Genome assembly of Genji firefly (Nipponoluciola cruciata) reveals novel luciferase-like luminescent proteins without peroxisome targeting signal.

The Genji firefly, Nipponoluciola cruciata, is an aquatic firefly endemic to Japan, inhabiting a wide area of the Japanese archipelago. The luminescence of fireflies is a scientifically interesting phenomenon, and many studies have evaluated this species in Japan. In this study, we sequenced the whole genome of male N. cruciata and constructed a high-quality genome assembly of 662 Mb with a BUSCO completeness of 99.1% in the genome mode. Using the detected set of 15,169 protein-coding genes, the genomic structures and genetic background of luminescence-related genes were also investigated. We found four new firefly luciferase-like genes in the genome. The highest bioluminescent activity was observed for LLa2, which originated from ancestral PDGY, a mitochondrial acyl-CoA synthetase. A thioesterase candidate, NcruACOT1, which is involved in d-luciferin biosynthesis, was expressed in the lantern. Two opsins were also detected and the absorption wavelength of the UV-type opsin candidate shifted from UV to blue. These findings provide an important resource for unravelling the adaptive evolution of fireflies in terms of luminescence and vision.

Transcriptomic comparison between populations selected for higher and lower mobility in the red flour beetle Tribolium castaneum.

Movement is an important behavior observed in a wide range of taxa. Previous studies have examined genes controlling movement using wing polymorphic insects and genes controlling wing size. However, few studies have investigated genes controlling movement activity rather than morphological traits. In the present study, we conducted RNA sequencing using populations with higher (WL) and lower (WS) mobility established by artificial selection in the red flour beetle Tribolium castaneum and compared gene expression levels between selected populations with two replicate lines. As a result, we found significant differences between the selected populations in 677 genes expressed in one replicate line and 1198 genes expressed in another replicate line, of which 311 genes were common to the two replicate lines. Furthermore, quantitative PCR focusing on 6 of these genes revealed that neuropeptide F receptor gene (NpF) was significantly more highly expressed in the WL population than in the WS population, which was common to the two replicate lines. We discuss differences in genes controlling movement between walking activity and wing polymorphism.

BRD9 determines the cell fate of hematopoietic stem cells by regulating chromatin state.

ATP-dependent chromatin remodeling SWI/SNF complexes exist in three subcomplexes: canonical BAF (cBAF), polybromo BAF (PBAF), and a newly described non-canonical BAF (ncBAF). While cBAF and PBAF regulate fates of multiple cell types, roles for ncBAF in hematopoietic stem cells (HSCs) have not been investigated. Motivated by recent discovery of disrupted expression of BRD9, an essential component of ncBAF, in multiple cancers, including clonal hematopoietic disorders, we evaluate here the role of BRD9 in normal and malignant HSCs. BRD9 loss enhances chromatin accessibility, promoting myeloid lineage skewing while impairing B cell development. BRD9 significantly colocalizes with CTCF, whose chromatin recruitment is augmented by BRD9 loss, leading to altered chromatin state and expression of myeloid-related genes within intact topologically associating domains. These data uncover ncBAF as critical for cell fate specification in HSCs via three-dimensional regulation of gene expression and illuminate roles for ncBAF in normal and malignant hematopoiesis.

Genomic and Secretomic Analyses of the Newly Isolated Fungus Perenniporia fraxinea SS3 Identified CAZymes Potentially Related to a Serious Pathogenesis of Hardwood Trees.

