Biology subjects

Nakano, H.

Publications and source records attributed to Nakano, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Causal contribution of motor-parietal network to conscious kinesthetic perception

Bodily awareness, including the conscious experience of limb movement (kinesthesia), is a fundamental form of human consciousness. Tendon vibration-induced kinesthetic illusions provide a tractable model for probing its neural basis. Although previous studies have reported coactivation of the primary motor cortex (M1), the right inferior parietal lobule (IPL), and the right inferior frontal gyrus (IFG) during illusory movement, their causal contributions to conscious kinesthetic experience remain unclear. Here, we combined functional MRI and regional facilitative dual-site transcranial direct current stimulation (tDCS) to establish a causal link between these cortices and illusory foot movement. fMRI revealed that the M1-IPL network supports kinesthetic illusion elicited by bottom-up sensory processing, whereas the IFG plays a role in top-down monitoring of the kinesthetic experience. Critically, concurrent facilitative dual-site tDCS applied to the M1 and IPL significantly enhanced the subjective strength of the illusion, compared with the sham and concurrent tDCS to the M1 and IFG. This effect was replicated without increasing muscle activity in another tDCS study and exceeded those observed with single-site (M1 or IPL) or concurrent higher-order IPL-IFG tDCS. Our findings provide the first compelling causal evidence that conscious kinesthetic perception can be augmented through coordinated interactions between the modality-specific kinesthetic-motor cortex (M1) and the higher-order right inferior parietal cortex (IPL). This coordinated interaction between modality-specific and higher-order cortical regions underlying conscious kinesthetic perception is consistent with the Global Neuronal Workspace hypothesis. Our findings have therefore extended the applicability of this framework from exteroceptive perception to bodily awareness.

neuroscience↗

bioRxiv2026

Interleukin-11 promotes the colonic epithelial organoid regeneration from mechanical disruption

The intestinal epithelium relies on rapid repair to maintain homeostasis after injury, and dysregulation of this process contributes to the pathogenesis of inflammatory bowel disease and colorectal cancer. Interleukin-11 (IL-11), a fibroblast-derived cytokine elevated in these diseases, has well-documented effects on stromal cells, but its direct action on intestinal epithelial cells remains poorly characterized. Here, we used mouse colon organoids as an isolated epithelial system to directly examine the effects of IL-11 on epithelial cells. IL-11 stimulation activated the canonical JAK/STAT3 pathway, as evidenced by increased STAT3 phosphorylation and Socs3 induction in a concentration-dependent manner. In a pipetting-based mechanical disruption model, IL-11 significantly increased the number of organoids recovered. Although mechanical disruption dominated the overall transcriptional landscape, RNA-seq analysis identified coordinated upregulation of STAT3 target genes and proliferation-related pathways specifically in response to IL-11. Pharmacological inhibition of STAT3 attenuated the IL-11-induced promotion of organoid recovery, indicating that STAT3 signaling mediates the epithelial response to IL-11 and maintains organoid size under basal conditions. Together, these findings demonstrate that IL-11 directly promotes intestinal epithelial repair after mechanical disruption through STAT3-dependent signaling, providing a mechanistic basis for its protective role in acute colonic injury.

cell biology↗

bioRxiv2026

Hmgb1 release kinetics shape its extracellular functions during regulated cell death

Danger-associated molecular patterns (DAMPs), such as high mobility group protein B1 (HMGB1), are released from dying cells, yet the kinetics and functional consequences of their release remain unclear. Using Hmgb1-mCherry transgenic mice and live-cell imaging, we visualize Hmgb1 and interleukin-1{beta} (IL-1{beta}) secretion at single-cell resolution. Hmgb1 exhibits two distinct release kinetics: short-duration completed within 1 min and long duration spanning several minutes. Mathematical modeling demonstrates that short-duration release generates substantially higher local Hmgb1 concentrations and steeper gradients near the dying cell than long-duration release. Such rapid release may enable one or a few dying cells to produce sufficient Hmgb1 to stimulate neighboring cells, consistent with its role as an alarmin. In vivo imaging of cisplatin-induced kidney injury reveals an inverse correlation between intracellular Hmgb1 levels and monocyte infiltration. Together, these findings suggest that Hmgb1 release kinetics shape its extracellular functions during regulated cell death.

