Anticancer Activity of Mannose-Specific Lectin, BPL2, from Marine Green Alga Bryopsis plumosa.

Lectin is a carbohydrate-binding protein that recognizes specific cells by binding to cell-surface polysaccharides. Tumor cells generally show various glycosylation patterns, making them distinguishable from non-cancerous cells. Consequently, lectin has been suggested as a good anticancer agent. Herein, the anticancer activity of Bryopsis plumosa lectins (BPL1, BPL2, and BPL3) was screened and tested against lung cancer cell lines (A549, H460, and H1299). BPL2 showed high anticancer activity compared to BPL1 and BPL3. Cell viability was dependent on BPL2 concentration and incubation time. The IC50 value for lung cancer cells was 50 µg/mL after 24 h of incubation in BPL2 containing medium; however, BPL2 (50 µg/mL) showed weak toxicity in non-cancerous cells (MRC5). BPL2 affected cancer cell growth while non-cancerous cells were less affected. Further, BPL2 (20 µg/mL) inhibited cancer cell invasion and migration (rates were ˂20%). BPL2 induced the downregulation of epithelial-to-mesenchymal transition-related genes (Zeb1, vimentin, and Twist). Co-treatment with BPL2 and gefitinib (10 µg/mL and 10 µM, respectively) showed a synergistic effect compared with monotherapy. BPL2 or gefitinib monotherapy resulted in approximately 90% and 70% cell viability, respectively, with concomitant treatment showing 40% cell viability. Overall, BPL2 can be considered a good candidate for development into an anticancer agent.

A strategic approach to apply bacterial substances for increasing metabolite productions of Euglena gracilis in the bioreactor.

Bacterial extracellular polymeric substances (EPS) are promising materials that have a role in enhancing growth, metabolite production, and harvesting efficiency. However, the validity of the EPS effectiveness in scale-up cultivation of microalgae is still unknown. Therefore, in order to verify whether the bacterial metabolites work in the scale-up fermentation of microalgae, we conducted a bioreactor fermentation following the addition of bacterial EPS derived from the marine bacterium, Pseudoalteromonas sp., to Euglena gracilis. Various culture strategies (i.e., batch, glucose fed-batch, and glucose and EPS fed-batch) were conducted to maximize metabolite production of E. gracilis in scale-up cultivation. Consequently, biomass and paramylon concentrations in the continuous glucose and EPS-treated culture were enhanced by 3.0-fold and 4.2-fold (36.1 ± 1.4 g L-1 and 25.6 ± 0.1 g L-1), respectively, compared to the non-treated control (12.0 ± 0.3 g L-1 and 6.1 ± 0.1 g L-1). Also, the supplementation led to the enhanced concentrations of α-tocopherols and total fatty acids by 3.7-fold and 2.8-fold, respectively. The harvesting efficiency was enhanced in EPS-supplemented cultivation due to the flocculation of E. gracilis. To the best of our knowledge, this is the first study that verifies the effect of bacterial EPS in scale-up cultivation of microalgae. Also, our results showed the highest paramylon productivity than any other previous reports. The results obtained in this study showed that the scale-up cultivation of E. gracilis using bacterial EPS has the potential to be used as a platform to guide further increases in scale and in the industrial environment. KEY POINTS: Effect of EPS on Euglena gracilis fermentation was tested in bioreactor scale. EPS supplement was effective for the paramylon, α-tocopherol, and lipid production. EPS supplement induced the flocculation of E. gracilis.

Cetaceans evolution: insights from the genome sequences of common minke whales.

BACKGROUND: Whales have captivated the human imagination for millennia. These incredible cetaceans are the only mammals that have adapted to life in the open oceans and have been a source of human food, fuel and tools around the globe. The transition from land to water has led to various aquatic specializations related to hairless skin and ability to regulate their body temperature in cold water. RESULTS: We present four common minke whale (Balaenoptera acutorostrata) genomes with depth of ×13 ~ ×17 coverage and perform resequencing technology without a reference sequence. Our results indicated the time to the most recent common ancestors of common minke whales to be about 2.3574 (95% HPD, 1.1521 - 3.9212) million years ago. Further, we found that genes associated with epilation and tooth-development showed signatures of positive selection, supporting the morphological uniqueness of whales. CONCLUSIONS: This whole-genome sequencing offers a chance to better understand the evolutionary journey of one of the largest mammals on earth.

