Biology subjects

Nakamura, A.

Publications and source records attributed to Nakamura, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Sex peptide-SPR-serotonin signaling regulates mating-induced Sex peptide replenishment in Drosophila, suggesting an ancestral role in males

Seminal fluid not only assists successful fertilization but also induces changes in female post-mating behavior and physiology. Consequently, males with depleted or inadequate seminal fluid produce fewer offspring. Males of iteroparous species therefore increase seminal fluid production to replenish exhausted supplies. However, it remains unclear how mating-induced replenishment of seminal fluid components is regulated at the molecular and cellular levels. Here, we show that replenishment of Sex peptide (SP) in male Drosophila melanogaster requires both SP and its receptor SPR. Mechanistically, mating-induced SP replenishment in the male accessory gland (MAG) is regulated by MAG-projecting serotonergic neurons, and this regulation depends on SPR, which is likely activated by SP as it passes through the reproductive tract during mating. This inter-tissue signaling pathway is not limited to SP but also coordinates the post-mating upregulation of an additional seminal fluid protein gene. While SP-SPR signaling is a well-known regulator of female post-mating responses, we demonstrate that it also modulates male physiology. Furthermore, comparative analyses across 12 drosophilid species are consistent with the hypothesis that SP-SPR signaling acquired a role in males before its role in canonical female post-mating responses evolved. We therefore propose that the role of SP-SPR signaling as a mating signal in males is ancestral to its more familiar but evolutionarily derived role in females.

physiology↗

bioRxiv2026

Biosurfactant polysaccharide drives dispersal of exopolysaccharide-coated myxospores in Myxococcus xanthus

Bacterial spores are highly resilient cell forms, yet the organization of their protective surface layers in diderm species remains largely unexplored. For the social predatory soil bacterium Myxococcus xanthus, myxospore formation within multicellular fruiting body structures is a key ecological adaptation to surviving conditions of nutrient deprivation. These myxospores were long thought to be outwardly coated with so-called major spore coat (MASC) polysaccharide. Using polysaccharide-deficient mutant strains, electron microscopy, surface hydrophobicity assays, dye binding, and functional swarm complementation, we show that MASC instead forms a protective sub-surface outer-cortex layer, while the exopolysaccharide (EPS) glycocalyx inherited from vegetative cells constitutes the true outermost myxospore coat. EPS mediates inter-myxospore adhesion and supports collective behaviors, whereas secretion of biosurfactant polysaccharide (BPS) reduces myxospore surface stickiness, preventing excessive aggregation. BPS-deficient myxospores form persistent clumps, while EPS-deficient myxospores fail to aggregate or restore EPS-dependent swarm motility of vegetative cells. These results reveal a mechanistic interplay in which MASC serves as a sub-surface protective layer, EPS defines the exterior interface, and BPS tunes surface accessibility to regulate dispersal. Our findings overturn long-standing assumptions about myxospore architecture and illustrate a polysaccharide-driven strategy for controlling aggregation and readiness for germination. More broadly, these results highlight how social diderm bacteria leverage inherited and dynamically remodeled polysaccharide surfaces to coordinate multicellular development, in contrast to the rigid protein-based spore surfaces of Gram-positive species.

microbiology↗

bioRxiv2026

High-speed atomic force-Raman microscopy

A comprehensive understanding of the mechanisms underlying biological systems requires correlative analysis of multiple complementary molecular properties through multidimensional measurements. High-speed atomic force microscopy (HS-AFM) is a powerful tool for elucidating biomolecular structural dynamics at the single-molecule level with high spatiotemporal resolution. However, structural information alone is often insufficient for fully understanding the biological function mechanisms. Here, we report high-speed atomic force-Raman microscopy (HS-AFRM), which enables multimodal measurements combining video-rate structural imaging with chemical-bond analysis. Raman spectroscopy is a powerful, non-invasive technique that probes molecular vibrations to provide chemical information. We achieved several key technical developments that facilitated the seamless integration of HS-AFM and micro-Raman spectroscopy, allowing reliable correlative measurements of structural and chemical information. We validated the versatility of the developed system using representative samples, including two-dimensional materials and a protein. Furthermore, we demonstrate probing of changes in the surrounding environment, which are inaccessible by HS-AFM alone. Multimodal measurements incorporating fluorescence spectroscopy were also demonstrated as an additional practical extension. This multimodal approach substantially enhances the analytical capability of HS-AFM, providing a powerful platform for revealing correlated structural and chemical properties across diverse research fields.

