ) were measured in the laboratory and mortality rate and embryonic development time (D _E ) were calculated. With M. minutum, Brachionus showed a typical growth curve (Monod-kinetics) dependent on food concentration. In contrast B. calyciflorus did not grow well on P. agardhii. With all food concentrations the measured growth rates were about r=0. At low food concentrations r was low with both food types, but the ER of B. calyciflorus was significantly higher with P. agardhii as food source. Furthermore the relative egg volume of females carrying one egg was higher with Planktothrix than with Monoraphidium. An addition of P. agardhii to M. minutum led to increasing growth rates. Highest growth rates were found with complementary food sources. High food concentrations of M. minutum shortened the juvenile development (D _J ) time, but D _j was uneffected by different P. agardhii food concentrations. A mixture of both algae did not shorten D _j compared with M. minutum as single food. The calculated D _E was not effected by different food qualities but the calculated mortality was nearly 3 times higher with P. agardhii as food.     

The effects of ultraviolet radiation on photosynthetic performance,growth and sunscreen compounds in aeroterrestrial biofilm algae isolated from building facades

The effects of artificial ultraviolet radiation [UVR; 8 W m⁻² ultraviolet-A (UVA), 0.4 W m⁻² ultraviolet-B (UVB)] on photosynthetic performance, growth and the capability to synthesise mycosporine-like amino acids (MAAs) was investigated in the aeroterrestrial green algae Stichococcus sp. and Chlorella luteoviridis forming biofilms on building facades, and compared with the responses of two green algae, from soil (Myrmecia incisa) and brackish water (Desmodesmus subspicatus). All species exhibited decreasing quantum efficiency (F _v/F _m) after 1–3 days exposure to UVR. After 8–12 days treatment, however, all aeroterrestrial isolates exhibited full recovery under UVA and UVA/B. In contrast, D. subspicatus showed only 80% recovery after treatment with UVB. While Stichococcus sp. and C. luteoviridis exhibited a broad tolerance in growth under all radiation conditions tested, M. incisa showed a significant decrease in growth rate after exposure to UVA and UVA/B. Similarly D. subspicatus grew with a reduced rate under UVA, but UVA/B led to full inhibition. Using HPLC, an UV-absorbing MAA (324 nm-MAA) was identified in Stichococcus sp. and C. luteoviridis. While M. incisa contained a specific 322 nm-MAA, D. subspicatus lacked any trace of such compounds. UV-exposure experiments indicated that all MAA-containing species are capable of synthesizing and accumulating these compounds, thus supporting their function as an UV-sunscreen. All data well explain the conspicuous ecological success of aeroterrestrial green algae in biofilms on facades. Biosynthesis and accumulation of MAAs under UVR seem to result in a reduced UV-sensitivity of growth and photosynthesis, which consequently may enhance survival in the environmentally harsh habitat.     

Biotreatment of Industrial Wastewater by Selected Algal‐Bacterial Consortia

A new approach for remediation processes in highly polluted environments is presented. The efficiency of algal‐bacterial associations for the remediation of industrial wastewater of a pond in Samara, Russia, was investigated. After screening of algae and bacteria for the resistance to the wastewater the following strains were selected: the algal strains Chlorella sp. ES‐13, Chlorella sp. ES‐30, Scenedesmus obliquus ES‐55, several Stichococcus strains (ES‐19, ES‐85, ES‐86, ES‐87, ES‐88), and Phormidium sp. ES‐90 and the bacterial strains Rhodococcus sp. Ac‐1267, Kibdelosporangium aridum 754 as well as two unidentified bacterial strains (St‐1, St‐2) isolated from the collector pond. All the strains listed above were immobilized onto various solid carriers (capron fibers for algae; ceramics, capron and wood for bacteria) and used for biotreatment in a pilot installation. The results showed that the selected algae and bacteria formed stable consortia during the degradation of the waste, which was demonstrated for the first time for the green alga Stichococcus. Stichococcus and Phormidium cells attached to capron fibers with the help of slime and formed a matrix. This matrix fixed the bacteria and eukaryotic algae and prevented them from being washed off. A significant decrease in the content of the pollutants was observed: phenols were removed up to 85 %, anionic surface active substances (anionic SAS) up to 73 %, oil spills up to 96 %, copper up to 62 %, nickel up to 62 %, zinc up to 90 %, manganese up to 70 %, and iron up to 64 %. The reduction of the biological oxygen demand (BOD₂₅) and the chemical oxygen demand COD amounted to 97 % and 51 %, respectively.     

