CREB5 promotes the proliferation and self-renewal ability of glioma stem cells.

Glioblastoma multiforme (GBM) is the most fatal form of brain cancer in humans, with a dismal prognosis and a median overall survival rate of less than 15 months upon diagnosis. Glioma stem cells (GSCs), have recently been identified as key contributors in both tumor initiation and therapeutic resistance in GBM. Both public dataset analysis and direct differentiation experiments on GSCs have demonstrated that CREB5 is more highly expressed in undifferentiated GSCs than in differentiated GSCs. Additionally, gene silencing by short hairpin RNA (shRNA) of CREB5 has prevented the proliferation and self-renewal ability of GSCs in vitro and decreased their tumor forming ability in vivo. Meanwhile, RNA-sequencing, luciferase reporter assay, and ChIP assay have all demonstrated the closely association between CREB5 and OLIG2. These findings suggest that targeting CREB5 could be an effective approach to overcoming GSCs.

Metagenomic Analysis of the Species Composition and Seasonal Distribution of Marine Dinoflagellate Communities in Four Korean Coastal Regions.

Biomonitoring of dinoflagellate communities in marine ecosystems is essential for efficient water quality management and limiting ecosystem disturbances. Current identification and monitoring of toxic dinoflagellates, which cause harmful algal blooms, primarily involves light or scanning electron microscopy; however, these techniques are limited in their ability to monitor dinoflagellates and plankton, leaving an incomplete analysis. In this study, we analyzed the species composition and seasonal distribution of the dinoflagellate communities in four Korean coastal regions using 18S rRNA amplicon sequencing. The results showed significantly high diversity in the dinoflagellate communities in all regions and seasons. Furthermore, we found seasonally dominant species and causative species of harmful algal blooms (Cochlodinium sp., Alexandrium sp., Dinophysis sp., and Gymnodinium sp.). Moreover, dominant species were classified by region and season according to the difference in geographical and environmental parameters. The molecular analysis of the dinoflagellate community based on metagenomics revealed more diverse species compositions that could not be identified by microscopy and revealed potentially harmful or recently introduced dinoflagellate species. In conclusion, metagenomic analysis of dinoflagellate communities was more precise and obtained results faster than microscopic analysis, and could improve the existing monitoring techniques for community analysis.

Development of a Method for Detecting Alexandrium pacificum Based on the Quantification of sxtA4 by Chip-Based Digital PCR.

Alexandrium pacificum, which produces the paralytic shellfish toxin (PST) saxitoxin (STX), is one of the causative species of paralytic shellfish poisoning outbreaks in coastal areas of Korea. In this study, we developed a chip-based digital PCR (dPCR) method for A. pacificum detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an A. pacificum strain isolated in 2017, species-specific primers targeting sxtA4 (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of A. pacificum. Cell abundance in field samples, estimated by a chip-based dPCR, was compared with the PST content, and measured using a mouse bioassay. A comparison with shellfish PST concentrations indicated that cell concentrations above 500 cells L-1, as measured using the dPCR assay, may cause shellfish PST concentrations to exceed the allowed limits for PSTs. Concordance rates between dPCR and PST results were 62.5% overall in 2018-2021, reaching a maximum of 91.7% in 2018-2019. The sensitivity of the dPCR assay was higher than that of microscopy and sxtA4-based qPCRs. Absolute quantification by chip-based dPCRs targeting sxtA4 in A. pacificum exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms.

Centrodinium punctatum (Dinophyceae) produces significant levels of saxitoxin and related analogs.

