Biology subjects

Kondo, H.

Publications and source records attributed to Kondo, H..

14 recordsLinked to original sources

bioRxiv2026

An integrated scalable process for adherent cultivated meat production: From proliferative cell selection to safety-verified product development

Cultivated meat can contribute to the global food supply; therefore, establishing efficient production processes is an urgent task to meet the growing demand for sustainable protein. Although cell culture technology has historically focused on suspension cells in the pharmaceutical and, subsequently, cultivated meat industries, the development of efficient processes for most adhesion-dependent cells has lagged, with limited examples reported to date. Therefore, this study used primary duck liver-derived adherent cells to develop and evaluate an integrated three-step meat production process involving pre-culture to select highly proliferative cells, packed-bed bioreactor expansion for mass production, and final processing, including packaging and heating. The established process allowed the efficient growth of selected cell populations while maintaining their proliferative characteristics. Moreover, the developed product met the microbiological and heavy metal safety criteria. Comprehensive compositional analysis (nutritional, amino acid, and fatty acid profiles) revealed that the product exhibited a protein profile distinct from that of conventional duck liver paste, along with a unique lower-fat signature. As productivity is dependent on cell doubling time, culture duration and monthly production were estimated for cells from various animal species. Overall, this study established a practical integrated production process for cultivated meat using adherent cells, providing a technological foundation for cellular agriculture applicable to diverse cell types and useful for future food supply diversification. GraphicalAbstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/698307v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@18b1148org.highwire.dtl.DTLVardef@11bbcecorg.highwire.dtl.DTLVardef@102d99org.highwire.dtl.DTLVardef@21af6f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Chromosome-scale genome assembly of Malcolmia littorea using long-read sequencing and single-pollen genotyping technologies

Malcolmia littorea, a member of the family Brassicaceae, is adapted to coastal and sandy environments and has become a model in studies of reproductive barriers. However, genomic resources for the species are limited. Here, with the aim of understanding the molecular mechanisms underlying key traits in M. littorea, including its survival under harsh conditions, we present a de novo genome assembly consisting of 10 chromosome-scale sequences. We employed a high-fidelity long-read sequencing technology for genome assembly. To anchor the sequences to chromosomes, we developed a single-pollen genotyping method to construct a genetic linkage map based on SNPs derived from transcriptomes of pollen grains, possessing recombinant haploid genomes. We built a genome assembly consisting of 10 chromosome-scale sequences (214 Mb in total) for M. littorea containing 30,861 predicted genes. A comparative genome analysis and gene prediction indicated that the genome of M. littorea is double the size of the Arabidopsis thaliana genome, consistent with a whole-genome duplication followed by gene subfunctionalization and/or neofunctionalization in M. littorea. This study provides a basis for research on M. littorea, an understudied species with ecological and evolutionary significance.

genomics↗

bioRxiv2024

Non-viral derivation of induced pluripotent stem cells from the canine umbilical cord

In our previous study, canine induced pluripotent stem cells (iPSCs) were successfully generated from skin-derived fibroblasts, without the use of viral vectors. However, for clinical application of canine iPSCs in veterinary regenerative medicine, iPSCs generated from less invasive cell sources would be desirable. Therefore, the purpose of this study was to generate iPSCs from canine umbilical cords discarded at fetal birth. Canine umbilical cords were cut into small pieces and cultured in Dulbeccos modified Eagles medium supplemented with 10% fetal bovine serum. Episomal vectors carrying 10 reprogramming gene sets were introduced into fibroblasts obtained from the umbilical cord using electroporation. When putative iPSCs colonies emerged, constitutive cell characterization was performed to evaluate cell morphology, proliferative potential, alkaline phosphatase staining, expression of stem cell markers, and the ability to differentiate into a trilineage following embryoid body and teratoma formation. Multiple putative iPSC colonies formed when reprogramming gene sets were introduced into fibroblasts obtained from the canine umbilical cord. The resulting colony cells stained positive for alkaline phosphatase, and showed expression of OCT4, SOX2, NANOG, SSEA1, and SSEA3 on fluorescent immunostaining for stem cell markers. Furthermore, the mRNA expression of canine endogenous OCT4, SOX2, and NANOG significantly increased, confirming the multi-differentiation potential of cells after embryoid and teratoma formation. In this study, iPSCs were successfully generated from canine umbilical cord without the use of a viral vector. Furthermore, canine umbilical cord-derived iPSCs were successfully cultured in a feeder-free manner. This study contributes to the development of veterinary regenerative medicine by using canine iPSCs.

