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Influenza A subtypes are categorized into group 1 and group 2 based on the hemagglutinin (HA) sequence. Owing to the phylogenetic distance of HAs in different groups, antibodies that bind multiple HA subtypes across different groups are extremely rare. In this study, we demonstrated that an immunization with acid-treated HA antigen elicits germinal center (GC) B cells that bind multiple HA subtypes in both group 1 and group 2. The cross-group GC B cells utilized mostly one VH gene (1S56) and exhibited a sign of clonal evolution within GCs. The 1S56-lineage IgGs derived from GC B cells were able to bind to HA protein on the infected cell surface but not to the native form of HA protein, suggesting the cryptic nature of the 1S56 epitope and its exposure in infected cells. Finally, the 1S56-lineage IgGs provided protection against lethal infection in an Fc-dependent manner, independent of the virus-neutralizing activity. Thus, we identified 1S56-lineage antibodies as a unique stereotype for achieving cross-group influenza specificity. The antigens exposing the 1S56 epitope may be good candidates for broadly protective immunogens.
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Linfocitos B/inmunología , Vacunas contra la Influenza/inmunología , Animales , Variación Antigénica/genética , Variación Antigénica/inmunología , Pollos , Vacunas contra la Influenza/genética , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos B/inmunologíaRESUMEN
Plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere, are notably different from non-root-associated soil environments. However, the microbial dynamics in these spatially divided compartments remain unexplored. In this study, we propose a combinational analysis of single-cell genomics with 16S rRNA gene sequencing. This method enabled us to understand the entire soil microbiome and individual root-associated microorganisms. We applied this method to soybean microbiomes and revealed that their composition was different between the rhizoplane and rhizosphere in the early growth stages, but became more similar as growth progressed. In addition, a total of 610 medium- to high-quality single-amplified genomes (SAGs) were acquired, including plant growth-promoting rhizobacteria (PGPR) candidates while genomes with high GC content tended to be missed by SAGs. The whole-genome analyses of the SAGs suggested that rhizoplane-enriched Flavobacterium solubilizes organophosphate actively and Bacillus colonizes roots more efficiently. Single-cell genomics, together with 16S rRNA gene sequencing, enabled us to connect microbial taxonomy and function, and assess microorganisms at a strain resolution even in the complex soil microbiome.
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Glycine max , Microbiota , Raíces de Plantas , ARN Ribosómico 16S , Rizosfera , Análisis de la Célula Individual , Microbiología del Suelo , Glycine max/microbiología , Raíces de Plantas/microbiología , ARN Ribosómico 16S/genética , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Bacterias/aislamiento & purificación , Flavobacterium/genética , Flavobacterium/clasificación , Flavobacterium/metabolismoRESUMEN
BACKGROUND: High lipoprotein (a) level is an established cardiovascular risk, but its association with non-cardiovascular diseases, especially cancer, is controversial. Serum lipoprotein (a) levels vary widely by genetic backgrounds and are largely determined by the genetic variations of apolipoprotein (a) gene, LPA. In this study, we investigate the association between SNPs in LPA region and cancer incidence and mortality in Japanese. METHODS: A genetic cohort study was conducted utilizing the data from 9923 participants in the Japan Public Health Center-based Prospective Study (JPHC Study). Twenty-five SNPs in the LPAL2-LPA region were selected from the genome-wide genotyped data. Cox regression analysis adjusted for the covariates and competing risks of death from other causes, were used to estimate the relative risk (hazard ratios (HR) with 95% confidence intervals (CI)) of overall and site-specific cancer incidence and mortality, for each SNP. RESULTS: No significant association was found between SNPs in the LPAL2-LPA region and cancer incidence or mortality (overall/site-specific cancer). In men, however, HRs for stomach cancer incidence of 18SNPs were estimated higher than 1.5 (e.g., 2.15 for rs13202636, model free, 95%CI: 1.28-3.62) and those for stomach cancer mortality of 2SNPs (rs9365171, rs1367211) were estimated 2.13 (recessive, 95%CI:1.04-4.37) and 1.61 (additive, 95%CI: 1.00-2.59). Additionally, the minor allele for SNP rs3798220 showed increased death risk from colorectal cancer (CRC) in men (HR: 3.29, 95% CI:1.59 - 6.81) and decreased CRC incidence risk in women (HR: 0.46, 95%CI: 0.22-0.94). Minor allele carrier of any of 4SNPs could have risk of prostate cancer incidence (e.g., rs9365171 dominant, HR: 1.71, 95%CI: 1.06-2.77). CONCLUSIONS: None of the 25 SNPs in the LPAL2-LPA region was found to be significantly associated with cancer incidence or mortality. Considering the possible association between SNPs in LPAL2-LPA region and colorectal, prostate and stomach cancer incidence or mortality, further analysis using different cohorts is warranted.