biophysics↗

bioRxiv2026

Neural mechanism of postural sway-related beta-band oscillations: a cortico-basal ganglia-thalamic network model of intermittent control

Electroencephalographic (EEG) studies of human quiet stance demonstrate beta-band event-related desynchronization (beta-ERD) during the micro-fall phase of postural sway, followed by event-related synchronization (beta-ERS; post-movement beta rebound) during the micro-recovery phase. These modulations correlate with intermittent ankle muscle inactivation that exploits the stable manifolds of an unstable upright equilibrium; however, the underlying neurocircuit mechanisms remain elusive. Here, we investigated this rhythmogenesis using an embodied spiking neural network model of the cortico-basal ganglia-thalamic (CBGT) circuitry integrated with a physical inverted pendulum. In this closed-loop system, continuous sensory feedback is integrated into the striatum, while the motor cortex executes decisions via drift-diffusion-like population competition, where the decision time (DT) represents the intermittent control-off period. We demonstrate that simulated cortical LFPs exhibit characteristic sway-phase-locked beta-ERD and beta-ERS exclusively when corticostriatal synaptic weights are functionally balanced to implement intermittent control. Conversely, forced-choice continuous control that ceaselessly generates feedback torque fails to replicate these modulations, sustaining flat network states devoid of control-off periods (DT). Structural dissections reveal that while sensory drive remains continuous, sway-phase-locked beta modulations are an emergent property generated fundamentally by bidirectional thalamocortical loops and the intrinsic dynamics of the GPe-STN pacemaker circuit. Our findings suggest that CBGT-mediated, sway-phase-locked beta activity serves as a hallmark of healthy intermittent motor selection. This computational framework provides a crucial bridge linking pathological alterations in basal ganglia dynamics and the loss of behavioral intermittency to the postural impairments observed in clinical populations such as Parkinsons disease. Significance StatementDuring quiet standing, human cortical beta oscillations dynamically modulate in phase with postural sway. Although this phenomenon is heavily linked to healthy motor control, the underlying neural mechanisms have remained unknown. This study resolves this ambiguity by developing a closed-loop computational model combining physical body dynamics with a cortico-basal ganglia-thalamic network. We demonstrate that human-like cortical beta modulations naturally emerge as an architectural property under an intermittent control strategy, driven fundamentally by the GPe-STN pacemaker circuit. Crucially, transitioning to a continuous control regime completely abolishes these oscillations. This framework establishes a direct neurocomputational link between the loss of behavioral intermittency and the pathological degradation of beta-band dynamics observed in patients with Parkinsons disease.

neuroscience↗

bioRxiv2026

Impaired Motor Awareness of Balance Control is Associated with Postural Instability in Parkinson's Disease

BackgroundBalance instability is a major contributor to disability and falls in people with Parkinsons disease (PwP) and is often insufficiently explained by motor impairment alone. Altered awareness of motor control has been suggested to contribute to sensorimotor dysfunction in PwP, but its relationship with balance performance is poorly understood. ObjectiveTo determine whether awareness of balance control, assessed using a control detection task (CDT), differs between healthy controls (HC) and PwP, and whether CDT performance is associated with balance-related measures. MethodsHealthy older adults (n=20) and PwP (n=22) performed a standing version of the CDT based on center-of-pressure (COP) control, using a force plate. CDT accuracy was used as the primary outcome measure. Static balance during quiet standing was assessed using the COP trajectory length and rectangular area. Dynamic standing balance was assessed using the Index of Postural Stability (IPS). Group differences were examined by independent-samples t-tests. Correlations between CDT accuracy and balance measures were analyzed. ResultsThe PwP group showed significantly lower CDT accuracy. Higher CDT accuracy was associated with better static balance in the HC group and the combined sample, and with higher IPS primarily in the PwP group. ConclusionsMotor awareness during postural tasks is altered in PwP and is associated with balance control. These findings suggest that balance instability in Parkinsons disease may involve altered balance-related action-outcome monitoring in addition to motor dysfunction.