cell biology↗

bioRxiv2023

Pulsed ultrasound promotes secretion of anti-inflammatory extracellular vesicles from skeletal myotubes via elevation of intracellular calcium level

The regulation of inflammatory responses is an important intervention in biological function and macrophages play an essential role during inflammation. Skeletal muscle is the largest organ in the human body and releases various factors which mediate anti-inflammatory/immune modulatory effects. Recently, the roles of extracellular vesicles (EVs) from a large variety of cells are reported. In particular, EVs released from skeletal muscle are attracting attention due to their therapeutic effects on dysfunctional organs and tissues. Also, ultrasound (US) promotes release of EVs from skeletal muscle. In this study, we investigated the output parameters and mechanisms of US-induced EV release enhancement and the potential of US-treated skeletal muscle-derived EVs in the regulation of inflammatory responses in macrophages. High-intensity US (3.0 W/cm2) irradiation increased EV secretion via elevating intracellular Ca2+ level without negative effects. Moreover, US-induced EVs suppressed expression levels of pro-inflammatory factors in macrophages. miRNA sequencing analysis revealed that miR-206-3p and miR-378a-3p were especially abundant in skeletal myotube-derived EVs. In this study we demonstrated that high-intensity US promotes the release of anti-inflammatory EVs from skeletal myotubes and exert anti-inflammatory effects on macrophages.

immunology↗

bioRxiv2023

Possible biological control of ash dieback using the parasitic Hymenoscyphus fraxineus mitovirus 2?

Invasive fungal diseases represent a major threat to forest ecosystems worldwide. As fungicides are often unfeasible and not a sustainable solution, only a few other control options are available, including biological control. In this context, the use of parasitic mycoviruses as biocontrol agents of fungal pathogens has recently gained particular attention. Since the 1990s, the Asian fungus Hymenoscyphus fraxineus has been causing lethal ash dieback across Europe. In the present study, we investigated the biocontrol potential of the mitovirus Hymenoscyphus fraxineus mitovirus 2 (HfMV2) previously identified in Japanese populations of the pathogen. HfMV2 could be successfully introduced via co-culturing into 16 out of 105 virus-free isolates. A virus infection had contrasting effects on fungal growth in vitro, from cryptic to detrimental or beneficial. Virus-infected H. fraxineus isolates whose growth was reduced by HfMV2 showed a lower virulence on ash (Fraxinus excelsior) saplings compared to their isogenic virus-free isolates. The results suggest that mycoviruses exist in the native populations of H. fraxineus in Asia that have the potential for biological control of ash dieback in Europe.

pathology↗

bioRxiv2023

Integrase-associated niche differentiation of endogenous large DNA viruses in crustaceans

