Biology subjects

Tanaka, M.

Publications and source records attributed to Tanaka, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2023

A high-sensitivity ELISA for detection of human FGF18 in culture supernatants from tumor cell lines

Fibroblast growth factor 18 (FGF18) is elevated in several human cancers, such as gastrointestinal and ovarian cancers, and stimulates the proliferation of tumor cells. This suggests that FGF18 may be a promising candidate biomarker in cancer patients. However, the lack of a high-sensitivity enzyme-linked immunosorbent assay (ELISA) does not permit testing of this possibility. In this study, we generated monoclonal antibodies against human FGF18 and developed a high-sensitivity ELISA to measure human FGF18 at concentrations as low as 10 pg/mL. Of the eight tumor cell lines investigated, we detected human FGF18 in culture supernatants from four tumor cell lines, including HeLa, OVCAR-3, BxPC-3, and SW620 cells, albeit the production levels were relatively low in the latter two cell lines. Moreover, the in-house ELISA could detect murine FGF18 in sera from mice overexpressing murine Fgf18 in hepatocytes, although the sensitivity in detecting murine FGF18 was relatively low. This FGF18 ELISA could be a valuable tool to validate FGF18 as a potential biomarker for cancer patients and to test the contribution of FGF18 for various disease models in vivo and in vitro.

cancer biology↗

bioRxiv2023

Reconstructing visual illusory experiences from human brain activity

Visual illusions provide significant insights into the brains interpretation of the world given sensory inputs. However, the precise manner in which brain activity translates into illusory experiences remains largely unknown. Here we leverage a brain decoding technique combined with deep neural network (DNN) representations to reconstruct illusory percepts as images from brain activity. The reconstruction model was trained on natural images to establish a link between brain activity and perceptual features and then tested on two types of illusions: illusory lines and neon color spreading. Reconstructions revealed lines and colors consistent with illusory experiences, which varied across the source visual cortical areas. This framework offers a way to materialize subjective experiences, shedding new light on the brains internal representations of the world.

neuroscience↗

bioRxiv2023

Genomic surveillance of antimicrobial-resistant Escherichia coli in fecal sludge and sewage in Uganda

The global increase of antimicrobial resistance (AMR) is a major public health concern. An effective AMR surveillance tool is needed to track the emergence and spread of AMR. Wastewater surveillance has been proposed as a resource-efficient tool for monitoring AMR carriage in the community. Here, we performed genomic surveillance of antimicrobial-resistant Escherichia coli obtained from fecal sludge and sewage in Uganda to gain insights into E. coli epidemiology and AMR burden in the underlying population. Selective media containing different antibiotic combinations (cefotaxime, ciprofloxacin, cefotaxime + ciprofloxacin + gentamicin) were used to obtain antimicrobial-resistant E. coli from fecal sludge and sewage. Short-read sequencing was performed for the obtained isolates, and a subset of isolates (selected from predominant sequence types (STs)) was also subjected to long-read sequencing. Genomic analysis of the obtained E. coli isolates (n = 181) revealed the prevalence of clonal complex 10, including ST167 (n = 43), ST10 (n = 28), ST1284 (n = 17), and ST617 (n = 4), in both fecal sludge and sewage, irrespective of antibiotics used for selection. We also detected global high-risk clones ST1193 (n = 10) and ST131 (n = 2 clade A, n = 3 subclade C1-M27, and n = 1 subclade C2). Diverse AMR determinants, including extended-spectrum {beta}-lactamase genes (mostly blaCTX-M-15) and mutations in gyrA and parC, were identified. Analysis of the completed genomes revealed that diverse IncF plasmids and chromosomal integration were the major contributors to the spread of AMR genes in the predominant STs. This study showed that a combination of sewage surveillance (or fecal sludge surveillance) and whole-genome sequencing can be a powerful tool for monitoring AMR carriage in the underlying population.

microbiology↗

bioRxiv2023

Tumor cell heterogeneity drives spatial organization of the intratumoral immune response in squamous cell skin carcinoma