neuroscience↗

bioRxiv2026

Transfer of symbolic numeral adaptation across eyes and hemifields

Visual perception of symbolic numerals is essential for everyday tasks; however, the neural and perceptual mechanisms underlying this ability remain unclear. Partially occluded digital numerals can elicit bistable perception, and adaptation to symbolic numerals alters the perception of these ambiguous stimuli. We aimed to examine how symbolic numeral adaptation is related to hierarchical visual processing by testing its interocular and interhemifield transfer. Experiment 1 tested interocular transfer by presenting the test stimulus to either the same or opposite eye as the adaptation stimulus. Experiment 2 assessed interhemifield transfer by presenting the test stimulus to either the same or opposite hemifield as the adaptation stimulus. Experiment 3 examined the interhemifield transfer of adaptation confined to the upper parts of digital numerals. Our results showed that adaptation to digital numerals induced shifted perceptual interpretations that transferred across eyes. In addition, we found that adaptation to digital numerals induced a relatively small but statistically significant interhemifield transfer. In contrast, adaptation restricted to the upper parts of digital numerals showed no significant interhemifield transfer. These findings suggest that the perceptual interpretation of symbolic numerals involves visual processing stages that integrate information across the eyes and hemifields.

neuroscience↗

bioRxiv2025

A dual-domain chitinase mechanism enables marine bacteria tosense and localize sparse crystalline chitin in the ocean

Chitin is one of the most abundant biopolymers in the ocean, and its remineralisation underpins major pathways of marine carbon cycling. A central challenge for chitinolytic bacteria is to locate and remain associated with sparsely distributed crystalline chitin particles. Here, we show that the GH18 chitinase VpChi1 from Vibrio parahaemolyticus possesses a dual-domain architecture that enhances particle encounter and retention. Full-length VpChi1 displayed much higher catalytic efficiency than a CBD-truncated mutant at low chitin concentrations, whereas substrate inhibition occurred at higher concentrations. Single-molecule fluorescence imaging demonstrated that the additional C-terminal chitin-binding domain (CBD) enables selective binding to the hydrophobic surface of crystalline chitin and substantially prolongs enzyme residence time. Kinetic simulations using experimentally determined dissociation rates showed that this extended residence arises from CBD-assisted rebinding of the catalytic domain. High-speed atomic force microscopy confirmed that the CBD does not alter stepping velocity or travel distance along individual crystals, indicating that substrate inhibition likely results from CBD engagement with neighbouring particles rather than impaired motility. Screening of seawater and marine sediments across multiple depths identified Vibrio and Pseudoalteromonas species as dominant chitin degraders, many encoding GH18 chitinases with dual CBDs, whereas terrestrial bacteria generally lacked this architecture. We propose that this two-domain configuration confers an ecological advantage by increasing enzyme retention on particulate chitin and enabling sustained production of chitobiose, a potent chemotactic signal for marine bacteria. These findings reveal a mechanistic basis for how bacteria sense and exploit particulate chitin and highlight an adaptive strategy for resource acquisition in the ocean.

molecular biology↗

bioRxiv2025

MAP7-driven cytoskeletal cross-talk establishes Sertoli cell polarity crucial for germ cell meiotic progression