Crustacean genomes harbor sequences originating from nimaviruses, a family of large double-stranded DNA viruses infecting crustaceans. In this study, we recovered metagenome-assembled genomes of 25 endogenous nimaviruses from crustacean genome data. Phylogenetic analysis revealed four major lineages within Nimaviridae, and for three of these lineages, we propose novel genera of endogenous nimaviruses: "Majanivirus" and "Pemonivirus" identified from penaeid shrimp genomes, and "Clopovirus" identified from terrestrial isopods. Majanivirus genomes contain multiple eukaryotic-like genes such as baculoviral inhibitor of apoptosis repeat-containing genes, innexins, and heat shock protein 70-like genes, some of which contain introns. An alignment of long reads revealed that that each endogenous nimavirus species specifically inserts into host microsatellites or within 28S rDNA. This insertion preference was associated with the type of virus-encoded DNA recombination enzymes, the integrases. Majaniviruses, pemoniviruses, some whispoviruses, and possibly clopoviruses specifically insert into the arthropod telomere repeat motif (TAACC/GGTTA)n and all possessed a specific tyrosine recombinase family. Pasiphaea japonica whipovirus and Portunus trituberculatus whispovirus, the closest relatives of white spot syndrome virus, integrate into the host 28S rDNA and are equipped with members of another family of tyrosine recombinases that are distantly related to telomere-specific tyrosine recombinases. Endogenous nimavirus genomes identified from sesarmid crabs, which lack tyrosine recombinases and are flanked by a 46-bp inverted terminal repeat, integrate into (AT/TA)n microsatellites through the acquisition of a Ginger2-like cut- and-paste DDE transposase. These results suggest that endogenous nimaviruses are giant transposable elements that occupy different sequence niches through the acquisition of different integrase families. ImportanceCrustacean genomes harbor sequences originating from a family of large DNA viruses called nimaviruses, but it is unclear why they are present. We show that endogenous nimaviruses selectively insert into repetitive sequences within the host genome, and this insertion specificity was correlated with different types of integrases, which are DNA recombination enzymes encoded by the nimaviruses themselves. This suggests that endogenous nimaviruses have colonized various genomic niches through the acquisition of integrases with different insertion specificities. Our results point to a novel survival strategy of endogenous large DNA viruses colonizing the host genomes. These findings may clarify the evolution and spread of nimaviruses in crustaceans and lead to measures to control and prevent the spread of pathogenic nimaviruses in aquaculture settings.

microbiology↗

bioRxiv2023

scRNA-seq analysis of hemocytes of penaeid shrimp under virus infection

The classification of cells in non-model organisms has lagged behind that of model organisms using established cluster of differentiation marker sets. To reduce fish diseases, research is needed to better understand immune-related cells, or hemocytes, in non-model organisms like shrimp and other marine invertebrates. In this study, we used Drop-seq to examine how virus infection affected the populations of hemocytes in kuruma shrimp, Penaeus japonicus, which had been artificially infected with a virus. The findings demonstrated that virus infection reduced particular cell populations in circulating hemolymph and inhibited the expression of antimicrobial peptides. We also identified the gene sets that are likely to be responsible for this reduction. Additionally, we identified functionally unknown genes as novel antimicrobial peptides, and we supported this assumption by the fact that these genes were expressed in the population of hemocytes that expressed other antimicrobial peptides. In addition, we aimed to improve the operability of the experiment by conducting Drop-seq with fixed cells as a template and discussed the impact of methanol fixation on Drop-seq data in comparison to previous results obtained without fixation. These results not only deepen our understanding of the immune system of crustaceans but also demonstrate that single-cell analysis can accelerate research on non-model organisms.

cell biology↗

bioRxiv2022

Pulmonary dust foci as rat pneumoconiosis lesion induced by titanium dioxide nanoparticles in 13-week inhalation study