Intratumoral heterogeneity (ITH)--defined as genetic and cellular diversity within a tumor--is linked to failure of immunotherapy and an inferior anti-tumor immune response. The underlying mechanism of this association is unknown. To address this question, we modeled heterogeneous tumors comprised of a pro-inflammatory ("hot") and an immunosuppressive ("cold") tumor population, labeled with YFP and RFP tags respectively to enable precise spatial tracking. The resulting mixed-population tumors exhibited distinct regions comprised of YFP+ (hot) cells, RFP+ (cold) cells, or a mixture. We found that tumor regions occupied by hot tumor cells (YFP+) harbored more total T cells and a higher frequency of Th1 cells and IFN{gamma}+ CD8 T cells compared to regions occupied by cold tumor cells (RFP+), whereas immunosuppressive macrophages showed the opposite spatial pattern. We identified the chemokine CX3CL1, produced at higher levels by our cold tumors, as a mediator of intratumoral macrophage accumulation, particularly immunosuppressive CD206Hi macrophages. Furthermore, we examined the response of heterogeneous tumors to a therapeutic combination of PD-1 blockade and CD40 agonist on a region-by-region basis. While the combination successfully increases Th1 abundance in "cold" tumor regions, it fails to bring overall T cell activity to the same level as seen in "hot" regions. The presence of the "cold" cells thus ultimately leads to a failure of the therapy to induce tumor rejection. Collectively, our results demonstrate that the organization of heterogeneous tumor cells has a profound impact on directing the spatial organization and function of tumor-infiltrating immune cells as well as on responses to immunotherapy.

cancer biology↗

bioRxiv2023

Hybridization and its impact on the ontogenetic allometry of skulls in macaques

The role of hybridization in morphological diversification is a fundamental topic in evolutionary biology. However, despite the accumulated knowledge on adult hybrid variation, how hybridization affects ontogenetic allometry is less well understood. Here, we investigated the effects of hybridization on postnatal ontogenetic allometry in the skulls of a putative hybrid population of introduced Taiwanese macaques (Macaca cyclopis) and native Japanese macaques (M. fuscata). Genomic analyses indicated that the population consisted of individuals with varying degrees of admixture, formed by male migration from Japanese to Taiwanese macaques. For overall skull shape, ontogenetic trajectories were shifted by hybridization in a nearly additive manner, with moderate transgressive variation observed throughout development. In contrast, for the maxillary sinus (hollow space in the face), hybrids grew as fast as Taiwanese macaques, diverging from Japanese macaques, which showed slow growth. Consequently, adult hybrids showed a mosaic pattern, i.e., the maxillary sinus is as large as that of Taiwanese macaques, while the overall skull shape is intermediate. Our findings suggest that the transgressive variation can be caused by prenatal shape modification and non-additive inheritance on regional growth rates, highlighting the complex genetic and ontogenetic bases underlying hybridization-induced morphological diversification.

evolutionary biology↗

bioRxiv2023

Sexually dimorphic dynamics of the microtubule network in medaka (Oryzias latipes) germ cells

Oogenesis is the process through which ovarian germ cells enter meiosis and acquire essential characteristics of an egg, such as an increase in follicular size and deposition of maternal factors. In vertebrates, however, the pathways that initiate oogenic processes after germ cell sex determination remain elusive. Here we report a dome-shaped microtubule structure that is maintained during oogenesis. This structure is established in primordial germ cells and maintained in gonocytes and stem-type germ cells in a foxl3-independent manner. dmrt1 is required for disruption of the dome structure in differentiating spermatogonia. In addition, the dome structure accumulates mitochondria and Golgi apparatus, and is replaced by the Balbiani body, an oocyte-specific aggregate producing animal-vegetal polarity, suggesting a role for this structure in organelle transfer and oocyte polarization. Our findings show the presence of a pathway regulating a sexually dimorphic microtubule network independent of germ cell feminization by foxl3. Summary StatementA dome-shaped microtubule structure leading to oocyte polarity was identified in germ cells. Genetic pathways involved in the formation and destabilization of the structure during germ cell sex determination were clarified.

