Urine Metabolite of Mice with Orientia tsutsugamushi Infection.

Scrub typhus is an acute febrile, mite-borne disease endemic to the Asia-Pacific region. In South Korea, it is a seasonal disease that occurs frequently in the autumn, and its incidence has increased steadily. In this study, we used a liquid chromatography and flow injection analysis-tandem mass spectrometry-based targeted urine metabolomics approach to evaluate the host response to Orientia tsutsugamushi infection. Balb/c mice were infected with O. tsutsugamushi Boryong, and their urine metabolite profile was examined. Metabolites that differed significantly between the experimental groups were identified using the Kruskal-Wallis test. Sixty-five differential metabolites were identified. The principal metabolite classes were acylcarnitines, glycerophospholipids, biogenic amines, and amino acids. An ingenuity pathway analysis revealed that several toxic (cardiotoxic, hepatotoxic, and nephrotoxic) metabolites are induced by scrub typhus infection. This is the first report of urinary metabolite biomarkers of scrub typhus infection and it enhances our understanding of the metabolic pathways involved.

Establishment of the large-scale longitudinal multi-omics dataset in COVID-19 patients: data profile and biospecimen.

Understanding and monitoring virus-mediated infections has gained importance since the global outbreak of the coronavirus disease 2019 (COVID-19) pandemic. Studies of high-throughput omics-based immune profiling of COVID-19 patients can help manage the current pandemic and future virus-mediated pandemics. Although COVID-19 is being studied since past 2 years, detailed mechanisms of the initial induction of dynamic immune responses or the molecular mechanisms that characterize disease progression remains unclear. This study involved comprehensively collected biospecimens and longitudinal multi-omics data of 300 COVID-19 patients and 120 healthy controls, including whole genome sequencing (WGS), single-cell RNA sequencing combined with T cell receptor (TCR) and B cell receptor (BCR) sequencing (scRNA(+scTCR/BCR)-seq), bulk BCR and TCR sequencing (bulk TCR/BCR-seq), and cytokine profiling. Clinical data were also collected from hospitalized COVID-19 patients, and HLA typing, laboratory characteristics, and COVID-19 viral genome sequencing were performed during the initial diagnosis. The entire set of biospecimens and multi-omics data generated in this project can be accessed by researchers from the National Biobank of Korea with prior approval. This distribution of largescale multi-omics data of COVID-19 patients can facilitate the understanding of biological crosstalk involved in COVID-19 infection and contribute to the development of potential methodologies for its diagnosis and treatment. [BMB Reports 2022; 55(9): 465-471].

The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters.

Solute carrier (SLC) transporters represent the second-largest fraction of the membrane proteome after G-protein-coupled receptors, but have been underutilized as drug targets and the function of many members of this family is still unknown. They are technically challenging to work with as they are difficult to express and highly dynamic, making them unstable in detergent solution. Many SLCs lack known inhibitors that could be utilized for stabilization. Furthermore, as they bind their physiological substrates with high micromolar to low millimolar affinities, binding and transport assays have proven to be particularly challenging to implement. Previously, we reported a GFP-based method for the overexpression and purification of membrane proteins in Saccharomyces cerevisiae. Here, we extend this expression platform with the GFP thermal shift (GFP-TS) assay, which is a simplified version of fluorescence-detection size-exclusion chromatography that combines the sample versatility of fluorescence-detection size-exclusion chromatography with the high-throughput capability of dye-based thermal shift assays. We demonstrate how GFP-TS can be used for detecting specific ligand interactions of SLC transporter fusions and measuring their affinities in crude detergent-solubilized membranes. We further show how GFP-TS can be employed on purified SLC transporter fusions to screen for specific lipid-protein interactions, which is an important complement to native mass spectrometry approaches that cannot cope easily with crude lipid-mixture preparations. This protocol is simple to perform and can be followed by researchers with a basic background in protein chemistry. Starting with an SLC transporter construct that can be expressed and purified from S. cerevisiae in a well-folded state, this protocol extension can be completed in ~4-5 d.

Blood transcriptome resources of chinstrap (Pygoscelis antarcticus) and gentoo (Pygoscelis papua) penguins from the South Shetland Islands, Antarctica.

