Biology subjects

Kojima, T.

Publications and source records attributed to Kojima, T..

18 recordsLinked to original sources

bioRxiv2026

Effects of periodic group housing opportunities on reproductive performance and welfare in sows

ObjectiveStall housing of pregnant sows raises welfare concerns, whereas conventional group housing systems often reduce space efficiency. This study evaluated the effects of periodic group housing (PG) on reproductive performance and welfare compared with continuous stall housing (CS). MethodsSows in the CS group (n = 15) were continuously housed in stalls. In the PG group (n = 15), sows were housed in groups of three and allocated 1 day of group housing and 6 days of stall housing per week over 10 weeks. During group housing sessions, the sows had access to a group housing area containing sawdust. Behavioral observations and salivary cortisol measurements were conducted on the first day of the stall housing session in weeks 1, 6, and 10. Behavioral indices were expressed as proportions based on 90 sampling points recorded at 1-min intervals. ResultsThe number of stillbirths was significantly lower in the PG group than in the CS group (0.63 vs. 1.49 piglets per litter). whereas other reproductive outcomes, including total litter size and average birth weight, did not differ. In older parity sows, the PG treatment markedly increased the proportion of time spent lying, suggesting reduced discomfort associated with restricted movement. Furthermore, the proportion of exploratory behavior decreased markedly, and drinking behavior showed a decreasing trend across parity levels in the PG group, suggesting partial satisfaction of motivations for environmental exploration and oral manipulation. The proportion of oral abnormal behavior showed a pronounced interaction between housing treatment and experimental week, increasing from week 1 to week 6 in the PG group. Salivary cortisol concentrations did not differ between the groups. ConclusionPG may improve reproductive performance and partially satisfy the behavioral motivations restricted under continuous stall housing. This system may represent a practical alternative for improving animal welfare while minimizing economic losses.

animal behavior and cognition↗

bioRxiv2026

Single-cell spatial multiomics identifies POSTN+ CAFs mediating chemoradiotherapy resistance in rectal cancer

Neoadjuvant chemoradiotherapy (CRT) is standard for locally advanced rectal cancer (LARC), yet many patients retain residual disease. To resolve CRT-associated remodeling of the tumor microenvironment, we generated a multimodal spatial atlas from serial sections of paired pretreatment and post-treatment specimens from 24 patients using Xenium single-cell spatial transcriptomics and PhenoCycler multiplex proteomics, profiling 2.8 million cells; matched Visium HD datasets were generated on adjacent serial sections. Resistance was most strongly associated with fibroblast and myeloid programs adjacent to residual tumor. We identify a periostin (POSTN)-expressing CAF subset selectively enriched around residual tumor cells in non-responders, displaying a myofibroblastic phenotype and activating extracellular matrix remodeling, noncanonical WNT signaling, and immunosuppressive pathways. Tumor cells neighboring POSTN+ CAFs show consistent epithelial-mesenchymal transition signatures. Together, this atlas enables interrogation of CRT-induced spatial remodeling and nominates POSTN+ CAFs as key mediators and targets of CRT resistance, with direct relevance to CRT-based combination strategies.

cancer biology↗

bioRxiv2025

Arabidopsis TITAN LIKE is required for U12-type intron splicing, especially for AT-AC subtypes

Many eukaryotes possess two types of spliceosomes: the U2-dependent and U12-dependent spliceosomes. The U2-dependent spliceosome processes more than 99% of all introns, whereas the U12-dependent spliceosome acts on only about 0.3% of introns, one-third of which start with AT and end with AC, with the remainder having GT-AG termini. How the U12-dependent spliceosome splices two types of introns with different terminal sequences remains poorly understood. Human CENATAC is a subunit of the U12-dependent spliceosome that is particularly required for the splicing of the AT-AC subtype. The Arabidopsis genome contains a single homolog, TITAN LIKE (TTL), but its function in splicing remains unknown. Here, we generated TTL mutants and isolated two viable alleles, of which we analyzed one, designated ttl-142, to investigate TTL function in splicing. ttl-142 carries a 42-nucleotide deletion that removes 14 amino acid residues from the predicted protein, and homozygous mutants exhibit morphological abnormalities. Most U12-dependent introns were less efficiently spliced in ttl-142 than in the wild-type, with the splicing of AT-AC introns particularly suppressed. Splicing suppression in ttl-142 was more extensive than that in a DROL1 mutant, which carries a mutation in a gene specifically required for AT-AC intron splicing. Conversely, fewer genes showed altered expression levels in ttl-142 than in drol1, and most differentially expressed genes differed between the two mutants. These results suggest that the phenotypes of ttl-142 and drol1 mutants may reflect the impairment of distinct spliceosomal functions.