cell biology↗

bioRxiv2025

Screening and machine-learning assisted prediction of translation-enhancing peptides reducing ribosomal stalling in Escherichia coli

We previously reported that the nascent SKIK peptide enhances translation and alleviates ribosomal stalling caused by arrest peptides (APs) such as SecM and polyproline when positioned immediately upstream of the APs in both Escherichia coli in vivo and in vitro translation systems. In this study, we performed a comprehensive screening of translation-enhancing peptides (TEPs) using a randomized artificial tetrapeptide library. The screening was based on the ability of the peptides to suppress SecM AP-induced translational stalling in E. coli cells. Various TEPs exhibiting a range of translation-enhancing activities were identified. In vitro translation analysis suggested that the fourth amino acid in the tetrapeptide plays a key role in reducing SecM AP-mediated stalling. Furthermore, we developed a machine learning model using a random forest algorithm to predict TEP activity. The predicted values showed a strong correlation with experimentally measured activities. These findings offer a compact peptide toolkit and a data-driven approach for mitigating AP-induced ribosome stalling, with potential applications in synthetic biology.

synthetic biology↗

bioRxiv2025

A nuclear CobW/WW-domain factor represses the CO2-concentrating mechanism in the green alga Chlamydomonas reinhardtii

Microalgae induce a CO2-concentrating mechanism (CCM) to maintain photosynthesis when dissolved CO2 is limited, but how this energy-intensive system is suppressed when CO2 levels rise has remained unclear. The CCM consumes 15-30% of photosynthetically-generated ATP, making its regulation critical for cellular energy balance. Here, we identify a nuclear repressor of the CCM in the green alga Chlamydomonas reinhardtii. A pull-down screen for interacting partners of the master activator CCM1/CIA5 revealed an uncharacterized protein that co-purifies with CCM1 even after high-salt washes. This protein, designated CCM1-binding protein 1 (CBP1), combines a CobW/CobW_C GTP-binding metallochaperone module with a WW domain characteristic of protein-protein interactions. CBP1 colocalizes with CCM1 in the nucleus regardless of CO2 conditions, and the two proteins interact in vivo. CRISPR/Cas9-based disruption of CBP1 does not affect growth or CCM induction under CO2 limitation but derepresses 27 of 41 CCM1-dependent low-CO2 inducible genes under high-CO2 conditions. These include the periplasmic and intracellular carbonic anhydrases (CAH1, LCIB) and inorganic carbon transporters/channels (LCIA, LCI1, BST1, BST3). Consistently, cbp1 mutants accumulate higher levels of CAH1 and LCIB proteins and exhibit a 40% increase in inorganic carbon affinity under high-CO2 conditions; this phenotype is rescued by CBP1 complementation or by acetazolamide treatment. These results demonstrate that CBP1 prevents unnecessary CCM activity when CO2 is abundant, acting upstream of both transporter/channel and carbonic anhydrase modules. Our findings suggest a regulatory mechanism potentially linking zinc-dependent protein chemistry to CCM gene repression, providing insights into energy-efficient CO2 sensing in aquatic photosynthetic organisms. Significance statementAlgae flourish by activating a CO2-concentrating mechanism (CCM) when dissolved CO2 is limited but must deactivate it when CO2 levels increase to conserve energy. We have identified the nuclear protein that functions as this long sought "off switch" in the model green alga. Deletion of this protein causes cells to overproduce CCM transporters and enzymes, maintaining CCM activity even under high CO2 conditions, demonstrating its essential role in suppressing the system when carbon is abundant. This finding illuminates how algae balance energy consumption with carbon capture and offers a new target for engineering strains that fix CO2 more efficiently for biofuel production or climate-mitigation technologies.

plant biology↗

bioRxiv2025

SMART platform for enzyme engineering: A novel single-molecule display approach for evolution and functional studies of D-amino acid oxidase