The Chinstrap (Pygoscelis antarcticus) and Gentoo (P. papua) penguins are distributed throughout Antarctica and the sub-Antarctic islands. In this study, high-quality de novo assemblies of blood transcriptomes from these penguins were generated using the Illumina MiSeq platform. A total of 22.2 and 21.8 raw reads were obtained from Chinstrap and Gentoo penguins, respectively. These reads were assembled using the Oases assembly platform and resulted in 26,036 and 21,854 contigs with N50 values of 929 and 933 base pairs, respectively. Functional gene annotations through pathway analyses of the Gene Ontology, EuKaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed for each blood transcriptome, resulting in a similar compositional order between the two transcriptomes. Ortholog comparisons with previously published transcriptomes from the Adélie (P. adeliae) and Emperor (Aptenodytes forsteri) penguins revealed that a high proportion of the four penguins' transcriptomes had significant sequence homology. Because blood and tissues of penguins have been used to monitor pollution in Antarctica, immune parameters in blood could be important indicators for understanding the health status of penguins and other Antarctic animals. In the blood transcriptomes, KEGG analyses detected many essential genes involved in the major innate immunity pathways, which are key metabolic pathways for maintaining homeostasis against exogenous infections or toxins. Blood transcriptome studies such as this may be useful for checking the immune and health status of penguins without sacrifice.

Antarctic blackfin icefish genome reveals adaptations to extreme environments.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

Transcriptome profiling suggests roles of innate immunity and digestion metabolism in purplish Washington clam.

The purplish Washington clam (Saxidomus purpuratus) in the family Veneridae is distributed widely along the intertidal zones of northeast Asia and is increasingly being utilized as a commercially important food resource. Bivalves maintain homeostasis by regulating their food intake and digestion, innate immunity, and biotransformation in a mollusk-specific organ, the digestive gland. To understand digestive gland-specific pathways, we generated a high-quality de novo assembly of the digestive gland transcriptome of this clam using the Illumina Miseq platform. A total of 9.9 million raw reads were obtained and assembled using the Oases assembly platform, resulting in 27,358 contigs with an N50 of 433 bp. Functional gene annotations were performed using Gene Ontology, Eukaryotic Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes pathway analyses. In the transcriptome, many crucial genes involved in innate immunity and digestion metabolism were detected. A number of enzymes associated with drug metabolism were annotated, as much as that identified from the whole transcriptome of the Pacific oyster Crassostrea gigas. We provide valuable sequence information of S. purpuratus to predict functional understandings of the bivalve-specific digestive gland. This resource will be valuable for researchers comparing gene compositions and their expression levels in the digestive glands of bivalves.

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.

Structural analyses of the MazEF4 toxin-antitoxin pair in Mycobacterium tuberculosis provide evidence for a unique extracellular death factor.

The bacterial toxin-antitoxin MazEF system in the tuberculosis (TB)-causing bacterium Mycobacterium tuberculosis is activated under unfavorable conditions, including starvation, antibiotic exposure, and oxidative stress. This system contains the ribonucleolytic enzyme MazF and has emerged as a promising drug target for TB treatments targeting the latent stage of M. tuberculosis infection and reportedly mediates a cell death process via a peptide called extracellular death factor (EDF). Although it is well established that the increase in EDF-mediated toxicity of MazF drives a cell-killing phenomenon, the molecular details are poorly understood. Moreover, the divergence in sequences among reported EDFs suggests that each bacterial species has a unique EDF. To address these open questions, we report here the structures of MazF4 and MazEF4 complexes from M. tuberculosis, representing the first MazEF structures from this organism. We found that MazF4 possesses a negatively charged MazE4-binding pocket in contrast to the positively charged MazE-binding pockets in homologous MazEF complex structures from other bacteria. Moreover, using NMR spectroscopy and biochemical assays, we unraveled the molecular interactions of MazF4 with its RNA substrate and with a new EDF homolog originating from M. tuberculosis The EDF homolog discovered here possesses a positively charged residue at the C terminus, making this EDF distinct from previously reported EDFs. Overall, our results suggest that M. tuberculosis evolved a unique MazF and EDF and that the distinctive EDF sequence could serve as a starting point for designing new anti-tuberculosis drugs. We therefore conclude that this study might contribute to the development of a new line of anti-tuberculosis agents.

Draft genome of the Antarctic dragonfish, Parachaenichthys charcoti.

