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This study aimed to identify the source of infection and medical costs for a respiratory infection outbreak in a facility for patients with severe motor and intellectual disabilities (SMID). Presenteeism refers to a situation wherein a person continues going to work despite being ill.The cohort included 1 healthcare worker and 17 patients who developed a fever of ≥37.5°C with respiratory symptoms for nearly a month. An outbreak investigation was conducted, which determined the initial case of the outbreak to be a single healthcare worker. We performed a univariate analysis to determine the association of the healthcare worker. From the medical records, we evaluated the costs of additional treatment and laboratory tests for the respiratory infection.The source of infection was a healthcare worker at the facility (Odds ratio, 17.5; 95% confidential interval, 3.0-101.8). The total medical cost for hospitalized patients due to this outbreak was $12,324.The source of a respiratory infection outbreak in a facility for SMID was suggested to be a healthcare worker's presenteeism.The cause of this outbreak was healthcare workers' presenteeism. To prevent outbreaks, such facilities should address the causative factors.

How mitochondria regulate the expression of their genes is poorly understood, partly because methods have not been developed for stably transforming mitochondrial genomes. In recent years, the disruption of mitochondrial genes has been achieved in several plant species using mitochondria-localized TALEN (mitoTALEN). In this study, we attempted to disrupt the NADH dehydrogenase subunit7 (NAD7) gene, a subunit of respiratory chain complex I, in Arabidopsis (Arabidopsis thaliana) using the mitoTALEN method. In some of the transformants, disruption of NAD7 was accompanied by severe growth inhibition and lethality, suggesting that NAD7 has an essential function in Arabidopsis. In addition, the mitochondrial genome copy number and overall expression of genes encoding mitochondrial proteins were generally increased by nad7 knockout. Similar increases were also observed in mutants with decreased NAD7 transcripts and with dysfunctions of other mitochondrial respiratory complexes. In these mutants, the expression of nuclear genes involved in mitochondrial translation or protein transport was induced in sync with mitochondrial genes. Mitochondrial genome copy number was also partly regulated by the nuclear stress-responsive factors NAC domain containing protein 17 and Radical cell death 1. These findings suggest the existence of overall gene-expression control through mitochondrial genome copy number in Arabidopsis and that disruption of single mitochondrial genes can have additional broad consequences in both the nuclear and mitochondrial genomes.

The precise control of cell growth and proliferation underpins the development of plants and animals. These factors affect the development and size of organs and the body. In plants, the growth and proliferation of cells are regulated by environmental stimuli and intrinsic signaling, allowing different cell types to have specific growth and proliferation characteristics. An increasing number of factors that control cell division and growth have been identified. However, the mechanisms underlying cell type–specific cell growth and proliferation characteristics in the normal developmental context are poorly understood. Here, we analyzed the rice mutant osmo25a1, which is defective in the progression of embryogenesis. The osmo25a1 mutant embryo developed incomplete embryonic organs, such as the shoot and root apical meristems. It showed a delayed progression of embryogenesis, associated with the reduced mitotic activity. The causal gene of this mutation encodes a member of the Mouse protein-25A (MO25A) family of proteins that have pivotal functions in a signaling pathway that governs cell proliferation and polarity in animals, yeasts and filamentous fungi. To elucidate the function of plant MO25A at the cellular level, we performed a functional analysis of MO25A in the moss Physcomitrium patens. Physcomitrium patens MO25A was uniformly distributed in the cytoplasm and functioned in cell tip growth and the initiation of cell division in stem cells. Overall, we demonstrated that MO25A proteins are conserved factors that control cell proliferation and growth.

The Closterium peracerosum-strigosum-littorale complex (Closterium, Zygnematophyceae) has an isogamous mating system. Members of the Zygnematophyceae are the closest relatives to extant land plants and are distantly related to chlorophytic models, for which a genetic basis of mating type (MT) determination has been reported. We thus investigated MT determination in Closterium. We sequenced genomes representing the two MTs, mt+ and mt-, in Closterium and identified CpMinus1, a gene linked to the mt- phenotype. We analyzed its function using reverse genetics methods. CpMinus1 encodes a divergent RWP-RK domain-containing-like transcription factor and is specifically expressed during gamete differentiation. Introduction of CpMinus1 into an mt+ strain was sufficient to convert it to a phenotypically mt- strain, while CpMinus1-knockout mt- strains were phenotypically mt+. We propose that CpMinus1 is the major MT determinant that acts by evoking the mt- phenotype and suppressing the mt+ phenotype in heterothallic Closterium. CpMinus1 likely evolved independently in the Zygnematophyceae lineage, which lost an egg-sperm anisogamous mating system. mt- specific regions possibly constitute an MT locus flanked by common sequences that undergo some recombination.

Chronic sleep disorders (CSD) comprise a potential risk factor for metabolic and cardiovascular diseases, obesity and stroke. Thus, the identification of biomarkers for CSD is an important step in the early prevention of metabolic dysfunctions induced by sleep dysfunction. Diagnostic saliva samples can be easily and noninvasively collected. Thus, we aimed to identify whole microRNA (miRNA) profiles of saliva in control and psychophysiologically stressed CSD mouse models and compare them at Zeitgeber time (ZT) 0 (lights on) and ZT12 (lights off). The findings of two-way ANOVA revealed that the expression of 342 and 109 salivary miRNAs was affected by CSD and the time of day, respectively. Interactions were found in 122 miRNAs among which, we identified 197 (ZT0) and 62 (ZT12) upregulated, and 40 (ZT0) and seven (ZT12) downregulated miRNAs in CSD mice. We showed that miR-30c-5p, which is elevated in the plasma of patients with hypersomnia, was upregulated in the saliva of CSD mice collected at ZT0. The miRNAs, miR-10a-5p, miR-146b-5p, miR-150-5p, and miR-25-3p are upregulated in the serum of humans with poor sleep quality, and these were also upregulated in the saliva of CSD mice collected at ZT0. The miRNAs miR-30c, miR146b-5p, miR150, and miR-25-5p are associated with cardiovascular diseases, and we found that plasma concentrations of brain natriuretic peptides were significantly increased in CSD mice. The present findings showed that salivary miRNA profiles could serve as useful biomarkers for predicting CSD.