Centrodinium punctatum is a fusiform dinoflagellate with a global marine distribution. Due to a close phylogenetic relationship of one C. punctatum strain to Alexandrium species, toxin production of this C. punctatum strain was assessed using liquid chromatography coupled to tandem mass spectrometry. The paralytic shellfish toxin (PST) profile of C. punctatum was dominated by six analogs, i.e. STX (30%), GTX-1 (20%) and neoSTX (24%), followed by GTX-2 (9%), GTX-4 (9%) and GTX-3 (8%); deoxy-STX was also putatively identified while no gymnodimines, spirolides or goniodomins were detected. This is the first record of C. punctatum producing saxitoxins. The estimated cellular toxicity was rather elevated, between 91 and 212 pg cell-1 (or 259 and 601 fmol cell-1). When considering the toxicity equivalent factors, results suggest that this species can produce high cellular toxicity compared to other STX-producing dinoflagellates. Morphological details of the sulcal area and the hypotheca of Centrodinium punctatum were re-examined by scanning electron microscopy (SEM); this revealed that in the sulcal area, the left posterior sulcal plate (Ssp) is larger and longer than the left posterior sulcal plate and extended into the hypotheca. Based on the morphological observation, a revised interpretation of the sulcus and hypotheca is proposed.

High diversity and potential translocation of DNA viruses in ballast water.

Ballast water is a common vector for the transport of invasive species to new marine and aquatic environments. We used a metagenomics approach to examine the composition and diversity of viral communities in ballast water from ships originating in Mexico, Saudi Arabia, New York, and Panama, and in water from the port of their destination in Busan, Korea. Myoviridae was the most abundant virus family in ballast water, followed Podoviridae and Siphoviridae. We also identified viruses that infect invertebrates, amoebas, and algae in ballast water and in the Busan port water. Interestingly, there were several viruses that infect humans or other animals (Swinepox virus, Raccoonpox virus, Suid herpesvirus, and Human endogenous retrovirus) in the samples from New York and Panama. In addition, there were giant viruses in all the ballast water samples, especially, identified Megavirus chilensis in New York and Panama, and Pandoravirus salinus in Mexico and Saudi Arabia. These results provide detailed descriptions of the characteristics of the viruses present in ballast water, document significant viral diversity, and indicate the potential translocation of viruses via ballast water.

Seasonal Dynamics and Metagenomic Characterization of Marine Viruses in Goseong Bay, Korea.

Viruses are the most abundant biological entities in the oceans, and account for a significant amount of the genetic diversity of marine ecosystems. However, there is little detailed information about the biodiversity of viruses in marine environments. Rapid advances in metagenomics have enabled the identification of previously unknown marine viruses. We performed metagenomic profiling of seawater samples collected at 6 sites in Goseong Bay (South Sea, Korea) during the spring, summer, autumn, and winter of 2014. The results indicated the presence of highly diverse virus communities. The DNA libraries from samples collected during four seasons were sequenced using Illumina HiSeq 2000. The number of viral reads was 136,850 during March, 70,651 during June, 66,165 during September, and 111,778 during December. Species identification indicated that Pelagibacter phage HTVC010P, Ostreococcus lucimarinus OIV5 and OIV1, and Roseobacter phage SIO1 were the most common species in all samples. For viruses with at least 10 reads, there were 204 species during March, 189 during June, 170 during September, and 173 during December. Analysis of virus families indicated that the Myoviridae was the most common during all four seasons, and viruses in the Polyomaviridae were only present during March. Viruses in the Iridoviridae were only present during three seasons. Additionally, viruses in the Iridoviridae, Herpesviridae, and Poxviridae, which may affect fish and marine animals, appeared during different seasons. These results suggest that seasonal changes in temperature contribute to the dynamic structure of the viral community in the study area. The information presented here will be useful for comparative analyses with other marine viral communities.

Differential gene expression patterns during embryonic development of sea urchin exposed to triclosan.

Triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) is a broad-spectrum antibacterial agent used in common industrial, personal care and household products which are eventually rinsed down the drain and discharged with wastewater effluent. It is therefore commonly found in the aquatic environment, leading to the continual exposure of aquatic organisms to TCS and the accumulation of the antimicrobial and its harmful degradation products in their bodies. Toxic effects of TCS on reproductive and developmental progression of some aquatic organisms have been suggested but the underlying molecular mechanisms have not been defined. We investigated the expression patterns of genes involved in the early development of TCS-treated sea urchin Strongylocentrotus nudus using cDNA microarrays. We observed that the predominant consequence of TCS treatment in this model system was the widespread repression of TCS-modulated genes. In particular, empty spiracles homeobox 1 (EMX-1), bone morphogenic protein, and chromosomal binding protein genes showed a significant decrease in expression in response to TCS. These results suggest that TCS can induce abnormal development of sea urchin embryos through the concomitant suppression of a number of genes that are necessary for embryonic differentiation in the blastula stage. Our data provide new insight into the crucial role of genes associated with embryonic development in response to TCS. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 426-433, 2017.

Detection of coat protein gene of nervous necrosis virus using loop-mediated isothermal amplification.

OBJECTIVE: To establish a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for the identification of nervous necrosis virus (NNV) infection. METHODS: A set of synthesized primers was used to match the sequences of a specific region of the nnv gene from the National Center for Biotechnology Information database, not originating from NNV-infected fish, the efficiency and specificity of LAMP were measured dependent on the concentration of DNA polymerase and the reaction temperature and time. In addition, to determine species-specific LAMP primers, cross reactivity testing was applied to the reaction between NVV and other virus families including viral hemorrhagic septicemia virus and marine birnavirus. RESULTS: The optimized LAMP reaction carried out at 64 °C for 60 min, and above 4 U Bst DNA polymerase. The sensitivity of LAMP for the detection of nnv was thus about 10 times greater than the sensitivity of polymerase chain reaction. The LAMP assay primers were specific for the detection NNV infection in Epinephelus septemfasciatus. CONCLUSIONS: The development of LAMP primers based on genetic information from a public database, not virus-infected samples, may provide a very simple and convenient method to identify viral infection in aquatic organisms.

Detection of the dinoflagellate, Cochlodinium polykrikoides, that forms algal blooms using sandwich hybridization integrated with nuclease protection assay.

OBJECTIVES: To detect Cochlodinum polykrikoides in long-term monitoring and high-throughput sampling projects using an integrated sandwich hybridization and nuclease protection assay (NPA-SH). RESULTS: The specificity of the probes was verified with individual and mixed cultures as well as field collection, and the quantity of C. polykrikoides determined by NPA-SH analysis showed a good correlation with that determined by cell-counting with a light microscope. In addition a standard curve for C. polykrikoides was established to represent the correlation between optical absorbance in the NPA-SH assay and cell density. CONCLUSIONS: This approach provides an efficient alternative to traditional, morphology-based methods for the rapid identification and quantification of harmful algal species and could be used to monitor phytoplankton in field surveys.

Seasonal Dynamics of Marine Microbial Community in the South Sea of Korea.

High-resolution 16S rRNA tag pyrosequencing was used to obtain seasonal snapshots of the bacterial diversity and community structure at two locations in Gosung Bay (South Sea, Korea) over a one year period. Seasonal sampling from the water column at each site revealed highly diverse bacterial communities containing up to 900 estimated Operational Taxonomic Units (OTUs). The Alphaproteobacteria and Gammaproteobacteria were the most abundant groups, and the most frequently recorded OTUs were members of Pelagibacter and Glaciecola. In particular, it was observed that Arcobacter, a genus of the Epsilonproteobacteria, dominated during summer. In addition, Psedoalteromonadaceae, Vibrionaceae and SAR11-1 were predominant members of the OTUs found in all sampling seasons. Environmental factors significantly influenced the bacterial community structure among season, with the phosphate and nitrate concentrations contributing strongly to the spatial distribution of the Alphaproteobacteria; the Gammaproteobacteria, Flavobacteria, and Actinobacteria all showed marked negative correlations with all measured nutrients, particularly silicon dioxide and chlorophyll-a. The results suggest that seasonal changes in environmental variables contribute to the dynamic structure of the bacterial community in the study area.

Fatty acid methyl ester profiles and nutritive values of 20 marine microalgae in Korea.

