Biology subjects

Yamada, S.

Publications and source records attributed to Yamada, S..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Immediate glucose signaling transmitted via the vagus nerve in gut-brain neural communication

Sucrose consumption is influenced by certain gut-brain signaling mechanisms. Among these, one pathway involves neuropod cells, which form synaptic connections with the vagus nerve, leading to the immediate activation of central dopaminergic pathways. This study explored the role of the frontal cortex in its process. We found that the vagus nerves immediate activation is mediated by the sodium-glucose cotransporter 1 (SGLT1) of neuropod cells after the intragastric glucose injection in mice. Also, we showed that the involvement of both astrocytes and neurons in the frontal cortex via D2 and D1 dopamine receptors, respectively, by in vivo Ca2+ imaging. Finally, we revealed that psychological stress, which induces a reduction in sucrose preference, significantly diminishes the activation levels of both the vagus nerve and the frontal cortex. These findings highlight the role of a comprehensive gut-brain network in modulating sucrose preference, involving neuropod cells, the vagus nerve, and the frontal cortex. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/586971v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1f28baeorg.highwire.dtl.DTLVardef@b053a9org.highwire.dtl.DTLVardef@449431org.highwire.dtl.DTLVardef@5d3ad9_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2024

Induction of NASH and vacuolar structures in the Nwd1-/- mouse liver via SERCA2-dependent endoplasmic reticulum stress

The endoplasmic reticulum (ER) stores Ca2+ and plays crucial roles in protein folding, lipid transfer, and its perturbations trigger an ER stress. In the liver, chronic ER stress is involved in the pathogenesis of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Previous studies revealed that dysfunction of sarco/endoplasmic reticulum calcium ATPase (SERCA2), a key regulator of Ca2+ transport from the cytosol to the ER, is associated with the induction of ER stress and lipid droplet formation. We previously identified NACHT and WD repeat domain-containing protein 1 (Nwd1), which is localized in the ER and mitochondria. However, the physiological significance of Nwd1 outside the central nervous system remains unclear. In this study, we revealed that Nwd1 knockout mice exhibited pathological manifestations comparable to NASH. Nwd1 interacts with SERCA2 near ER membranes. Nwd1-/- livers exhibited reduced SERCA2 ATPase activity and a smaller Ca2+ pool in the ER, leading to an exacerbated state of ER stress. These findings highlight the importance of SERCA2 activity mediated by Nwd1 in the pathogenesis of NASH. HighlightsO_LINwd1-/- mice exhibited NASH-like liver steatosis. C_LIO_LIElevated ER stress, fibrosis, and pyroptosis were observed in Nwd1-/- livers. C_LIO_LINwd1 interacts with SERCA2, an ER membrane Ca2+ pump. C_LIO_LINwd1-/- livers exhibited reduced SERCA2 activity and smaller Ca2+ pools in the ER. C_LI

molecular biology↗

bioRxiv2024

Dysregulated expanded endocannabinoid system as therapeutic targets of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the selective loss of upper and lower motor neurons. ALS patients often manifest systemic metabolic abnormalities such as glucose intolerance. Herein, to elucidate the systemic metabolic changes related to ALS progression, we performed metabolomics analysis on the serum of ALS patients and identified several metabolites associated with the disease progression, including metabolites involved in the expanded endocannabinoid system (ECS). In particular, the levels of N-acyl taurines (NAT) were correlated with the longitudinal change in the revised ALS functional rating scale (ALSFRS-R) rating. In vitro experiments with ALS cell models and in vivo studies with SOD1G93A transgenic mice revealed that PF-04457845, a fatty amide acid hydrolase (FAAH) inhibitor, up-regulated the expanded ECS, particularly the levels of NATs and N-acyl ethanolamine and ameliorates motor neuron degeneration through the regulation of microglial polarization, synapse plasticity, and neuronal development. Our study indicates that dysregulation of the expanded ECS is associated with ALS progression and a target for novel disease-modifying therapies.

neuroscience↗

bioRxiv2023

An in vivo platform to identify clinically relevant pathogenic loci using Drosophila melanogaster