Serpentinization of ultramafic rocks provides molecular hydrogen (H2) that can support lithotrophic metabolism of microorganisms, but also poses extremely challenging conditions, including hyperalkalinity and limited electron acceptor availability. Investigation of two serpentinization-active systems reveals that conventional H2-/CO2-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO2 levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, “Ca. Lithacetigenota”, that exclusively inhabits serpentinite-hosted systems and harbors genes encoding alternative modes of H2-utilizing lithotrophy. Rather than CO2, these putative metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein—the glycine reductase. A survey of microbiomes shows that glycine reductases are diverse and nearly ubiquitous in serpentinite-hosted environments. “Ca. Lithacetigenota” glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation by Terrabacteria for gaining energy from geogenic H2 even under hyperalkaline, CO2-poor conditions. Unique non-CO2-reducing metabolisms presented here shed light on potential strategies that extremophiles may employ for overcoming a crucial obstacle in serpentinization-associated environments, features potentially relevant to primordial lithotrophy in early Earth.

To quantify the biases introduced during human gut microbiome studies, analyzing an artificial mock community as the reference microbiome is indispensable. However, there are still limited resources for a mock community which well represents the human gut microbiome. Here, we constructed a novel mock community comprising the type strains of 18 major bacterial species in the human gut and assessed the influence of experimental and bioinformatics procedures on the 16S rRNA gene and shotgun metagenomic sequencing. We found that DNA extraction methods greatly affected the DNA yields and taxonomic composition of sequenced reads, and that some of the commonly used primers for 16S rRNA genes were prone to underestimate the abundance of some gut commensal taxa such as Erysipelotrichia, Verrucomicrobiota and Methanobacteriota. Binning of the assembled contigs of shotgun metagenomic sequences by MetaBAT2 produced phylogenetically consistent, less-contaminated bins with varied completeness. The ensemble approach of multiple binning tools by MetaWRAP can improve completeness but sometimes increases the contamination rate. Our benchmark study provides an important foundation for the interpretation of human gut microbiome data by providing means for standardization among gut microbiome data obtained with different methodologies and will facilitate further development of analytical methods.

Background Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, partly due to the complexity of long ssDNA preparation, it remains unclear whether ssDNA is the optimal type of HDR donors for insertion of long transgenes such as fluorescent reporters in human cells. Results In this study, we established a nuclease-based simple method for the preparation of long ssDNA with high yield and purity, and comprehensively compared the performance of ssDNA and dsDNA donors with 90 bases of homology arms for endogenous gene tagging with long transgenes in human diploid RPE1 and HCT116 cells. Quantification using flow cytometry revealed lower efficiency of endogenous fluorescent tagging with ssDNA donors than with dsDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was lower with ssDNA donors than with dsDNA. Moreover, in off-target integration analyses using donors without homology arms, ssDNA and dsDNA were comparably prone to non-homologous integration. Conclusions These results indicate that ssDNA is not superior to dsDNA as long HDR donors with relatively short homology arms for gene knock-in in human RPE1 and HCT116 cells.

In the Japanese hagfish,Eptatretus burgeri, approximately 21% of the genomic DNA in germ cells (2n=52) consists of 16 chromosomes (eliminated [E]-chromosomes) that are eliminated from presumptive somatic cells (2n=36). To uncover the eliminated genome (E-genome), we have identified 16 eliminated repetitive DNA families from eight hagfish species, with 11 of these repeats being selectively amplified in the germline genome ofE. burgeri. Furthermore, we have demonstrated that six of these sequences, namely EEEb1–6, are exclusively localized on all 16 E-chromosomes. This has led to the hypothesis that the eight pairs of E-chromosomes are derived from one pair of ancestral chromosomes via multiple duplication events over a prolonged evolutionary period. NGS analysis has recently facilitated the re-assembly of two distinct draft genomes ofE. burgeri, derived from the testis and liver. This advancement allows for the prediction of not only nonrepetitive eliminated sequences but also over 100 repetitive and eliminated sequences, accomplished through K-mer-based analysis. In this study, we report four novel eliminated repetitive DNA sequences (designated as EEEb7–10) and confirm the relative chromosomal localization of all eliminated repeats (EEEb1–10) by fluorescencein situhybridization (FISH). With the exception of EEEb10, all sequences were exclusively detected on EEEb1-positive chromosomes. Surprisingly, EEEb10 was detected as an intense signal on EEEb1-positive chromosomes and as a scattered signal on other chromosomes in germ cells. The study further divided the eight pairs of E-chromosomes into six groups based on the signal distribution of each DNA family, and fiber-FISH experiments showed that the EEEb2–10 family was dispersed in the EEEb1-positive extended chromatin fiber. These findings provide new insights into the mechanisms underlying chromosome elimination and the evolution of E-chromosomes, supporting our previous hypothesis.