Perenniporia fraxinea can colonize living trees and cause severe damage to standing hardwoods by secreting a number of carbohydrate-activate enzymes (CAZymes), unlike other well-studied Polyporales. However, significant knowledge gaps exist in understanding the detailed mechanisms for this hardwood-pathogenic fungus. To address this issue, five monokaryotic P. fraxinea strains, SS1 to SS5, were isolated from the tree species Robinia pseudoacacia, and high polysaccharide-degrading activities and the fastest growth were found for P. fraxinea SS3 among the isolates. The whole genome of P. fraxinea SS3 was sequenced, and its unique CAZyme potential for tree pathogenicity was determined in comparison to the genomes of other nonpathogenic Polyporales. These CAZyme features are well conserved in a distantly related tree pathogen, Heterobasidion annosum. Furthermore, the carbon source-dependent CAZyme secretions of P. fraxinea SS3 and a nonpathogenic and strong white-rot Polyporales member, Phanerochaete chrysosporium RP78, were compared by activity measurements and proteomic analyses. As seen in the genome comparisons, P. fraxinea SS3 exhibited higher pectin-degrading activities and higher laccase activities than P. chrysosporium RP78, which were attributed to the secretion of abundant glycoside hydrolase family 28 (GH28) pectinases and auxiliary activity family 1_1 (AA1_1) laccases, respectively. These enzymes are possibly related to fungal invasion into the tree lumens and the detoxification of tree defense substances. Additionally, P. fraxinea SS3 showed secondary cell wall degradation capabilities at the same level as that of P. chrysosporium RP78. Overall, this study suggested mechanisms for how this fungus can attack the cell walls of living trees as a serious pathogen and differs from other nonpathogenic white-rot fungi. IMPORTANCE Many studies have been done to understand the mechanisms underlying the degradation of plant cell walls of dead trees by wood decay fungi. However, little is known about how some of these fungi weaken living trees as pathogens. P. fraxinea belongs to the Polyporales, a group of strong wood decayers, and is known to aggressively attack and fell standing hardwood trees all over the world. Here, we report CAZymes potentially related to plant cell wall degradation and pathogenesis factors in a newly isolated fungus, P. fraxinea SS3, by genome sequencing in conjunction with comparative genomic and secretomic analyses. The present study provides insights into the mechanisms of the degradation of standing hardwood trees by the tree pathogen, which will contribute to the prevention of this serious tree disease.

Single-cell transcriptomics of the goldfish retina reveals genetic divergence in the asymmetrically evolved subgenomes after allotetraploidization.

The recent whole-genome duplication (WGD) in goldfish (Carassius auratus) approximately 14 million years ago makes it a valuable model for studying gene evolution during the early stages after WGD. We analyzed the transcriptome of the goldfish retina at the level of single-cell (scRNA-seq) and open chromatin regions (scATAC-seq). We identified a group of genes that have undergone dosage selection, accounting for 5% of the total 11,444 ohnolog pairs. We also identified 306 putative sub/neo-functionalized ohnolog pairs that are likely to be under cell-type-specific genetic variation at single-cell resolution. Diversification in the expression patterns of several ohnolog pairs was observed in the retinal cell subpopulations. The single-cell level transcriptome analysis in this study uncovered the early stages of evolution in retinal cell of goldfish after WGD. Our results provide clues for understanding the relationship between the early stages of gene evolution after WGD and the evolution of diverse vertebrate retinal functions.

Profiling a single-stranded DNA region within an rDNA segment that affects the loading of bacterial condensin.

Bacterial condensin preferentially loads onto single-stranded DNA (ssDNA) in vitro and onto rDNA in vivo to support proper chromosome compaction. Thus, the actively transcribing rDNA would provide the ssDNA region for the loading of bacterial condensin. We attempted to detect the ssDNA region in the rrnI gene in situ. Non-denaturing sodium bisulfite treatment catalyzed the conversion of cytosines to thymines (CT-conversion) at the melted DNA of a genome. Using next-generation sequencing, we generated an average of 11,000 reads covering each cytosine on the rDNA segment. In principle, the CT-conversion rate is an accurate guide to detect ssDNA segment. We detected multiple ssDNA segments throughout the rDNA. The deletion mutations of the rDNA hindered the ssDNA formation at the 100-500 bp segment downstream of the promoter. These data support the idea that the ssDNA segment plays a crucial role as the condensin-loading site and suggest the mechanism of condensin loading onto rDNA.

Horizontal transfer of a plasmid possessing mcr-1 marked with a single nucleotide mutation between Escherichia coli isolates from community residents.

OBJECTIVES: The widespread dissemination of phenotypic colistin-resistant (COR) bacteria in the community threatens public health. The horizontal gene transfer of the mobile colistin resistance gene via plasmids is thought to be one of the main mechanisms for dissemination. However, genotypic evidence to prove this in community settings is limited. This study used genome analysis to demonstrate the direct horizontal colistin resistance gene transfer via plasmids in isolates from the community. RESULTS: A total of 19 isolates of COR Escherichia coli from stool specimens of 23 residents from seven households in the Vietnamese community were assessed in this study. The whole-genome sequence data of isolates were acquired using a combination of DNBSEQ short-reads and Nanopore long-read sequencing. Analysis of genomic data was performed using online tools such as Geneious. Analysis of the genomic information of COR E. coli isolates revealed that the isolates from two residents of different households had a similar IncP1 plasmid possessing mcr-1.1, marked with a single nucleotide mutation at the same position. The study provided direct evidence to prove that mcr was horizontally transmitted among bacteria in community residents.