OBJECTIVE: To screen the fatty acid (FA) composition of 20 marine microalgae species, including seven Diophyceae, six Bacillariophyceae, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species. METHODS: Microalgal cells cultured at the Korea Institute of Ocean Science & Technology were harvested during the late exponential growth phase and the FA composition analyzed. RESULTS: The FA composition of microalgae was species-specific. For example, seven different species of Dinophyceae were composed primarily of C14:0, C16:0, C18:0, C20:4n-6, C20:5n-3 and C22:6n-3, while C14:0, C16:0, C16:1, C18:0, C20:5n-3 and C22:6n-3 were abundant FAs in six species of Bacillariophyceae. In addition, four Chlorophyceae, two Haptophyceae and one Raphidophyceae species all contained a high degree of C16:1n-7 [(9.28-34.91)% and (34.48-35.04)%], C14:0 [(13.34-25.96)%] and [(26.69-28.24)%], and C16:0 [(5.89-29.15)%] and [(5.70-16.81)%]. Several factors contribute to the nutritional value of microalgae, including the polyunsaturated FA content and n-3 to n-6 FA ratio, which could be used to assess the nutritional quality of microalgae. CONCLUSIONS: This study is the first comprehensive assessment of the FA composition and nutritional value of microalgae species in South Korea, and identifies the potential utility of FAs as species-specific biomarkers.

Anti-inflammation activities of mycosporine-like amino acids (MAAs) in response to UV radiation suggest potential anti-skin aging activity.

Certain photosynthetic marine organisms have evolved mechanisms to counteract UV-radiation by synthesizing UV-absorbing compounds, such as mycosporine-like amino acids (MAAs). In this study, MAAs were separated from the extracts of marine green alga Chlamydomonas hedleyi using HPLC and were identified as porphyra-334, shinorine, and mycosporine-glycine (mycosporine-Gly), based on their retention times and maximum absorption wavelengths. Furthermore, their structures were confirmed by triple quadrupole MS/MS. Their roles as UV-absorbing compounds were investigated in the human fibroblast cell line HaCaT by analyzing the expression levels of genes associated with antioxidant activity, inflammation, and skin aging in response to UV irradiation. The mycosporine-Gly extract, but not the other MAAs, had strong antioxidant activity in the 2,2-diphenyl-1-picryhydrazyl (DPPH) assay. Furthermore, treatment with mycosporine-Gly resulted in a significant decrease in COX-2 mRNA levels, which are typically increased in response to inflammation in the skin, in a concentration-dependent manner. Additionally, in the presence of MAAs, the UV-suppressed genes, procollagen C proteinase enhancer (PCOLCE) and elastin, which are related to skin aging, had increased expression levels equal to those in UV-mock treated cells. Interestingly, the increased expression of involucrin after UV exposure was suppressed by treatment with the MAAs mycosporine-Gly and shinorine, but not porphyra-334. This is the first report investigating the biological activities of microalgae-derived MAAs in human cells.

Distinct patterns of marine bacterial communities in the South and North Pacific Oceans.

The study of oceanic microbial communities is crucial for our understanding of the role of microbes in terms of biomass, diversity and ecosystem function. In this study, 16S rRNA gene tag pyrosequencing was used to investigate change in bacterial community structure between summer and winter water masses from Gosung Bay in the South Sea of Korea and Chuuk in Micronesia, located in the North and South Pacific Oceans, respectively. Summer and winter sampling from each water mass revealed highly diverse bacterial communities, containing ~900 Operational Taxonomic Units (OTUs). The microbial distribution and highly heterogeneous composition observed at both sampling sites were different from those of most macroorganisms. The bacterial communities in the seawater at both sites were most abundant in Proteobacteria during the summer in Gosung and in Bacterioidetes during the winter. The proportion of Cyanobacteria was higher in summer than in winter in Chuuk and similar in Gosung. Additionally, the microbial community during summer in Gosung was significantly different from other communities observed based on the unweighted UniFrac distance. These data suggest that in both oceanic areas sampled, the bacterial communities had distinct distribution patterns with spatially- and temporally-heterogeneous distributions.