Rare genetic disease discovery efforts typically lead to the identification of new disease genes. PreMIER (Precision Medicine Integrated Experimental Resources) is a collaborative platform designed to facilitate functional evaluation of human genetic variants in model systems, and to date the PreMIER Consortium has evaluated over 50 variants in patients with genetic disorders. To understand if Drosophila could be used to identify pathogenic disease loci as part of the PreMIER Consortium, we used tissue-specific gene knockdown in the fly as a proof of principle experiment. Tissue-specific knockdown of seven conserved disease genes caused significant changes in viability, longevity, behavior, motor function, and neuronal survival arguing a set of defined assays can be used to determine if a gene of uncertain significance (GUS) regulates specific physiological processes. This study highlights the utility of a tissue-specific knockdown platform in Drosophila to characterize GUS, which may provide the first genephenotype correlations for patients with idiopathic genetic disorders

physiology↗

bioRxiv2023

Energy metabolism modulates the regulatory impact of activators on gene expression

Gene expression is a regulated process fueled by ATP consumption. Therefore, regulation must be coupled to constraints imposed by the level of energy metabolism. Here, we explore this relationship both theoretically and experimentally. A stylized mathematical model predicts that activators of gene expression have variable impact depending on metabolic rate. Activators become less essential when metabolic rate is reduced and more essential when metabolic rate is enhanced. We find that in the Drosophila eye, expression dynamics of the yan gene are less affected by loss of EGFR-mediated activation when metabolism is reduced, and the opposite effect is seen when metabolism is enhanced. The effects are also seen at the level of pattern regularity in the adult eye, where loss of EGFR-mediated activation is mitigated by lower metabolism. We propose that gene activation is tuned by energy metabolism to allow for faithful expression dynamics in the face of variable metabolic conditions.

developmental biology↗

bioRxiv2023

Mechanisms of the FTO locus association with obesity: Irx3 controls a sumoylation-dependent switch between adipogenesis and osteogenesis

BackgroundIRX3 is implicated in genetic predisposition to obesity via the FTO variant locus. IRX3 shows FTO risk allele-dependent upregulation specifically during early adipogenesis, leading to a shift from energy-dissipation to fat storage in mature adipocytes. However, how changes in IRX3 expression at one developmental stage affect cellular phenotype at a later stage remains unclear. We here hypothesize that IRX3 regulates adipocyte development via transcriptional modulation of epigenetic reprogramming factors. MethodsWe combined ChIP-, ATAC- and RNA-sequencing to map direct Irx3 target genes in regions of open chromatin during early adipogenesis of wild-type and Irx3-KO preadipocytes. Gene ontology analyses was performed to identify significantly enriched biological pathways. Denaturing western blotting was used to assess sumoylation levels, and the inhibitor ML-792 was used to specifically block sumoylation. Luciferase assays were performed to estimate effects of ML-792 on Ppar{gamma} activity. Bodipy lipid staining, immunofluorescence and qPCR were employed to assess adipogenic differentiation in 3D culture. Alkaline phosphatase and Alizarine Red S staining, as well as immunofluorescence and qPCR were used to assess osteogenic differentiation in 3D culture. ResultsWe identified more than 300 Irx3 binding sites in preadipocytes, and these were almost exclusively restricted to promoter regions, with a strong enrichment of genes related to sumoylation, histone modifications and chromatin remodeling. Genes from every step of the sumoylation cycle were bound by Irx3 and differentially expressed in response to Irx3-KO, leading to increased global sumoylation levels in the KO cells. Irx3 ablation and elevated sumoylation inhibited Ppar{gamma} activity and adipogenic differentiation in preadipocytes, both of which could be restored by pharmacological inhibition of sumoylation. The Irx3-KO cells demonstrated reduced epigenetic suppression against osteogenesis, resulting in increased osteogenesis in 3D culture. Finally, osteogenesis induced by Irx3 ablation could partially be reversed by inhibition of sumoylation. ConclusionsOur study has uncovered IRX3 as a novel upstream regulator of sumoylation, and a potent controller of epigenetic regulators, both directly and indirectly via suppressing global sumoylation levels. This study indicates that the FTO locus promotes obesity via IRX3-mediated suppression of sumoylation, which promotes adipogenic commitment and differentiation through epigenetic programming. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=61 SRC="FIGDIR/small/562662v1_figu1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@115feaborg.highwire.dtl.DTLVardef@102a6c0org.highwire.dtl.DTLVardef@111bd1corg.highwire.dtl.DTLVardef@1969061_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Spatiotemporal regulation of de novo and salvage purine synthesis during brain development