plant biology↗

bioRxiv2024

Chemotherapy resistance due to epithelial-to-mesenchymal transition is caused by abnormal lipid metabolic balance

Invasive cancer is defined by the loss of epithelial cell traits resulting from the ectopic expression of epithelial-mesenchymal transition (EMT)-related transcription factors such as Snail. Although EMT is known to impart chemoresistance to cancer cells, the precise molecular mechanisms remain elusive. We found that Snail expression confers chemoresistance by upregulating the cholesterol efflux pump ABCA1 as a countermeasure to the excess of cytotoxic free cholesterol relative to its major interaction partner in cellular membranes, sphingomyelin. This imbalance is introduced by the transcriptional repression of enzymes involved in the biosynthesis of sphingomyelin by Snail. Inhibiting esterification of cholesterol, which renders it inert, selectively suppresses growth of a xenograft model of Snail-positive kidney cancer. Our findings offer a new perspective on lipid-targeting strategies for invasive cancer therapy.

cell biology↗

bioRxiv2024

PDPN/CLEC-2 axis modulates megakaryocyte subtypes in a hematopoietic stem cell-regulating megakaryocyte-dominant manner

IntroductionMegakaryocytes are classified into several subtypes including LSP1-positive immune-skewed, MYLK4-positive hematopoietic stem cell (HSC)-regulating, and BMAL1-positive platelet-producing megakaryocytes. Podoplanin (PDPN)-expressing stromal cells generate a microenvironment that promotes megakaryopoiesis in the bone marrow. In this context, PDPN interacts with C-type lectin-like receptor-2 (CLEC-2) on megakaryocyte progenitors, which induces megakaryocyte proliferation. However, the megakaryocyte subtypes developed by the regulation of the PDPN/CLEC-2 axis have not yet been elucidated. Materials and MethodsWe established an immortalized bone marrow PDPN-expressing stromal cell line and a PDPN-knockout line (PDPN WT and KO feeder cells, respectively). Bone marrow hematopoietic progenitors were committed to megakaryocytes in co-culture with PDPN WT or KO feeder cells. The number and ploidy of megakaryocytes, resultant platelets, and the polarization of megakaryocyte subtypes were investigated. ResultsThe number of megakaryocytes was significantly increased in the co-culture with PDPN WT feeder cells compared to that with PDPN KO feeder cells. The megakaryocytes on the PDPN WT and KO feeders showed their main ploidy at 16N[~]32N and 8N[~]16N, respectively. The number of platelets decreased in the co-culture with the PDPN WT feeder compared to those in the co-culture with the PDPN KO feeder. Megakaryocyte subtypes were immunocytochemically detected in in vitro differentiated CD41-positive megakaryocytes. For each megakaryocyte subtype, the percentage of MYLK4-positive megakaryocytes significantly increased and the percentage of BMAL1-positive megakaryocytes significantly decreased when co-cultured with the PDPN WT feeder. ConclusionThe PDPN/CLEC-2 axis modulates megakaryocyte subtype differentiation, with a predominance of HSC-regulating megakaryocytes.

cell biology↗

bioRxiv2024

Tissue growth associated transition of podoplanin-expressing stromal cells in the postnatal mouse femur