Nintedanib induces gene expression changes in the lung of induced-rheumatoid arthritis-associated interstitial lung disease mice.

Nintedanib is a multi-tyrosine kinase inhibitor widely used to treat progressive fibrosing interstitial lung diseases because it slows the reduction in forced vital capacity. However, the prognosis for patients treated with nintedanib remains poor. To improve nintedanib treatment, we examined the effects of nintedanib on gene expression in the lungs of induced-rheumatoid arthritis-associated interstitial lung disease model mice, which develop rheumatoid arthritis and subsequent pulmonary fibrosis. Using next-generation sequencing, we identified 27 upregulated and 130 downregulated genes in the lungs of these mice after treatment with nintedanib. The differentially expressed genes included mucin 5B and heat shock protein 70 family genes, which are related to interstitial lung diseases, as well as genes associated with extracellular components, particularly the myocardial architecture, suggesting unanticipated effects of nintedanib. Of the genes upregulated in the nintedanib-treated lung, expression of regulatory factor X2, which is suspected to be involved in cilia movement, and bone morphogenetic protein receptor type 2, which is involved in the pathology of pulmonary hypertension, was detected by immunohistochemistry and RNA in situ hybridization in peripheral airway epithelium and alveolar cells. Thus, the present findings indicate a set of genes whose expression alteration potentially underlies the effects of nintedanib on pulmonary fibrosis. It is expected that these findings will contribute to the development of improved nintedanib strategies for the treatment of progressive fibrosing interstitial lung diseases.

Relationship Between the Structure of Alkylpsoralens and Their Antiproliferative Activity in HL60 Cells.

BACKGROUND/AIM: As part of our continuing investigation in coumarin derivatives as potential anticancer substances, a series of alkylpsoralens were synthesized, and their antiproliferative activity was evaluated in leukemic HL60 cells. MATERIALS AND METHODS: Alkylpsoralens were systematically synthesized from the combination of several chloroketones and 7-hydroxycoumarin derivatives. RESULTS: Among the compounds synthesized, 4,4',8-trimethylpsoralen demonstrated the most potent activity (IC50=6.6 µM). CONCLUSION: The correlation between the alkylation pattern and antiproliferative activity showed the importance of the C4-methyl and C8-methyl moieties in the psoralen nucleus as well as the importance of lipophilicity for their antiproliferative activity.

Complete Genome Sequences of Three Phocaeicola vulgatus Strains Isolated from a Healthy Japanese Individual.

Phocaeicola vulgatus (formerly Bacteroides vulgatus) is a pathogenic anaerobic bacterium frequently involved in human infections. We present the complete genome sequences of three Phocaeicola vulgatus strains isolated from the same healthy person, determined by hybrid assembly using Nanopore long-read sequencing and DNBseq short-read sequencing.

MoG+: a database of genomic variations across three mouse subspecies for biomedical research.

Laboratory mouse strains have mosaic genomes derived from at least three major subspecies that are distributed in Eurasia. Here, we describe genomic variations in ten inbred strains: Mus musculus musculus-derived BLG2/Ms, NJL/Ms, CHD/Ms, SWN/Ms, and KJR/Ms; M. m. domesticus-derived PGN2/Ms and BFM/Ms; M. m. castaneus-derived HMI/Ms; and JF1/Ms and MSM/Ms, which were derived from a hybrid between M. m. musculus and M. m. castaneus. These strains were established by Prof. Moriwaki in the 1980s and are collectively named the "Mishima Battery". These strains show large phenotypic variations in body size and in many physiological traits. We resequenced the genomes of the Mishima Battery strains and performed a comparative genomic analysis with dbSNP data. More than 81 million nucleotide coordinates were identified as variant sites due to the large genetic distances among the mouse subspecies; 8,062,070 new SNP sites were detected in this study, and these may underlie the large phenotypic diversity observed in the Mishima Battery. The new information was collected in a reconstructed genome database, termed MoG+ that includes new application software and viewers. MoG+ intuitively visualizes nucleotide variants in genes and intergenic regions, and amino acid substitutions across the three mouse subspecies. We report statistical data from the resequencing and comparative genomic analyses and newly collected phenotype data of the Mishima Battery, and provide a brief description of the functions of MoG+, which provides a searchable and unique data resource of the numerous genomic variations across the three mouse subspecies. The data in MoG+ will be invaluable for research into phenotype-genotype links in diverse mouse strains.

