Biology subjects

Matsumoto, T.

Publications and source records attributed to Matsumoto, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Continuous Large Maf Activity Maintains Fast Myofiber Identity and Multicellular Homeostasis in Adult Skeletal Muscle

Fast myofibers are preferentially affected during aging, exhibiting atrophy and shifts toward slower myofiber programs, yet the mechanisms preserving established fast-fiber identity remain unclear. Here, we show that the large Maf transcription factors Mafa, Mafb, and Maf are continuously required to maintain type IIb myofiber identity in adult skeletal muscle. Inducible deletion of large Mafs caused near-complete loss of type IIb fibers, a transition toward type IIx/IIa states, reduced strength, and increased fatigue resistance. RNA-seq and ATAC-seq revealed transcriptional and chromatin accessibility remodeling across the fast myosin locus, characterized by persistent loss of Myh4 expression and increased accessibility around Myh1. Single-nucleus RNA-seq identified transient myonuclei co-expressing multiple myosin isoforms before emergence of a Myh1-dominant state, accompanied by remodeling of non-myonuclear cell populations. These changes shared features with aging- and denervation-associated muscle remodeling. Thus, continuous large Maf activity maintains adult fast myofiber identity and coordinates skeletal muscle tissue homeostasis.

cell biology↗

bioRxiv2026

Determination of effective meropenem and gentamicin doses in a silkworm infection model using a clinical Klebsiella aerogenes isolate

Klebsiella aerogenes, a member of the Enterobacteriaceae, is a causative agent of healthcare-associated infections, and outbreaks caused by drug-resistant K. aerogenes have been reported worldwide. The range of antimicrobial agents available for treating infections caused by carbapenem-resistant K. aerogenes is limited. While in vivo animal experiments using clinical K. aerogenes isolates to evaluate antimicrobial therapy could facilitate selection of the most effective treatment, conducting infection experiments involving large numbers of mammals such as mice is challenging due to ethical concerns related to animal welfare. Silkworms are invertebrates increasingly used as experimental models for infectious disease research to evaluate antimicrobial efficacy. In this study, we aimed to establish a silkworm infection model using a clinical K. aerogenes isolate to evaluate its utility for determining effective antimicrobial doses. K. aerogenes strains were isolated from a patient at a Japanese hospital, and a silkworm infection model was established using the clinical isolate. The non-metallo-beta-lactamase-producing strain K. aerogenes TUM25562, isolated from a patient with a complicated urinary tract infection, was susceptible to meropenem (MEPM) and gentamicin (GM) in vitro. During treatment, additional isolates with increased resistance to MEPM and subsequently to both MEPM and GM emerged. K. aerogenes TUM25562 caused dose-dependent mortality in silkworms. Treatment with clinically equivalent weight-based doses of MEPM or GM did not cure the infected silkworms. The median effective (ED50) doses of MEPM and GM were therefore investigated using the silkworm infection model. Administration of higher doses corresponding to four times the ED50 significantly prolonged the survival of infected silkworms. These results suggest that a silkworm infection model using clinical K. aerogenes isolates may provide a practical approach for evaluating antimicrobial efficacy and determining effective antimicrobial doses.

microbiology↗

bioRxiv2026

Dysregulation of the p16-CDK6 axis characterizes HPV-unrelated p16-positive oropharyngeal carcinoma