In vertebral long bones, such as the femur, bone formation involves endochondral ossification. Endochondral ossification first occurs in the central region of the fetal diaphysis, the primary ossification center. Podoplanin (PDPN) is a transmembrane mucin-like glycoprotein, and PDPN-positive cells play key roles in various organ/tissue development. In adult mice, osteolineage PDPN-positive cells are associated with femoral microarchitecture. However, specific roles of PDPN in fetal bone development remain unclear. Therefore, in this study, we aimed to investigate the spatiotemporal dynamics and physiological functions of PDPN-positive cells during fetal femur development. In the fetal femur, PDPN-positive cells first emerged in the primitive cortical bone, termed the bone collar, concurrently with primary ossification center initiation. Several PDPN-positive cells in the bone collar migrated to the marrow cavity and populated the metaphyseal trabecular bone. Most PDPN-positive cells in both the bone collar and trabeculae exhibited osteolineage features, such as osterix expression. Pdpn knockout fetuses exhibited abnormal recruitment of osterix-positive cells and mineral deposition in the dorsal bone collar. Overall, our results suggest that PDPN-positive cells constitute a spatially regulated osteolineage population that contributes to coordinated fetal femur development.

cell biology↗

bioRxiv2024

Functional studies of deafness-associated pendrin and prestin variants

Pendrin and prestin are evolutionary conserved membrane proteins that are essential for normal hearing. Pendrin is an anion transporter required for normal development and maintenance of ion homeostasis in the inner ear, while prestin is a voltage-dependent motor responsible for cochlear amplification essential for high sensitivity and frequency selectivity of mammalian hearing. Dysfunction of these proteins result in hearing loss in humans, and numerous deafness-associated pendrin and prestin variants have been identified in patients. However, the pathogenic impacts of many of these variants are ambiguous. Here we report results from our ongoing efforts in experimentally characterizing pendrin and prestin variants using in vitro functional assays, providing invaluable information regarding their pathogenicity.

pathology↗

bioRxiv2023

Basement membrane extract potentiates the endochondral ossification phenotype of bone marrow-derived mesenchymal stem cell-based cartilage organoids

Endochondral ossification is a developmental process in the skeletal system and bone marrow of vertebrates. During endochondral ossification, primitive cartilaginous anlages derived from mesenchymal stem cells (MSCs) undergo vascular invasion and ossification. In vitro regeneration of endochondral ossification is beneficial for research on the skeletal system and bone marrow development as well as their clinical aspects. However, to achieve the regeneration of endochondral ossification, a stem cell-based artificial cartilage (cartilage organoid, Cart-Org) that possesses an endochondral ossification phenotype is required. Here, we modified a conventional 3D culture method to create stem cell-based Cart-Org by mixing it with a basement membrane extract (BME) and further characterized its chondrogenic and ossification properties. BME enlarged and matured the bone marrow MSC-based Cart-Orgs without any shape abnormalities. Histological analysis using Alcian blue staining showed that the production of cartilaginous extracellular matrices was enhanced in Cart-Org treated with BME. Transcriptome analysis using RNA sequencing revealed that BME altered the gene expression pattern of Cart-Org to a dominant chondrogenic state. BME triggered the activation of the SMAD pathway and inhibition of the NK-{kappa}B pathway, which resulted in the upregulation of SOX9, COL2A1, and ACAN in Cart-Org. BME also facilitated the upregulation of genes associated with hypertrophic chondrocytes (IHH, PTH1R, and COL10A1) and ossification (SP7, ALPL, and MMP13). Our findings indicate that BME promotes cartilaginous maturation and further ossification of bone marrow MSC-based Cart-Org, suggesting that Cart-Org treated with BME possesses the phenotype of endochondral ossification. HighlightsO_LIBasement membrane extract (BME) enlarges MSC-based Cart-Org. C_LIO_LIBME activates the SMAD pathway and inhibits the NK-kB pathway of the Cart-Org. C_LIO_LIBME promotes cartilaginous maturation and further ossification of Cart-Org. C_LI

cell biology↗

bioRxiv2023

Rapid and cost-effective epitope mapping using PURE ribosome display coupled with next-generation sequencing and bioinformatics