Genome-wide DNA methylation studies suggest distinct DNA methylation patterns in pediatric embryonal and alveolar rhabdomyosarcomas

Rhabdomyosarcoma is the most common soft-tissue sarcoma in children. While cytogenetic abnormalities have been well characterized in this disease, aberrant epigenetic events such as DNA hypermethylation have not been described in genome-wide studies. We have analyzed the methylation status of 25,500 promoters in normal skeletal muscle, and in cell lines and tumor samples of embryonal and alveolar rhabdomyosarcoma from pediatric patients. We identified over 1,900 CpG islands that are hypermethylated in rhabdomyosarcomas relative to skeletal muscle. Genes involved in tissue development, differentiation, and oncogenesis such as DNAJA4, HES5, IRX1, BMP8A, GATA4, GATA6, ALX3, and P4HTM were hypermethylated in both RMS cell lines and primary samples, implicating aberrant DNA methylation in the pathogenesis of rhabdomyosarcoma. Furthermore, cluster analysis revealed embryonal and alveolar subtypes had distinct DNA methylation patterns, with the alveolar subtype being enriched in DNA hypermethylation of polycomb target genes. These results suggest that DNA methylation signatures may aid in the diagnosis and risk stratification of pediatric rhabdomyosarcoma and help identify new targets for therapy.     

GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION1

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μ g genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A ₂₆₀/ A ₂₈₀ ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum . The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.     

Potential importance of algae in the diet of adult Cyclops vicinus

• 1 The potential importance of phytoplankton to the nutrition of adult Cyclops vicinus was studied.
• 2 The flagellate Chlamydomonas reinhardii was ingested and digested at a higher rate than the coccale green alga Monoraphidium minutum.
• 3 The predation rate on the rotifer Brachionus rubens decreased if C. reinhardii was also available as food. No significant reduction of predation was found when M. minutum was offered together with B. rubens.
• 4 The species of available phytoplankton influenced egg production. Females which were allowed to feed on B. rubens and C. reinhardii produced more eggs than females which fed on rotifers only or a diet containing rotifers and M. minutum. Egg production was also possible when rotifers were absent from the diet.
• 5 Production efficiency was higher when C. reinhardii was the only food resource than on a diet containing rotifers only.

     

Microalgae for phosphorus removal and biomass production: a six species screen for dual‐purpose organisms

Microalgae biofuel production can be feasible when a second function is added, such as wastewater treatment. Microalgae differ in uptake of phosphorus (P) and growth, making top performer identification fundamental. The objective of this screen was to identify dual‐purpose candidates capable of high rates of P removal and growth. Three freshwater – Chlorella sp., Monoraphidium minutum sp., and Scenedesmus sp. – and three marine – Nannochloropsis sp., N. limnetica sp., and Tetraselmis suecica sp. – species were batch cultured in 250 mL flasks over 16 days to quantitate total phosphorus (TP) removal and growth as a function of P loads (control, and 5, 10, and 15 mg L^?1 enrichment of control). Experimental design used 100 μmol m^?2 s^?1 of light, a light/dark cycle of 14/10 h, and no CO₂ enrichment. Phosphorus uptake was dependent on species, duration of exposure, and treatment, with significant interaction effects. Growth was dependant on species and treatment. Not all species showed increased P removal with increasing P addition, and no species demonstrated higher growth. Nannochloropsis sp and N. limnetica sp. performed poorly across all treatments. Two dual‐purpose candidates were identified. At the 10 mg L^?1 treatment Monoraphidium minutum sp. removed 67.1% (6.66 mg L^?1 ± 0.60 SE) of TP at day 8, 79.3% (7.86 mg L^?1 ± 0.28 SE) at day 16, and biomass accumulation of 0.63 g L^?1 ± 0.06 SE at day 16. At the same treatment Tetraselmis suecica sp. removed 79.4% (6.98 mg L^?1 ± 0.24 SE) TP at day 8, 83.0% (7.30 mg L^?1 ± 0.60 SE) at day 16, and biomass of 0.55 g L^?1 ± 0.02 SE at day 16. These species merit further study using high‐density wastewater cultures and lipid profiling to assess suitability for a nutrient removal and biomass/biofuel production scheme.     

Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites,including expanded (CAG)n/(CTG)n repeats

Most epigenetic studies assess methylation of 5'-CpG-3' sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5'-CHG-3', where H = A, C or T, and it preferentially occurs at 5'-CpA-3' and 5'-CpT-3' sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5'-GpC-3' methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83?(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500?(CTG)500 or (CAG)800?(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5'-GpC-3' methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n?(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.     

