Biology subjects

Yoshida, K.

Publications and source records attributed to Yoshida, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Exitron-type splicing of intrinsically disordered regions recurs across jawed vertebrates despite sequence divergence: donor-proximal cis control in TARDBP

Intrinsically disordered regions (IDRs) mediate molecular interactions but can also promote aberrant self-association. Using donors embedded within IDR-coding sequences, exitron-type splicing can remove these sequences, giving them both protein-coding and splice-regulatory roles. Yet IDR amino-acid sequences often diverge rapidly, raising the question of whether and how their coding sequences support IDR-removing splicing across species. Using RNA-seq data from jawed vertebrates, we identified events predicted to remove most of an IDR while preserving annotated structured regions. Across species, donor-flanking nucleotide identity exceeded expectations from synonymous alternatives encoding the same amino-acid sequence. TARDBP, which encodes TDP-43, provided a model for examining how coding sequences both encode IDRs and regulate exitron-type splicing. Sequence-divergent vertebrate TARDBP terminal regions supported this splicing in human cells. In teleosts, TARDBP ohnologs (tardbpa and tardbpb) encoded compositionally similar disordered tails but differed markedly in IDR-removing exitron-type splicing. Reciprocal donor-proximal substitutions in zebrafish minigenes redirected donor choice and altered the balance between IDR-retaining and IDR-removing transcripts. In corresponding reporters, these substitutions altered protein-product balance and cytoplasmic puncta formation. These findings show that IDR-removing exitron-type splicing can recur despite sequence divergence and that donor-proximal TARDBP sequences both encode IDR segments and regulate IDR inclusion, linking RNA processing to protein behavior.

molecular biology↗

bioRxiv2026

Adaptations of the pregnant murine heart to pre-existing hypertension

2-5% of pregnancies are complicated by chronic hypertension [1], increasing the risk of peripartum cardiomyopathy and maternal morbidity [2, 3]. Hypertension reduces stroke volume, whereas pregnancy increases stroke volume. The combined impact of these opposing loads on cardiac function and strain remains unclear. We used a mouse model of pregnancy with pre-existing hypertension to characterize cardiac remodeling. We hypothesized that pregnancy may counteract hypertension-related reductions in cardiac function. Nulliparous female C57Bl6/J mice (7-11 weeks old) were assigned into pregnant (PREG, n=11), angiotensin II-treated (ANGII, n=7), and ANGII+PREG (n=8) groups. Hypertension was induced with subcutaneous angII infusion (1000 ng/kg/min). Cardiac function was measured with high-frequency ultrasound. Ejection fraction (EF) was maintained in PREG animals at 65% but decreased significantly by day 36 in ANGII animals (44.85{+/-}3.27%). In ANGII+PREG mice, EF initially decreased but trended towards PREG values on postpartum day 1. Left ventricle strain was lower in ANGII and ANGII+PREG groups compared to PREG. ANGII and ANGII+PREG animals showed decreased early longitudinal diastolic strain rate at mid-pregnancy compared to baseline (0.82{+/-}0.09% ANGII; 0.73{+/-}0.09% ANGII+PREG), while increasing in PREG (1.76{+/-}0.14%). Minimal physiologically relevant fibrosis was noted across groups via immunohistochemistry. Heart mass-to-body mass was lowest in PREG (6.20{+/-}0.22 mg/g), greatest in ANGII (8.95{+/-}0.39 mg/g), and intermediate in ANGII+PREG (6.92{+/-}0.25 mg/g). Cardiomyocyte cross-sectional area was similar between groups. Overall, pregnancy altered the functional and hypertrophic response to elevated blood pressure. Findings from this work set the foundation for future efforts that may eventually improve cardiovascular care for pregnant patients.

bioengineering↗

bioRxiv2026

Genotype-dependent reductions of competition are associated with yield maintenance in a modern common wheat Triticum aestivum L. cultivar

Modern crop breeding has increased yield under monoculture stands, but the mechanisms underlying this improvement remain incompletely understood. We tested whether genotype-dependent reduction of competition, whereby plants adjust competitive responses according to neighbor genetic identity, contributes to yield maintenance in modern common wheat cultivars. We conducted a root-exudate application experiment and two competition experiments using three common wheat (Triticum aestivum L.) landraces and one modern cultivar, Norin 61. Norin 61 exhibited little change in shoot-to-root allocation in response to root exudates from the same cultivar compared with distilled water, whereas it increased shoot-to-root allocation exposed to root exudate from different cultivars. Moreover, shoot-to-root allocation increased with decreasing genetic relatedness between neighboring cultivars in common wheat plants. In competition experiments, greater shoot-to-root allocation was associated with stronger suppression of competitors. Norin 61 maintained grain biomass under the same-cultivar competition compared to solitary conditions but exhibited substantial yield reductions when competing with different cultivars, whereas these patterns were weaker or absent in landraces. These results suggest that genotype-dependent reduction of competition may contribute to yield maintenance in modern wheat cultivars and provide a new perspective on how crop breeding has shaped plant-plant interactions in monoculture stands.

ecology↗

bioRxiv2026

A patient-derived LMX1B variant causes tissue-specific manifestations of nail-patella syndrome in mice