Human papillomavirus (HPV)-related and HPV-unrelated oropharyngeal squamous cell carcinomas (OPCs) are distinct entities with different clinical outcomes. While p16 immunohistochemistry (IHC) is widely used as a surrogate marker for HPV-driven OPC, a subset of HPV-unrelated OPCs also overexpress p16, and the biological basis of this discordance remains unclear. Here, we performed integrated clinicopathological, transcriptomic, genomic, and functional analyses of OPCs and demonstrated that dysregulation of the p16-CDK6 axis characterizes HPV-unrelated p16-positive OPCs. Although these tumors closely resembled HPV-unrelated p16-negative OPCs in their clinicopathological and transcriptomic characteristics, they exhibited a more favorable prognosis. CDK6 was recurrently upregulated in HPV-unrelated OPC regardless of p16 status and was already detectable in high-grade dysplastic leukoplakia, suggesting that CDK6 activation is an early event in HPV-unrelated tumorigenesis. In experimental models, CDK6 overexpression induced compensatory p16 upregulation, creating selective pressure for subsequent CDKN2A inactivation. Consistent with this model, homozygous CDKN2A loss predominated in p16-negative tumors. We further identified CDKN2A frameshift mutations generating p14ARF-p16 chimeric proteins that retain p16 immunoreactivity despite functional loss of wild-type p16, revealing a previously unrecognized diagnostic pitfall of p16 IHC. These findings provide a biological framework for p16 overexpression in HPV-unrelated OPC and suggest that assessment of the p16-CDK6 axis may refine molecular classification and risk stratification beyond p16 IHC alone.

cancer biology↗

bioRxiv2026

Mapping human-macaque encounter potential by integrating visitor-use and macaque-use intensity in Kamikochi, Japan

Managing visitor-wildlife interactions in high-use protected areas requires identifying not only where visitors concentrate but also where visitor use overlaps with wildlife space use. We estimated relative human- macaque encounter potential in Kamikochi, Chubu Sangaku National Park, Japan, where Japanese macaques (Macaca fuscata) and visitors use the same frontcountry landscape. We integrated Mobile Spatial Statistics derived from mobile phone network data, trail-camera counts, and direct-follow location data from macaques along a frontcountry-backcountry gradient. Broad-scale 500-m grid estimates of daytime visitor presence for the 2023 and 2024 open seasons were allocated to 50-m trail sections and adjusted using counts from 10 trail cameras. Automated video counts based on YOLO11 and ByteTrack showed high agreement with manual counts of 100 validation videos (MAE = 0.10 persons/video, MAPE = 1.26%, exact agreement rate = 92.0%). Visitor-use and macaque-use data were converted into kernel density surfaces, and encounter potential was calculated as their normalized product. High encounter potential was concentrated in the central frontcountry: 87.1% of areas with index values [≥] 0.9 fell within the frontcountry and 12.9% within the backcountry. Areas with high visitor use or high macaque use alone did not necessarily show high encounter potential when the two distributions were spatially offset. These results show that spatial overlap provides management information not captured by visitor counts alone. Relative encounter potential can help prioritize patrols, visitor messaging, viewing management, and other interventions in protected areas where wildlife does not consistently avoid heavily used visitor spaces.

zoology↗

bioRxiv2026

Therapeutic Potential of the Novel Monoclonal Antibody 7-4G against HBsAg in a Mouse Model of Chronic Hepatitis B Virus Infection

Chronic hepatitis B virus (HBV) infection remains a major global health challenge because current antiviral therapies rarely achieve complete viral clearance. We evaluated the antiviral activity of a newly developed monoclonal antibody, 7-4G mAb, directed against the hepatitis B surface antigen (HBsAg). The antibody showed specific binding to the p27 component of HBsAg and efficiently removed HBV-related particles from patient serum in vitro. Significant HBV neutralizing activity of 7-4G mAb was observed in primary human hepatocytes and in humanized mouse models. In persistently HBV-infected PXB mice, a single administration of 7-4G mAb, either alone or in combination with another monoclonal antibody, reduced serum HBsAg levels and HBV DNA levels by two orders magnitude for two weeks after the treatment. In half of the treated mice, suppression of both viral markers was maintained through the study period, whereas rebound occurred within three to four weeks in the remaining animals. In addition, 7-4G mAb enhanced the HBV DNA-lowering effect of entecavir, a known anti-HBV drug. These findings support the further development of 7-4G mAb as part of an innovative HBV treatment strategy, addressing limitations of existing therapies and advancing toward a more effective HBV management solution.

immunology↗

bioRxiv2026

A senescent iCAF-like fibroblast state governs therapy resistance in rheumatoid arthritis