The Antarctic bathydraconid dragonfish, Parachaenichthys charcoti, is an Antarctic notothenioid teleost endemic to the Southern Ocean. The Southern Ocean has cooled to -1.8ºC over the past 30 million years, and the seawater had retained this cold temperature and isolated oceanic environment because of the Antarctic Circumpolar Current. Notothenioids dominate Antarctic fish, making up 90% of the biomass, and all notothenioids have undergone molecular and ecological diversification to survive in this cold environment. Therefore, they are considered an attractive Antarctic fish model for evolutionary and ancestral genomic studies. Bathydraconidae is a speciose family of the Notothenioidei, the dominant taxonomic component of Antarctic teleosts. To understand the process of evolution of Antarctic fish, we select a typical Antarctic bathydraconid dragonfish, P. charcoti. Here, we have sequenced, de novo assembled, and annotated a comprehensive genome from P. charcoti. The draft genome of P. charcoti is 709 Mb in size. The N50 contig length is 6145 bp, and its N50 scaffold length 178 362 kb. The genome of P. charcoti is predicted to contain 32 712 genes, 18 455 of which have been assigned preliminary functions. A total of 8951 orthologous groups common to 7 species of fish were identified, while 333 genes were identified in P. charcoti only; 2519 orthologous groups were also identified in both P. charcoti and N. coriiceps, another Antarctic fish. Four gene ontology terms were statistically overrepresented among the 333 genes unique to P. charcoti, according to gene ontology enrichment analysis. The draft P. charcoti genome will broaden our understanding of the evolution of Antarctic fish in their extreme environment. It will provide a basis for further investigating the unusual characteristics of Antarctic fishes.

The genome of the Antarctic-endemic copepod, Tigriopus kingsejongensis.

Background: The Antarctic intertidal zone is continuously subjected to extremely fluctuating biotic and abiotic stressors. The West Antarctic Peninsula is the most rapidly warming region on Earth. Organisms living in Antarctic intertidal pools are therefore interesting for research into evolutionary adaptation to extreme environments and the effects of climate change. Findings: We report the whole genome sequence of the Antarctic-endemic harpacticoid copepod Tigriopus kingsejongensi . The 37 Gb raw DNA sequence was generated using the Illumina Miseq platform. Libraries were prepared with 65-fold coverage and a total length of 295 Mb. The final assembly consists of 48 368 contigs with an N50 contig length of 17.5 kb, and 27 823 scaffolds with an N50 contig length of 159.2 kb. A total of 12 772 coding genes were inferred using the MAKER annotation pipeline. Comparative genome analysis revealed that T. kingsejongensis -specific genes are enriched in transport and metabolism processes. Furthermore, rapidly evolving genes related to energy metabolism showed positive selection signatures. Conclusions: The T. kingsejongensis genome provides an interesting example of an evolutionary strategy for Antarctic cold adaptation, and offers new genetic insights into Antarctic intertidal biota.

First Insights into the Subterranean Crustacean Bathynellacea Transcriptome: Transcriptionally Reduced Opsin Repertoire and Evidence of Conserved Homeostasis Regulatory Mechanisms.

Bathynellacea (Crustacea, Syncarida, Parabathynellidae) are subterranean aquatic crustaceans that typically inhabit freshwater interstitial spaces (e.g., groundwater) and are occasionally found in caves and even hot springs. In this study, we sequenced the whole transcriptome of Allobathynella bangokensis using RNA-seq. De novo sequence assembly produced 74,866 contigs including 28,934 BLAST hits. Overall, the gene sequences were most similar to those of the waterflea Daphnia pulex. In the A. bangokensis transcriptome, no opsin or related sequences were identified, and no contig aligned to the crustacean visual opsins and non-visual opsins (i.e. arthropsins, peropsins, and melaopsins), suggesting potential regressive adaptation to the dark environment. However, A. bangokensis expressed conserved gene family sets, such as heat shock proteins and those related to key innate immunity pathways and antioxidant defense systems, at the transcriptional level, suggesting that this species has evolved adaptations involving molecular mechanisms of homeostasis. The transcriptomic information of A. bangokensis will be useful for investigating molecular adaptations and response mechanisms to subterranean environmental conditions.

Characterization of Two Antimicrobial Peptides from Antarctic Fishes (Notothenia coriiceps and Parachaenichthys charcoti).