How specialized apical membrane domains are assembled in vivo remains poorly understood. In the mouse testis, Sertoli cells build a luminal apical domain after birth that organizes the seminiferous epithelium and supports germ cell differentiation, distinct from fetal polarity established during cord formation. Here, we identify the microtubule-associated protein MAP7 as a key regulator of cytoskeletal organization during apical domain formation. MAP7 preferentially localizes to apical microtubules in Sertoli cells, showing limited overlap with stable microtubule markers. Native-tissue imaging indicates that MAP7-decorated microtubules, initially lacking obvious directionality, become increasingly aligned along the tubule axis as the apical domain matures. In Map7-deficient testes, microtubule abundance is maintained, but higher-order organization is disrupted and accompanied by persistent luminal F-actin accumulation. These defects compromise apical domain maturation and lumen formation during the first wave of spermatogenesis and are associated with altered tight-junction patterning. Proteomic analysis identifies the non-muscle myosin II heavy chains MYH9 and MYH10 among MAP7-associated proteins. MYH9 becomes enriched at luminal regions where microtubules and F-actin converge, but remains diffuse in Map7-deficient Sertoli cells despite ectopic luminal cytoskeletal assemblies, suggesting that MAP7 helps establish a cytoskeletal context that supports spatially restricted NMII enrichment. Single-cell RNA sequencing shows that Sertoli transcriptional differentiation largely proceeds without MAP7, whereas germ cells exhibit delayed meiotic progression with expansion of pachytene-stage subpopulations. Together, these findings establish MAP7-dependent microtubule remodeling as an organizing mechanism for postnatal apical domain maturation and suggest how epithelial cytoskeletal architecture shapes a niche that helps pace developmental progression of neighboring germ cells.

cell biology↗

bioRxiv2025

NudC moonlights in ribosome biogenesis and homeostasis in Drosophila melanogaster polyploid cells

Ribosomes, the cellular machinery responsible for protein synthesis, are fundamental across all kingdoms of life. Disruption in ribosome biogenesis (RiBi) can cause severe ribosomopathies, underscoring the critical need for precise regulatory mechanisms governing RiBi. In this study, we identified the gene NudC (nuclear distribution C, dynein complex regulator), a previously unrecognized regulator of RiBi in polyploid cells of Drosophila melanogaster larvae. RNAi-mediated depletion of NudC in polyploid salivary gland cells led to a significant reduction in ribosome abundance, accompanied by the loss of ribosome-binding sites on rough endoplasmic reticulum and impaired translation. These defects are linked to decreased levels of nucleolar ribosomal RNA. Notably, NudC knockdown also triggered a homeostatic response, characterized by increased transcription and translation of both ribosome biogenesis factors (RBFs) and ribosomal proteins. This response parallels that seen in RBF-deficient cells, suggesting that NudC and RBFs cooperate to maintain RiBi homeostasis. Meanwhile, NudC-deficient cells exhibited chromosome abnormalities, activated JNK signaling, and underwent autophagy, closely resembling the defects observed upon loss of RBFs. Finally, our findings suggest that the role of NudC in RiBi is independent of its established function in dynein regulation, indicating the moonlighting role in RiBi played by this gene. Together, these results uncover a new, fundamental function for NudC in maintaining RiBi and homeostasis in polyploid cells, with broader implications for understanding conserved mechanisms of NudC function and RiBi across species.

cell biology↗

bioRxiv2025

The I-BAR domain protein baiap2l1a is required for protrusion and lateral elongation of epithelial microridge structures