Genetic diversity and differentiation of the Korean starry flounder (Platichthys stellatus) between and within cultured stocks and wild populations inferred from microsatellite DNA analysis.

The Korean starry flounder, Platichthys stellatus, is economically valuable coastal resident fish species. However, the annual catch of this fish has fluctuated and suffered major declines in Korea. We examined the genetic diversity and population structure for four wild populations and three hatchery stocks of Korean starry flounder to protect its genetic integrity using nine microsatellites. A group of 339 genotypes belonging to seven populations were screened. High degrees of polymorphism at the microsatellite loci were observed within both the wild and hatchery populations. Compared to the wild populations, genetic changes, including reduced genetic diversity and highly significant differentiation, have occurred in cultured stocks. Significant population differentiation was also observed in wild starry flounder populations. Similar degrees of inbreeding and significant Hardy-Weinberg equilibrium deviations were detected in both the wild and the hatchery populations. The genetic connectivity pattern identified four distinct metapopulations of starry flounder in Korea by clustering in the phylogenetic tree, Bayesian analyses, molecular variance analysis, PCA and multidimensional scaling analysis. A pattern of isolation-by-distance was not significant. This genetic differentiation may be the result of the co-effects of various factors, such as historic dispersal, local environment or anthropogenic activities. These results provide useful information for the genetic monitoring of P. stellatus hatchery stocks, for the genetic improvement of this species by selective breeding and for designing suitable management guidelines for the conservation of this species.

Genetic structure of the Korean black scraper Thamnaconus modestus inferred from microsatellite marker analysis.

The Korean black scraper, Thamnaconus modestus, is one of the most economically important maricultural fish species in Korea. However, the annual catch of this fish has been continuously declining over the past several decades. In this study, the genetic diversity and relationships among four wild populations and two hatchery stocks of Korean black scraper were assessed based on 16 microsatellite (MS) markers. A total of 319 different alleles were detected over all loci with an average of 19.94 alleles per locus. The hatchery stocks [mean number of alleles (N(A)) = 12, allelic richness (A(R)) = 12, expected heterozygosity (He) = 0.834] showed a slight reduction (P > 0.05) in genetic variability in comparison with wild populations (mean N(A) = 13.86, A(R) = 12.35, He = 0.844), suggesting a sufficient level of genetic variation in the hatchery populations. Similarly low levels of inbreeding and significant Hardy-Weinberg equilibrium deviations were detected in both wild and hatchery populations. The genetic subdivision among all six populations was low but significant (overall F(ST) = 0.008, P < 0.01). Pairwise F(ST), a phylogenetic tree, and multidimensional scaling analysis suggested the existence of three geographically structured populations based on different sea basin origins, although the isolation-by-distance model was rejected. This result was corroborated by an analysis of molecular variance. This genetic differentiation may result from the co-effects of various factors, such as historical dispersal, local environment and ocean currents. These three geographical groups can be considered as independent management units. Our results show that MS markers may be suitable not only for the genetic monitoring of hatchery stocks but also for revealing the population structure of Korean black scraper populations. These results will provide critical information for breeding programs, the management of cultured stocks and the conservation of this species.

Development of microsatellite markers for the Korean Mussel, Mytilus coruscus (Mytilidae) using next-generation sequencing.