A novel, efficient and cost-effective approach for epitope identification of an antibody has been developed using a ribosome display platform. This platform, known as PURE ribosome display, utilizes an Escherichia coli-based reconstituted cell-free protein synthesis system (PURE system). It stabilizes the mRNA-ribosome-peptide complex via a ribosome-arrest peptide sequence. This system was complemented by next-generation sequencing (NGS) and an algorithm for analyzing binding epitopes. To showcase the effectiveness of this method, selection conditions were refined using the anti-PA tag monoclonal antibody with the PA tag peptide as a model. Subsequently, a random peptide library was constructed using 10 NNK triplet oligonucleotides via the PURE ribosome display. The resulting random peptide library-ribosome-mRNA complex was selected using a commercially available anti-HA (YPYDVPDYA) tag monoclonal antibody, followed by NGS and bioinformatic analysis. Our approach successfully identified the "DVPDY" sequence as an epitope within the hemagglutinin amino acid sequence, which was then experimentally validated. This platform provided a valuable tool for investigating continuous epitopes in antibodies.

bioengineering↗

bioRxiv2023

Molecular mechanisms underlying the formation of larval green color and camouflage patterns in swallowtail butterfly, Papilio memnon

Insects have various strategies like mimicry or camouflage to avoid predation. Swallowtail butterfly larvae switch from a black and white pattern mimicking bird droppings to a green camouflage pattern in the fifth (final) instar. This larval pattern switch is regulated during the juvenile hormone (JH)-sensitive period, when JH titer declines rapidly, and clawless (cll), abdominal-A (abd-A), and Abdominal-B (Abd-B) function during this period. However, the molecular mechanism behind the background green color, a crucial aspect of the camouflage pattern, remains poorly understood. Here, we used Papilio memnon, which switches to the camouflage pattern in the fifth instar but is greenish from the third instar, to investigate the mechanism of camouflage pattern formation, particularly the larval green coloration. Through RNA sequencing, we found that BBPs forming a gene cluster are upregulated in the green regions of P. memnon larvae during the fourth instar, whereas P. xuthus larvae, which have not yet turned green, showed minimal BBPs expression. When BBP1 and BBP2, which were particularly highly expressed, were knocked down by RNAi, there was a phenotypic change in green to yellow in both fourth and fifth instar larvae. Expression analysis and knockdown experiments were conducted also for JHBP, which had been previously reported, and confirmed that it is involved in the synthesis of yellow pigment. Furthermore, knockdown of Ubx resulted in no phenotypic change in fourth instar larvae, but in fifth instar larvae, the eyespots pattern characteristic of the camouflage pattern almost entirely disappeared, suggesting that Ubx is also functional only during JH-sensitive period. Our results indicate that the switch from mimetic to camouflage patterns resulted from the function of cll, abd-A, Abd-B, and Ubx prepatterning genes during the JH-sensitive period. And the increased expression of BBPs and JHBPs, independent of the JH-sensitive period, contributed to the development of green coloration.

molecular biology↗

bioRxiv2023

AtMYB50 regulates root cell elongation by upregulating PECTIN METHYLESTERASE INHIBITOR 8 in Arabidopsis thaliana

Plant root development is regulated by several signal transduction pathways. Among them, plant phytohormones, like auxin and cytokinin, are well characterized for their molecular mechanisms of action. Reactive oxygen species (ROS) play important roles as signaling molecules in controlling root development. Under these signal transduction pathways, the gene regulatory network, which is controlled by transcription factors, is the key to regulating root growth. We have previously reported an important transcription factor, UP BEAT1 (UPB1), that regulates ROS homeostasis at the root tip, further controlling the transition from cell proliferation to differentiation. Although UPB1 directly regulates the expression of several peroxidases that control ROS homeostasis, UPB1 still targets genes other than peroxidases. This indicates that UPB1 may regulate root growth through different ROS signals. Here, we investigated the function of the transcription factor MYB50, a direct target of UPB1, in Arabidopsis thaliana. We then examined whether UPB1 regulates MYB50 expression in the roots using an induction expression system and imaging of multiple fluorescent proteins. We also performed RNA-Seq analysis using MYB50 estradiol induction lines and ChIP-seq analysis to identify the MYB50 regulatory gene network. Integrating these analyses with UPB1 regulatory network revealed that MYB50 regulates the expression of PECTIN METHYLESTERASE INHIBITOR 8 (PMEI8). These data suggest that MYB50 is a new root growth regulator under the UPB1 gene regulatory network, which differs from the control of ROS homeostasis. Our study presents a model including a new transcriptional network under MYB50 into UPB1 regulatory root growth system and will provide novel insights into the cell elongation controlled by pectin modification.