ObjectivesRheumatoid arthritis (RA) synovitis frequently persists despite cytokine-targeted therapies, suggesting the existence of pathogenic stromal programs that sustain chronic inflammation independently of canonical immune pathways. Although synovial fibroblasts (SF) are increasingly implicated in treatment resistance, the pathogenic fibroblast states driving refractory disease and their therapeutic vulnerabilities remain poorly defined. MethodsWe integrated multimodal single-cell and spatial profiling of synovial tissue from 54 patients with RA with prospective treatment-response data and functional studies in human fibroblasts and experimental arthritis models. ResultsWe identified a C-X-C motif chemokine 12 (CXCL12)hi Apolipoprotein C1 (APOC1)+ fibroblast population selectively enriched in treatment-refractory synovitis. Spatial analyses demonstrated that these fibroblasts establish CXCL12-dependent plasmablast niches within inflamed synovium, resembling inflammatory cancer-associated fibroblasts (iCAF) that orchestrate immune cell recruitment in the tumor microenvironment. CXCL12hi APOC1+ fibroblasts exhibited a senescence-associated iCAF-like transcriptional program characterized by STAT3-C/EBP activation and APOC1 expression and were associated with poor response to TNF and IL-6 pathway inhibition. Mechanistically, APOC1 knockdown in RA-SF attenuated invasive mesenchymal behavior and disrupted senescence-associated inflammatory programs, identifying APOC1 as a central regulator of pathogenic fibroblast reprogramming. Importantly, genetic or pharmacological elimination of senescent cells ameliorated experimental arthritis and enhanced the efficacy of TNF blockade. ConclusionsThese findings implicate iCAF-like fibroblasts with senescent properties as a mechanistic driver of refractory RA synovitis and highlight stromal senescence programs as preclinically actionable therapeutic targets beyond cytokine inhibition.

immunology↗

bioRxiv2025

2-Mercaptoethanol/DMSO workflow enables highly reproducible quantitative proteomics

Proteomics provides a systematic and high-throughput approach to comprehensively characterize protein networks, enabling insights into cellular functions and disease mechanisms. Carbamidomethylation using iodoacetamide (IAA), a common method for cysteine alkylation, is known to cause nonspecific modifications that increase MS spectral complexity and reduce quantitative accuracy. Here, we extended our 2-mercaptoethanol (2-ME) adduction method, enhanced by dimethyl sulfoxide (DMSO), from single-protein applications to a proteome-wide workflow. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by LC-MS/MS analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6-1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage. Quantitative reproducibility was also enhanced, with peptides quantified coefficient of variation (CV) [≤] 20% increasing from 61.5% (IAA) to 86.1% (2-ME), and proteins from 80.6% to 93.5%. Application of the workflow to an ovarian clear cell carcinoma reliably detected cisplatin-induced alterations. The 2-ME/DMSO workflow offers a simple and highly reproducible proteomics strategy for accurate quantitative proteomics.

biochemistry↗

bioRxiv2025

Efficacy of vocal cord injection of dedifferentiated fat cells in treating glottis closure insufficiency: Insights from a rat model of recurrent laryngeal nerve resection

Glottic insufficiency results from impaired vocal cord contact, leading to a gap between the cords and manifesting as hoarseness and respiratory difficulties. Vocal cord injection is a commonly utilized therapeutic approach to rectify this gap by augmenting vocal cord volume; however, the optimal injectable material remains undetermined. Dedifferentiated fat cells (DFATs), derived from mature adipocytes, exhibit robust proliferative capacity and multipotency, establishing them as potential candidates for treating glottic insufficiency. This study investigated the therapeutic efficacy of DFATs in a rat model of recurrent laryngeal nerve paralysis. In the experimental design, the recurrent laryngeal nerve of each rat was resected unilaterally, and 5 weeks later, the atrophied vocal cord muscles were injected with either 10 l of saline (control), 1.0 x 106 DFATs in 10 l (DFAT group), or 1.0 x 106 DFATs combined with 500 ng of basic fibroblast growth factor (bFGF) in 10 l (DFAT + bFGF group). At 4 weeks post-injection, laryngeal endoscopy was performed to evaluate the glottic gap, followed by histological analyses to assess vocal muscle atrophy, collagen deposition, and markers of cellular proliferation (Ki-67) and apoptosis (TUNEL). To confirm DFAT engraftment, GFP-labeled DFATs were evaluated at 2, 4, and 6 weeks post-injection. Results indicated that both the DFAT and DFAT + bFGF groups exhibited significantly reduced glottic gaps, increased collagen deposition, and decreased TUNEL-positive apoptotic cells compared to controls. Notably, the DFAT + bFGF group displayed superior outcomes, including a greater vocal muscle area and enhanced Ki-67-positive cell proliferation, indicating mitigation of muscle atrophy. GFP-positive DFATs were detectable in the tissue for up to 6 weeks, confirming engraftment. These findings underscore the potential of DFATs, particularly in combination with bFGF, as an innovative and effective therapeutic approach for glottic insufficiency secondary to recurrent laryngeal nerve paralysis.