Mytilus coruscus (family Mytilidae) is one of the most important marine shellfish species in Korea. During the past few decades, this species has become endangered due to the loss of habitats and overfishing. Despite this species' importance, information on its genetic background is scarce. In this study, we developed microsatellite markers for M. coruscus using next-generation sequencing. A total of 263,900 raw reads were obtained from a quarter-plate run on the 454 GS-FLX titanium platform, and 176,327 unique sequences were generated with an average length of 381 bp; 2569 (1.45%) sequences contained a minimum of five di- to tetra-nucleotide repeat motifs. Of the 51 loci screened, 46 were amplified successfully, and 22 were polymorphic among 30 individuals, with seven of trinucleotide repeats and three of tetranucleotide repeats. All loci exhibited high genetic variability, with an average of 17.32 alleles per locus, and the mean observed and expected heterozygosities were 0.67 and 0.90, respectively. In addition, cross-amplification was tested for all 22 loci in another congener species, M. galloprovincialis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 22 newly developed microsatellites will be useful in future conservation genetic studies of this species.

Application of novel polymorphic microsatellite loci identified in the Korean Pacific Abalone (Haliotis diversicolor supertexta (Haliotidae)) in the genetic characterization of wild and released populations.

The small abalone, Haliotis diversicolor supertexta, of the family Haliotidae, is one of the most important species of marine shellfish in eastern Asia. Over the past few decades, this species has drastically declined in Korea. Thus, hatchery-bred seeds have been released into natural coastal areas to compensate for the reduced fishery resources. However, information on the genetic background of the small abalone is scarce. In this study, 20 polymorphic microsatellite DNA markers were identified using next-generation sequencing techniques and used to compare allelic variation between wild and released abalone populations in Korea. Using high-throughput genomic sequencing, a total of 1516 (2.26%; average length of 385 bp) reads containing simple sequence repeats were obtained from 86,011 raw reads. Among the 99 loci screened, 28 amplified successfully, and 20 were polymorphic. When comparing allelic variation between wild and released abalone populations, a total of 243 different alleles were observed, with 18.7 alleles per locus. High genetic diversity (mean heterozygosity = 0.81; mean allelic number = 15.5) was observed in both populations. A statistical analysis of the fixation index (F(ST)) and analysis of molecular variance (AMOVA) indicated limited genetic differences between the two populations (F(ST) = 0.002, p > 0.05). Although no significant reductions in the genetic diversity were found in the released population compared with the wild population (p > 0.05), the genetic diversity parameters revealed that the seeds released for stock abundance had a different genetic composition. These differences are likely a result of hatchery selection and inbreeding. Additionally, all the primer pair sets were effectively amplified in another congeneric species, H. diversicolor diversicolor, indicating that these primers are useful for both abalone species. These microsatellite loci may be valuable for future aquaculture and population genetic studies aimed at developing conservation and management plans for these two abalone species.

Genetic characterization of five hatchery populations of the Pacific Abalone (Haliotis discus hannai) using microsatellite markers.

The Pacific abalone, Haliotis discus hannai, is a popular food in Eastern Asia. Aquacultural production of this species has increased because of recent resource declines, the growing consumption, and ongoing government-operated stock release programs. Therefore, the genetic characterization of hatchery populations is necessary to maintain the genetic diversity of this species and to develop more effective aquaculture practices. We analyzed the genetic structures of five cultured populations in Korea using six microsatellite markers. The number of alleles per locus ranged from 15 to 64, with an average of 23.5. The mean observed and expected heterozygosities were 0.797 and 0.904, respectively. The inbreeding coefficient F(IS) ranged from 0.054 to 0.184 (mean F(IS) = 0.121 ± 0.056). The genetic differentiation across all populations was low but significant (overall F(ST) = 0.009, P < 0.01). Pairwise multilocus F(ST) tests, estimates of genetic distance, and phylogenetic and principal component analyses did not show a consistent relationship between geographic and genetic distances. These results could reflect extensive aquaculture, the exchange of breeds and eggs between hatcheries and/or genetic drift due to intensive breeding practices. Thus, for optimal resource management, the genetic variation of hatchery stocks should be monitored and inbreeding controlled within the abalone stocks that are being released every year. This genetic information will be useful for the management of both H. discus hannai fisheries and the aquaculture industry.

