Synthetic pro-peptide design to enhance the secretion of heterologous proteins by Saccharomyces cerevisiae.

Heterologous protein production in Saccharomyces cerevisiae is a useful and effective strategy with many advantages, including the secretion of proteins that require posttranslational processing. However, heterologous proteins in S. cerevisiae are often secreted at comparatively low levels. To improve the production of the heterologous protein, human granulocyte colony-stimulating factor (hG-CSF) in S. cerevisiae, a secretion-enhancing peptide cassette including an hIL-1ß-derived pro-peptide, was added and used as a secretion enhancer to alleviate specific bottlenecks in the yeast secretory pathway. The effects of three key parameters-N-glycosylation, net negative charge balance, and glycine-rich flexible linker-were investigated in batch cultures of S. cerevisiae. Using a three-stage design involving screening, selection, and optimization, the production and secretion of hG-CSF by S. cerevisiae were significantly increased. The amount of extracellular mature hG-CSF produced by the optimized pro-peptide after the final stage increased by 190% compared to that of the original pro-peptide. Although hG-CSF was used as the model protein in the current study, this strategy is applicable to the enhanced production of other heterologous proteins, using S. cerevisiae as the host.

Trophic Ecology of Endangered Gold-Spotted Pond Frog in Ecological Wetland Park and Rice Paddy Habitats.

The gold-spotted pond frog (Pelophylax chosenicus) is an endangered amphibian species in South Korea. In order to obtain ecological information regarding the gold-spotted pond frog's habitat environment and biological interactions, we applied stable isotope analysis to quantify the ecological niche space (ENS) of frogs including black-spotted pond frogs (P. nigromaculatus) and bullfrogs (Lithobates catesbeianus) within the food web of two different habitats-an ecological wetland park and a rice paddy. The gold-spotted pond frog population exhibited a broader ENS in the ecological wetland park than in the rice paddy. According to the carbon stable isotope ratios, gold-spotted pond frogs mainly fed on insects, regardless of habitat type. However, the results comparing the range of both carbon and nitrogen stable isotopes showed that gold-spotted pond frogs living in the rice paddy showed limited feeding behavior, while those living in the ecological wetland park fed on various food sources located in more varied trophic positions. Although the ENS of the gold-spotted pond frog was generally less likely to be overlapped by that of other frog species, it was predicted to overlap with a high probability of 87.3% in the ecological wetland park. Nevertheless, gold-spotted pond frogs in the ecological wetland park were not significantly affected by the prey competition with competitive species by feeding on other prey for which other species' preference was low. Since these results show that a habitats' food diversity has an effect on securing the ENS of gold-spotted pond frogs and prey competition, we recommend that the establishment of a food environment that considers the feeding behavior of gold-spotted pond frogs is important for the sustainable preservation of gold-spotted pond frogs and their settlement in alternative habitats.

Application of Next-Generation Sequencing for the Determination of the Bacterial Community in the Gut Contents of Brackish Copepod Species (Acartia hudsonica, Sinocalanus tenellus, and Pseudodiaptomus inopinus).

The gut bacterial communities of copepods can affect metabolic processes, and consequently, their activity can be related to the release of organic substances to the environment. Hence, they are important for organic matter cycling in marine coast food webs. However, information regarding the variation in gut bacterial communities based on copepod species and environmental variations is limited. We analysed the differences in gut bacterial communities from dominant copepod species, i.e., Acartia hudsonica, Sinocalanus tenellus, and Pseudodiaptomus inopinus, in a brackish reservoir. The core bacteria among the copepod species and locations consisted of the following main operational taxonomic units (OTUs): Novosphingobium capsulatum and the family Rhodobacteraceae belonging to Alphaproteobacteria, which is abundant in seawater and freshwater aquatic ecosystems as a zooplankton-associated bacterial community. The bacterial community composition of each copepod (except the core species) showed high variability. The bacterial community diversity differed depending on the copepod species and the sites' environmental conditions, especially salinity, e.g., compositional variations in the bacterial community of P. inopinus were high at sites with low salinity. Therefore, the gut bacterial community of each copepod species responds differently to the environment.