Hypoxia-modulated gene expression profiling in sea urchin (Strongylocentrotus nudus) immune cells.

Hypoxia is an issue that affects ocean coastal waters worldwide. It has severe consequences for marine organisms, including death and rapid adaptive changes in metabolic organization. Although some aquatic animals are routinely exposed and resistant to severe environmental hypoxia, others such as sea urchins (Strongylocentrotus nudus) have a limited capacity to withstand this stress. In this study, hypoxia induced a significant increase in the number of red spherule cells among coelomocytes, which function as immune cells. This suggests that sea urchin immune cells could be used as a biological indicator of hypoxic stress. In the current study, we used cDNA microarrays to investigate the differential expression patterns of hypoxia-regulated genes to better understand the molecular mechanisms underlying the response of immune cells to hypoxia. Surprisingly, the predominant major effect of hypoxia was the widespread suppression of gene expression. In particular, the expression of RNA helicase and GATA-4/5/6 was decreased significantly in response to hypoxia, even in field conditions, suggesting that they could be utilized as sensitive bioindicators of hypoxic stress in the sea urchin.

Phenol content, antioxidant and tyrosinase inhibitory activity of mangrove plants in Micronesia.

OBJECTIVE: To find out and compare the in vitro antioxidant and tyrosinase inhibitory activities of two species of mangrove plants. METHODS: Mangrove samples were harvested at the shoreline on the island of Weno, Chuuk State in Micronesia. The phenol content, antioxidant activity (based on DPPH-free radical scavenging) and tyrosinase inhibitory activity in different tissues (leaves, barks and roots) of Rhizophora stylosa (R. stylosa) and Sonneratia alba (S. alba), collected from the island of Weno. RESULTS: Total phenol content ranged from 4.87 to 11.96 mg per g of freeze dried samples. The highest antioxidant activity was observed in R. stylosa bark (85.5%). The highest tyrosinase inhibitory activity was found in S. alba bark. Also, total phenol content and antioxidant activity were higher in methanol extracts than in aqueous extracts. CONCLUSIONS: Taken together, the results of this study proved that mangroves can be excellent sources of antioxidant compounds.

Effects of triclosan on reproductive prarmeters and embryonic development of sea urchin, Strongylocentrotus nudus.

Triclosan (TCS, 2,4,4'-trichloro-2'-hydroxydiphenyl ether), a broad-spectrum antibacterial agent, is commonly found in the aquatic environment. In this study, we investigated TCS toxicity with pertaining to gamete viability, fertilization, and embryogenesis up to pluteus stage of the sea urchin, (Strongylocentrotus nudus). When the sperm and eggs were exposed to TCS (0-3.0 µM), the viability of sperm was significantly decreased at molarities higher than 1 µM of TCS. In addition, for exposure of 2.0 µM TCS the viability of eggs was not influenced and none of the sperm was viable. Fertilization rate was significantly decreased when sperm were exposed to 0.5 and 1 µM of TCS (p<0.001) and no fertilization was observed for the exposure of 1.5 µM of TCS. In embryonic development, embryos are treated with higher than 1.0 µM levels of TCS displayed arrested development. For TCS, the EC50 and LOECs values were 1.8, 1.49 and 0.99 µM and 0.53, 0.62 and 0.39 µM for sperm viability, fertilization rate, and larval development to pluteus, respectively. In the recovery test regarding normal development of arrested embryos based upon TCS exposure time, it was observed that embryos exposed to 1 µM TCS for 15 h were normally recovered for normal development, while embryos with more than 30 h exposure were not recovered to normal larvae. Overall, the results of this study strongly suggest that the gametes and embryos of S. nudus can provide the basis for an effective bioassay, with a fast and sensitive means of evaluating TCS contamination in the marine ecosystem.