Wild and hatchery populations of Korean starry flounder (Platichthys stellatus) compared using microsatellite DNA markers.

Starry flounder (Platichthys stellatus) is an important sport and food fish found around the margins of the North Pacific. Aquaculture production of this species in Korea has increased because of its commercial value. Microsatellite DNA markers are a useful DNA-based tool for monitoring the genetic variation of starry flounder populations. In this study, 12 polymorphic microsatellite DNA markers were identified from a partial genomic starry flounder DNA library enriched in CA repeats, and used to compare allelic variation between wild and hatchery starry flounder populations in Korea. All loci were readily amplified and demonstrated high allelic diversity, with the number of alleles ranging from 6 to 18 in the wild population and from 2 to 12 in the farmed population. A total of 136 alleles were detected at the 12 microsatellite loci in the two populations. The mean observed and expected heterozygosities were 0.62 and 0.68, respectively, in the hatchery samples and 0.67 and 0.75, respectively, in the wild samples. These results indicate lower genetic variability in the hatchery population as compared to the wild population. Significant shifts in allelic frequencies were detected at eight loci, which resulted in a small but significant genetic differences between the wild and hatchery populations (F(ST) = 0.043, P < 0.05). Further studies with additional starry flounder sample collections are needed for comprehensive determinations of the genetic varieties between the wild and hatchery populations. These microsatellite loci may be valuable for future population genetic studies, monitoring the genetic variation for successful aquaculture management and the preservation of aquatic biodiversity.

Novel polymorphic microsatellite loci for the Korean Black Scraper (Thamnaconus modestus), and their application to the genetic characterization of wild and farmed populations.

In this study, we developed 20 polymorphic microsatellite markers for the Korean black scraper, Thamnaconus modestus (Günther, 1877), Monacanthidae, and used them to compare allelic variation between wild and hatchery populations in Korea. All loci were readily amplified and demonstrated allelic variability, with the number of alleles ranging from 5-35 in the wild population and 5-22 in the farmed population. The average observed and expected heterozygosities were estimated, respectively, as 0.74 and 0.80 in the hatchery samples and 0.78 and 0.81 in the wild ones. These results indicate lower genetic variability in the hatchery population than in the wild population and minor, but significant, genetic differentiation between the two populations (F(ST) = 0.005, P < 0.01). Additionally, cross-amplification was tested in another monacanthid species, Stephanolepis cirrhifer; many loci were found that yielded useful information. The high degree of polymorphism exhibited by the 20 microsatellites will be useful in future aquaculture and population genetic studies for developing conservation and management plans.

Comparison between wild and hatchery populations of Korean pen shell (Atrina pectinata) using microsatellite DNA markers.

Pen shell (Atrina pectinata) is a popular food source with a high commercial value in a number of Asian Pacific areas. The natural A. pectinata population has been declining continuously over the past several decades. Microsatellite DNA markers are a useful DNA-based tool for monitoring the genetic variation of pen shell populations. In this study, 20 polymorphic microsatellite (MS) DNA markers were identified from a partial genomic pen shell DNA library enriched in CA repeats, and used to compare allelic variation between wild and hatchery pen shell populations in Korea. A total of 438 alleles were detected at the 20 MS loci in the two populations. All loci were easily amplified and demonstrated allelic variability, with the number of alleles ranging from 5 to 35 in the wild population and from 5 to 22 in the farmed population. The average observed and expected heterozygosities were 0.69 and 0.82, respectively, in the hatchery samples and 0.69 and 0.83, respectively, in the wild samples. Statistical analysis of fixation index (F(ST)) and analysis of molecular variance (AMOVA) showed minor, but significant, genetic differences between the wild and hatchery populations (F(ST) = 0.0106, CI(95%) = 0.003-0.017). These microsatellite loci may be valuable for future aquaculture and population genetic studies for developing conservation and management plans. Further studies with additional pen shell samples are needed to conclusively determine the genetic diversity between the wild and hatchery populations.