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Apolipoproteína A-II , Polimorfismo de Nucleótido Simple , Neoplasias Gástricas , Femenino , Humanos , Masculino , Estudios de Cohortes , Pueblos del Este de Asia , Incidencia , Japón/epidemiología , Lipoproteína(a)/sangre , Estudios Prospectivos , Salud Pública , Factores de Riesgo , Neoplasias Gástricas/epidemiología , Neoplasias Gástricas/genética , Apolipoproteína A-II/genéticaRESUMEN
Background: The house cricket, Acheta domesticus, is one of the most farmed insects worldwide and the foundation of an emerging industry using insects as a sustainable food source. Edible insects present a promising alternative for protein production amid a plethora of reports on climate change and biodiversity loss largely driven by agriculture. As with other crops, genetic resources are needed to improve crickets for food and other applications. Methods: We present the first high quality annotated genome assembly of A. domesticus from long read data and scaffolded to chromosome level, providing information needed for genetic manipulation. Results: Gene groups related to immunity were annotated and will be useful for improving value to insect farmers. Metagenome scaffolds in the A. domesticus assembly, including Invertebrate Iridescent Virus 6 (IIV6), were submitted as host-associated sequences. We demonstrate both CRISPR/Cas9-mediated knock-in and knock-out of A. domesticus and discuss implications for the food, pharmaceutical, and other industries. RNAi was demonstrated to disrupt the function of the vermilion eye-color gene producing a useful white-eye biomarker phenotype. Conclusions: We are utilizing these data to develop technologies for downstream commercial applications, including more nutritious and disease-resistant crickets, as well as lines producing valuable bioproducts, such as vaccines and antibiotics.
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Gryllidae , Animales , Gryllidae/genética , Gryllidae/metabolismo , Agricultura , Productos Agrícolas , Alérgenos/metabolismo , Ingeniería GenéticaRESUMEN
To understand the role of the skin commensal bacterial community in skin health and the spread of pathogens, it is crucial to identify genetic differences in the bacterial strains corresponding to human individuals. A culture-independent genomics approach is an effective tool for obtaining massive high-quality bacterial genomes. Here we present a single-cell genome sequencing to obtain comprehensive whole-genome sequences of uncultured skin bacteria from skin swabs. We recovered 281 high-quality (HQ) and 244 medium-quality single-amplified genomes (SAGs) of multiple skin bacterial species from eight individuals, including cohabiting group. Single-cell sequencing outperformed in the genome recovery from the same skin swabs, showing 10-fold non-redundant strain genomes compared to the shotgun metagenomic sequencing and binning approach. We then focused on the abundant skin bacteria and identified intra-species diversity, especially in 47 Moraxella osloensis derived HQ SAGs, characterizing the strain-level heterogeneity at mobile genetic element profiles, including plasmids and prophages. Even between the cohabiting individual hosts, they have unique skin bacterial strains in the same species, which shows microdiversity in each host. Genetic and functional differences between skin bacterial strains are predictive of in vivo competition to adapt bacterial genome to utilize the sparse nutrients available on the skin or produce molecules that inhibit the colonization of other microbes or alter their behavior. Thus, single-cell sequencing provides a large number of genomes of higher resolution and quality than conventional metagenomic analysis and helps explore the skin commensal bacteria at the strain level, linking taxonomic and functional information.