Microridges are laterally elongated membrane protrusions from the apical surface of epithelial cells. Microridges are arranged in striking maze-like patterns. They are found on various mucosal epithelia in many animals, including the skin of zebrafish, where they are required to maintain mucus on the skin surface. Recent studies have revealed molecular mechanisms of how microridiges formation involving actin and actin-regulatory proteins. However, the molecular mechanism that deforms epithelial membranes to create microridge protrusions remain unknown. We have found that one of the I-BAR domain proteins, baiap2l1a, which is known to regulate membrane curvature, is required for microridge morphogenesis. CRISPR/Cas9 knockdown showed that baiap2l1a mutant zebrafish had defects in microridge morphogenesis. Baiap2l1a mutant zebrafish had shorter and wider microridges than WT microridges. Baiap2l1a localized to microridges, and its localization proceeded microridge actin formation. Furthermore, the baiap2l1a I-BAR domain, which binds and curves membranes, was sufficient to localize to microridges in zebrafish skin cells. Structure/function experiments revealed that the I-BAR domain alone could partially rescued microridge length in baiap2l1a mutants. A 39 amino acid deletion in the I-BAR domain, which caused the loss of one -helix according to AlphaFold2 simulations, is sufficient to impair microridge localization and failed to rescue microridge elongation in baiap2l1a mutants. These results suggest that the I-BAR domain is required for baiap2l1a microridge localization and function. Eps8like1a, a member of the Eps8 family proteins known as an actin capping and bundling protein genetically interacted with baiap2l1a in microridge elongation. Together, we found that the membrane curvature protein baiap2l1a plays an important role in generating microridges in zebrafish epithelia.

cell biology↗

bioRxiv2025

Faster relaxation of nonphotochemical quenching (NPQ) in C4 than in C3 species

Acceleration of photoprotective non-photochemical quenching (NPQ) responses to changes in light intensity has been suggested as a strategy to enhance crop yield. Despite many key crops utilising C4 photosynthesis, our current understanding of NPQ overwhelmingly comes from C3 species. Using a series of experiments on three phylogenetically controlled C3 and C4 comparisons, we show that NPQ relaxation is faster in C4 species. Temporal analysis of NPQ relaxation in leaves infiltrated with inhibitors to block proton motive force formation or xanthophyll de-epoxidation showed that the faster relaxation observed in C4 species is driven by a greater contribution of energy-dependent quenching (qE) to overall NPQ. We show that the C4-associated enhancement of qE is linked to altered regulation of lumen pH in C4 species, reflecting increases in cyclic electron flow and membrane proton conductivity to meet the increased ATP demands of the C4 pathway. Indeed, in two of the three tested C4 species, NPQ relaxation became significantly slower and statistically indistinguishable from paired C3 species when ATP and NADPH consumption was suppressed by performing measurements in CO2-free air. Altogether, our results suggest that NPQ responses in C4 species may already be optimised to maintain high photosynthetic efficiency in the fluctuating light conditions typically found within C4 canopies. Given the intrinsically faster NPQ in C4 photosynthesis, further acceleration of NPQ may have limited scope to enhance crop photosynthetic efficiency. Significance StatementAcceleration of non-photochemical quenching has been proposed as a means to enhance crop photosynthetic efficiency in C3 species but whether this strategy has potential in C4 species, which include several major crops, remains unclear. We use three phylogenetically paired C3 and C4 species to show that NPQ relaxation is significantly faster in species with the C4 pathway, possibly aiding the maintenance of photosynthetic efficiency in fluctuating light environments. As a result, accelerating the rate of NPQ relaxation in C4 crops may have a more limited scope to enhance photosynthesis.

plant biology↗

bioRxiv2025

Global Identification of Highly Expandable and Shrinkable Proteins via Intramolecular Distance Scoring Analysis

Experimental and analytical characterizations of protein structural changes are essential for understanding various biological functions. It is well known that each protein or protein complex undergoes shape changes in a specific manner and to a certain extent, influenced by factors such as molecular interactions and the solvent environment. To enable quantitative comparisons of shape changes among multiple proteins, we previously introduced an index called the "UnMorphness Factor" (UMF), which is derived using a simple, original method-- intramolecular distance scoring analysis (DSA). Using this approach, we have accumulated data for over 15,000 proteins. Here, we show that among the proteins exhibiting significant structural changes, some are found to undergo substantial isotropic volume expansion or contraction. The uniformity of three-dimensional size changes can be represented by a linear correlation between the average and standard deviation of all intramolecular C-C distances across a proteins structural set. Packing analysis of such proteins reveals that the void volume within the same polypeptide chain can increase by up to 50% relative to the reference state. Our study clearly demonstrates that this type of structural change plays a crucial role in understanding the detailed processes of ligand binding, catalytic reaction cycles, and protein folding pathways.