Genetic analysis and population genetic structure of hard-shelled mussel, Mytilus coruscus Gould 1861 (Mytiloida: Mytilidae) from the coasts of South Korea based on mitochondrial cytochrome oxidase (COI) gene sequences.

BACKGROUND: Mytilus coruscus Gould, 1861 is a mussel species in the family Mytilidae, native to the Northwest Pacific Ocean, ranging from the East China Sea, the Yellow Sea, and as far as the Peter the Great Gulf in the East Sea. In Korea, this species has been heavily exploited for nutrient-rich food resources and experienced severe reduction in their population. OBJECTIVE: The aim of this study was to investigate the genetic diversity and population structure and to provide baseline data to facilitate the conservation and sustainable use of the vulnerable species M. coruscus in South Korea. METHODS: Mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) sequences of 91 adult individuals from four islands and one coastal localities in South Korea were sequenced. We then compared genetic diversity and haplotype data with previously published Chinese wild populations. RESULTS: Mytilus coruscus populations on Korean coasts were found to exhibit high genetic diversity despite concerns regarding recent population reduction. A total of 42 haplotypes were defined by 56 polymorphic sites. High-level genetic diversity was observed on four Island sites (Hd = 0.906-0.955, π = 0.0068-0.0090). The other seashore site represented relatively lower genetic diversity (Hd = 0.529, π = 0.0011) and was genetically differentiated from the others. In a previous study, wild populations on the East China Sea exhibited similarly high genetic diversity as that observed in our study. Additionally, Chinese M. coruscus populations exhibit a distinct regional haplotype distribution pattern while sharing six haplotypes with Korean populations. CONCLUSIONS: The results of this study provide insights that further the current understanding regarding the evolution of M. coruscus species and provides comprehensive genetic data to facilitate the development of an effective conservation strategy.

Generation of mmp15b Zebrafish Mutant to Investigate Liver Diseases.

Upon gene inactivation in animal models, the zebrafish (Danio rerio) has become a useful model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. Genome editing is a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced in the genome of a living organism. Mainly, CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 (CRISPR-associated protein 9) is a technology that enables geneticists to edit parts of the genome. In this study, we utilized this technology to generate an mmp15b mutant by using zebrafish as an animal model. MMP15 is the membrane-type MMP (MT-MMP) which is a recently identified matrix metalloproteinase (MMP) capable of degrading all kinds of extracellular matrix proteins as well as numerous bioactive molecules. Although the newly-established mmp15b zebrafish mutant didn't exhibit morphological phenotypes in the developing embryos, it might be further utilized to understand the role of MMP15 in liver-related diseases, such as liver fibrosis, and associated pathogeneses in humans.

Complete mitochondrial genome of Cheilio inermis (Labriformes, Labridae).

In the present report, we describe the first sequencing and assembly of the complete mitochondrial genome of Cheilio inermis. The mitochondrial genome of C. inermis, with 16,494 bp in length, has the typical vertebrate mitochondrial gene arrangement. It contains 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region. All the tRNA genes typically formed a cloverleaf secondary structure. Phylogenetic analysis using mitochondrial genomes of 11 species showed that C. inermis formed monophyletic group with other Labridae species.

Correction to: Development of 21 polymorphic microsatellite markers for the black-banded sea krait, Laticauda semifasciata (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species.

Unfortunately, one of the co-author's family name has been incorrectly published in the original online publication. The correct family name should be Tsai.

Development of 21 polymorphic microsatellite markers for the black-banded sea krait, Laticauda semifasciata (Elapidae: Laticaudinae), and cross-species amplification for two other congeneric species.