developmental biology↗

bioRxiv2023

SAM68-regulated ALE selection of Pcdh15 maintains proper synapse development and function

Thousands of mammalian genes encode alternatively spliced isoforms in their 3 untranslated region (3UTR). Alternative 3UTR diversity may contribute to several neurological processes in developing and adult brains. SAM68 is the key splicing regulator for the diversity of neuronal 3UTR isoforms through alternative last exon (ALE) selection. However, the mechanisms underlying the control of splicing at the 3 end and its function in the nervous system remain unclear. Here, we show that neuronal SAM68-dependent ALE splicing is regulated depending on its target transcripts. For example, the selection of the ALE of protocadherin-15 (Pcdh15), a gene implicated in Usher syndrome and several neuropsychiatric disorders, is largely dependent on the expression of SAM68, partially regulated via the CaMK pathway, but independent of the U1 small nuclear ribonucleoprotein. We found that the aberrant ALEs of Pcdh15 caused membrane-to-soluble isoform conversion of the produced protein and disrupted its localization into excitatory and inhibitory synapses. In addition, the neuronal expression of the soluble form of PCDH15 (sPCDH15) preferentially affected the number of inhibitory synapses. sPCDH15 further reduced neuroligin-2-induced inhibitory, but not excitatory, synapses in artificial synapse formation assays. Our findings provide insights into the role of alternative 3UTR isoform selections in synapse development.

neuroscience↗

bioRxiv2022

A newly-recognized population of residual neural crest cells in the adult leptomeninges is re-activated for vascular repair

The neural crest (NC) is a transient structure in vertebrate embryogenesis comprising highly migratory multipotent stem cells that give rise to a diverse array of cell types in organs throughout the body, including initiating neurovascular patterning. It is assumed that neural crest stem cells (NCSCs) disappear after development. Unexpectedly, using single-nucleus RNA-sequencing, we discovered residual quiescent NCSCs in the adult mouse meninges which are activated by injury and contribute to the brains homeostatic response. RNA velocity, pathway, and transcription factor analyses in a murine stroke model (combined with in vivo imaging) show that these adult NCSCs migrate towards the perivascular spaces of the infarct and undergo a perivascular stromal cell transition that is regulated by Ptp1b, Ghr, and Stat3. Loss- and gain-of-function experiments show that these "vestigial" NCSCs are required for restoring vascular endothelial barrier function via {beta}-catenin and Stat3 signaling. These findings suggest that, in the adult, an unexpected reservoir of cells -- once pivotal to embryogenesis and vascular morphogenesis -- are re-invoked for neurovascular repair.

neuroscience↗

bioRxiv2022

Haploid-resolved and chromosome-scale genome assembly in hexa-autoploid sweetpotato (Ipomoea batatas (L.) Lam)

Sweetpotato (Ipomoea batatas (L.) Lam) is the worlds seventh most important food crop by production quantity. Cultivated sweetpotato is a hexaploid (2n = 6x = 90), and its genome (B1B1B2B2B2B2) is quite complex due to polyploidy, self-incompatibility, and high heterozygosity. Here we established a haploid-resolved and chromosome-scale de novo assembly of autohexaploid sweetpotato genome sequences. Before constructing the genome, we created chromosome-scale genome sequences in I. trifida using a highly homozygous accession, Mx23Hm, with PacBio RSII and Hi-C reads. Haploid-resolved genome assembly was performed for a sweetpotato cultivar, Xushu18 by hybrid assembly with Illumina paired-end (PE) and mate-pair (MP) reads, 10X genomics reads, and PacBio RSII reads. Then, 90 chromosome-scale pseudomolecules were generated by aligning the scaffolds onto a sweetpotato linkage map. De novo assemblies were also performed for chloroplast and mitochondrial genomes in I. trifida and sweetpotato. In total, 34,386 and 175,633 genes were identified on the assembled nucleic genomes of I. trifida and sweetpotato, respectively. Functional gene annotation and RNA-Seq analysis revealed locations of starch, anthocyanin, and carotenoid pathway genes on the sweetpotato genome. This is the first report of chromosome-scale de novo assembly of the sweetpotato genome. The results are expected to contribute to genomic and genetic analyses of sweetpotato.