BackgroundMost toxicological studies on titanium dioxide (TiO2) particles to date have concentrated on carcinogenicity and acute toxicity, with few studies focusing of pneumoconiosis, which is a variety of airspace and interstitial lung diseases caused by particle-laden macrophages. The present study examined rat pulmonary lesions associated with pneumoconiosis after inhalation exposure to TiO2 nanoparticles (NPs). MethodsMale and female F344 rats were exposed to 6.3, 12.5, 25, or 50 mg/m3 anatase type TiO2 NPs for 6 hours/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. After the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. ResultsNumerous milky white spots were present in the lungs after exposure 25 and 50 mg/m3 TiO2 NPs. Histopathological analysis revealed that the spots were alveolar lesions, characterized predominantly by the agglomeration of particle-laden macrophages and the presence of reactive alveolar epithelial type 2 cell (AEC2) hyperplasia. We defined this characteristic lesion as pulmonary dust foci (PDF). The PDF is an inflammatory niche, with decreased vascular endothelial cells in the interstitium, and proliferating AEC2 transformed into alveolar epithelial progenitor cells. The AEC2 in the PDF had acquired DNA damage. Based on PDF induction, the lowest observed adverse effect concentration for pulmonary disorders in male and female rats in this study was 12.5 mg/m3 and 6.3 mg/m3, respectively. The no observed adverse effect concentration for male rats was 6.3 mg/m3. There was a sex difference in lung lesion development, with females showing more pronounced lesion parameters than males. ConclusionsInhalation exposure to TiO2 NPs caused PDF, an air-space lesion which is an alveolar inflammatory niche containing particle-laden macrophages and proliferating AEC2. This PDF histopathologically resembles some pneumoconiosis lesions (pulmonary siderosis and hard metal pneumoconiosis) in workers and lung disease in smokers, suggesting that it is an early pneumoconiosis lesion caused by exposure to TiO2 NPs in rats and a common alveolar reaction in mammals.

pathology↗

bioRxiv2021

Cortical glutamatergic projection neuron types contribute to distinct functional subnetworks

The cellular basis of cerebral cortex functional architecture remains not well understood. A major challenge is to monitor and decipher neural network dynamics across broad cortical areas yet with projection neuron (PN)-type resolution in real time during behavior. Combining genetic targeting and wide-field imaging, we monitored activity dynamics of subcortical-projecting (PTFezf2) and intratelencephalic-projecting (ITPlxnD1) types across dorsal cortex of mice during different brain states and behaviors. ITPlxnD1 and PTFezf2 neurons showed distinct activation patterns during wakeful resting, spontaneous movements, and upon sensory stimulation. Distinct ITPlxnD1 and PTFezf2 subnetworks were dynamically tuned to different sensorimotor components of a naturalistic feeding behavior, and optogenetic inhibition of ITsPlxnD1 and PTsFezf2 in subnetwork nodes disrupted distinct components of this behavior. Lastly, ITPlxnD1 and PTFezf2 projection patterns are consistent with their subnetwork activation patterns. Our results show that, in addition to the concept of columnar organization, dynamic areal and PN type-specific subnetworks are a key feature of cortical functional architecture linking microcircuit components with global brain networks.

neuroscience↗

bioRxiv2021

Dose-response relationship of pulmonary disorders by inhalation exposure to cross-linked water-soluble acrylic acid polymers in F344 rats

BackgroundIn Japan, six workers handling cross-linked water-soluble acrylic acid polymer (CWAAP) at a chemical plant suffered from lung diseases, including fibrosis, interstitial pneumonia, emphysema, and pneumothorax. We recently demonstrated that inhalation of CWAAP-A, one type of CWAAP, causes pulmonary disorders in rats. It is important to investigate dose-response relationships and recoverability from exposure to CWAAPs for establishing occupational health guidelines, such as setting threshold limit value for CWAAPs in the workplace. MethodsMale and female F344 rats were exposed to 0.3, 1, 3, or 10 mg/m3 CWAAP-A for 6 hours/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. At 1 hour, 4 weeks, and 13 weeks after the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. In a second experiment, male rats were pre-treated with clodronate liposome or polymorphonuclear leukocyte-neutralizing antibody to deplete macrophages or neutrophils, respectively, and exposed to CWAAP-A for 6 hours/day for 2 days. ResultsCWAAP-A exposure damaged only the alveoli. The lowest observed adverse effect concentration (LOAEC) was 1 mg/m3 and the no observed adverse effect concentration (NOAEC) was 0.3 mg/m3. Rats of both sexes were able to recover from the tissue damage caused by 13 weeks exposure to 1 mg/m3 CWAAP-A. In contrast, tissue damage caused by exposure to 3 and 10 mg/m3 was irreversible due to the development of interstitial lung lesions. There was a gender difference in the recovery from CWAAP-A induced pulmonary disorders, with females recovering less than males. Finally, acute lung effects caused by CWAAP-A were significantly reduced by depletion of alveolar macrophages. ConclusionsPulmonary damage caused by inhalation exposure to CWAAP-A was dose-dependent, specific to the lung and lymph nodes, and acute lung damage was ameliorated by depleting macrophages in the lungs. CWAAP-A had both a LOAEC and a NOAEC, and tissue damage caused by exposure to 1 mg/m3 CWAAP-A was reversible: recovery in female rats was less than for males. These findings indicate that concentration limits for CWAAPs in the workplace can be determined.