The levels of purines, essential molecules to sustain eukaryotic cell homeostasis, are regulated by the coordination of the de novo and salvage synthesis pathways. In the embryonic central nervous system (CNS), the de novo pathway is considered crucial to meet the requirements for the active proliferation of neural stem/progenitor cells (NSPCs). However, how these two pathways are balanced or separately utilized during CNS development remains poorly understood. In this study, we showed a dynamic shift in pathway utilization, with greater reliance on the de novo pathway during embryonic stages and on the salvage pathway at postnatal-adult stages. The pharmacological effects of various purine synthesis inhibitors in vitro and the expression profile of purine synthesis enzymes indicated that NSPCs in the embryonic cerebrum mainly utilize the de novo pathway. Simultaneously, NSPCs in the cerebellum require both the de novo and the salvage pathways. In vivo administration of de novo inhibitors resulted in severe hypoplasia of the forebrain cortical region, indicating a gradient of purine demand along the anteroposterior axis of the embryonic brain, with cortical areas of the dorsal forebrain having higher purine requirements than ventral or posterior areas such as the striatum and thalamus. This histological defect of the neocortex was accompanied by strong downregulation of the mechanistic target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase (S6K)/S6 signaling cascade, a crucial pathway for cell metabolism, growth, and survival. These findings indicate the importance of the spatiotemporal regulation of both purine pathways for mTORC1 signaling and proper brain development. Significance StatementBrain development requires a balance of de novo and salvage purine synthetic pathways. However, the utilization of these pathways during brain development remains poorly understood. This study provides evidence that the spatiotemporal regulation of these two purine synthesis pathways is essential for normal brain development. We revealed that inhibition of de novo purine synthesis results in the downregulation of mammalian/mechanistic target of rapamycin (mTOR) signaling, leading to malformations in specific embryonic brain regions such as the cerebral neocortex. These results suggest a temporal and spatial gradient of purine demand during embryonic brain development. These findings could improve our understanding of neurological diseases caused by defects in purine metabolism.

neuroscience↗

bioRxiv2022

Phase transition of tensin-1 during the focal adhesion disassembly and cell division

Biomolecular condensates are non-membranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here we report that GFP-tagged tensin-1 (TNS1) proteins at physiological levels phase separate to form biomolecular condensates in TNS1 knockout cells. Live cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while post-mitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.

cell biology↗

bioRxiv2022

Impact of reinfection with SARS-CoV-2 Omicron variants in previously infected hamsters

The diversity of SARS-CoV-2 mutations raises the possibility of reinfection of individuals previously infected with earlier variants, and this risk is further increased by the emergence of the B.1.1.529 Omicron variant. In this study, we used an in vivo, hamster infection model to assess the potential for individuals previously infected with SARS-CoV-2 to be reinfected with Omicron variant and we also investigated the pathology associated with such infections. Initially, Syrian hamsters were inoculated with a lineage A, B.1.1.7, B.1.351, B.1.617.2 or a subvariant of Omicron, BA.1 strain and then reinfected with the BA.1 strain 5 weeks later. Subsequently, the impact of reinfection with Omicron subvariants (BA.1 and BA.2) in individuals previously infected with the BA.1 strain was examined. Although viral infection and replication were suppressed in both the upper and lower airways, following reinfection, virus-associated RNA was detected in the airways of most hamsters. Viral replication was more strongly suppressed in the lower respiratory tract than in the upper respiratory tract. Consistent amino acid substitutions were observed in the upper respiratory tract of infected hamsters after primary infection with variant BA.1, whereas diverse mutations appeared in hamsters reinfected with the same variant. Histopathology showed no acute pneumonia or disease enhancement in any of the reinfection groups and, in addition, the expression of inflammatory cytokines and chemokines in the airways of reinfected animals was only mildly elevated. These findings are important for understanding the risk of reinfection with new variants of SARS-CoV-2. IMPORTANCEThe emergence of SARS-CoV-2 variants and the widespread use of COVID-19 vaccines has resulted in individual differences in immune status against SARS-CoV-2. A decay in immunity over time and the emergence of variants that partially evade the immune response can also lead to reinfection. In this study, we demonstrated that, in hamsters, immunity acquired following primary infection with previous SARS-CoV-2 variants was effective in preventing the onset of pneumonia after reinfection with the Omicron variant. However, viral infection and multiplication in the upper respiratory tract were still observed after reinfection. We also showed that more diverse nonsynonymous mutations appeared in the upper respiratory tract of reinfected hamsters that had acquired immunity from primary infection. This hamster model reveals the within-host evolution of SARS-CoV-2 and its pathology after reinfection, and provides important information for countermeasures against diversifying SARS-CoV-2 variants.