MetaPlatanus: a metagenome assembler that combines long-range sequence links and species-specific features.

De novo metagenome assembly is effective in assembling multiple draft genomes, including those of uncultured organisms. However, heterogeneity in the metagenome hinders assembly and introduces interspecies misassembly deleterious for downstream analysis. For this purpose, we developed a hybrid metagenome assembler, MetaPlatanus. First, as a characteristic function, it assembles the basic contigs from accurate short reads and then iteratively utilizes long-range sequence links, species-specific sequence compositions, and coverage depth. The binning information was also used to improve contiguity. Benchmarking using mock datasets consisting of known bacteria with long reads or mate pairs revealed the high contiguity MetaPlatanus with a few interspecies misassemblies. For published human gut data with nanopore reads from potable sequencers, MetaPlatanus assembled many biologically important elements, such as coding genes, gene clusters, viral sequences, and over-half bacterial genomes. In the benchmark with published human saliva data with high-throughput nanopore reads, the superiority of MetaPlatanus was considerably more evident. We found that some high-abundance bacterial genomes were assembled only by MetaPlatanus as near-complete. Furthermore, MetaPlatanus can circumvent the limitations of highly fragmented assemblies and frequent interspecies misassembles obtained by the other tools. Overall, the study demonstrates that MetaPlatanus could be an effective approach for exploring large-scale structures in metagenomes.

Three genomes in the algal genus Volvox reveal the fate of a haploid sex-determining region after a transition to homothallism.

Transitions between separate sexes (dioecy) and other mating systems are common across eukaryotes. Here, we study a change in a haploid dioecious green algal species with male- and female-determining chromosomes (U and V). The genus Volvox is an oogamous (with large, immotile female gametes and small, motile male gametes) and includes both heterothallic species (with distinct male and female genotypes, associated with a mating-type system that prevents fusion of gametes of the same sex) and homothallic species (bisexual, with the ability to self-fertilize). We date the origin of an expanded sex-determining region (SDR) in Volvox to at least 75 Mya, suggesting that homothallism represents a breakdown of dioecy (heterothallism). We investigated the involvement of the SDR of the U and V chromosomes in this transition. Using de novo whole-genome sequences, we identified a heteromorphic SDR of ca 1 Mbp in male and female genotypes of the heterothallic species Volvox reticuliferus and a homologous region (SDLR) in the closely related homothallic species Volvox africanus, which retained several different hallmark features of an SDR. The V. africanus SDLR includes a large region resembling the female SDR of the presumptive heterothallic ancestor, whereas most genes from the male SDR are absent. However, we found a multicopy array of the male-determining gene, MID, in a different genomic location from the SDLR. Thus, in V. africanus, an ancestrally female genotype may have acquired MID and thereby gained male traits.

Comparative genomic analyses illuminate the distinct evolution of megabats within Chiroptera.

The revision of the sub-order Microchiroptera is one of the most intriguing outcomes in recent mammalian molecular phylogeny. The unexpected sister-taxon relationship between rhinolophoid microbats and megabats, with the exclusion of other microbats, suggests that megabats arose in a relatively short period of time from a microbat-like ancestor. In order to understand the genetic mechanism underlying adaptive evolution in megabats, we determined the whole-genome sequences of two rousette megabats, Leschenault's rousette (Rousettus leschenaultia) and the Egyptian fruit bat (R. aegyptiacus). The sequences were compared with those of 22 other mammals, including nine bats, available in the database. We identified that megabat genomes are distinct in that they have extremely low activity of SINE retrotranspositions, expansion of two chemosensory gene families, including the trace amine receptor (TAAR) and olfactory receptor (OR), and elevation of the dN/dS ratio in genes for immunity and protein catabolism. The adaptive signatures discovered in the genomes of megabats may provide crucial insight into their distinct evolution, including key processes such as virus resistance, loss of echolocation, and frugivorous feeding.