Temperature responses of growth, photosynthesis, fatty acid and nitrate reductase in Antarctic and temperate Stichococcus

Stichococcus, a genus of green algae, distributes in ice-free areas throughout Antarctica. To understand adaptive strategies of Stichococcus to permanently cold environments, the physiological responses to temperature of two psychrotolerants, S. bacillaris NJ-10 and S. minutus NJ-17, isolated from rock surfaces in Antarctica were compared with that of one temperate S. bacillaris FACHB753. Two Antarctic Stichococcus strains grew at temperature from 4 to 25°C, while the temperate strain could grow above 30°C but could not survive at 4°C. The photosynthetic activity of FACHB753 at lower than 10°C was less than that of Antarctic algae. Nitrate reductase in NJ-10 and NJ-17 had its optimal temperature at 20°C, in comparison, the maximal activity of nitrate reductase in FACHB753 was found at 25°C. When cultured at 4–15°C a large portion of unsaturated fatty acids in the two Antarctic species was detected and the regulation of the degree of unsaturation of fatty acids by temperature was observed only above 15°C, though the content of the major unsaturated fatty acid αC18:3 in FACHB753 decreased with the temperatures elevated from 10 to 25°C. Elevated nitrate reductase activity and photosynthetic rates at low temperatures together with the high proportion of unsaturated fatty acids contribute to the ability of the Antarctic Stichococcus to thrive.     

Genome-scale analysis of DNA methylation in colorectal cancer using Infinium HumanMethylation450 BeadChips

Illumina’s Infinium HumanMethylation450 BeadChip arrays were used to examine genome-wide DNA methylation profiles in 22 sample pairs from colorectal cancer (CRC) and adjacent tissues and 19 colon tissue samples from cancer-free donors. We show that the methylation profiles of tumors and healthy tissue samples can be clearly distinguished from one another and that the main source of methylation variability is associated with disease status. We used different statistical approaches to evaluate the methylation data. In general, at the CpG-site level, we found that common CRC-specific methylation patterns consist of at least 15,667 CpG sites that were significantly different from either adjacent healthy tissue or tissue from cancer-free subjects. Of these sites, 10,342 were hypermethylated in CRC, and 5,325 were hypomethylated. Hypermethylated sites were common in the maximum number of sample pairs and were mostly located in CpG islands, where they were significantly enriched for differentially methylated regions known to be cancer-specific. In contrast, hypomethylated sites were mostly located in CpG shores and were generally sample-specific. Despite the considerable variability in methylation data, we selected a panel of 14 highly robust candidates showing methylation marks in genes SND1, ADHFE1, OPLAH, TLX2, C1orf70, ZFP64, NR5A2, and COL4A. This set was successfully cross-validated using methylation data from 209 CRC samples and 38 healthy tissue samples from The Cancer Genome Atlas consortium (AUC = 0.981 [95% CI: 0.9677–0.9939], sensitivity = 100% and specificity = 82%). In summary, this study reports a large number of loci with novel differential methylation statuses, some of which may serve as candidate markers for diagnostic purposes.     

Longitudinal epigenetic drift in mice perinatally exposed to lead

An understanding of the natural change in DNA methylation over time, defined as “epigenetic drift,” will inform the study of environmental effects on the epigenome. This study investigates epigenetic drift in isogenic mice exposed perinatally to lead (Pb) acetate at four concentrations, 0 ppm (control), 2.1 ppm (low), 16 ppm (medium), and 32 ppm (high) prior to conception through weaning, then followed until 10 months of age. Absolute values of DNA methylation in a transposon-associated metastable locus, Cdk5-activator binding protein (Cabp^IAP), and three imprinted loci (Igf2, Igf2r, and H19) were obtained from tail tissue in paired samples. DNA methylation levels in the controls increased over time at the imprinted Igf2 and Igf2r loci (both P = 0.0001), but not at the imprinted H19 locus or the Cabp^IAP metastable epiallele. Pb exposure was associated with accelerated DNA hypermethylation in Cabp^IAP (P = 0.0209) and moderated hypermethylation in Igf2r (P = 0.0447), and with marginally accelerated hypermethylation at H19 (P = 0.0847). In summary, the presence and magnitude of epigenetic drift was locus-dependent, and enhancement of drift was mediated by perinatal Pb exposure, in some, but not all, loci.     