microbiology↗

bioRxiv2022

Plap-1/Aspn lineage tracing and single-cell transcriptomics reveals cellular dynamics in the periodontal ligament

Periodontal tissue supports teeth in the alveolar bone socket via fibrous attachment of the periodontal ligament (PDL). The PDL contains periodontal fibroblasts and stem/progenitor cells, collectively known as PDL cells (PDLCs), on top of osteoblasts and cementoblasts on the surface of alveolar bone and cementum, respectively. However, the characteristics and lineage hierarchy of each cell type remain poorly defined. This study identified periodontal ligament associated Protein-1 (Plap-1/Aspn) as a PDL-specific extracellular matrix. We generated knock-in mice expressing CreERT2 and GFP specifically in Plap-1-positive PDLCs. Genetic lineage tracing confirmed the long-standing hypothesis that PDLCs differentiated into osteoblasts and cementoblasts. A PDL single-cell atlas defined cementoblasts and osteoblasts as Plap-1-Ibsp+Sparcl1+ and Plap-1-Ibsp+Col11a2+, respectively. Other populations such as Nes+ mural cells, S100B+ Schwann cells, and other non-stromal cells were also identified. RNA velocity analysis suggested that Plap-1highLy6a+ cell population was the source of PDLCs. Lineage tracing of Plap-1+ PDLCs during the periodontal injury model showed periodontal tissue regeneration by PDLCs. Our study defines diverse cell populations in PDL and clarifies the role of PDLCs in periodontal tissue homeostasis and repair.

developmental biology↗

bioRxiv2022

Diagnosis and early prediction of lymphoma using high-throughput clonality analysis of bovine leukemia virus-infected cells

Bovine leukemia virus (BLV), a retrovirus, infects into B cells of ruminants and causes aggressive leukemia or lymphoma in cattle, enzootic bovine leukosis (EBL). Clonal expansion of BLV-infected cells is a promising marker for early detection and diagnosis of EBL. Recently, we developed rapid amplification of the integration site without interference by genomic DNA contamination (RAISING) and CLOVA, a software to analyze clonality. RAISING-CLOVA could assess the risk of adult T-cell leukemia/lymphoma development in human T-cell leukemia virus-I-infected individuals through its clonality analysis. Thus, we herein examined the performance of RAISING-CLOVA for the clonality analysis of BLV-infected cells and conducted a comprehensive clonality analysis by RAISING-CLOVA in EBL and non-EBL cattle. RAISING-CLOVA successfully distinguished EBL from non-EBL cattle with high sensitivity and specificity. A longitudinal clonality analysis in BLV-infected sheep, an EBL model, also confirmed the effectiveness of BLV clonality analysis with RAISING-CLOVA for early detection of EBL development. Therefore, our study emphasizes the usefulness of RAISING-CLOVA as a routine clinical test for monitoring virus-related cancers.