This study introduces SMART (Single Molecule Assay on Ribonucleic Acid by Translated Product), an innovative in vitro platform integrating mRNA display, next-generation sequencing, and bioinformatics to address several key challenges in enzyme engineering. The characteristic feature of SMART is its auxiliary unit, attached to the mRNA-displayed enzyme library to mediate the biotinylation of reactive enzyme variants and enable their enrichment by streptavidin beads pull-down. The unit comprises a hairpin single-stranded DNA that hybridizes with the mRNA and anchors a DNA-binding protein, single-chain Cro, which carries functional auxiliary molecules specific to the enzyme of interest. Here, we report on the establishment of SMART for the engineering of oxidases, specifically D-amino acid oxidase (DAAO) from Schizosaccharomyces pombe, with engineered ascorbate peroxidase 2 as its auxiliary enzyme. In a single selection round of a DAAO library with randomized catalytic residue Y232 and D-Alanine as the substrate, we identified several catalytically active enzyme variants with altered substrate specificity in a matter of days. This surpasses traditional enzyme engineering methods in speed, cost, library capacity, and precision. This research underscores the potential of SMART for the engineering of various oxidases, and other enzymes after the corresponding adjustment of the auxiliary unit.

bioengineering↗

bioRxiv2024

Human formin FHOD3-mediated actin elongation is required for sarcomere integrity in cardiomyocytes

Contractility and cell motility depend on accurately controlled assembly of the actin cytoskeleton. Formins are a large group of actin assembly proteins that nucleate and elongate new actin filaments. Some formins may cap filaments while others sever or bundle filaments. The Formin HOmology Domain-containing protein (FHOD)-family of formins is critical to the formation of the fundamental contractile unit in muscle, the sarcomere. Specifically, mammalian FHOD3L plays an essential role in cardiomyocytes. Despite our knowledge of FHOD3Ls importance in cardiomyocytes, its biochemical and cellular activities remain poorly understood. It was proposed that FHOD-family formins act by capping and bundling, as opposed to assembling new filaments. Here, we demonstrate that FHOD3L nucleates actin and rapidly but briefly elongates filaments after temporarily pausing elongation. We designed function-separating mutants that enabled us to distinguish which biochemical roles are required in the cell. We found that human FHOD3Ls elongation activity, but not its nucleation, capping, or bundling activity, is necessary for proper sarcomere formation and contractile function in neonatal rat ventricular myocytes. The results of this work provide new insight into the mechanisms by which formins build specific structures and will contribute to knowledge regarding how cardiomyopathies arise from defects in sarcomere formation and maintenance.

cell biology↗

bioRxiv2024

Improvement of Antibody Affinity and Using PURE Ribosome Display and Microbial Secretion System

A method has been developed for efficiently enriching and analyzing high-affinity antibody variants by combining the PURE ribosome display (ribosome display using purified cell-free protein synthesis components) with next-generation sequencing (NGS) and the Brevibacillus choshinensis secretion system, using the NZ-1 antibody, which targets the PA tag peptide (GVAMPGAEDDVV), as a model antibody. An artificial DNA encoding the single-chain fragment of binding (scFab) of the NZ-1 antibody was synthesized and actively expressed by cell-free protein synthesis system (CFPS). Region-specific saturation mutations were introduced into the scFab gene based on its structural information. The resulting scFab library was selected against the PA tag through two rounds of PURE ribosome display, followed by Illumina sequencing to identify potential scFab variants with higher affinity. The candidates were expressed as Fab fragments using the B. choshinensis secretion system. These Fab fragments were then purified from the culture supernatant using two-step column chromatography. The binding affinity of the purified Fab was evaluated using a biolayer interferometry assay, revealing a variant Fab with higher affinity than the wild-type Fab. These results demonstrate that integrating PURE ribosome display with NGS analysis and the B. choshinensis secretion expression system enables the rapid identification and analysis of high-affinity antibody variants.

synthetic biology↗

bioRxiv2024

Investigation of Intestinal Bacteria-binding Monoclonal Antibodies Derived from Rabbit Single B Cells

Immunoglobulins in the intestine have been reported to play a pivotal role in regulating the composition and function of intestinal microbiota. However, the relationship between monoclonal antibodies (mAbs) and intestinal bacteria remains elusive due to the lack of analytical methods. Here we report the utilization of Ecobody technology, a single B cell technique developed by our group, for obtaining and investigating intestinal bacteria-binding mAbs derived from single B cells of a non-immunized rabbit. Bacteroides cellulosilyticus isolated from the rabbit stool was used as the antigen for selecting B cells, followed by antibody gene amplification, mAb expression in cell-free protein synthesis (CFPS), and activity evaluation. For large-scale and cost-effective production of the selected mAb, we employed the secretion expression system for antigen-binding fragment (Fab) using Brevibacillus choshinensis, and successfully yielded [~]2.0 mg/L-culture of purified antibody with high affinity to the target bacterium. Purified Fab showed binding activity towards various intestinal bacteria, suggesting the broad specificity toward intestinal bacteria-associated antigens.