Genome sizes are known to vary within and among closely related species, but the knowledge about genomic factors contributing to the variation and their impacts on gene functions is limited to only a small number of species. This study identified a more than twofold heritable genome size variation among the unicellular Zygnematophycean alga,Closterium peracerosum-strigosum-littorale(C. psl.) complex, based on short-read sequencing analysis of 22 natural strains and F1segregation analysis. Sixde novoassembled genomes revealed that genome size variation is largely attributable to genome-wide copy number variation (CNV) among strains rather than mating type-linked genomic regions or specific repeat sequences such as rDNA. Notably, about 30% of genes showed CNV even between strains that can mate with each other. Transcriptome and gene ontology analysis demonstrated that CNV is distributed nonrandomly in terms of gene functions, such that CNV was more often observed in the gene set with stage-specific expression. Furthermore, in about 30% of these genes with CNV, the expression level does not increase proportionally with the gene copy number, suggesting presence of dosage compensation, which was overrepresented in genes involved in basic biological functions, such as translation. Nonrandom patterns in gene duplications and corresponding expression changes in terms of gene functions may contribute to maintaining the high level of CNV associated with extensive genome size variation in theC. psl. complex, despite its possible detrimental effects.

SummaryThe formation of RAD51/DMC1 filaments on single-stranded (ss)DNAs, which is essential for homology search and strand exchange in DNA double-strand break (DSB) repair and for the protection of stalled DNA replication forks, is tightly regulated in time and space. FIGNL1 AAA+++ ATPase plays positive and negative roles in the RAD51-mediated recombination in human cells. However, the role of FIGNL1 in gametogenesis remains unsolved. Here, we characterized a germ-line-specific conditional knockout (cKO) mouse ofFIGNL1. TheFignl1cKO male mice showed defective chromosome synapsis and impaired meiotic DSB repair with the accumulation of RAD51/DMC1 on chromosomes in mid-meiotic prophase I, supporting a role of FIGNL1 in a post-assembly stage of RAD51/DMC1 filaments.Fignl1cKO spermatocytes accumulate RAD51 and DMC1 ensembles on chromosomes not only in early meiotic prophase I but also in meiotic S-phase. These RAD51/DMC1 assemblies are independent of meiotic DSB formation. Finally, we showed that purified FIGNL1 dismantles RAD51 filament on double-stranded (ds)DNA as well as ssDNA. These results suggest a critical role of FIGNL1 to limit the uncontrolled assembly of RAD51 and DMC1 on native dsDNAs during the meiotic S-phase and meiotic prophase I.

Hepatic encephalopathy (HE) is the neuropsychiatric complication of liver cirrhosis (LC). The influence of gut microbiota on HE pathogenesis has been suggested but not precisely elucidated. Here, we investigate how the gut microbial profile changed in patients with HE to clarify the functional gut microbial species associated with HE. We focused on their responses to rifaximin (RFX), a nonabsorbable antibiotic used in HE therapy. Feces samples were collected from patients with decompensated LC (all HE), patients with compensated LC, and healthy controls, and fecal gut microbial profiles were compared using 16S ribosomal RNA gene amplicon and metagenomic sequencing. The linear discriminant analysis effect size was used to identify specific species. Urease-positive Streptococcus salivarius, which can produce ammonia, was identified as the most significantly abundant gut microbiota in the HE group, and its ability to elevate the levels of blood ammonia as well as brain glutamine was experimentally verified in mice. Urease-negative Ruminococcus gnavus was also identified as a significantly abundant species in patients with RFX-nonresponsive HE after RFX administration. Interestingly, R. gnavus enhanced urease activity of recombinant urease itself, implying that R. gnavus could amplify ammonia production of surrounding urease-positive microbiota. Furthermore, the sensitivity of S. salivarius and R. gnavus to RFX depended on conjugated secondary bile acid levels, suggesting a therapeutic potential of the combined use of secondary bile acid levels with RFX for enhancing the efficacy of RFX. This study identified specific gut bacterial species abundant in patients with HE and verified their functions linked to HE pathophysiology. Targeting these bacteria could be a potentially effective strategy to treat HE.

Basidiomycetous yeast Mrakia gelida MGH-2 has been reported from Surikogi Ike in the Skarvsnes ice-free area, East Antarctica. Here, we report on the high-quality genome sequence of the Mrakia gelida MGH-2 strain analyzed by PacBio Sequel and HiSeq 2500 instruments.

Transposable elements (TEs) are among the most dynamic parts of genomes. Since TEs are potentially deleterious, eukaryotes silence them through epigenetic mechanisms such as DNA methylation and RNAi. We have previously reported that Arabidopsis TEs, calledVANDALs, counteract epigenetic silencing through a group of sequence-specific anti-silencing proteins, VANCs. VANC proteins bind to noncoding regions of specificVANDALcopies and induce a loss of silent chromatin marks. Sequence-specific anti-silencing allows these TEs to proliferate with minimum host damage. Here, we show that RNAi efficiently targets noncoding regions ofVANDALTEs to silence themde novo. Target motifs of VANC, in turn, evolved to escape RNAi. Escaping RNAi could be the primary event leading to the differentiation of sequence-specific anti-silencing systems. We propose that this selfish behaviour of TEs paradoxically could make them less harmful to the host.

A high-quality genome assembly is imperative to explore the evolutionary basis of characteristic attributes that define chemotype and provide essential resources for a molecular breeding strategy for enhanced production of medicinal metabolites. Here, using single-molecule high-fidelity (HiFi) sequencing reads, we report chromosome-scale genome assembly for Chinese licorice (Glycyrrhiza uralensis), a widely used herbal and natural medicine. The entire genome assembly was achieved in eight chromosomes, with contig and scaffold N50 as 36.02 and 60.2 Mb, respectively. With only 17 assembly gaps and half of the chromosomes having no or one assembly gap, the presented genome assembly is among the best plant genomes to date. Our results showed an advantage of using highly accurate long-read HiFi sequencing data for assembling a highly heterozygous genome including its complexed repeat content. Additionally, our analysis revealed that G. uralensis experienced a recent whole-genome duplication at approximately 59.02 million years ago post a gamma (γ) whole-genome triplication event, which contributed to its present chemotype features. The metabolic gene cluster analysis identified 355 gene clusters, which included the entire biosynthesis pathway of glycyrrhizin. The genome assembly and its annotations provide an essential resource for licorice improvement through molecular breeding and the discovery of valuable genes for engineering bioactive components and understanding the evolution of specialized metabolites biosynthesis.