genomics↗

bioRxiv2022

2-Thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes significant morbidity and mortality worldwide, seriously impacting not only human health but also the global economy. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these and many other viruses have been approved, they continue to be responsible for large-scale epidemics and global pandemics. Thus, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral nucleoside analogue that exhibited antiviral activity against SARS-CoV-2 and its variants of concern, including the Delta and Omicron variants, as well as a number of other positive-sense single-stranded RNA (ssRNA+) viruses, including DENV. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with SARS-CoV-2 or DENV in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against SARS-CoV-2 and DENV but other ssRNA+ viruses.

microbiology↗

bioRxiv2022

Transcriptional lability of brain oxytocin receptor (Oxtr) generates diversity in brain OXTR distribution and social behaviors

Although oxytocin (OXT) exhibits a highly conserved neuroanatomical pattern among vertebrates, the distribution of OXT receptor (OXTR) in brain varies considerably across species and is associated with species-typical social behavior. To investigate the genomic basis of the phylogenetic plasticity in brain Oxtr expression and its social behavioral consequences, we generated transgenic mice carrying a bacteria artificial chromosome (BAC) harboring the entire prairie vole Oxtr locus and flanking intergenic regulatory regions. We established eight independent "volized" mouse lines expressing prairie vole Oxtr (pvOxtr). Strikingly, despite conserved Oxtr expression in mammary gland of all transgenic mouse lines, each line displayed a unique pattern of brain expression distinct from both mice and prairie voles. Together with topologically associating domain (TAD) structure analysis with mouse genome, our findings suggest that unlike Oxt, Oxtr expression patterns in brain, involve contributions of distal regulatory elements beyond the BAC insert. In contrast, Oxtr expression in peripheral tissues appears resistant to such distal influences. Moreover, the "volized" mouse lines with different brain Oxtr expression patterns showed differences in partner preference and maternal behaviors, providing direct functional evidence that variation in brain Oxtr expression can drive differences in social behaviors. We propose that brain Oxtr expression is transcriptionally sensitive to long-range interactions with distal genomic elements, rendering it more susceptible to diverse regulatory influences. This supports a model in which regulatory flexibility facilitates the evolutionary diversification of social behavior, while maintaining essential peripheral Oxtr expression.

neuroscience↗

bioRxiv2022

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in enteric nervous system

Inositol pyrophosphates (PP-IPs) regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled PP-IP levels in mammalian organs using a novel HILIC-MS/MS protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of IP7 and its precursor IP6. Although their absolute levels in the GIT is diet-dependent, elevated IP7 metabolism still exists under dietary regimes devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. Additionally, HILIC-MS/MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition induced the gene sets associated with mature neurons such as inhibitory, GABAergic and dopaminergic neurons, concomitantly with suppression of those for neural progenitor/stem cells and glial cells. In addition, IP6K2 inhibition explicitly affected transcript levels of certain genes modulating neuronal differentiation and functioning, implying critical roles of IP6K2-IP7 axis in developmental and functional regulation of enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7--a highly active IP6K2-IP7 pathway is conducive to enteric nervous system.

molecular biology↗

bioRxiv2022

Human Dectin-1 is O-glycosylated and serves as a ligand for C-type lectin receptor CLEC-2