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Coral symbionts are important members of the coral holobiont, and coral bacterial flora are essential in host health maintenance and coral conservation. Coral symbionts are affected by various environmental factors, such as seawater temperature, pH, and salinity. Although physicochemical and chemical factors have been highlighted as possible causes of these effects, the effects of water flow and the co-culture of different species corals have not been elucidated. In this study, we designed an artificial rearing environment to examine the impact of environmental and biological factors on Acropora tenuis, one of the major coral species in Okinawa, and Montipora digitata, during their co-culture. We intervened with the water flow to reveal that the movement of the rearing environment alters the bacterial flora of A. tenuis. During the rearing under captive environment, the alpha diversity of the coral microbiota increased, suggesting the establishment of rare bacteria from the ocean. No differences in the bacterial composition between the control and water flow groups were observed under the rearing conditions. However, the structure of the bacterial flora was significantly different in the co-culture group. Comparison of bacterial community succession strongly suggested that the differences observed were due to the suppressed transmission of bacteria from the ocean in the co-culture group. These results enhance our understanding of interactions between corals and shed light on the importance of regional differences and bacterial composition of coral flora.
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Antozoos , Animales , Antozoos/microbiología , Bacterias/genética , Factores Biológicos , Técnicas de Cocultivo , Arrecifes de Coral , ARN Ribosómico 16S/genética , AguaRESUMEN
Single-cell genomics is applied to environmental samples as a method to solve the problems of current metagenomics. However, in the fluorescence-activated cell sorting-based cell isolation and subsequent whole genome amplification, the sorting efficiency and the sequence quality are greatly affected by the type of target environment, limiting its adaptability. Here, we developed an improved single-cell genomics platform, named SAG-gel, which utilizes gel beads for single-cell isolation, lysis, and whole genome amplification. To validate the versatility of SAG-gel, single-cell genome sequencing was performed with model bacteria and microbial samples collected from eight environmental sites, including soil and seawater. Gel beads enabled multiple lysis treatments. The genome coverage with model bacteria was improved by 9.1-25%. A total of 734 single amplified genomes were collected from the diverse environmental samples, and almost full-length 16S rRNA genes were recovered from 57.8% of them. We also revealed two marine Rhodobacter strains harboring nearly identical 16S rRNA genes but having different genome contents. In addition, searching for viral sequences elucidated the virus-host linkage over the sampling sites, revealing the geographic distribution and diverse host range of viruses.
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BACKGROUND: Endozoicomonas bacteria symbiosis with various marine organisms is hypothesized as a potential indicator of health in corals. Although many amplicon analyses using 16S rRNA gene have suggested the diversity of Endozoicomonas species, genome analysis has been limited due to contamination of host-derived sequences and difficulties in culture and metagenomic analysis. Therefore, the evolutionary and functional potential of individual Endozoicomonas species symbiotic with the same coral species remains unresolved. RESULTS: In this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genomes (SAGs) for uncultured coral-associated Endozoicomonas spp. We obtained seven novel Endozoicomonas genomes and quantitative bacterial composition from Acropora tenuis corals at four sites in Japan. Our quantitative 16S rRNA gene and comparative genomic analysis revealed that these Endozoicomonas spp. belong to different lineages (Clade A and Clade B), with widely varying abundance among individual corals. Furthermore, each Endozoicomonas species possessed various eukaryotic-like genes in clade-specific genes. It was suggested that these eukaryotic-like genes might have a potential ability of different functions in each clade, such as infection of the host coral or suppression of host immune pathways. These Endozoicomonas species may have adopted different host adaptation strategies despite living symbiotically on the same coral. CONCLUSIONS: This study suggests that coral-associated Endozoicomonas spp. on the same species of coral have different evolutional strategies and functional potentials in each species and emphasizes the need to analyze the genome of each uncultured strain in future coral-Endozoicomonas relationships studies. Video Abstract.