bioengineering↗

bioRxiv2024

Theoretical proposal for restoration of hand motor function utilizing plasticity of motor-cortical interhemispheric interaction

After stroke, the poorer recovery of motor function of upper extremities compared to other body parts is a longstanding problem. Based on our recent functional MRI evidence on healthy volunteers, this perspective paper proposes systematic hand motor rehabilitation utilizing the plasticity of interhemispheric interaction between motor cortices and following its developmental rule. We first discuss the effectiveness of proprioceptive intervention on the paralyzed (immobile) hand synchronized with voluntary movement of the intact hand to induce muscle activity in the paretic hand. In healthy participants, we show that this bilateral proprioceptive-motor coupling intervention activates the bilateral motor cortices (= bilaterally active mode), facilitates interhemispheric motor-cortical functional connectivity, and augments muscle activity of the passively-moved hand. Next, we propose training both hands to perform different movements, which would be effective for stroke patients who become able to manage to move the paretic hand. This bilaterally different movement training may guide the motor cortices into left-right independent mode to improve interhemispheric inhibition and hand dexterity, because we have shown in healthy older adults that this training reactivates motor-cortical interhemispheric inhibition (= left-right independent mode) declined with age, and can improve hand dexterity. Transition of both motor cortices from the bilaterally active mode to the left-right independent mode is a developmental rule of hand motor function and a common feature of motor function recovery after stroke. Hence, incorporating the brains inherent capacity for spontaneous recovery and adhering to developmental principles may be crucial considerations in designing effective rehabilitation strategies.

neuroscience↗

bioRxiv2024

Effect of translation enhancing nascent SKIK peptide on the arrest peptides containing consecutive Proline

Ribosome arrest peptides (RAPs) such as SecM arrest peptide (SecM AP) and WPPP with consecutive Pro residues, are known to induce translational stalling in Escherichia coli. We demonstrate that the translation enhancing SKIK peptide tag, which consisted of four amino acid residues Ser-Lys-Ile-Lys, effectively alleviate translational arrest caused by WPPP. Moreover, the proximity between SKIK and WPPP significantly influences the extent of this alleviation, observed in both PURE cell-free protein synthesis and in vivo protein production systems, resulting in a substantial increase in the yield of proteins containing such RAPs. Furthermore, we unveil that nascent SKIK peptide tag and translation elongation factor P (EF-P) which alleviate ribosome stalling in consecutive-Pro rich protein, synergistically promote translation. A kinetic analysis based on the generation of super folder green fluorescent protein under in vitro translation reaction reveals that the ribosome turnover is enhanced by more than 10-fold when the SKIK peptide tag is positioned immediately upstream of the SecM AP sequence. Our findings provide valuable insights into optimizing protein production processes, which are essential for advancing synthetic biology applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/582505v2_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@c97150org.highwire.dtl.DTLVardef@12e3fb1org.highwire.dtl.DTLVardef@157af4forg.highwire.dtl.DTLVardef@1b67da9_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

Substrate profiling of human Transglutaminase 1 using cDNA display and next-generation sequencing

Human transglutaminase 1 regulates skin development and is linked with several disease conditions with yet fully unknown mechanisms. To uncover all of its roles in health and disease, the understanding of protein substrates and their reactivity with transglutaminase 1 is necessary. To gain insight into the substrate profile of human transglutaminase 1, this study uses an in vitro selection system based on the cDNA display technology to screen two displayed peptide libraries differing in the number and position of mutated sites relative to the reactive glutamine, in terms of their reactivity to transglutaminase 1. Analysis of the selected DNA pools of by next-generation sequencing and in-house bioinformatics methods revealed a detailed transglutaminase 1 substrate profile indicating preferred and non-preferred amino acid sequences. We have identified a peptide with sequence AEQHKLPSKWPF showing high reactivity to human transglutaminase 1 and low reactivity to transglutaminase 2 and transglutaminase 3. The position weight matrix consisting of per residue amino acid enrichment factors of all selected peptides was used to search human proteins by our in-house search algorithm and identify highly scoring sequence motifs in their primary structure. The search identified six already known transglutaminase 1 substrate proteins as highly scored hits as well as a list of candidate substrates that are under investigation.