Depression is associated with taste disorders; however, the mechanisms by which mental stress affects taste perception are not well understood. This study aimed to elucidate the effects of psychosocial stress on peripheral taste-sensing systems using a mouse depression model. Male mice were subjected to subchronic and mild social defeat stress (sCSDS). Results showed that sCSDS significantly increased body weight, food and water intake, and social avoidance behavior and that sCSDS did not change reward-seeking behavior on sucrose preference but tended to decrease pheromonal preference for female urine. Furthermore, sCSDS downregulated the mRNA levels of sweet and umami taste receptor subunits, i.e., sweet taste receptor type 1 members 2 and 3 (T1R2 and T1R3), but not the umami taste receptor subunit, i.e., taste receptor type 1 member 1 (T1R1), in the circumvallate papillae of mice. It is known that sucrose preference is mediated by the gut–brain axis without taste perception; thus, it was considered that sCSDS affected the peripheral taste-sensing systems, rather than the central reward systems, which mediate sucrose preference. This is the first study to report that psychosocial stress affects peripheral sweet and umami taste-sensing systems.

Dosage compensation (DC) on the X Chromosome counteracts the deleterious effects of gene loss on the Y Chromosome. However, DC is not efficient if the X Chromosome also degenerates. This indeed occurs in Drosophila miranda, in which both the neo-Y and the neo-X are under accelerated pseudogenization. To examine the generality of this pattern, we investigated the evolution of two additional neo-sex chromosomes that emerged independently in D. albomicans and D. americana and reanalyzed neo-sex chromosome evolution in D. miranda. Comparative genomic and transcriptomic analyses revealed that the pseudogenization rate on the neo-X is also accelerated in D. albomicans and D. americana although to a lesser extent than in D. miranda. In males, neo-X-linked genes whose neo-Y-linked homologs are pseudogenized tended to be up-regulated more than those whose neo-Y-linked homologs remain functional. Moreover, genes under strong functional constraint and genes highly expressed in the testis tended to remain functional on the neo-X and neo-Y, respectively. Focusing on the D. miranda and D. albomicans neo-sex chromosomes that emerged independently from the same autosome, we further found that the same genes tend to become pseudogenized in parallel on the neo-Y. These genes include Idgf6 and JhI-26, which may be unnecessary or even harmful in males. Our results indicate that neo-sex chromosomes in Drosophila share a common evolutionary trajectory after their emergence, which may prevent sex chromosomes from being an evolutionary dead end.

Mammalian sex chromosomes are highly conserved, and sex is determined by

Two strictly anaerobic, Gram-stain-positive, non-motile bacteria (strains OPF53T and TOC12T) were isolated from mouse intestines. Strains OPF53T and TOC12T grew at pH 5.5–9.0 and 5.0–9.0, respectively, and at temperatures of 30–45 °C. The cell morphologies of these strains were short rods and rods, respectively, and the cells possessed intracellular granules. The major cellular fatty acids of OPF53T were C18 : 1 cis 9 and C18 : 1 cis 9 dimethyl acetal, whereas those of TOC12T were C18 : 0 and C18 : 1 cis 9. In OPF53T, the main end-products of modified peptone–yeast extract–glucose (PYG) fermentation were lactate, formate and butyrate, whereas, in addition to these acids, TOC12T also produced hydrogen. The genomes of OPF53T and TOC12T were respectively 2.2 and 2.0 Mbp in size with a DNA G+C contents of 69.1 and 58.7 %. The 16S rRNA gene sequences of OPF53T and TOC12T showed the highest similarity to members of the family Atopobiaceae , namely, Olsenella phocaeensis Marseille-P2936T (94.3 %) and Olsenella umbonata KCTC 15140T (93.2 %), respectively. Phylogenetic analyses revealed that both isolates formed distinct lineages from other genera of the family Atopobiaceae . In addition, the two strains were characterized by relatively low 16S rRNA gene sequence similarity (93.4 %) and can be distinguished by their distinctive traits (including cell shape, DNA G+C content, and major fatty acids profiles). On the basis of their polyphasic taxonomic properties, these isolates represent two noel species of two novel genera within the family Atopobiaceae , for which the names Granulimonas faecalis gen. nov., sp. nov. (OPF53T=JCM 35015T=KCTC 25474T) and Leptogranulimonas caecicola gen. nov., sp. nov. (TOC12T=JCM 35017T=KCTC 25472T) are proposed.

The intestinal microbiome is closely related to host health, and metatranscriptomic analysis can be used to assess the functional activity of microbiomes by quantifying microbial gene expression levels, helping elucidate the interactions between the microbiome and the environment. However, the functional changes in the microbiome along the host intestinal tract remain unknown, and previous analytical methods have limitations, such as potentially overlooking unknown genes due to dependence on existing databases. The objective of this study is to develop a computational pipeline combined with next-generation sequencing for spatial covariation analysis of the functional activity of microbiomes at multiple intestinal sites (biogeographic locations) within the same individual. This method reconstructs a reference metagenomic sequence across multiple intestinal sites and integrates the metagenome and metatranscriptome, allowing the gene expression levels of the microbiome, including unknown bacterial genes, to be compared among multiple sites. When this method was applied to metatranscriptomic analysis in the intestinal tract of common marmosets, a New World monkey, the reconstructed metagenome covered most of the expressed genes and revealed that the differences in microbial gene expression among the cecum, transverse colon, and feces were more dynamic and sensitive to environmental shifts than the abundances of the genes. In addition, metatranscriptomic profiling at three intestinal sites of the same individual enabled covariation analysis incorporating spatial relevance, accurately predicting the function of a total of 10,856 unknown genes. Our findings demonstrate that our proposed analytical method captures functional changes in microbiomes at the gene resolution level.