plant biology↗

bioRxiv2023

Biochemical reactions between intra-articular tissues and joint instability in a rat model of osteoarthritis

AimsJoint instability is associated with various joint conditions including osteoarthritis (OA) and inflammation, and we have developed model which is determined to role of knee instability. Investigating cartilage maintenance factors such as hyaluronic acid (HA) and glycosaminoglycans (GAGs) can provide insights into the effect of the mechanical stress and the inhibitor used, with the following aims: 1) whether cartilage degeneration is inhibited in the new model, 2) whether combination TGF-{beta}1 inhibition mitigates cartilage degeneration, and to determine the role of TGF-{beta}1 in synovitis using fibroblasts. Main methodsWe used this novel model to investigate inhibition of OA progression with a focus on HA and GAGs, which help maintain the cartilage and synovial membrane. In detail, mechanical tests, X-ray, histological, and protein and mRNA expression analyses were used to determine the role of joint stability using in vivo model or fibroblast from synovial membrane. Key findingsJoint stability mitigated cartilage degeneration loss, decreased osteophytes, increased the expression levels of HA and GAGs in the synovial membrane, and decreased the release of pro-inflammatory factors in rats. Moreover, injection of TGF-{beta}1 inhibitor in an inflammatory synovial membrane promoted HA and GAGs expression. In synovial fibroblast cells, inhibition of TGF-{beta}1 over expression significantly inhibited the downregulation of pro-inflammatory factors and promoted the upregulation of lubrification for cartilage. SignificanceOur results suggest that joint instability is an independent mechanical factor for OA progression. The results provide novel insights into the association between OA and joint instability, which has significant human sciences implications. Research Highlights* Established a new experimental rat model of the different joint instability for elucidate osteoarthritis onset/progression * Using Histological staining to investigated the osteoarthritis including synovitis and osteophytes of the novel model * Using fibroblast from synovial membrane to investigated the fibrosis * Joint instability exacerbates articular cartilage degeneration and decreases HA and GAGs protein expressions in synovial membrane * TGF-{beta}1 inhibitor on early osteoarthritis joints may suppress synovial inflammation

pathology↗

bioRxiv2023

Contribution of default mode network to game and delayed-response task performance: power and connectivity analyses of theta oscillation in the monkey

Neuroimaging studies have demonstrated the presence of a default mode network (DMN) which shows greater activity during rest, and an executive network (EN) which is activated during cognitive tasks. DMN and EN are thought to have competing functions. However, recent studies reported that the two networks show coactivation during some cognitive tasks. To clarify how DMN works and how DMN interacts with EN for cognitive control, we recorded EEG activities in the medial prefrontal (anterior DMN: aDMN), posterior cingulate/precuneus (posterior DMN: pDMN), and lateral prefrontal (EN) areas in the monkey. As cognitive tasks, we employed a monkey-monkey competitive video game (GAME) and a delayed-response (DR) task. We focused on theta oscillation because of its importance in cognitive control. We also examined theta band connectivity among the three network areas using the Granger causality analysis. DMN and EN were found to work cooperatively in both tasks. In all the three network areas, we found GAME-task-related, but no DR-task-related, increase in theta power from the resting level, maybe because of the higher cognitive demand associated with the GAME task performance. The information flow conveyed by the theta oscillation was directed more to aDMN than from aDMN for both tasks. The GAME-task-related increase in theta power in aDMN is supposed to be supported by more information flow conveyed by the theta oscillation from EN and pDMN.

neuroscience↗

bioRxiv2022

Nascent MSKIK peptide prevents or releases translation arrest in Escherichia coli