bioengineering↗

bioRxiv2023

Rtf1-dependent transcriptional pausing regulates cardiogenesis

Transcriptional pause-release critically regulates cellular RNA biogenesis, yet how dysregulation of this process impacts embryonic development is not fully understood. Rtf1 is a multifunctional transcription regulatory protein involved in modulating promoter-proximal pausing of RNA polymerase II. Using zebrafish and mouse as model systems, we show that Rtf1 activity is essential for the differentiation of the myocardial lineage from mesoderm. Ablation of rtf1 impairs the formation of nkx2.5+/tbx5a+ cardiac progenitor cells, resulting in the development of embryos without cardiomyocytes. Structure-function analysis demonstrates that Rtf1s cardiogenic activity requires its Plus3 domain, which confers interaction with the pausing/elongation factor Spt5. In Rtf1-deficient embryos, the occupancy of RNA Pol II at transcription start sites was reduced relative to downstream occupancy, suggesting a reduction in transcriptional pausing. Intriguingly, attenuating pause release by pharmacological inhibition or morpholino targeting of CDK9 improved RNA polymerase II occupancy at the transcription start sites of key cardiac genes and restored cardiomyocytes in Rtf1-deficient embryos. Thus, our findings demonstrate the crucial role that Rtf1-mediated transcriptional pausing plays in controlling the precise spatio-temporal transcription programs that govern early heart development.

developmental 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

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

Atlas of fetal metabolism during mid-to-late gestation and diabetic pregnancy

Mounting evidence supports an instructive role for metabolism in stem cell fate decisions. However, much is yet unknown about how fetal metabolism changes during mammalian development and how altered maternal metabolism shapes fetal metabolism. Here, we present a descriptive atlas of in vivo fetal murine metabolism during mid-to-late gestation in normal and diabetic pregnancy. Using 13C-glucose and LC-MS, we profiled the metabolism of fetal brains, hearts, livers, and placentas harvested from pregnant dams between embryonic days (E)10.5 and 18.5. Comparative analysis of our large metabolomics dataset revealed metabolic features specific to fetal tissues developed under a hyperglycemic environment as well as metabolic signatures that may denote developmental transitions during euglycemic development. We observed sorbitol accumulation in fetal tissues and altered neurotransmitter levels in fetal brains isolated from dams with maternal hyperglycemia. Tracing 13C-glucose revealed disparate nutrient sourcing in fetuses depending on maternal glycemic states. Regardless of glycemic state, histidine-derived metabolites accumulated during late development in fetal tissues and maternal plasma. Our rich dataset presents a comprehensive overview of in vivo fetal tissue metabolism and alterations occurring as a result of maternal hyperglycemia.

cell biology↗

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

Mechanical Stimulation Shapes the Immunoregulatory Properties of Dendritic Cells

Motile cells such as immune and cancer cells experience large deformation events that result from the physical constraints they encounter while migrating within tissues or circulating between organs. It has become increasingly clear that these cells can survive and adapt to these changes in cell shape using dedicated shape sensing pathways. However, how shape sensing impacts their function and fate remains largely unknown. Here we identify a shape sensing mechanism that couples cell motility to expression of CCR7, the chemokine receptor that guides immune cells to lymph nodes. We found that this mechanism is controlled by the lipid metabolism enzyme cPLA2, requires an intact nuclear envelop and exhibits an exquisitely sensitive activation threshold tuned by ARP2/3 and its inhibitor Arpin. We further show that shape sensing through the ARP2/3-cPLA2 axis controls Ikk{beta}-NF{kappa}B-dependent transcriptional reprogramming of dendritic cells, which instructs them to migrate to lymph nodes in an immunoregulatory state compatible with their homeostatic tolerogenic function. These results highlight that the cell shape changes experienced by motile cells evolving within the complex environment of tissues can dictate their behavior and fate.

cell biology↗