The Genetic Basis of Morphological Diversity in Domesticated Goldfish.

Although domesticated goldfish strains exhibit highly diversified phenotypes in morphology, the genetic basis underlying these phenotypes is poorly understood. Here, based on analysis of transposable elements in the allotetraploid goldfish genome, we found that its two subgenomes have evolved asymmetrically since a whole-genome duplication event in the ancestor of goldfish and common carp. We conducted whole-genome sequencing of 27 domesticated goldfish strains and wild goldfish. We identified more than 60 million genetic variations and established a population genetic structure of major goldfish strains. Genome-wide association studies and analysis of strain-specific variants revealed genetic loci associated with several goldfish phenotypes, including dorsal fin loss, long-tail, telescope-eye, albinism, and heart-shaped tail. Our results suggest that accumulated mutations in the asymmetrically evolved subgenomes led to generation of diverse phenotypes in the goldfish domestication history. This study is a key resource for understanding the genetic basis of phenotypic diversity among goldfish strains.

Whole-genome sequencing and comparative analysis of the genomes of Bacteroides thetaiotaomicron and Escherichia coli isolated from a healthy resident in Vietnam.

OBJECTIVES: The aim of this study was to report the draft genome sequences of two multidrug-resistant bacteria (Bacteroides thetaiotaomicron F9-2 and Escherichia coli 09-02E) isolated from stool samples of a healthy resident in Vietnam. METHODS: Genome sequences were determined using MiSeq and MinION platforms. Genome assembly was performed using Platanus Assembler v.1.2.4 and Canu v.1.7. The DDBJ Fast Annotation and Submission Tool were used for genome annotation. RESULTS: The genome of B. thetaiotaomicron F9-2 comprised 6 283 774 bp with a GC content of 42.7% and 4802 protein coding sequences (CDS), whereas the genome of E. coli 09-02E comprised 5 246 320 bp with a GC content of 50.6% and 4991 protein CDS. Both strains harboured common antimicrobial resistance genes, such as those for sulfonamides (sul2) and aminoglycosides (strA, strB). However, the sul2-strA-strB cassette was located on the chromosome of B. thetaiotaomicron F9-2, whereas it was located on a plasmid in E. coli 09-02E. These genes were flanked by different insertion sequences. CONCLUSION: Considering their diversities in the human gut resistome, these strains would be of considerable interest for detailed comparative genomic analysis. Notably, the same sul2 cassette was found in facultative and obligate anaerobic bacterial isolates (resident in humans). However, the different location of the cassette indicates a possible mechanism of gene transfer among gut microbes.

Anisogamy evolved with a reduced sex-determining region in volvocine green algae.

Male and female gametes differing in size-anisogamy-emerged independently from isogamous ancestors in various eukaryotic lineages, although genetic bases of this emergence are still unknown. Volvocine green algae are a model lineage for investigating the transition from isogamy to anisogamy. Here we focus on two closely related volvocine genera that bracket this transition-isogamous Yamagishiella and anisogamous Eudorina. We generated de novo nuclear genome assemblies of both sexes of Yamagishiella and Eudorina to identify the dimorphic sex-determining chromosomal region or mating-type locus (MT) from each. In contrast to the large (>1 Mb) and complex MT of oogamous Volvox, Yamagishiella and Eudorina MT are smaller (7-268 kb) and simpler with only two sex-limited genes-the minus/male-limited MID and the plus/female-limited FUS1. No prominently dimorphic gametologs were identified in either species. Thus, the first step to anisogamy in volvocine algae presumably occurred without an increase in MT size and complexity.

Exploring the Limits and Causes of Plastid Genome Expansion in Volvocine Green Algae.