cell biology↗

bioRxiv2025

Conserved and Unique Features of Terminal Telomeric Sequences in ALT-Positive Cancer Cells

A significant portion of human cancers utilize a recombination-based pathway, Alternative Lengthening of Telomeres (ALT), to maintain telomere length. Targeting the ALT is of growing interest as a cancer therapy, yet a substantial knowledge gap remains regarding the basic features of telomeres in ALT-positive cells. To address this, we adopted END-seq, an unbiased sequencing-based approach, to define the identity and regulation of the terminal sequences of chromosomes in ALT cells. Our data reveal that the terminal portions of chromosomes in ALT cells contain canonical telomeric sequences with the same 5 terminus bias (-ATC) observed in non-ALT cells. Furthermore, as reported for non-ALT cells, POT1 is required to preserve the precise regulation of the 5 end in cells that maintain telomere length using the ALT pathway. Thus, the regulation of the terminal 5 of chromosomes occurs independently of the mechanism of telomere elongation. Additionally, we employed an S1 endonuclease-based sequencing method to determine the presence and origin of single-stranded regions within ALT telomeres. These data shed light on conserved and unique features of ALT telomeres.

molecular biology↗

bioRxiv2024

Improvement of D-lactic acid production from methanol by metabolically engineered Komagataella phaffii via ultra-violet mutagenesis

Methanol has attracted attention as an alternative carbon source to petroleum. Komagataella phaffii, a methanol-assimilating yeast, is a useful host for the chemical production from methanol. A previous study successfully constructed a metabolically engineered K. phaffii GS115/S8/Z3 strain capable of producing D-lactic acid from methanol. In this study, we aimed to develop a strain with improved D-lactic acid production by applying ultra-violet mutagenesis to the D-lactic acid-producing strain, GS115/S8/Z3. The resulting mutant strain DLac_Mut2_221 produced 5.38 g/L of D-lactic acid from methanol, a 1.52-fold increase compared to the parent strain GS115/S8/Z3. Transcriptome analysis revealed 167 differentially expressed genes in DLac_Mut2_221, comprising 104 upregulated and 63 downregulated genes. These results suggest that the improvement in D-lactic acid production from methanol in the mutant strain involves three important mechanisms: (1) avoiding excessive formaldehyde accumulation, (2) activating the glyoxylate pathway, and (3) reducing the expression of O-glycosylated proteins required for cell wall stability. Metabolic engineering strategies for K. phaffii based on the knowledge gained from this study will contribute to improving the productivity of various useful chemicals from methanol.

bioengineering↗

bioRxiv2024

Integrated hepatic ferroptosis gene signature dictates pathogenic features of ferroptosis