pharmacology and toxicology↗

bioRxiv2021

Lack of pulmonary fibrogenicity and carcinogenicity of titanium dioxide nanoparticles in 26-week inhalation study in rasH2 mouse model

BackgroundWith the rapid development of alternative methods based on the spirit of animal welfare, the publications of animal studies evaluating endpoints such as cancer have been extremely reduced. There have been no systemic inhalation exposure studies of titanium dioxide nanoparticles (TiO2 NPs) using CByB6F1-Tg(HRAS)2Jic (rasH2) 26-week study mice model for detecting carcinogenicity. MethodsMale and female rasH2 mice were exposed to 2, 8 or 32 mg/m3 of TiO2 NPs for 6 hours/day, 5 days/week for 26 weeks using a whole-body inhalation exposure system, with reference to the Organization for Economic Co-operation and Development principles of Good Laboratory Practice. All tissues including lungs, and blood were collected and subjected to biological and histopathological analyses. Additionally, Ki67 positive index were evaluated in mice lung alveolar epithelial type 2 cell (AEC2). ResultsThis study established a stable method for generating and exposing TiO2 NPs aerosol, and clarified the dose-response relationship by TiO2 NPs inhalation to rasH2 mice. TiO2 NPs exposure induced deposition of particles in lungs and mediastinal lymph nodes in a dose-dependent manner in each exposure group. Additionally, alveolar inflammation was only observed in 32 mg/m3 exposure group in both the sexes. Exposure to TiO2 NPs, as well as other organs, did not increase the incidence of lung tumors in any group, and pulmonary fibrosis and pre-neoplastic lesions were not observed in all groups. Finally, the cell proliferative activity of AEC2 was examined, and it was not increased by exposure to TiO2 NPs. ConclusionsThis is the first report showing the lack of pulmonary fibrogenicity and carcinogenicity (no evidence of carcinogenic activity) of TiO2 NPs in 26-week inhalation study in rasH2 mice exposed up to 32 mg/m3, which is considered to be a high concentration. Macrophages undergoing phagocytosis due to TiO2 NPs exposure formed inflammatory foci in the alveolar regions of exposed mice but did not develop fibrosis or hyperplasia or tumors. Moreover, the cell proliferative ability of AEC2 in lesions was not increased. In addition, no carcinogenicity was observed for any organs other than the lungs in this study.

pharmacology and toxicology↗

bioRxiv2021

Pathological characteristics of pulmonary toxicity in F344 rats exposed by inhalation to cross-linked water-soluble acrylic acid polymers