The paper nautilus or greater argonaut, Argonauta argo, is a species of octopods which is characterized by its pelagic lifestyle and by the presence of a protective spiral-shaped shell-like eggcase in females. To reveal the genomic background of how the species adapted to the pelagic lifestyle and acquired its shell-like eggcase, we sequenced the draft genome of the species. The genome size was 1.1 Gb, which is the smallest among the cephalopods known to date, with the top 215 scaffolds (average length 5,064,479 bp) covering 81% (1.09 Gb) of the total assembly. A total of 26,433 protein-coding genes were predicted from 16,802 assembled scaffolds. From these, we identified nearly intact HOX, Parahox, Wnt clusters, and some gene clusters that could probably be related to the pelagic lifestyle, such as reflectin, tyrosinase, and opsin. The gene models also revealed several homologous genes related to calcified shell formation in Conchiferan mollusks, such as Pif-like, SOD, and TRX. Interestingly, comparative genomics analysis revealed that the homologous genes for such genes were also found in the genome of the shell-less octopus, as well as Nautilus, which has a true outer shell. Therefore, the draft genome sequence of Arg. argo presented here has helped us to gain further insights into the genetic background of the dynamic recruitment and dismissal of genes to form an important, converging extended phenotypic structure such as the shell and the shell-like eggcase. Additionally, it allows us to explore the evolution of from benthic to pelagic lifestyles in cephalopods and octopods.

The Iberian ribbed newt (Pleurodeles waltl) constitutes a central model for probing the basis of regeneration. Here, we present the sequencing and chromosome-scale assembly of the 20.3GbP.waltlgenome, which exhibits the highest level of contiguity and completeness among giant genomes. We uncover that DNA transposable elements are the major contributors to its expansion, with hAT transposons comprising a large portion of repeats. Several hATs are actively transcribed and differentially expressed during adultP.waltllimb regeneration, along with domesticated hAT transposons of the ZBED transcription factor family. Despite its size, syntenic relationships are conserved across the genome. As an example we show the high degree of conservation of the regeneration-associated Tig1 locus with several neighbouring genes. Together, theP.waltlgenome provides a fundamental resource for the study of regenerative, developmental and evolutionary principles.

Cystobasidium tubakii has been reported as a new basidiomycetous yeast species from East Ongul Island, East Antarctica. This species does not require amino acids and vitamins for growth and can grow at subzero temperatures. Here, we report a high-quality genome sequence of Cystobasidium tubakii strain JCM 31526 T , which was isolated from East Antarctica.

The molecular subtypes of pancreatic cancer (PC), either classical/progenitor-like or basal/squamous-like, are currently a major topic of research because of their direct association with clinical outcomes. Some transcription factors (TFs) have been reported to be associated with these subtypes. However, the mechanisms by which these molecular signatures of PCs are established remain unknown. Epigenetic regulatory processes, supported by dynamic changes in the chromatin structure, are essential for transcriptional profiles. Previously, we reported the importance of open chromatin profiles in the biological features and transcriptional status of PCs. Here, we aimed to analyze the relationships between three-dimensional (3D) genome structures and the molecular subtypes of human PCs using Hi-C analysis. We observed a correlation of the specific elements of 3D genome modules, including compartments, topologically associating domains, and enhancer-promoter loops, with the expression of related genes. We focused on HNF1B, a TF that is implicated in the progenitor subtype. Forced expression of HNF1B in squamous-type PC organoids induced the upregulation and downregulation of genes associated with progenitor and squamous subtypes, respectively. Long-range genomic interactions induced by HNF1B were accompanied by compartment modulation and H3K27ac redistribution. We also found that these HNF1B-induced changes in subtype-related gene expression required an intrinsically disordered region, suggesting a possible involvement of phase separation in compartment modulation. Thus, mapping of 3D structural changes induced by TFs, such as HNF1B, may become a useful resource for further understanding the molecular features of PCs.

Cystobasidium tubakii has been reported as a new basidiomycetous yeast species from East Ongul Island, East Antarctica. This species does not require amino acids and vitamins for growth and can grow at subzero temperatures. Here, we report a high-quality genome sequence of Cystobasidium tubakii strain JCM 31526

We report the complete genome sequence of Staphylococcus aureus strain JP025, which was isolated from a furunculosis sample from a Japanese patient. The strain carried two hyaluronate lyase genes, JP025 hysA and JP025 hysB , on the chromosome and was classified as sequence type 30.

The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.