Background & AimsFerroptosis, a distinctive form of cell death induced by iron-dependent lipid peroxidation, is implicated in various biological processes, including liver diseases. Establishing an iron overload-induced ferroptosis model and identifying hepatic gene signatures associated with ferroptosis are crucial for understanding its role in liver pathogenesis. MethodsF-box and leucine-rich repeat protein 5 (FBXL5) is a substrate-recognition component of the SCF E3 ligase complex that restricts intracellular iron levels. In this study, we used liver-specific Fbxl5-null mice to establish an iron overload-induced ferroptosis model. Transcriptome analysis identified genes involved in hepatic ferroptosis. Integrating these gene signatures with another ferroptosis model enabled the assessment of ferroptosis-related pathology in murine liver injury models and in 174 patients undergoing liver resection surgery. ResultsIron overload induced severe liver damage in liver-specific Fbxl5-null mice, characterized by elevated liver enzymes, histopathological changes, and lipid peroxidation. Transcriptome analysis revealed a distinct set of genes associated with hepatic ferroptosis response. Generating a gene signature for evaluating ferroptosis enhanced the understanding of ferroptosis-related pathologies in liver diseases. Iron overload exacerbated liver damage in murine ischemia-reperfusion injury models via ferroptosis induction. In human patients, elevated serum iron levels correlated with sustained post-operative liver damage, indicating heightened susceptibility to ferroptosis. ConclusionHere, a murine model of iron overload-induced hepatic ferroptosis was established, and a gene signature indicative of hepatic ferroptosis response in both mice and humans was identified. These findings underscore the role of ferroptosis in liver injury progression and suggest potential therapeutic targets for liver disease intervention. HIGHLIGHTSO_LILiver-specific Fbxl5 knockout mice provide an iron-induced ferroptotic injury model C_LIO_LIIntegrated gene signature of iron- and acetaminophen-induced liver injury dictates ferroptosis C_LIO_LIIron overload aggravates hepatic ischemia-reperfusion injury in mice C_LIO_LIPatients with high iron levels show delayed post-operative liver damage recovery C_LI IMPACT AND IMPLICATIONSOur study elucidated the critical role of iron in liver disease pathogenesis and ischemia-reperfusion injury (IRI). By establishing a murine model of iron overload-induced ferroptosis, we confirmed that iron overload exacerbated hepatic IRI, underscoring the importance of ferroptosis in liver damage. Additionally, the development of an integrated gene signature for hepatic ferroptosis response provides a valuable tool for evaluating ferroptosis in liver diseases. Via analysis of patient data, we also highlighted the clinical relevance of ferroptosis in post-operative liver damage, offering insights into potential therapeutic strategies targeting iron and ferroptosis to improve outcomes in patients with liver diseases.

molecular biology↗

bioRxiv2024

Increase trajectories of tendon micro vibration intensity during ankle plantar flexion: A longitudinal data analysis using latent curve models

We focus on fine vibrations originating from tendons (Mechanotendography: MTG) as a novel method for quantifying muscle activity. Quantifying muscle activity using MTG can enable daily and long-term continuous measurements, which have been challenging for electromyography (EMG) and mechanomyography (MMG). However, the detailed trajectory of MTG increase relative to exerted muscle strength has not been clarified, nor has the mechanism of MTG generation. Our research has two objectives. The first is to clarify the detailed relationship between exerted muscle strength levels and MTG through statistical modeling. The second is to establish a highly accurate hypothesis concerning the mechanism of MTG generation based on the modeling results and physiological knowledge. We focused on the Achilles tendon to study these two objectives. Experiments were conducted on 62 participants, and MTG data were obtained at various levels of exerted muscle strength. The obtained data were structured into a longitudinal data format representing the trajectory of MTG increase with increasing exerted muscle strength. We used latent curve models (LCM) to identify this structure. By applying various LCMs to explore an optimal model, we found that the quadratic LCM received the best fit for females, while the piecewise linear LCM with a breakpoint at 50% exerted muscle strength received the best fit for males. Notably, a significant sex difference was observed in the rate of increase in MTG at low levels of exerted muscle strength. These results suggest that MTG is caused by fine vibrations generated by muscle fiber contractions, and these fine vibrations are transmitted to the tendons connected to the muscles, where they are observed. Future research will focus on verifying this hypothesis through increased time points and physiological experiments.