biophysics↗

bioRxiv2025

Gamma-aminobutyric acid treatment reduces estradiol-stimulated breast cancer cell growth and estradiol-induced breast tumorigenesis

Gamma-aminobutyric acid (GABA) is a non-protein amino acid and contained in various foods, such as fruits and vegetables. Although various health benefits of GABA have been reported, GABA seems to have unidentified functions. In this study, we first show increase in intracellular GABA level in GABA-treated breast cancer cell lines, MCF-7 and T-47D, in amino acid analyses and immunofluorescence. In our experimental condition, GABA treatment reduced estradiol-stimulated cell growth in these cell lines. In addition, we performed mouse early breast cancer induction model system to investigate GABA function in mammary dysplasia formation. In the results, GABA administration reduced dysplasia formation, suggesting that GABA prevents breast tumorigenesis. This study demonstrates a novel function of GABA to maintain normal breast tissues.

bioengineering↗

bioRxiv2024

Stress-induced organismal death is genetically regulated by the mTOR-Zeste-Phae1 axis

All organisms are exposed to various stressors, which can sometimes lead to organismal death, depending on their intensity. While stress-induced organismal death has been observed in many species, the underlying mechanisms remain unclear. In this study, we investigated the molecular mechanisms of stress-induced organismal death in the fruit fly Drosophila melanogaster. We identified a chymotrypsin-like serine protease Phaedra1 (Phae1) as a death mediator in D. melanogaster larvae. Phae1 expression was upregulated by lethal heat stress (40 {degrees}C) but not non-lethal heat stress (38 {degrees}C or lower). The most prominent induction of Phae1 occurred in the central nervous system (CNS). We found neuro-specific knockdown of Phae1 increased survival and reduced neuronal caspase activity following exposure to lethal heat stress, suggesting that the transcriptional upregulation of Phae1 in the CNS is essential for stress-induced organismal death. We next found via bioinformatic and biochemical analyses that the transcription factor Zeste (Z) bound the Phae1 enhancer region and that z loss-of-function impaired Phae1 induction in the CNS, increasing survival following lethal heat stress. In addition, we found via chemical screening that rapamycin, a chemical inhibitor of mechanistic target of rapamycin (mTOR), suppressed Phae1 expression. Neuro-specific knockdown of mTor reduced the protein levels of both Phae1 and Z, leading to an increase in survival following lethal heat stress. Together, these results indicate that heat stress-induced organismal death in D. melanogaster larvae is regulated by a genetically encoded transcriptional signaling pathway involving the mTOR-Z-Phae1 axis.

physiology↗

bioRxiv2024

Secretory leukocyte protease inhibitor influences periarticular joint inflammation in B. burgdorferi-infected mice

Lyme disease, caused by Borrelia burgdorferi, is the most common tick-borne infection in the United States. Arthritis is a major clinical manifestation of infection, and synovial tissue damage has been attributed to the excessive pro-inflammatory responses. The secretory leukocyte protease inhibitor (SLPI) promotes tissue repair and exerts anti-inflammatory effects. The role of SLPI in the development of Lyme arthritis in C57BL/6 mice, which can be infected with B. burgdorferi, but only develop mild joint inflammation, was therefore examined. SLPI-deficient C57BL/6 mice challenged with B. burgdorferi had a higher infection load in the tibiotarsal joints and marked periarticular swelling, compared to infected wild type control mice. The ankle joint tissues of B. burgdorferi-infected SLPI-deficient mice contained significantly higher percentages of infiltrating neutrophils and macrophages. B. burgdorferi-infected SLPI-deficient mice also exhibited elevated serum levels of IL-6, neutrophil elastase, and MMP-8. Moreover, using a recently developed BASEHIT (BActerial Selection to Elucidate Host-microbe Interactions in high Throughput) library, we found that SLPI directly interacts with B. burgdorferi. These data demonstrate the importance of SLPI in suppressing periarticular joint inflammation in Lyme disease.