Reconstruction of metagenome-assembled genomes (MAGs) has become a fundamental approach in microbial ecology. However, a MAG is hardly complete and overlooks genomic microdiversity because metagenomic assembly fails to resolve microvariants among closely related genotypes. Aiming at understanding the universal factors that drive or constrain prokaryotic genome diversification, we performed an ecosystem-wide high-resolution metagenomic exploration of microdiversity by combining spatiotemporal (2 depths × 12 months) sampling from a pelagic freshwater system, high-quality MAG reconstruction using long- and short-read metagenomic sequences, and profiling of single nucleotide variants (SNVs) and structural variants (SVs) through mapping of short and long reads to the MAGs, respectively. We reconstructed 575 MAGs, including 29 circular assemblies, providing high-quality reference genomes of freshwater bacterioplankton. Read mapping against these MAGs identified 100 to 101, 781 SNVs/Mb and 0 to 305 insertions, 0 to 467 deletions, 0 to 41 duplications, and 0 to 6 inversions for each MAG. Nonsynonymous SNVs were accumulated in genes potentially involved in cell surface structural modification to evade phage recognition. Most (80.2%) deletions overlapped with a gene coding region, and genes of prokaryotic defense systems were most frequently (>8% of the genes) overlapped with a deletion. Some such deletions exhibited a monthly shift in their allele frequency, suggesting a rapid turnover of genotypes in response to phage predation. MAGs with extremely low microdiversity were either rare or opportunistic bloomers, suggesting that population persistency is key to their genomic diversification. The results concluded that prokaryotic genomic diversification is driven primarily by viral load and constrained by a population bottleneck., 環境微生物のゲノム多様性を高解像度に検出 --「似て非なるゲノム」から生物多様性の源泉に迫る--. 京都大学プレスリリース. 2022-08-10., Same same but different. 京都大学プレスリリース. 2022-09-22.

Onions are one of the most widely cultivated vegetables worldwide; however, the development and utilization of molecular markers have been limited because of the large genome of this plant. We present a genome-wide marker design workflow for onions and its application in a high-throughput genotyping method based on target amplicon sequencing. The efficiency of the method was evaluated by genotyping of F2 populations. In the marker design workflow, unigene and genomic sequence data sets were constructed, and polymorphisms between parental lines were detected through transcriptome sequence analysis. The positions of polymorphisms detected in the unigenes were mapped onto the genome sequence, and primer sets were designed. In total, 480 markers covering the whole genome were selected. By genotyping an F2 population, 329 polymorphic sites were obtained from the estimated positions or the flanking sequences. However, missing or sparse marker regions were observed in the resulting genetic linkage map. We modified the markers to cover these regions by genotyping the other F2 populations. The grouping and order of markers on the linkages were similar across the genetic maps. Our marker design workflow and target amplicon sequencing are useful for genome-wide genotyping of onions owing to their reliability, cost effectiveness, and flexibility.

Depressive disorders are partially attributed to chronic inflammation associated with the tryptophan (Trp)-kynurenine (Kyn) pathway. Recent evidence suggests that anti-inflammatory agents may reduce the risk of depression. The present study aimed to elucidate the potential of the citrus flavonoid hesperidin, which exhibits anti-inflammatory activity, in suppressing the Trp-Kyn pathway in the brain, using a lipopolysaccharide (LPS)-induced inflammation mouse model. Dietary hesperidin was found to suppress activation of the Trp-Kyn pathway in the prefrontal cortex. In addition, it reduced systemic LPS-induced signs of illness, such as low skin temperature and enhanced leukocyte count in the blood. However, dietary supplementation with hesperidin did not improve body weight loss, food intake, water intake, or splenic increases in leukocyte numbers in the LPS model. Collectively, the results suggest that dietary hesperidin can partially regulate central and peripheral events linked to inflammation in LPS mouse models.

Deficiency of the functional amino acid-like compound taurine induced in cats by taurine-depleted food was previously shown to significantly decrease the levels of taurine-conjugated bile acids (BAs) and significantly increase the levels of unconjugated BAs, with a significant decrease in total BA concentration in the bile. Because the ratios of primary BAs (cholic acid [CA] and chenodeoxycholic acids [CDCA]) have also been shown to be altered in the bile, taurine has been suggested to play an important role in BA synthesis in the liver. The present study showed that in the liver of taurine-deficient cats, CYP7A1 protein expression and its metabolites (7α-hydroxycholesterol and α-hydroxy-4-cholesten-3-one) were significantly increased and, therefore, the ratio of the CA product in this pathway was decreased. On the other hand, the expression of the mitochondrial CYP27A1 protein and its metabolite 27-hydroxycholesterol (27HC) were significantly decreased in the taurine-deficient liver. Thus, a significantly decreased ratio of CDCA, which is the main product of 27HC, was found. The decreased activity of the CDCA-producing pathway might be related to mitochondrial dysfunction induced by taurine deficiency. In addition, a significant decrease in cholesterol levels in the liver was induced by a decrease in intestinal cholesterol absorption because of decreased hepatic-intestinal circulation of taurine-conjugated BAs. The results of this study showed that taurine deficiency alters both the quality and quantity of BAs through inactivity of the mitochondrial CDCA production pathway caused by impaired mitochondrial function and inhibited the absorption of cholesterol in the intestine.

Although targeted genome editing technology has become a powerful reverse genetic approach for accelerating functional genomics, conventional mutant libraries induced by chemical mutagens remain valuable for plant studies. Plants containing chemically induced mutations are simple yet effective genetic tools that can be grown without regard for biosafety issues. Whole-genome sequencing of mutant individuals reduces the effort required for mutant screening, thereby increasing their utility. In this study, we sequenced members of a mutant library of Oryza sativa cv. Nipponbare derived from treating single fertilized egg cells with N-methyl-N-nitrosourea (MNU). By whole-genome sequencing 266 M1 plants in this mutant library, we identified a total of 0.66 million induced point mutations. This result represented one mutation in every 146-kb of genome sequence in the 373 Mb assembled rice genome. These point mutations were uniformly distributed throughout the rice genome, and over 70,000 point mutations were located within coding sequences. Although this mutant library was a small population, nonsynonymous mutations were found in nearly 61% of all annotated rice genes, and 8.6% (3248 genes) had point mutations with large effects on gene function, such as gaining a stop codon or losing a start codon. WGS showed MNU-mutagenesis using rice fertilized egg cells induces mutations efficiently and is suitable for constructing mutant libraries for an in silico mutant screening system. Expanding this mutant library and its database will provide a useful in silico screening tool that facilitates functional genomics studies with a special emphasis on rice.