physiology↗

bioRxiv2024

Recent codon preference reversals in the Drosophila melanogaster lineage

We employ fine-scale population genetic analyses to reveal dynamics among interacting forces that act at synonymous sites and introns among closely related Drosophila species. Synonymous codon usage bias has proven to be well-suited for population genetic inference. Under major codon preference, translationally superior "major" codons confer fitness benefits relative to their less efficiently and/or accurately decoded synonymous counterparts. Our codon family and lineage-specific analyses expand on previous findings in the Drosophila simulans lineage; patterns in naturally occurring polymorphism demonstrate fixation biases toward GC-ending codons that are consistent in direction, but heterogeneous in magnitude, among synonymous families. These forces are generally stronger than fixation biases in intron sequences. In contrast, population genetic analyses reveal unexpected evidence of codon preference reversals in the Drosophila melanogaster lineage. Codon family-specific polymorphism patterns support reduced efficacy of natural selection in most synonymous families but indicate reversals of favored states in the four codon families encoded by NAY. Accelerated synonymous fixations in favor of NAT and greater differences for both allele frequencies and fixation rates among X-linked, relative to autosomal, loci bolster support for fitness effect reversals. The specificity of preference reversals to codons whose cognate tRNAs undergo wobble position queuosine modification is intriguing. However, our analyses reveal prevalent dinucleotide preferences for ApT over ApC that act in opposition to GC favoring forces in both coding and intron regions. We present evidence that changes in the relative efficacy of translational selection and dinucleotide preference underlie apparent codon preference reversals.

evolutionary biology↗

bioRxiv2024

Co-utilization of microalgae and heterotrophic microorganisms improves wastewater treatment efficiency

Wastewater treatment using co-culture systems of microalgae and heterotrophic microorganisms is expected to be useful under atmospheric dilute carbon dioxide conditions. In this study, we investigated the combination of microalgae and heterotrophic microorganisms to improve the efficiency of wastewater treatment. Furthermore, to elucidate the cause of the changes in wastewater treatment efficiency in the co-culture system, changes in gene expression were revealed through transcriptome analysis. Three types of microalgae and five heterotrophic microorganisms were used in combination for wastewater treatment. The combination of Chlamydomonas reinhardtii NIES-2238 and Saccharomyces cerevisiae SH-4 showed the highest wastewater treatment efficiency. Using this combination for artificial wastewater treatment, the removal rates of TOC (Total organic carbon), PO43-, and NH4+ reached 80%, 93%, and 63%, respectively, after 18 h of treatment. Transcriptome analysis revealed that the combined wastewater treatment altered the expression of 1371 and 692 genes in C. reinhardtii and S. cerevisiae, respectively. The genes upregulated in C. reinhardtii included those related to molecular and ion transport. Genes upregulated in S. cerevisiae included those related to cell protection from various types of damage and stress. To the best of our knowledge, this is the first study to show that a combination of green algae and yeast improves the efficiency of wastewater treatment. As both the green alga C. reinhardtii and the yeast S. cerevisiae are highly safe microorganisms, the establishment of their effective combination for wastewater treatment is highly significant.

bioengineering↗

bioRxiv2024

Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTORs play a role in leaf senescence regulation

ACTIN DEPOLYMERIZING FACTORs (ADFs) regulate the organization and dynamics of actin microfilaments (AFs) by cleavage and depolymerization of AFs. The Arabidopsis thaliana genome encodes 11 ADF genes grouped into four subclasses. Subclass I ADFs, ADF1, -2, -3, and -4, are expressed in all vegetative tissues and are reportedly involved in the regulation of plant growth, and biotic and abiotic stress responses. Furthermore, the nuclear localization of ADF4 is seemingly important in disease responses. Here, we present data that indicate a previously unknown regulatory role of subclass I ADFs in leaf senescence. ADF4 knockout mutants (adf4) and transgenic lines in which the expression of all members of subclass I ADFs was downregulated (ADF1-4Ri) showed acceleration of both dark-induced and age-dependent leaf senescence. Among the eight ACTIN genes encoded in A. thaliana, ACT2, -7, and -8 are expressed in vegetative tissues. In contrast to adf4 and ADF1-4Ri, neither ACT2 and ACT8 double mutant (act2/8), nor ACT7 knockout mutant (act7), showed accelerated leaf senescence. Upregulation of the expression of senescence-associated genes occurred earlier in adf4 and ADF1-4Ri lines than in wild type. Examination of the expression of subclass I ADFs genes during senescence revealed a reduced expression of ADF4 but not of other subclass I members. Additionally, we showed that nuclear localization of ADF4 was important for regulating leaf senescence. Altogether, our data indicate that subclass I ADFs, particularly ADF4, play an important role in the regulation of leaf senescence.