Occurrence, distribution, and potential exposure risk of organophosphate flame retardants in house dust in South Korea.

Concentrations of organophosphate flame retardants (OPFRs), which are used in various plastic products, were analyzed in house dust samples collected from three Korean cities (Suwon, n = 23; Jeonju, n = 20; Kunsan, n = 42). OPFRs, including tris (2-chloroethyl) phosphate (TCEP), tris (2-chloroisopropyl) phosphate (TCPP), and tris (1,3-dichloro-2-propyl) phosphate (TDCPP), were detected in 95%-100% of the samples analyzed, suggesting the widespread use of these compounds in Korea. The levels of TCEP, TCPP, and TDCPP in Suwon, Jeonju, and Kunsan ranged from the limit of quantitation to 46,000, 28,000, and 2400 ng/g, respectively. The concentrations of all OPFRs were significantly higher in house dust samples from Suwon than from Jeonju and Kunsan; this is likely due to the increased use of these compounds in Suwon, which may be associated with the number, volume, and variety of household products in homes. In Korean homes, the estimated daily intake (EDI) of OPFRs through house dust ingestion was lower than the guideline values; however, the EDI of OPFRs for toddlers was 30-fold greater than for adults, suggesting a limited risk to human health. This is the first comprehensive study of the occurrence and distribution of OPFRs in house dust in Korea.

Trophic response to ecological conditions of habitats: Evidence from trophic variability of freshwater fish.

To adapt to ecological and environmental conditions, species can change their ecological niche (e.g., interactions among species) and function (e.g., prey-predation, diet competition, and habitat segregation) at the species and guild levels. Stable isotope analysis of bulk carbon and nitrogen of organisms has conventionally been used to evaluate such adaptabilities in the scenopoetic and bionomic views as the isotopic niche width.Compound-specific stable isotope analysis (CSIA) of nitrogen within amino acids provides trophic information without any disruption of scenopoetic views in the isotope ratios, unlike conventional bulk isotope analysis provides both information and therefore frequently hinders its usefulness for trophic information.We performed CSIA of amino acids to understand the trophic variability of the pike gudgeon Pseudogobio esocinus and largemouth bass Micropterus salmoides as representative specialist and generalist fish species, respectively, from 16 ecologically variable habitats in the four major rivers of Korea.There was little variation (1σ) in the trophic position (TP) among habitats for P. esocinus (± 0.2); however, there was considerably large variation for M. salmoides (± 0.6). The TP of M. salmoides was negatively correlated with the benthic invertebrate indices of the habitats, whereas the TP of P. esocinus showed no significant correlation with any indices. Thus, these two representative fish species have different trophic responses to ecological conditions, which is related to known differences in the trophic niche between specialists (i.e., small niche width) and generalists (i.e., large niche width).Over the past four decades, the conventional bulk isotope analysis has not been capable of deconvoluting "scenopoetic" and "bionomic" information. However, in the present study, we demonstrated that the CSIA of amino acids could isolate trophic niches from the traditional ecological niche composed of trophic and habitat information and evaluated how biological and ecological indices influence the trophic response of specialists and generalists.

Spatiotemporal variability in a copepod community associated with fluctuations in salinity and trophic state in an artificial brackish reservoir at Saemangeum, South Korea.