GHSR DNA hypermethylation is a new epigenetic biomarker for gastric adenocarcinoma and beyond

Aberrations of DNA methylation are early events in the development of tumors. In this study, we investigated the DNA methylation status of growth hormone secretagogue receptor (GHSR), a promising pan-cancer biomarker, in gastric cancer (GC). Initially, data sets from DNA methylation and gene expression studies available at Gene Expression Omnibus (GEO) were analyzed. Confirmation was done on primary tumor specimens and adjacent normal stomach tissue samples. Both analyses showed significant hypermethylation of GHSR. For further validation, The Cancer Genome Atlas data on stomach cancer was used. A receiver operating characteristic curve analysis yielded an area under the curve value of 0.85, corroborating its usefulness as a diagnostic marker. A genome-wide comethylation analysis revealed several correlated genes. CREB1 was found to act as an upstream regulator of this gene network. Furthermore, GHSR methylation was found to be a biomarker in several other tumor entities, namely cancers of the bladder, endometrium, esophagus, head and neck, liver, thyroid, kidney, and ovary. Our findings along with previous reports on other types of cancer suggest a high potential of GHSR gene methylation as a pan-cancer biomarker, which could be considered for liquid biopsy applications.     

A simplified protocol for preparing DNA from filamentous cyanobacteria

The preparation of good quality genomic DNA from microalgae and plants is often time-consuming because of the need to remove contaminants that may interfere with the downstream enzymatic manipulation of the DNA. Simpler protocols have been reported but these are applicable only to a few species and in many cases are not effective for removing trace contaminants. In this report, we describe a modification of existing protocols that significantly simplified the preparation of genomic DNA from cyanobacteria and plants. A key step in our protocol is the precipitation of DNA in a high concentration of salt (2–2.5 M NaCl) in the presence of isopropanol, immediately following phenol and chloroform extractions. The preparation and enzymatic digestion of the DNA can be performed in a single day. The DNA was easily digested in 2 h at normal restriction enzyme concentrations, and is highly suitable for PCR and Southern hybridization. We successfully used this simplified protocol to prepare genomic DNA from several filamentous cyanobacteria, such asAnabaena sp. PCC 7120,Anabaena siamensis, andSpirulina strains M2 and Kenya. This protocol may also be useful for preparing genomic DNA from other algae and from higher plants.     

Enhancement of epigenetic reprogramming status of porcine cloned embryos with zebularine,a DNA methyltransferase inhibitor

Aberrant epigenetic reprogramming is known to be a major cause of inefficient somatic cell nuclear transfer (SCNT) in pigs, and use of epigenetic modification agents, such as DNA methyltransferase inhibitors (DNMTis), is a promising approach for enhancing SCNT efficacy. Here, we attempted to find the optimal condition of zebularine (Zb), a DNMTi, treatment on porcine SCNT embryos during in vitro culture (IVC). As results, treatment with 5 nM Zb for 24 hr showed the highest rate of embryo development to blastocyst compared to other groups (p < .05). Also, the relative intensities of global DNA methylation levels of anti‐5‐methylcytosine in pseudo‐pronuclear (PNC), 2‐cell and 4‐cell stages were significantly lower in the Zb‐treated group (p < .05), however, changes in methylation levels of centromeric satellite repeat were noted only in PNC and blastocyst stages. In addition, significant positive alterations in the relative expression of genes related to pluripotency (OCT4 and SOX2), histone acetylation (HAT1, HDAC1, HDAC2, and HDAC3) and DNA methylation (DNMT1 and DNMT3a) were observed compared to the control (p < .05). In conclusion, we found that Zb could modify DNA methylation levels in the early stages of porcine SCNT embryos and promote their developmental competence.     

Mutations in the dim-1 gene of Neurospora crassa reduce the level of DNA methylation

Mutants that show reduced DNA methylation were identified in a mutant screen based on the assumptions that (i) the nucleoside analog 5-azacytidine (5-azaC) promotes the formation of potentially lethal DNA-methyltransferase adducts; (ii) reduction in DNA methyltransferase will decrease the sensitivity of cells to 5-azaC; and (iii) this potential selective advantage will be enhanced in mutants that are deficient in the repair of 5-azaC-induced DNA damage. Of fifteen potential repair mutants screened for sensitivity to 5-azaC, five (mus-9, mus-10, mus-11, mus-18, and uvs-3) showed moderately increased sensitivity and two (mus-20, mei-3) showed highly increased sensitivity. A mus-20 mutation was used to isolate three non-complementing methylation mutants. The mutations, named dim-1 (defective in methylation), reduced female fertility, reduced methylation by 40–50%, and altered patterns of methylation. In wild-type strains hypomethylation perse fails to alter methylation specificity. We demonstrate a growth-phase-dependent change in methylation patterns, detectable only in hypomethylated DNA from dim ⁺ cultures. This may represent a growth-phase-dependent change in the relative amounts of distinct species of methyltransferase, one of which may be encoded by the dim-1 gene. Received: 3 January 1998 / Accepted: 26 March 1998