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Antozoos , Gammaproteobacteria , Animales , Antozoos/microbiología , ARN Ribosómico 16S/genética , Adaptación al Huésped , Gammaproteobacteria/genética , Simbiosis , Bacterias , Genómica , Arrecifes de CoralRESUMEN
The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain 'Entotheonella' symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, 'Candidatus Poriflexus aureus' from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.
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Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 µm) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at two years after fixation and embedding and detected approximately 7000 genes in micro-punched tissue-spots (thickness: 10 µm), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.
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MicroARNs , Ratones , Animales , Adhesión en Parafina/métodos , Fijación del Tejido/métodos , Formaldehído , Análisis de Secuencia de ARN/métodos , Perfilación de la Expresión Génica/métodos , Transcriptoma , ARN/genética , ARN/análisisRESUMEN
BACKGROUND: Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once. RESULTS: Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (>97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing >90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing >90% completeness), respectively. Conventional metagenomics recovered one Staphylococcus hominis genome, whereas sc-metagenomics recovered two S. hominis genomes from identical skin microbiota sample. Single-cell sequencing revealed that these S. hominis genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional S. hominis MAGs had a substantial lack or excess of genome sequences and contamination from other Staphylococcus species (S. epidermidis). CONCLUSIONS: SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at https://github.com/kojiari/smaglinker . Video abstract.
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Metagenómica , Microbiota , Genoma Microbiano , Humanos , Metagenoma , Microbiota/genética , FilogeniaRESUMEN
Humanized mouse models are attractive experimental models for analyzing the development and functions of human dendritic cells (DCs) in vivo. Although various types of DC subsets, including DC type 3 (DC3s), have been identified in humans, it remains unclear whether humanized mice can reproduce heterogeneous DC subsets. CD14, classically known as a monocyte/macrophage marker, is reported as an indicator of DC3s. We previously observed that some CD14+ myeloid cells expressed CD1c, a pan marker for bona fide conventional DC2 (cDC2s), in humanized mouse models in which human FLT3L and GM-CSF genes were transiently expressed using in vivo transfection (IVT). Here, we aimed to elucidate the identity of CD14+CD1c+ DC-like cells in humanized mouse models. We found that CD14+CD1c+ cells were phenotypically different from cDC2s; CD14+CD1c+ cells expressed CD163 but not CD5, whereas cDC2s expressed CD5 but not CD163. Furthermore, CD14+CD1c+ cells primed and polarized naïve CD4+ T cells toward IFN-γ+ Th1 cells more profoundly than cDC2s. Transcriptional analysis revealed that CD14+CD1c+ cells expressed several DC3-specific transcripts, such as CD163, S100A8, and S100A9, and were clearly segregated from cDC2s and monocytes. When lipopolysaccharide was administered to the humanized mice, the frequency of CD14+CD1c+ cells producing IL-6 and TNF-α was elevated, indicating a pro-inflammatory signature. Thus, humanized mice are able to sustain development of functional CD14+CD1c+ DCs, which are equivalent to DC3 subset observed in humans, and they could be useful for analyzing the development and function of DC3s in vivo.