We identified two antimicrobial peptides (AMPs) with similarity to moronecidin in Antarctic fishes. The characteristics of both AMPs were determined using moronecidin as a control. Moronecidin, which was first isolated from hybrid striped bass, is highly salt-resistant, and possesses broad-spectrum activity against various microbes. The moronecidin-like peptide from Notothenia coriiceps exhibited a narrower spectrum of activity and a higher salt sensitivity than moronecidin. The AMP from Parachaenichthys charcoti exhibited similar antimicrobial activity to moronecidin, and similar salt sensitivity. In an experiment to identify toxic effects, both of the moronecidin-like peptides from the Antarctic fishes exhibited lower hemolytic activity than moronecidin. In spite of its low toxicity, the AMP from N. coriiceps is unlikely to be considered as a candidate for antibiotic development, owing to its narrow spectrum of activity and high salt sensitivity. In contrast, the high salt resistance and broad-spectrum activity of the AMP from P. charcoti could be more advantageous for clinical use than moronecidin, since it could kill bacteria under physiological conditions with low toxicity. A further comparison of these two AMPs from Antarctic fishes with other AMPs could help to reduce the toxicity of AMPs for clinical use.

Transcriptome analysis of immune response genes induced by pathogen agonists in the Antarctic bullhead notothen Notothenia coriiceps.

Fish are a representative population of lower vertebrates that serve as an essential link to early vertebrate evolution, and this has fueled academic interest in studying ancient vertebrate immune defense mechanisms in teleosts. Notothenia coriiceps, a typical Antarctic notothenioid teleost, has evolved to adapt to the cold and thermally stable Antarctic sea. In this study, we examined adaptive signaling pathways and immune responses to bacterial and viral pathogenic exposure in N. coriiceps. Using RNA sequencing, we investigated transcriptional differences in the liver tissues of N. coriiceps challenged with two pathogen-mimicking agonists, a bacterial ligand (heat-killed Escherichia coli, HKEB) and a viral ligand (polyinosinic:polycytidylic acid, Poly I:C). We found that 567 unique genes were up-regulated two-fold in the HKEB-exposed group, whereas 392 unique genes, including 124 immune-relevant genes, were up-regulated two-fold in the Poly I:C-exposed group. A KEGG pathway analysis of the 124 immune-relevant genes revealed that they exhibited major features of antigen processing and presentation bacterial ligand exposure, but they were down-regulated after viral ligand exposure. A quantitative real time RT-PCR analysis revealed that TNFα and TNF2, major inducers of apoptosis, were highly up-regulated after exposure to the viral ligand but not the bacterial ligand. The results suggest that the bacterial and viral ligands up-regulate inducers of different immune mechanisms in N. coriiceps liver tissue. N. coriiceps has an immune response defense strategy that uses antigen presentation against bacterial infection, but it may use a different defense, such as TNF-mediated apoptosis, against viral infection. The specific immune responses of N. coriiceps may be adaptations to the Antarctic environment and pathogens. These results will help define the characteristics of Antarctic fish and increase our understanding of their immune response mechanisms.

Development of a cost-effective metabarcoding strategy for analysis of the marine phytoplankton community.

We developed a cost-effective metabarcoding strategy to analyze phytoplankton community structure using the Illumina MiSeq system. The amplicons (404-411 bp) obtained by end-pairing of two reads were sufficiently long to distinguish algal species and provided barcode data equivalent to those generated with the Roche 454 system, but at less than 1/20th of the cost. The original universal primer sequences targeting the 23S rDNA region and the PCR strategy were both modified, and this resulted in higher numbers of eukaryotic algal sequences by excluding non-photosynthetic proteobacterial sequences supporting effectiveness of this strategy. The novel strategy was used to analyze the phytoplankton community structure of six water samples from the East/Japan Sea: surface and 50 m depths at coastal and open-sea sites, with collections in May and July 2014. In total, 345 operational taxonomic units (OTUs) were identified, which covered most of the prokaryotic and eukaryotic algal phyla, including Dinophyta, Rhodophyta, Ochrophyta, Chlorophyta, Streptophyta, Cryptophyta, Haptophyta, and Cyanophyta. This highlights the importance of plastid 23S primers, which perform better than the currently used 16S primers for phytoplankton community surveys. The findings also revealed that more efforts should be made to update 23S rDNA sequences as well as those of 16S in the databases. Analysis of algal proportions in the six samples showed that community structure differed depending on location, depth and season. Across the six samples evaluated, the numbers of OTUs in each phylum were similar but their relative proportions varied. This novel strategy would allow laboratories to analyze large numbers of samples at reasonable expense, whereas this has not been possible to date due to cost and time. In addition, we expect that this strategy will generate a large amount of novel data that could potentially change established methods and tools that are currently used in the realms of oceanography and marine ecology.