BackgroundRecently in Japan, six workers at a chemical plant that manufactures resins developed interstitial lung diseases after being involved in loading and packing cross-linked water-soluble acrylic acid polymers (CWAAPs). Since CWAAPs are not on the list of occupational diseases, the present study examined the lung damage potential of two CWAAPs (CWAAP-A and CWAAP-B) in rats, investigated pathological mechanisms, and established a method to rapidly evaluate the harmfulness of CWAAPs. MethodsUsing a whole-body inhalation exposure system, male F344 rats were exposed once to 40 or 100 mg/m3 of CWAAP-A for 4 hours or to 15 or 40 mg/m3 of CWAAP-A for 4 hours per day once per week for 2 months (a total of 9 exposures). In a separate set of experiments, male F344 rats were administered 1 mg/kg CWAAP-A or CWAAP-B by intratracheal instillation once every two weeks for 2 months (a total of five doses). Lung tissues, mediastinal lymph nodes, and bronchoalveolar lavage fluid were collected and subjected to biological and histopathological analyses. ResultsA single 4-hour exposure to CWAAP caused alveolar injury, and repeated exposures resulted in regenerative changes in the alveolar epithelium with activation of TGF{beta} signaling. During the recovery period after the last exposure, some alveolar lesions were partially healed, but other lesions developed into alveolitis with fibrous thickening of the alveolar septum. Rats administered CWAAP-A by intratracheal instillation developed qualitatively similar pulmonary pathology as rats exposed to CWAAP-A by inhalation. At 2 weeks after intratracheal instillation, rats administered CWAAP-B appeared to have a slightly higher degree of lung lesions compared to rats administered CWAAP-A, however, there was no difference in these lesions of CWAAP-A and CWAAP-B in rats examined 18 weeks after administration of these materials. ConclusionsThe present study provides evidence of rat lung pathogenesis after inhalation exposure to CWAAP-A. This study also demonstrates that the lung pathology of rats exposed to CWAAP-A by systemic inhalation was qualitatively similar to that of rats administered CWAAP-A by intratracheal instillation. The use of intratracheal instillation as an adjunct to systemic inhalation is expected to significantly accelerate the risk assessment for a variety of CWAAPs.

pharmacology and toxicology↗

bioRxiv2020

Cellular Anatomy of the Mouse Primary Motor Cortex

An essential step toward understanding brain function is to establish a cellular-resolution structural framework upon which multi-scale and multi-modal information spanning molecules, cells, circuits and systems can be integrated and interpreted. Here, through a collaborative effort from the Brain Initiative Cell Census Network (BICCN), we derive a comprehensive cell type-based description of one brain structure - the primary motor cortex upper limb area (MOp-ul) of the mouse. Applying state-of-the-art labeling, imaging, computational, and neuroinformatics tools, we delineated the MOp-ul within the Mouse Brain 3D Common Coordinate Framework (CCF). We defined over two dozen MOp-ul projection neuron (PN) types by their anterograde targets; the spatial distribution of their somata defines 11 cortical sublayers, a significant refinement of the classic notion of cortical laminar organization. We further combine multiple complementary tracing methods (classic tract tracing, cell type-based anterograde, retrograde, and transsynaptic viral tracing, high-throughput BARseq, and complete single cell reconstruction) to systematically chart cell type-based MOp input-output streams. As PNs link distant brain regions at synapses as well as host cellular gene expression, our construction of a PN type resolution MOp-ul wiring diagram will facilitate an integrated analysis of motor control circuitry across the molecular, cellular, and systems levels. This work further provides a roadmap towards a cellular resolution description of mammalian brain architecture.

neuroscience↗

bioRxiv2018

An F-actin shell ruptures the nuclear envelope by sorting pore-dense and pore-free membranes in meiosis of starfish oocytes

The nucleus of oocytes, traditionally referred to as the germinal vesicle, is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) stockpiled for embryonic development. We have shown that breakdown of this specialized NE during meiosis of starfish oocytes is mediated by an Arp2/3-nucleated F-actin shell, in contrast to microtubule-driven tearing in somatic cells. The detailed mechanism of how the cytoskeletal forces disrupt the NE remains poorly understood in any system. Here, we address the mechanism of F-actin-driven NE rupture by using live-cell and correlated super-resolution light and electron microscopy. We show that actin is nucleated within the lamina and sprouts filopodia-like spikes towards the nuclear membranes forcing lamina and nuclear membranes apart. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining membrane stretches accumulate packed NPCs associated with the still-intact lamin network. NPC conglomerates sort into a distinct tubular-vesicular membrane network, while breaks appear in pore-free, ER-like regions. Together, our work reveals a novel function for Arp2/3-mediated membrane shaping in NE rupture that is likely to have broad relevance in regulating NE dynamics in diverse other contexts such as nuclear rupture frequently observed in cancer cells.

cell biology↗