Most chromosomal aberrations revealed by chromosomal microarray testing (CMA) are simple; however, very complex chromosomal structural rearrangements can also be found. Although the mechanism of structural rearrangements has been gradually revealed, not all mechanisms have been elucidated. We analyzed the breakpoint-junctions (BJs) of two or more clustered copy number variations (CNVs) in the same chromosome arms to understand their conformation and the mechanism of complex structural rearrangements. Combining CMA with long-read whole-genome sequencing (WGS) analysis, we successfully determined all BJs for the clustered CNVs identified in four patients. Multiple CNVs were intricately intertwined with each other, and clustered CNVs in four patients were involved in global complex chromosomal rearrangements. The BJs of two clustered deletions identified in two patients showed microhomologies, and their characteristics were explained by chromothripsis. In contrast, the BJs in the other two patients, who showed clustered deletions and duplications, consisted of blunt-end and nontemplated insertions. These findings could be explained only by alternative nonhomologous end-joining, a mechanism related to polymerase theta. All the patients had at least one inverted segment. Three patients showed cryptic aberrations involving a disruption and a deletion/duplication, which were not detected by CMA but were first identified by WGS. This result suggested that complex rearrangements should be considered if clustered CNVs are observed in the same chromosome arms. Because CMA has potential limitations in genotype-phenotype correlation analysis, a more detailed analysis by whole genome examination is recommended in cases of suspected complex structural aberrations.

Recent studies have revealed a surprising diversity of sex chromosomes in vertebrates. However, the detailed mechanism of their turnover is still elusive. To understand this process, it is necessary to compare closely related species in terms of sex-determining genes and the chromosomes harboring them. Here, we explored the genus Takifugu, in which one strong candidate sex-determining gene, Amhr2, has been identified. To trace the processes involved in transitions in the sex-determination system in this genus, we studied 12 species and found that while the Amhr2 locus likely determines sex in the majority of Takifugu species, three species have acquired sex-determining loci at different chromosomal locations. Nevertheless, the generation of genome assemblies for the three species revealed that they share a portion of the male-specific supergene that contains a candidate sex-determining gene, GsdfY, along with genes that potentially play a role in male fitness. The shared supergene spans ∼100 kb and is flanked by two duplicated regions characterized by CACTA transposable elements. These results suggest that the shared supergene has taken over the role of sex-determining locus from Amhr2 in lineages leading to the three species, and repeated translocations of the supergene underlie the turnover of sex chromosomes in these lineages. These findings highlight the underestimated role of a mobile supergene in the turnover of sex chromosomes in vertebrates.

Significance Although turnover of sex chromosomes is very common in many vertebrate lineages, the transition process is still elusive. We studied the sex-determining region (SDR) of 12 congeneric fish species of Takifugu fish that compose an important model for the study of genomics and sex determination. We found that while nine species retained their ancestral SDR, three species had acquired derived SDRs. Although the derived SDRs resided in three different chromosomes, they harbored a shared supergene flanked by two putative transposable elements. The results highlight the underestimated role of a mobile supergene in turnover of sex chromosomes in vertebrates.

Recent advances in CRISPR technology have enabled us to perform gene knock-in in various species and cell lines. CRISPR-mediated knock-in requires donor DNA which serves as a template for homology-directed repair (HDR). For knock-in of short sequences or base substitutions, ssDNA donors are frequently used among various other forms of HDR donors, such as linear dsDNA. However, for insertion of long transgenes such as fluorescent reporters in human cells, the optimal type of HDR donors remains unclear. In this study, we established a simple and efficient CRISPR-mediated knock-in method for long transgenes using linear dsDNA and ssDNA donors, and systematically compared the performance of these two donors for endogenous gene tagging in human non-transformed diploid cells. Quantification using flow cytometry revealed higher efficiency of fluorescent tagging with dsDNA donors than with ssDNA. By analyzing knock-in outcomes using long-read amplicon sequencing and a classification framework, a variety of mis-integration events were detected regardless of the donor type. Importantly, the ratio of precise insertion was higher with dsDNA donors than with ssDNA. Moreover, in off-target integration analyses, dsDNA and ssDNA were comparably prone to non-homologous integration. These results indicate that ssDNA is not superior to dsDNA as long HDR donors for gene knock-in in human cells.

Populations or species exploiting different habitats can differ in sensory perception as a result of divergent adaptation. In bony fish, the water current is perceived via neuromasts, the end organ of the lateral line system. Although fish in different habitats are known to vary in neuromasts, we know little about the genetic basis for such variation. Here, we investigate the genetic basis for variation in supraorbital neuromasts in a hybrid zone between the Japanese gobies Gymnogobius breunigii and Gymnogobius castaneus. The former has supraorbital canal neuromasts with six cephalic pores, whereas the latter has only superficial neuromasts with no canals or pores in the supraorbital region. Our genomic analysis showed that G. breunigii and G. castaneus occur mainly in the lower and mid/upper reaches, respectively. In a river in northern Japan, hybrids were found at the sites between the habitats of the two species. These hybrids exhibited anomalies of cephalic pores. Using this hybrid zone, we conducted genome-wide association studies and identified one locus significantly associated with the number of pores. Genomic cline analysis in the hybrid zone demonstrated that this locus exhibited a higher introgression rate compared with the genomic background, indicating the possibility of adaptive introgression.