microbiology↗

bioRxiv2024

Identification of Ephrin type-B receptor 4 as a critical mediator of tissue fibrosis

Pulmonary fibrosis (PF) is a pathology associated with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). Fibrosis promotes continual secretion of extracellular matrix (ECM), producing non-functional scar tissue and causing organ failure. This study investigated the tyrosine kinase receptor Ephrin type-B receptor 4 (EphB4) as a mediator of PF. To this end, we generated mice with conditional Col1a2-driven deletion of Ephb4 and used a preclinical mouse model of PF, total and single nuclei RNA (snRNA) sequencing, NanoString, previously published single cell data, computational analysis, and functional assays of mouse and human healthy control and IPF lung fibroblasts. Col1a2-CreERT-driven Ephb4 deletion, or EphB4 inhibition via NVP-BHG712, markedly protected against bleomycin-induced PF. Total RNA-sequencing of fibroblasts isolated from Ephb4-deficient fibrotic mouse lungs exhibited reduced expression of ECM, ER Cargo, and protein trafficking-related genes. NVP-BHG712 reduced expression of these identified genes in mouse lung fibroblasts under fibrotic conditions in vitro. SnRNA sequencing of mouse lungs treated with NVP-BHG712 identified transcriptomic changes of ECM genes in specific fibroblast subpopulations. RNA sequencing, computational, and functional assays using mouse and human IPF fibroblasts identified elastin as a key mediator involved in EphB4 signaling. Combined, our data show that EphB4 is a crucial mediator of PF.

pathology↗

bioRxiv2024

AistSeq: An In-House Tn5-Based Plasmid Sequencing Platform Using A Compact Benchtop Sequencer

Sequence verification of plasmids is a fundamental process in synthetic biology. For plasmid sequence verification using next-generation sequencing (NGS) library preparation, Tn5 transposase is widely used. Streamlined sequencing workflow for laboratory-scale applications is important; however, recombinant Tn5 production in-house can be laborious. In this study, we demonstrated that the addition of a large soluble tag was not essential for purification and that the fusion of a His10 tag and protein A was sufficient to purify sufficient amounts of active Tn5 transposase. In addition, we evaluated exonuclease-based genomic DNA digestion for plasmid sequencing from an E. coli lysate and the data analysis pipeline of sequences derived from the Illumina iSeq100 platform for de novo assembly, reference mapping, and annotation. This study proposes a simple workflow of an in-house Tn5-based plasmid Sequencing platform using a compact benchtop sequencer (AistSeq). Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=37 SRC="FIGDIR/small/618112v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@f1546corg.highwire.dtl.DTLVardef@1dd947aorg.highwire.dtl.DTLVardef@3efb78org.highwire.dtl.DTLVardef@efbde1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Becker muscular dystrophy mice showed site-specific decay of type IIa fibers with capillary change in skeletal muscle

Becker muscular dystrophy (BMD), an X-linked muscular dystrophy, is mostly caused by an in-frame deletion of DMD. BMD severity varies from asymptomatic to severe, associated with the genotype of DMD. However, the underlying mechanisms remain unclear. We established BMD mice carrying three representative exon deletions: ex45-48 del., ex45-47 del., and ex45-49 del. (d45-48, d45-47 and d45-49), with high frequencies and different severities in the human BMD hotspot. All three BMD mice showed muscle weakness, muscle degeneration, and fibrosis, but these changes appeared at different times for each exon deletion, consistent with the severities obtained by the natural history study of BMD. BMD mice showed site-specific muscle changes, unlike mdx mice, which showed diffuse muscle changes, and we demonstrated selective type IIa fiber reduction in BMD mice. Furthermore, BMD mice showed sarcolemmal neuronal nitric oxide synthase (nNOS) reduction and morphological capillary changes around type IIa fibers. These results suggest that capillary changes caused by nNOS reduction may be associated with the mechanism of skeletal muscle degeneration and type IIa fiber reduction in BMD mice. BMD mice may be useful in elucidating the pathomechanisms and developing vascular targeted therapies for human BMD.

genetics↗