The insertion of the sequence encoding SKIK peptide adjacent to the M start codon of a difficult-to-express protein enhances protein production in Escherichia coli. In this report, we show that the increased production of the SKIK-tagged protein is not due to codon usage of the SKIK sequence. In addition, insertion of SKX, KKX, and AKX (X = G, L, H, Y, E, and F) at the N-terminus increased protein production. Furthermore, insertion of MSKIK just before the SecM arrest peptide (FSTPVWISQAQGIRAGP), which causes translational stalling on mRNA, greatly increased the production of the protein containing the SecM arrest peptide in the E. coli reconstituted cell-free protein synthesis system (PURE system). A similar phenomenon was observed for CmlA leader whose arrest is induced by chloramphenicol. These results suggest that the nascent MSKIK peptide prevents or releases translational stalling immediately following its generation during the translation process.

biochemistry↗

bioRxiv2022

The draft genome sequence of Japanese rhinoceros beetle Trypoxylus dichotomus

Beetles are the largest insect order and one of the most successful animal groups in terms of number of species. The Japanese rhinoceros beetle Trypoxylus dichotomus (Coleoptera, Scarabaeidae, Dynastini) is a giant beetle with distinctive exaggerated horns present on the head and prothoracic regions of the male. T. dichotomus has been used as research model in various fields such as evolutionary developmental biology, ecology, ethology, biomimetics, and drug discovery. In this study, de novo assembly of 615 Mb, representing 80% of the genome estimated by flow cytometry, was obtained using the 10x Chromium platform. The scaffold N50 length of the genome assembly was 8.02 Mb, with repetitive elements predicted to comprise 49.5% of the assembly. In total, 23,987 protein-coding genes were predicted in the genome. In addition, de novo assembly of the mitochondrial genome yielded a contig of 20,217 bp. We also analyzed the transcriptome by generating 16 RNA-seq libraries from a variety of tissues of both sexes and developmental stages, which allowed us to identify 13 co-expressed gene modules. The detailed genomic and transcriptomic information of T. dichotomus is the most comprehensive among those reported for any species of Dynastinae. This genomic information will be an excellent resource for further functional and evolutionary analyses, including the evolutionary origin and genetic regulation of beetle horns and the molecular mechanisms underlying sexual dimorphism.

genomics↗

bioRxiv2021

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage; this induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in a perivascular microenvironment, and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in the postnatal bone marrow generation. We found that periosteum-derived PDPN-expressing stromal cells regulate vascularization during postnatal epiphyseal marrow development. Our findings suggest that these cells act as pericytes on the primitive vasculature of the nascent marrow. They invade the cartilaginous epiphysis and regulate marrow development and homeostasis by maintaining vascular integrity. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.

developmental biology↗

bioRxiv2021

Protocell Arrays for Simultaneous Detection of Diverse Analytes

Simultaneous detection of multiple analytes from a single sample (multiplexing), particularly when at the point of need, can guide complex decision-making without increasing the required sample volume or cost per test. Despite recent advances, multiplexing still typically faces the critical limitation of measuring only one type of molecule per assay platform - for example, only small molecules or only nucleic acids. In this work, we address this bottleneck with a customizable platform that integrates cell-free expression (CFE) with a polymer-based aqueous two-phase system (ATPS) to produce membrane-less "protocells" containing transcription and translation machinery used for analyte detection. Multiple protocells are arrayed in microwells where each protocell droplet performs distinct reactions to detect chemically diverse targets including small molecules, minerals, and nucleic acid sequences, all from the same sample. We demonstrate that these protocell arrays can measure analytes in a human biofluid matrix, maintain function after lyophilization and rehydration, and produce visually interpretable readouts, illustrating its potential for application as a minimal-equipment, field-deployable, multi-analyte detection tool.

synthetic biology↗

bioRxiv2020

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over PCR-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a dsDNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based Protection of LET) method effectively increases sfGFP expression levels from LETs in Escherichia coli CFPS reactions by 6-fold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from non-model organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.

synthetic biology↗