Theory predicts that local adaptation should favor the evolution of a concentrated genetic architecture, where the alleles driving adaptive divergence are tightly clustered on chromosomes. Adaptation to marine versus freshwater environments in threespine stickleback has resulted in an architecture that seems consistent with this prediction: divergence among populations is mainly driven by a few genomic regions harboring multiple quantitative trait loci for environmentally adapted traits, as well as candidate genes with well-established phenotypic effects. One theory for the evolution of these “genomic islands” is that rearrangements remodel the genome to bring causal loci into tight proximity, but this has not been studied explicitly. We tested this theory using synteny analysis to identify micro- and macro-rearrangements in the stickleback genome and assess their potential involvement in the evolution of genomic islands. To identify rearrangements, we conducted a de novo assembly of the closely related tubesnout (Aulorhyncus flavidus) genome and compared this to the genomes of threespine stickleback and two other closely related species. We found that small rearrangements, within-chromosome duplications, and lineage-specific genes (LSGs) were enriched around genomic islands, and that all three chromosomes harboring large genomic islands have experienced macro-rearrangements. We also found that duplicates and micro-rearrangements are 9.9× and 2.9× more likely to involve genes differentially expressed between marine and freshwater genotypes. While not conclusive, these results are consistent with the explanation that strong divergent selection on candidate genes drove the recruitment of rearrangements to yield clusters of locally adaptive loci.

De novo mutations accumulate with zygotic cell divisions. However, the occurrence of these mutations and the way they are inherited by somatic cells and germ cells remain unclear. Here, we present a novel method to reconstruct cell lineages. We identified mosaic mutations in mice using deep whole-genome sequencing and reconstructed embryonic cell lineages based on the variant allele frequencies of the mutations. The reconstructed trees were confirmed using nuclear transfer experiments and the genotyping of approximately 50 offspring of each tree. The most detailed tree had 32 terminal nodes and showed cell divisions from the fertilized egg to germ cell– and somatic cell–specific lineages, indicating at least five independent cell lineages that would be selected as founders of the primordial germ cells. The contributions of each lineage to germ cells and offspring varied widely. At the emergence of the germ cell–specific lineages, 10–15 embryonic mutations had accumulated, suggesting that the pregastrulation mutation rate is 1.0 mutation per mitosis. Subsequent mutation rates were 0.7 for germ cells and 13.2 for tail fibroblasts. Our results show a new framework to assess embryonic lineages; further, we suggest an evolutionary strategy for preserving heterogeneity owing to postzygotic mutations in offspring.

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.

DNA methylation is important for silencing transposable elements (TEs) in diverse eukaryotes, including plants. In plant genomes, TEs are silenced by methylation of histone H3 lysine 9 (H3K9) and cytosines in both CG and non-CG contexts. The role of RNA interference (RNAi) in establishing TE-specific silent marks has been extensively studied, but the importance of RNAi-independent pathways remains largely unexplored. Here, we directly investigated transgenerational de novo DNA methylation of TEs after the loss of silent marks. Our analyses uncovered potent and precise RNAi-independent pathways for recovering non-CG methylation and H3K9 methylation in most TE genes (that is, coding regions within TEs). Characterization of a subset of TE genes without the recovery revealed the effects of H3K9 demethylation, replacement of histone H2A variants and their interaction with CG methylation, together with feedback from transcription. These chromatin components are conserved among eukaryotes and may contribute to chromatin reprogramming in a conserved manner.

Different evolutionary interests between males and females can lead to the evolution of sexual dimorphism. However, intersex genetic correlations due to the shared genome can constrain the evolution of sexual dimorphism, resulting in intra-locus sexual conflict. One of the mechanisms resolving this conflict is sex linkage, which allows males and females to carry different alleles on sex chromosomes. Another is a regulatory mutation causing sex-biased gene expression, which is often mediated by gonadal steroids in vertebrates. How do these two mechanisms differ in the contributions to the resolution of intra-locus sexual conflict? The magnitude of sexual conflict often varies between the juvenile and adult stages. Because gonadal steroids change in titre during development, we hypothesized that gonadal steroids play a role in sexual dimorphism expression only at certain developmental stages, whereas sex linkage is more important for sexual dimorphism expressed throughout life. Our brain transcriptome analysis of juvenile and adult threespine sticklebacks showed that the majority of genes that were sex-biased in both stages were sex-linked. The relative contribution of androgen-dependent regulation to the sex-biased transcriptome increased and that of sex linkage declined in adults compared to juveniles. The magnitude of the sex differences was greater in sex-linked genes than androgen-responsive genes, suggesting that sex linkage is more effective than androgen regulation in the production of large sex differences in gene expression. Overall, our data are consistent with the hypothesis that sex linkage is effective in resolving sexual conflict throughout life, whereas androgen-dependent regulation can contribute to temporary resolution of sexual conflict.

In this study, we investigated the effects of dietary supplementation of Citrus tumida hort. ex Tanaka on food intake, body and fat tissue weights, and metabolic profiles of plasma and liver in mice. Supplementation with 5% (w/w) of peels of immature C. tumida (PIC) for 4 weeks significantly suppressed body weight gain and decreased adipose tissue weight in epididymal, perirenal, and subcutaneous fats. Metabolome analyses showed that 2-hydroxyvaleric acid levels were reduced in the blood plasma of mice fed with PIC. PIC supplementation significantly elevated dipeptide (Thr-Asp, Ser-Glu, and Ala-Ala), glucuronic acid, and S-methylglutathione levels, and significantly reduced betaine aldehyde levels in the liver. In conclusion, PIC supplementation affects the metabolism of fatty acids, pectin, glutathione, and choline, showing potential beneficial effects for metabolic syndrome and obesity. PIC may be developed as a functional food and used in the treatment of these diseases.