C-type lectin receptors (CLRs) elicit immune responses upon recognition of glycoconjugates present on pathogens and self-components. While Dectin-1 is the best-characterized CLR recognizing {beta}-glucan on pathogens, the endogenous targets of Dectin-1 are not fully understood. Herein, we report that human Dectin-1 is a ligand for CLEC-2, another CLR expressed on platelets. Biochemical analyses revealed that Dectin-1 is a mucin-like protein as its stalk region is highly O-glycosylated. A sialylated core 1 glycan attached to the EDxxT motif of human Dectin-1, which is absent in mouse Dectin-1, provides a ligand moiety for CLEC-2. Strikingly, the expression of human Dectin-1 in mice rescued the lethality and lymphatic defect resulting from a deficiency of Podoplanin, a known CLEC-2 ligand. This finding is the first example of an innate immune receptor also functioning as a physiological ligand to regulate ontogeny upon glycosylation.

immunology↗

bioRxiv2022

Cell-surface protein YwfG of Lactococcus lactis binds to α-1,2-linked mannose

Lactococcus lactis strains are used as starter cultures in the production of fermented dairy and vegetable foods, but the species also occurs in other niches such as plant material. Lactococcus lactis subsp. lactis G50 (G50) is a plant-derived strain and potential candidate probiotics. Western blotting of cell-wall proteins using antibodies generated against whole G50 cells detected a 120-kDa protein. MALDI-TOF MS analysis identified it as YwfG, a Leu-Pro-any-Thr-Gly cell-wall-anchor-domain-containing protein. Based on a predicted domain structure, a recombinant YwfG variant covering the N-terminal half (aa 28-511) of YwfG (YwfG28-511) was crystallized and the crystal structure was determined. The structure consisted of an L-type lectin domain, a mucin-binding protein domain, and a mucus-binding protein repeat. Recombinant YwfG variants containing combinations of these domains (YwfG28-270, YwfG28-311, YwfG28-511, MubR4) were prepared and their interactions with monosaccharides were examined by isothermal titration calorimetry; the only interaction observed was between YwfG28-270, which contained the L-type lectin domain, and D-mannose. Among four mannobioses, -1,2-mannobiose had the highest affinity for YwfG28-270 (dissociation constant = 34 M). YwfG28-270 also interacted with yeast mannoproteins and yeast mannan. Soaking of the crystals of YwfG28-511 with mannose or -1,2-mannobiose revealed that both sugars bound to the L-type lectin domain in a similar manner, although the presence of the mucin-binding protein domain and the mucus-binding protein repeat within the recombinant protein inhibited the interaction between the L-type lectin domain and mannose residues. Three of the YwfG variants (except MubR4) induced aggregation of yeast cells. Strain G50 also induced aggregation of yeast cells, which was abolished by deletion of ywfG from G50, suggesting that surface YwfG contributes to the interaction with yeast cells. These findings provide new structural and functional insights into the interaction between L. lactis and its ecological niche via binding of the cell-surface protein YwfG with mannose.

microbiology↗

bioRxiv2022

Phenotypic screening using waveform analysis of calcium dynamics in primary cortical cultures

At present, in vitro phenotypic screening methods are widely used for drug discovery. In the field of epilepsy research, measurements of neuronal activity have been utilized for predicting efficacy of anti-epileptic drugs. Fluorescence measurements of calcium oscillations in neurons are commonly used for measurement of neuronal activities, and some anti-epileptic drugs have been evaluated using this assay technique. However, changes in waveforms were not quantified in previous reports. Here, we have developed a high-throughput screening system containing a new analysis method for quantifying waveforms, and our method has successfully enabled simultaneous measurement of calcium oscillations in a 96-well plate. Features of waveforms were extracted automatically and allowed the characterization of some anti-epileptic drugs using principal component analysis. Moreover, we have shown that trajectories in accordance with the concentrations of compounds in principal component analysis plots were unique to the mechanism of anti-epileptic drugs. We believe that an approach that focuses on the features of calcium oscillations will lead to better understanding of the characteristics of existing anti-epileptic drugs and allow prediction of the mechanism of action of novel drug candidates.