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Células Dendríticas/clasificación , Células Dendríticas/inmunología , Receptores de Lipopolisacáridos/análisis , Animales , Antígenos CD1/análisis , Linfocitos T CD4-Positivos/inmunología , Perfilación de la Expresión Génica , Glicoproteínas/análisis , Humanos , Mediadores de Inflamación/análisis , RatonesRESUMEN
Species of infraorder Gryllidea, or crickets, are useful invertebrate models for studying developmental biology and neuroscience. They have also attracted attention as alternative protein sources for human food and animal feed. Mitochondrial genomic information on related invertebrates, such as katydids, and locusts, has recently become available in attempt to clarify the controversial classification schemes, although robust phylogenetic relationships with emphasis on crickets remain elusive. Here, we report newly sequenced complete mitochondrial genomes of crickets to study their phylogeny, genomic rearrangements, and adaptive evolution. First, we conducted de novo assembly of mitochondrial genomes from eight cricket species and annotated protein-coding genes and transfer and ribosomal RNAs using automatic annotations and manual curation. Next, by combining newly described protein-coding genes with public data of the complete Gryllidea genomes and gene annotations, we performed phylogenetic analysis and found gene order rearrangements in several branches. We further analyzed genetic signatures of selection in ant-loving crickets (Myrmecophilidae), which are small wingless crickets that inhabit ant nests. Three distinct approaches revealed two positively selected sites in the cox1 gene in these crickets. Protein 3D structural analyses suggested that these selected sites could influence the interaction of respiratory complex proteins, conferring benefits to ant-loving crickets with a unique ecological niche and morphology. These findings enhance our understanding of the genetic basis of cricket evolution without relying on estimates based on a limited number of molecular markers.
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Hormigas , Genoma Mitocondrial , Gryllidae , Animales , Hormigas/genética , Evolución Molecular , Gryllidae/genética , Insectos/genética , FilogeniaRESUMEN
Rickettsiales-like organisms are important for the survival and functioning of corals, prompting an investigation of their complete genomes. Earlier reports of the genomes of these organisms remain incomplete. Here, we report a novel draft genome of Rickettsiales bacterial strain SESOKO1, found in Acropora tenuis coral, using single-cell genome technology.
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BACKGROUND: The gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel single-cell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. RESULTS: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as high- and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin-responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. CONCLUSIONS: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota and could be exploited to improve human and animal health. Video abstract.
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Bacterias/clasificación , Fibras de la Dieta/metabolismo , Microbioma Gastrointestinal , Genómica/métodos , Inulina/metabolismo , Análisis de la Célula Individual , Animales , Bacterias/metabolismo , Fibras de la Dieta/administración & dosificación , Heces/microbiología , Fermentación , Genoma Bacteriano , Inulina/administración & dosificación , Masculino , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos BALB C , Prebióticos/análisis , Ácido Succínico/metabolismoRESUMEN
Bioprinting technology is expected to be applied in the fields of regenerative medicine and drug discovery. There are several types of bioprinters, especially inkjet-based bioprinter, which can be used not only as a printer for arranging cells but also as a precision cell-dispensing device with controlled cell numbers similar to a fluorescence activated cell sorter (FACS). Precise cell dispensers are expected to be useful in the fields of drug discovery and single-cell analysis. However, there are enduring concerns about the impacts of cell dispensers on cell integrity, particularly on sensitive cells, such as stem cells. In response to the concerns stated above, we developed a stress-free and media-direct-dispensing inkjet bioprinter. In the present study, in addition to conventional viability assessments, we evaluated the gene expression using RNA-seq to investigate whether the developed bioprinter influenced cell integrity in mouse embryonic stem cells. We evaluated the developed bioprinter based on three dispensing methods: manual operation using a micropipette, FACS and the developed inkjet bioprinter. According to the results, the developed inkjet bioprinter exhibited cell-friendly dispensing performance, which was similar to the manual dispensing operation, based not only on cell viability but also on gene expression levels.
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Bioimpresión/métodos , Supervivencia Celular , Análisis de Secuencia de ARN/métodos , Animales , Proliferación Celular , Separación Celular , Citometría de Flujo/métodos , Expresión Génica , RatonesRESUMEN
The genus Okeania is a globally distributed group of microorganisms that live in shallow seabed regions. These organisms play several environmentally important roles and are also known producers of several active secondary metabolites with potential human applications. Here, we present a draft genome of Okeania sp. strain KiyG1 (92.7% completeness) that was assembled from four single-amplified genomes.