Saemangeum Reservoir in South Korea is an estuarine system enclosed by a dyke construction, where seawater inflow and retained water outflow are managed by the opening/closing of sluice gates installed in the southern part of the dyke. An exchange of the reservoir water can cause spatiotemporal fluctuations in the salinity and trophic state, which are major drivers determining variation in the composition of biological communities in estuarine systems. Here, we investigated the seasonal and spatial variability in the copepod community and environmental conditions (water temperature, salinity, transparency, chlorophyll a concentration, total nitrogen, total phosphorus and Carlson's trophic state index) based on seasonally conducted field monitoring in the Saemangeum Reservoir from July 2013 to January 2018. In addition to the role of temperature, salinity and chlorophyll a concentration in structuring the copepod community and diversity, the biological indices of copepods with respect to salinity range and trophic state, were evaluated. The spatiotemporal variability in the salinity and trophic state variables showed contrasting patterns, and chlorophyll a concentration was negatively affected by salinity, indicating that the reservoir water was being highly exchanged with opening of the sluice gates. The mean trophic state index values, however, were constant in the eutrophic state (50-70). Dominant copepods were Acartia (A. hudsonica, A. sinjiensis, Acartia spp.) and Oithona (O. davisae and Oithona spp.), which are common species in eutrophic neritic water. Variation in the copepod community was mainly associated with the seasonal succession of the dominant species rather than a spatial gradient (from around the estuary to the sluice gates); however, site-specific differences in frequencies of several non-dominant species could be detected around the estuary (Sinocalanus tenellus) and the sluice gates (Centropages spp., Tigriopus spp. and Labidocera rotunda). The copepod diversity increased with species-richness from around the estuary to the sluice gates, which could result from variation in the site-specific location of non-dominant species. The frequency of particular species was also able to discriminate in terms of the salinity range (oligohaline: A. pacifica, S. tenellus and A. sinjiensis; mesohaline: Pseudodiaptomus inopinus; and polyhaline: C. abdominalis and Centropages spp.) and the trophic state (mesotrophic: C. abdominalis, Calanus sinicus and Centropages spp.; and hypereutrophic: S. tenellus, P. inopinus and Sinocalanus spp.). The findings from this study not only identify the factors determining spatiotemporal variation in the copepod community in the Saemangeum Reservoir, but also expand the applicability of copepods as biological indicators of conditions associated with salinity range and trophic state in other enclosed estuarine systems.

Comparison of taxon-based and trophi-based response patterns of rotifer community to water quality: applicability of the rotifer functional group as an indicator of water quality.

Rotifer community is often used as a taxon-based bioindicator for water quality. However, studies of the planktonic community from the viewpoint of functional groups in freshwater ecosystems have been limited, particularly for rotifers. Because rotifers have various trophi types determining their feeding strategies, thereby representing an ecological niche, their functional feeding groups can act as biological and ecological indicators in lakes and reservoirs where planktonic communities are dominant. We analyzed the patterns of spatial distribution of the rotifer community in various reservoirs and then its relationship with water quality through redundancy and regression analyses. Compared with taxon-based composition, the response of trophi-based composition appears simplistic and showed clearer tendency in relation with water-quality variables. Each trophi responded differently by the degree of eutrophication indicating that each trophi group is possibly affected by environments such as the combinations of water-quality variables in different ways.

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae.

O-linked ß-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

Heat Shock RNA 1, Known as a Eukaryotic Temperature-Sensing Noncoding RNA, Is of Bacterial Origin.

Heat shock RNA 1 (HSR1) is described as a "eukaryotic heat-sensing noncoding RNA" that regulates heat shock response in human and other eukaryotic cells. Highly conserved HSR1 sequences have been identified from humans, hamsters, Drosophila, Caenorhabditis elegans, and Arabidopsis. In a previous study, however, it was suggested that HSR1 had originated from a bacterial genome. HSR1 showed no detectible nucleotide sequence similarity to any eukaryotic sequences but harbored a protein coding region that showed amino-acid sequence similarity to bacterial voltage-gated chloride channel proteins. The bacterial origin of HSR1 was not convincible because the nucleotide sequence similarity was marginal. In this study, we have found that a genomic contig sequence of Comamonas testosteroni strain JL14 contained a sequence virtually identical to that of HSR1, decisively confirming the bacterial origin of HSR1. Thus, HSR1 is an exogenous RNA, which can ectopically trigger heat shock response in eukaryotes. Therefore, it is no longer appropriate to cite HSR1 as a "eukaryotic functional noncoding RNA."