neuroscience↗

bioRxiv2022

Human blastoid from primed human embryonic stem cells

Artificial human blastoids are used to investigate early embryo development, pregnancy failures, and birth deficiencies, previously impossible without human embryos. Recent methods generating blastoids used human naive pluripotent stem cells, which are prone to genomic instability during in vitro culturing. Here, we introduce a simple, robust, and scalable method for generating human blastoids from more stable, primed human pluripotent stem cells (hPSC). Using a non-cell-adhesive hydrogel, hPSC aggregates formed an asymmetric blastoid structure with a cellular distribution similar to that of a human blastocyst, with in vitro implantation capability. Single-cell RNA-seq followed by cellular trajectory analysis revealed that hydrogel promoted differentiation to tri-lineage cells associated with a blastocyst. This model will allow studies on the underlying mechanisms of human pre- and postimplantation processes, consider elaborating on the potential implications of the model on assistive reproductive technology. One Sentence SummaryNo more than 125 characters and spaces.

bioengineering↗

bioRxiv2022

MGTdb: A web service and database for studying the global and local genomic epidemiology of bacterial pathogens

Multilevel genome typing (MGT) enables the genomic characterization of bacterial isolates and the relationships among them. The MGT system describes an isolate using multiple multilocus sequence typing (MLST) schemes, referred to as levels. Thus, for a new isolate, sequence types (STs) assigned at multiple precisely defined levels can be used to type isolates at multiple resolutions. The MGT designation for isolates is stable, and assignment is faster than existing approaches. MGTs utility has been demonstrated in multiple species. This paper presents a publicly accessible web service called MGTdb, which enables the assignment of MGT sequence types to isolates, along with their storage, retrieval and analysis. The MGTdb web service enables upload of genome data as sequence reads or alleles, which are processed and assigned MGT identifiers. Additionally, any newly sequenced isolates deposited in NCBI Sequence Read Archive are also regularly retrieved (currently daily), processed, assigned MGT and made publicly available in MGTdb. Interactive visualisation tools are presented to assist analysis, along with capabilities to download publicly available isolates and assignments for use with external software. MGTdb is currently available for Salmonella enterica serovars Typhimurium and Enteritidis, and Vibrio cholerae. We demonstrate the usability of MGTdb through three case studies to study the long-term national surveillance of S. Typhimurium, and the local epidemiology and outbreaks of S. Typhimurium, and the global epidemiology of V. cholerae. Thus, MGTdb enables epidemiological and microbiological investigations at multiple levels of resolution for all publicly available isolates of these pathogens. Database URLhttps://mgtdb.unsw.edu.au

bioinformatics↗

bioRxiv2022

Chromosomal integration of blaCTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan

Occurrence of extended-spectrum {beta}-lactamase (ESBL)-producing Escherichia coli (ESBLEC) in environmental waters is of great concern. However, unlike clinical ESBLEC, their genetic characteristics, in particular the genetic contexts of ESBL genes, are not well understood. In this study, we sequenced and analyzed the genomes of CTX-M-producing E. coli isolates recovered from river water to fully characterize the genetic contexts of blaCTX-M genes. Among the 14 isolates with completed genomes, blaCTX-M genes were detected on the chromosome in nine isolates. All but one chromosomal blaCTX-M genes were associated with ISEcp1 and were carried on different transposition units ranging in size from 2,855 bp to 11,093 bp; the exception, blaCTX-M-2, was associated with ISCR1. The remaining five isolates carried blaCTX-M genes on epidemic IncI1 plasmids of different sequence types (STs) (ST3, ST16, ST113, and ST167) (n = 4) or on an IncB/O/K/Z plasmid (n = 1). This study revealed that environmental E. coli carry blaCTX-M genes in diverse genetic contexts. Apparent high prevalence of chromosomal blaCTX-M potentially indicates that some E. coli can stably maintain blaCTX-M genes in environmental waters, though further studies are needed to confirm this.

microbiology↗