microbiology↗

bioRxiv2022

TDP 2 modulates the expression of estrogen-responsive oncogenes

With its ligand estrogen, the estrogen receptor (ER) stimulates tumor cell growth by activating a global transcriptional program. This activation involves topoisomerase 2 (TOP2), which resolves topological problems by transiently creating and re-ligating DNA double-strand breaks (DSBs). Recent studies have uncovered the involvement of DNA repair proteins in the repair of TOP2-induced DSBs. These noncanonical repair pathways may serve as backup processes when TOP2 is halted and fails to re-ligate DSBs, but their impact on transcription remains elusive. In this study, we investigated the role of tyrosyl-DNA phosphodiesterase 2 (TDP2), an enzyme that acts for the removal of halted TOP2 from the 5-end of the DNA, in the estrogen-induced transcriptome. Using TDP2-deficient ER-positive cells and mice, we showed that TDP2 regulates the expression of oncogene MYC. MYC induction by estrogen was a very early event (1 hour) and TOP2-dependent. In TDP2-deficient cells, the induction of MYC by estrogen became prolonged and volatile. Bulk and single-cell RNA-seq identified the oncogenes MYC and CCND1 as genes whose estrogen response was regulated by TDP2. These results suggest that TDP2 may play a role in the repair of TOP2-induced DSBs in specific genomic loci and tightly regulates the expression of oncogenes.

molecular biology↗

bioRxiv2022

Inka2, a novel Pak4 inhibitor, regulates actin dynamics in dendritic spine development

The actin filament is a fundamental part of the cytoskeleton defining cell morphology and regulating various physiological processes, including filopodia formation and dendritic spinogenesis of neurons. Serine/threonine-protein kinase Pak4, an essential effector, links Rho GTPases to control actin polymerization. Previously, we identified the Inka2 gene, a novel mammalian protein exhibiting sequence similarity to Inka1, which serves as a possible inhibitor for Pak4. Although Inka2 is dominantly expressed in the nervous system and involved in focal-adhesion dynamics, its molecular role remains unclear. Here, we found that Inka2-iBox directly binds to Pak4 catalytic domain to suppress actin polymerization. Inka2 promoted actin depolymerization and inhibited the formation of cellular protrusion caused by Pak4 activation. We further generated the conditional knockout mice of the Inka2 gene. The beta-galactosidase reporter indicated the preferential Inka2 expression in the dorsal forebrain neurons. Cortical pyramidal neurons of Inka2-/- mice exhibited decreased density and aberrant morphology of dendritic spines with marked activation/phosphorylation of downstream molecules of Pak4 signal cascade, including LIMK and cofilin. These results uncovered the unexpected function of endogenous Pak4 inhibitor in neurons. Unlike Inka1, Inka2 is a critical mediator for actin reorganization required for dendritic spine development.

neuroscience↗

bioRxiv2022

Uncertainty quantification in the cerebral circulation simulation focusing on the collateral flow: Surrogate model approach with machine learning

Collateral circulation in the circle of Willis (CoW), closely associated with disease mechanisms and treatment outcomes, can be effectively investigated using one-dimensional-zero-dimensional hemodynamic simulations. As the entire cardiovascular system is considered in the simulation, it captures the systemic effects of local arterial changes, thus reproducing collateral circulation that reflects biological phenomena. The simulation facilitates rapid assessment of clinically relevant hemodynamic quantities under patient-specific conditions by incorporating clinical data. During patient-specific simulations, the impact of clinical data uncertainty on the simulated quantities should be quantified to obtain reliable results. However, as uncertainty quantification (UQ) is time-consuming and computationally expensive, its implementation in time-sensitive clinical applications is considered impractical. Therefore, we constructed a surrogate model based on machine learning using simulation data. The model accurately predicts the flow rate and pressure in the CoW in a few milliseconds. This reduced computation time enables the UQ execution with 100 000 predictions in a few minutes on a single CPU core and in less than a minute on a GPU. We performed UQ to predict the risk of cerebral hyperperfusion (CH), a life-threatening condition that can occur after carotid artery stenosis surgery if collateral circulation fails to function appropriately. We predicted the statistics of the postoperative flow rate increase in the CoW, which is a measure of CH, considering the uncertainties of arterial diameters, stenosis parameters, and flow rates measured using the patients clinical data. A sensitivity analysis was performed to clarify the impact of each uncertain parameter on the flow rate increase. Results indicated that CH occurred when two conditions were satisfied simultaneously: severe stenosis and when arteries of small diameter serve as the collateral pathway to the cerebral artery on the stenosis side. These findings elucidate the biological aspects of cerebral circulation in terms of the relationship between collateral flow and CH. Author summaryCerebral arteries generate a ring-like network that provides alternative routes for blood supply in the case of carotid artery stenosis. This collateral circulation is closely associated with the potential risk of stroke and treatment outcomes in patients with stenosis. In this study, we propose a method to elucidate the cerebral circulation of individual patients using a blood flow simulation that incorporates the patients clinical data. A key feature of our approach is its capability to obtain the probability of the different outputs using simulation, considering the uncertainty of patient conditions. Although this capability is essential for obtaining reliable results, the process is time-consuming and requires numerous computer resources. We solved this problem by combining the blood flow simulation with machine learning to perform predictions 43 000 times faster than conventional simulations. We applied the proposed method to predict cerebral circulation following surgery in three patients with stenosis and verified that the method can assess the surgical risk almost in real-time, even on a desktop computer. Additionally, extensive prediction results (100 000 cases for each patient) obtained using this method clarify the relationship between collateral circulation and life-threatening surgical outcomes.