The genus Laticauda (Reptilia: Elapidae), commonly known as sea kraits, is venomous marine amphibious snakes distributed throughout the south and southeast Asian islands and mostly found in coastal waters. To facilitate genetic studies, we have developed microsatellite loci for L. semifasciata using the 454 GS-FLX pyrosequencing technique. A total of 65,680 sequences containing a minimum of five repeat motifs were identified from 451,659 reads. Among 80 loci containing more than nine repeat units, 34 primer sets (42.5%) produced strong PCR products, of which 21 were polymorphic among 36 samples of L. semifasciata. All loci exhibited high genetic variability, with an average of 7.38 alleles per locus, and the mean observed and expected heterozygosities were 0.73 and 0.76, respectively. The cross-species amplification of these loci in two laticaudine species, L. colubrina and L. laticaudata, revealed a high transferability (78.6%) and polymorphism (59.5%) of the loci. Our work demonstrated the utility of next-generation 454 sequencing as the rapid and cost-effective method for development of microsatellite markers. The high level of polymorphism in these microsatellite loci will be useful for the detection of population subdivision and the study of migration, gene flow, relatedness and philopatry of L. semifasciata and other laticaudine species.

The genome of common long-arm octopus Octopus minor.

Background: The common long-arm octopus (Octopus minor) is found in mudflats of subtidal zones and faces numerous environmental challenges. The ability to adapt its morphology and behavioral repertoire to diverse environmental conditions makes the species a promising model for understanding genomic adaptation and evolution in cephalopods. Findings: The final genome assembly of O. minor is 5.09 Gb, with a contig N50 size of 197 kb and longest size of 3.027 Mb, from a total of 419 Gb raw reads generated using the Pacific Biosciences RS II platform. We identified 30,010 genes; 44.43% of the genome is composed of repeat elements. The genome-wide phylogenetic tree indicated the divergence time between O. minor and Octopus bimaculoides was estimated to be 43 million years ago based on single-copy orthologous genes. In total, 178 gene families are expanded in O. minor in the 14 bilaterian species. Conclusions: We found that the O. minor genome was larger than that of closely related O. bimaculoides, and this difference could be explained by enlarged introns and recently diversified transposable elements. The high-quality O. minor genome assembly provides a valuable resource for understanding octopus genome evolution and the molecular basis of adaptations to mudflats.

Complete mitochondrial genome of Parupeneus barberinus (Perciformes, Mullidae): mitogenome characterization and phylogenetic analysis.

Parupeneus barberinus is a tropical/subtropical reef-dwelling marine fish belonging to the family Mullidae. Herein, we report the first sequencing and assembly of the complete mitochondrial genome of P. barberinus. The complete mitochondrial genome is 16,560 bp long and has the typical vertebrate mitochondrial gene arrangement, consisting of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region. Phylogenetic analysis using mitochondrial genomes of 18 species showed that P. barberinus is clustered with P. multifasciatus and P. chrysopleuron and rooted with other Mullidae species. This mitochondrial genome provides potentially important resources for addressing taxonomic issues and studying molecular evolution.

Complete mitochondrial genome of the Pacific cod, Gadus macrocephalus (Gadiformes, Gadidae).

The Pacific cod Gadus macrocephalus is a commercially important species belonging to the family Gadidae. In this study, we performed the first sequencing and assembly of the complete mitochondrial genome of G. macrocephalus. The complete mitochondrial genome is 16,567 bp long, consisting of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. It has the typical vertebrate mitochondrial gene arrangement. Phylogenetic analysis using the mitochondrial genomes of 15 species showed that G. macrocephalus clusters with G. ogac. This mitochondrial genome provides potentially important resources for performing population genetic analysis and addressing phylogenetic issues.

The complete mitochondrial genome of Gymnogobius heptacanthus (Perciformes, gobiidae).

Gymnogobius heptacanthus is a small intertidal species belonging to the family Gobiidae. Herein, we report the first sequencing and assembly of the complete mitochondrial genome of G. heptacanthus. The complete mitochondrial genome is 16,529 bp long and has the typical vertebrate mitochondrial gene arrangement, consisting of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. Phylogenetic analysis using mitochondrial genomes of 12 species showed that G. heptacanthus is clustered with G. urotaenia and G. petschiliensis and rooted with other Gobiidae species. This mitochondrial genome provides potentially important resources for addressing taxonomic issues and studying molecular evolution.