Complete genome sequence of Hymenobacter sp. strain PAMC26554, an ionizing radiation-resistant bacterium isolated from an Antarctic lichen.

A Gram-negative, rod-shaped, red-pink in color, and UV radiation-resistant bacterium Hymenobacter sp. strain PAMC26554 was isolated from Usnea sp., an Antarctic lichen, and belongs to the class of Cytophagia and the phylum of Bacteroidetes. The complete genome of Hymenobacter sp. PAMC26554 consists of one chromosome (5,244,843bp) with two plasmids (199,990bp and 6421bp). The genomic sequence indicates that Hymenobacter sp. strain PAMC26554 possesses several genes involved in the nucleotide excision repair pathway that protects damaged DNA. This complete genome information will help us to understand its adaptation and novel survival strategy in the Antarctic extreme cold environment.

Complete genome sequence of Burkholderia sp. strain PAMC28687, a potential octopine-utilizing bacterium isolated from Antarctica lichen.

We report the complete genome sequence of Burkholderia sp. PAMC28687, which was isolated from the Antarctica lichen Useea sp., for better understanding of its catabolic traits in utilizing octopine as a source of carbon/nitrogen between Burkholderia and lichen. The genome consists of three circular chromosomes with five circular plasmids for the total 6,881,273bp sized genome with a G+C content of 58.14%.

Complete genome sequence of ionizing radiation-resistant Hymenobacter sp. strain PAMC26628 isolated from an Arctic lichen.

Ionizing radiation-resistant Hymenobacter sp. strain PAMC26628 was isolated from Stereocaulon sp., an Arctic lichen. Complete genome sequencing of Hymenobacter sp. PAMC26628 revealed one chromosome (5,277,381 bp), one plasmid (89,596 bp), and several genes involved in nucleotide excision repair, a DNA damage removal pathway. An analysis of the Hymenobacter sp. PAMC26628 genome will help us understand its evolution and provide novel insight into the adaptations that allow this organism to survive in the extreme cold of the Arctic.

Complete genome sequence of carotenoid-producing Microbacterium sp. strain PAMC28756 isolated from an Antarctic lichen.

Microbacterium sp. strain PAMC28756, of the family Microbacteriaceae, was isolated from Stereocaulon sp., an Antarctic lichen. Complete genome sequencing of Microbacterium sp. PAMC28756 revealed, for the first time in the genus Microbacterium, a series of key genes involved in C50 carotenoid biosynthesis. An analysis of the Microbacterium sp. PAMC28756 genome will lead to a better understanding of the carotenoid biosynthesis pathway. Furthermore, the sequence data will provide novel insight into UV radiation resistance in extremely cold environments.

Comparative Analysis of Lacinutrix Genomes and Their Association with Bacterial Habitat.

The genus Lacinutrix, which belongs to the family Flavobacteriaceae, consists of seven bacterial species that were mainly isolated from marine life and sediments. As most bacteria in the family Flavobacteriaceae favor aerobic conditions, the seven bacterial species in the genus Lacinutrix also showed aerobic growth. We selected four monophyletic bacterial species living in a polar environment. Two of these species were isolated from sediment and two types were isolated from algae. In a comparative analysis, we investigated how these different environments were related to genomic features of these four species in the genus Lacinutrix. We found that the gene sets for glycolysis, the Krebs cycle, and oxidative phosphorylation were conserved in these four type strains. However, the presence of nitrous oxide reductase for denitrification and the absence of essential components related to thiamin biosynthesis for aerobic respiration were only found in isolates from sediment. Elevated bacterial metabolism on the surface of marine sediments might limit the oxygen penetration into sediment, and such an environment might affect the genomes of bacteria isolated from these habitats.