Loss of gene function and evolution of human phenotypes.

Humans have acquired many distinct evolutionary traits after the human-chimpanzee divergence. These phenotypes have resulted from genetic changes that occurred in the human genome and were retained by natural selection. Comparative primate genome analyses reveal that loss-of-function mutations are common in the human genome. Some of these gene inactivation events were revealed to be associated with the emergence of advantageous phenotypes and were therefore positively selected and fixed in modern humans (the "less-ismore" hypothesis). Representative cases of human gene inactivation and their functional implications are presented in this review. Functional studies of additional inactive genes will provide insight into the molecular mechanisms underlying acquisition of various human-specific traits.

Frequent loss and alteration of the MOXD2 gene in catarrhines and whales: a possible connection with the evolution of olfaction.

The MOXD2 gene encodes a membrane-bound monooxygenase similar to dopamine-ß-hydroxylase, and has been proposed to be associated with olfaction. In this study, we analyzed MOXD2 genes from 64 mammalian species, and identified loss-of-function mutations in apes (humans, Sumatran and Bornean orangutans, and five gibbon species from the four major gibbon genera), toothed whales (killer whales, bottlenose dolphins, finless porpoises, baijis, and sperm whales), and baleen whales (minke whales and fin whales). We also identified a shared 13-nt deletion in the last exon of Old World cercopithecine monkeys that results in conversion of a membrane-bound protein to a soluble form. We hypothesize that the frequent inactivation and alteration of MOXD2 genes in catarrhines and whales may be associated with the evolution of olfaction in these clades.

Retroduplication and loss of parental genes is a mechanism for the generation of intronless genes in Ciona intestinalis and Ciona savignyi.

Tunicates, the sister clade of vertebrates, have miniature genomes and numerous intronless genes compared to other animals. It is still unclear how the tunicates acquired such a large number of intronless genes. Here, we analyzed sequences and intron-exon organizations of homologous genes from two closely related tunicates, Ciona intestinalis and Ciona savignyi. We found seven cases in which ancestral introns of a gene were completely lost in a species after their divergence. In four cases, both the intronless copy and the intron-containing copy were present in the genome, indicating that the intronless copy was generated by retroduplication. In the other three cases, the intron-containing copy was absent, implying it was lost after retroduplication. This result suggests that retroduplication and loss of parental genes is a major mechanism for the accumulation of intronless genes in tunicates.

Plant RNA virus sequences identified in kimchi by microbial metatranscriptome analysis.

Plant pathogenic RNA viruses are present in a variety of plant-based foods. When ingested by humans, these viruses can survive the passage through the digestive tract, and are frequently detected in human feces. Kimchi is a traditional fermented Korean food made from cabbage or vegetables, with a variety of other plant-based ingredients, including ground red pepper and garlic paste. We analyzed microbial metatranscriptome data from kimchi at five fermentation stages to identify plant RNA virus-derived sequences. We successfully identified a substantial amount of plant RNA virus sequences, especially during the early stages of fermentation: 23.47% and 16.45% of total clean reads on days 7 and 13, respectively. The most abundant plant RNA virus sequences were from pepper mild mottle virus, a major pathogen of red peppers; this constituted 95% of the total RNA virus sequences identified throughout the fermentation period. We observed distinct sequencing read-depth distributions for plant RNA virus genomes, possibly implying intrinsic and/or technical biases during the metatranscriptome generation procedure. We also identified RNA virus sequences in publicly available microbial metatranscriptome data sets. We propose that metatranscriptome data may serve as a valuable resource for RNA virus detection, and a systematic screening of the ingredients may help prevent the use of virus-infected low-quality materials for food production.