Plastid genomes are not normally celebrated for being large. But researchers are steadily uncovering algal lineages with big and, in rare cases, enormous plastid DNAs (ptDNAs), such as volvocine green algae. Plastome sequencing of five different volvocine species has revealed some of the largest, most repeat-dense plastomes on record, including that of Volvox carteri (∼525 kb). Volvocine algae have also been used as models for testing leading hypotheses on organelle genome evolution (e.g., the mutational hazard hypothesis), and it has been suggested that ptDNA inflation within this group might be a consequence of low mutation rates and/or the transition from a unicellular to multicellular existence. Here, we further our understanding of plastome size variation in the volvocine line by examining the ptDNA sequences of the colonial species Yamagishiella unicocca and Eudorina sp. NIES-3984 and the multicellular Volvox africanus, which are phylogenetically situated between species with known ptDNA sizes. Although V. africanus is closely related and similar in multicellular organization to V. carteri, its ptDNA was much less inflated than that of V. carteri. Synonymous- and noncoding-site nucleotide substitution rate analyses of these two Volvox ptDNAs suggest that there are drastically different plastid mutation rates operating in the coding versus intergenic regions, supporting the idea that error-prone DNA repair in repeat-rich intergenic spacers is contributing to genome expansion. Our results reinforce the idea that the volvocine line harbors extremes in plastome size but ultimately shed doubt on some of the previously proposed hypotheses for ptDNA inflation within the lineage.

Direct estimation of de novo mutation rates in a chimpanzee parent-offspring trio by ultra-deep whole genome sequencing.

Mutations generate genetic variation and are a major driving force of evolution. Therefore, examining mutation rates and modes are essential for understanding the genetic basis of the physiology and evolution of organisms. Here, we aim to identify germline de novo mutations through the whole-genome surveyance of Mendelian inheritance error sites (MIEs), those not inherited through the Mendelian inheritance manner from either of the parents, using ultra-deep whole genome sequences (>150-fold) from a chimpanzee parent-offspring trio. We identified such 889 MIEs and classified them into four categories based on the pattern of inheritance and the sequence read depth: [i] de novo single nucleotide variants (SNVs), [ii] copy number neutral inherited variants, [iii] hemizygous deletion inherited variants, and [iv] de novo copy number variants (CNVs). From de novo SNV candidates, we estimated a germline de novo SNV mutation rate as 1.48 × 10-8 per site per generation or 0.62 × 10-9 per site per year. In summary, this study demonstrates the significance of ultra-deep whole genome sequencing not only for the direct estimation of mutation rates but also for discerning various mutation modes including de novo allelic conversion and de novo CNVs by identifying MIEs through the transmission of genomes from parents to offspring.

Chromosomal Aneuploidy Improves the Brewing Characteristics of Sake Yeast.

The effect of chromosomal aneuploidy on the brewing characteristics of brewery yeasts has not been studied. Here we report that chromosomal aneuploidy in sake brewery yeast (Saccharomyces cerevisiae) leads to the development of favorable brewing characteristics. We found that pyruvate-underproducing sake yeast, which produces less off-flavor diacetyl, is aneuploid and trisomic for chromosomes XI and XIV. To confirm that this phenotype is due to aneuploidy, we obtained 45 haploids with various chromosomal additions and investigated their brewing profiles. A greater number of chromosomes correlated with a decrease in pyruvate production. Especially, sake yeast haploids with extra chromosomes in addition to chromosome XI produced less pyruvate than euploids. Mitochondrion-related metabolites and intracellular oxygen species in chromosome XI aneuploids were higher than those in euploids, and this effect was canceled in their "petite" strains, suggesting that an increase in chromosomes upregulated mitochondrial activity and decreased pyruvate levels. These findings suggested that an increase in chromosome number, including chromosome XI, in sake yeast haploids leads to pyruvate underproduction through the augmentation of mitochondrial activity. This is the first report proposing that aneuploidy in brewery yeasts improves their brewing profile.IMPORTANCE Chromosomal aneuploidy has not been evaluated in development of sake brewing yeast strains. This study shows the relationship between chromosomal aneuploidy and brewing characteristics of brewery yeast strains. High concentrations of pyruvate during sake storage give rise to α-acetolactate and, in turn, to high concentrations of diacetyl, which is considered an off-flavor. It was demonstrated that pyruvate-underproducing sake yeast is trisomic for chromosome XI and XIV. Furthermore, sake yeast haploids with extra chromosomes produced reduced levels of pyruvate and showed metabolic processes characteristic of increased mitochondrial activity. This novel discovery will enable the selection of favorable brewery yeasts by monitoring the copy numbers of specific chromosomes through a process that does not involve generation/use of genetically modified organisms.