bioengineering↗

bioRxiv2022

Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergences impact on choosing optimal drug treatment.

microbiology↗

bioRxiv2022

Triple-helix potential of the mouse genome

Certain DNA sequences, including mirror-symmetric polypyrimidine/polypurine runs, are capable of folding into a triple-helix-containing non-B-form DNA structure called H-DNA. Such H-DNA-forming sequences are frequent in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)-specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally-positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides a new approach for studying DNA secondary structure genome wide at high spatial resolution.

molecular biology↗

bioRxiv2021

Cell Adhesion-Dependent Biphasic Axon Outgrowth Elucidated by Femtosecond Laser Impulse

Axon outgrowth is promoted by the mechanical coupling between F-actin and adhesive substrates via clutch and adhesion molecules in an axonal growth cone. In this study, we utilized a femtosecond laser-induced impulse to break the coupling between the growth cone and the substrate, enabling us to evaluate the strength of the binding between the growth cone and a laminin on the substrate, and also determine the contribution of adhesion strength to neurite outgrowth and traction force for the outgrowth. We found that the adhesion strength of axonal L1 cell adhesion molecule (L1CAM)-laminin binding increased with the laminin density on the substrate. In addition, fluorescent speckle microscopy revealed that the retrograde flow of F-actin in the growth cone was dependent on the laminin density such that the flow speed reduced with increasing L1CAM-laminin binding. However, neurite outgrowth and the traction force did not increase monotonically with increased L1CAM-laminin binding but rather exhibited biphasic behavior, in which the outgrowth was suppressed by excessive L1CAM-laminin binding. Our quantitative evaluations suggest that the biphasic outgrowth is regulated by the balance between traction force and adhesion strength. These results imply that adhesion modulation is key to the regulation of neurite outgrowth.

cell biology↗

bioRxiv2021

Guided Axon Outgrowth of Neurons by Real-Time Femtosecond Laser Penetration on a 4 μm Thick Thin-Glass Sheet

Transplantation of scaffold-embedded guided neurons has been reported to increase neuronal regeneration following brain injury. However, precise axonal integration between host and transplant neurons to form functional synapses remains a major problem. This study aims to develop a real-time femtosecond (fs) laser penetration on a 4 m thick thin-glass sheet to promote guided axon outgrowth influenced by molecular gradients in a microfluidic device. The device enables the introduction of the guidance molecule (i.e., netrin-1), neuronal culture, and manipulation by fs laser. After fabricating multiple micro-holes on the thin-glass sheet using fs laser, netrin-1 gradients with radial concentrations are generated in the chamber, affecting axon outgrowth and guidance. A majority of axons (~92%) experiences guided outgrowth with positive angular changes towards netrin-1 gradients. These results demonstrate the capability of the precise and real-time manipulation system based on a fs laser and a microfluidic device to control the growth of neurons.

bioengineering↗