plant biology↗

bioRxiv2024

Enrichment of allelic editing outcomes by Prime Editing in iPS cells

Gene editing in human induced pluripotent stem (iPS) cells with programmable nucleases facilitates reliable disease models, but methods using double-strand break repair often produce random on-target by-products. Prime editing (PE) combines Cas9 nickase with reverse transcriptase (RT) and a prime editing guide RNA (pegRNA) encoding a repair template to reduce by-products. We implemented a GMP-compatible protocol for transfecting Cas9- or PE-2A-mCherry plasmids to track and fractionate human iPS cells based on PE expression level. We compared the editing outcomes of Cas9- and PE-based methods in a GFP-to-BFP conversion assay, at the HEK3 benchmark locus, and at the APOE Alzheimers risk locus, revealing superior precision of PE at high expression levels. Moreover, sorting cells for PE expression level influenced allelic editing outcomes at the target loci. We expect that our findings will aid in the creation of gene-edited human iPS cells with intentional heterozygous and homozygous genotypes. HighlightsO_LIDelivered large plasmids to human iPS cells under GMP-compliant conditions C_LIO_LIDeveloped a flow cytometry-based approach to enrich for PE in human iPS cells C_LIO_LIDemonstrated few on-target indels in cells regardless of PE expression C_LIO_LISorted iPS cells based on PE expression level to influence mono- or bi-allelic editing C_LI

bioengineering↗

bioRxiv2024

Development of a metabolic engineering technology to simultaneously suppress the expression of multiple genes in yeast and application in carotenoid production

In yeast metabolic engineering, there is a need for technologies that simultaneously suppress and regulate the expression of multiple genes and improve the production of target chemicals. In this study, we aimed to develop a novel technology that simultaneously suppresses the expression of multiple genes by combining RNA interference with global metabolic engineering technology (GMES). Furthermore, using {beta}-carotene as the target chemical, we attempted to improve its production by using the technology. First, we developed a technology to suppress the expression of the target genes with various strengths using RNA interference. Using this technology, total carotenoid production was successfully improved by suppressing the expression of a single gene out of 10 candidate genes. Then, using this technology, RNA interference strain targeting 10 candidate genes for simultaneous suppression was constructed. The total carotenoid production of the constructed RNA interference strain was 1.7 times compared with the parental strain. In the constructed strain, the expression of eight out of the 10 candidate genes was suppressed. We developed a novel technology that can simultaneously suppress the expression of multiple genes at various intensities and succeeded in improving carotenoid production in yeast. Because this technology can suppress the expression of any gene, even essential genes, using only gene sequence information, it is considered a useful technology that can suppress the formation of by-products during the production of various target chemicals by yeast.

bioengineering↗

bioRxiv2023

Highly efficient transgenic mouse production using piggyBac and its application to rapid phenotyping at the founder generation

Pronuclear microinjection is the most popular method for producing transgenic (Tg) animals. Because the production efficiency is typically less than 20%, phenotypic characterization of Tg animals is generally performed on the next generation (F1) onwards. However, apart from in rodents, in many animal species with long generation times, it is desirable to perform phenotyping in the founder (F0) generation. In this study, we attempted to optimize a method of Tg mouse production to achieve higher Tg production efficiency using piggyBac transposon systems and established optimal conditions under which almost all individuals in the F0 generation were Tg. We also succeeded in generating bacterial artificial chromosome Tg mice with efficiency of approximately 70%. By combining this method with genome editing technology, we established a new strategy to perform phenotyping of mice with tissue-specific knockout using the F0 generation. Taking the obtained findings together, by using this method, experimental research using Tg animals can be carried out more efficiently.

bioengineering↗