Nail-patella syndrome (NPS) is a multisystem disorder caused by pathogenic variants in LMX1B and is characterized by dysplasia of the nails and patellae as well as extraskeletal complications such as progressive nephropathy and glaucoma. We generated a CRISPR/Cas9 knock-in mouse carrying the R252Q substitution, corresponding to a human LMX1B variant associated with renal-predominant disease. Phenotypic analysis revealed that homozygous mice were viable, but they displayed marked growth retardation and severe bilateral ocular opacity. Interestingly, while this model exhibited clear skeletal and ocular defects, the renal phenotype was relatively mild, although increased urinary albumin excretion, focal glomerular basement membrane abnormalities, and subtle changes in renal gene expression were detected. Beyond the classical NPS hallmarks, mutant mice also displayed midbrain morphological abnormalities, suggesting broader developmental consequences of this LMX1B variant. This patient-derived variant model not only recapitulates the pleiotropic features of NPS but also demonstrates organ-specific susceptibility to the R252Q substitution, providing a foundation for elucidating the complex molecular mechanisms underlying multisystem disease.

genetics↗

bioRxiv2026

Evaluation of Locomotor Activity in Group-Housed Hamsters: Effects of Cage Size and Running Wheel Availability

While hamsters are commonly housed in groups within pet shops in Japan, small cages are often thought to restrict their physical activity, leading to arguments that larger cages should be provided. Although previous research has investigated how cage size and running wheel availability influence activity levels in individually housed hamsters, no studies to date have examined these specific effects in a social housing context. Therefore, this study investigated how cage size and the presence of a running wheel affect the activity levels of individual hamsters using the group-housing conditions with five hamsters. Video recordings were captured for 24 hours across four distinct cage environments using a camera installed directly above each cage. From these recordings, the distances traveled on both the cage floor and the running wheel were calculated for each hamster and statistically analyzed. The results revealed that overall activity levels were significantly higher in cages equipped with a running wheel than in those without. Although cage size did not yield a statistically significant difference, a marginal trend toward higher activity in larger cages was observed.

animal behavior and cognition↗

bioRxiv2026

EV-Tracer enables lineage-resolved detection and molecular profiling of extracellular vesicle-associated signals in cancer-fibroblast co-culture

Extracellular vesicles (EVs) facilitate intercellular communication by transferring diverse bioactive molecules from donor to recipient cells. However, EVs released by distinct cellular lineages become difficult to distinguish when mixed in multicellular experimental models, limiting the analysis of how cell-cell interactions affect EV-associated molecular profiles. To address this, EV-Tracer, a CD63-based dual-fluorescence tracing and capture system for detecting, isolating, and profiling lineage-associated EV fractions, was developed. Achilles or mScarlet was inserted into the small extracellular loop of CD63, enabling tracer-specific EV detection by digital counting, antibody-based isolation, and live-cell visualisation. Exploratory EV RNA sequencing suggested that physical cell-cell contact was associated with distinct EV RNA profiles, including interferon-related signals, which were supported by targeted cellular and EV-associated RNA analyses. EV-Tracer provides a practical framework for investigating lineage-associated EV dynamics and molecular signals in mixed-cell systems.

cell biology↗

bioRxiv2026

Model based analysis of the orderly size-wise activation observed with sinusoidal low frequency alternating current stimulation of peripheral nerves

Extracellular sinusoidal low frequency alternating current (LFAC) stimulation of peripheral motor nerves has been observed to induce size wise activation of nerve fibers, unlike the inverse recruitment order typically seen in extracellular pulsed stimulation. This study aims to explore potential biophysical mechanisms responsible for this phenomenon using computational modeling. Volume conductor model was utilized with a bipolar cuff electrode encasing a single rat-sized fascicle. The extracellular potentials generated by LFAC and pulse stimulation were projected onto the McIntyre-Richardson-Grill models of myelinated motor nerve fibers to examine the activation of fibers ranging from 5.7 to 16m in diameter. Intracellular and extracellular stimulation were compared for strength-frequency relationships with LFAC (1-20Hz) and strength-duration curves for pulse stimulation. The threshold tracking technique was used to study membrane electrotonus and threshold electrotonus of different fibers to examine subthreshold accommodation in response to LFAC and prolonged pulse stimulation. The simulations revealed that the inverse order of fiber recruitment is an inherent characteristic of extracellular stimulation and is theoretically independent of the stimulation waveform. LFAC showed an inverse strength-frequency relationship (higher frequency, lower threshold current), similar to the inverse strength-duration relationship for pulsed stimulation. Analysis of subthreshold accommodation showed that larger fibers exhibit greater accommodation than smaller fibers, leading to increased activation thresholds as fast Na+ activation factor m3h decreases while slow K+ activation increases, supporting accommodation as a contributor to orderly recruitment. With increasing LFAC frequency (up to 20Hz), these accommodation characteristics were reduced and large-fiber state dynamics shifted toward those of smaller fibers. LFAC was also found to induce subthreshold oscillations that promoted spike initiation during slow depolarization. These findings suggest that LFAC provides a controlled and optimized method for achieving orderly recruitment without the need for complex selective blocking protocols. By leveraging intrinsic membrane properties, LFAC offers a neuromodulation strategy that preserves physiological recruitment order, with direct implications for selective nerve stimulation in clinical and neuroprosthetic applications. Author summaryElectrical stimulation is widely used to activate peripheral nerves in motor rehabilitation and neuroprosthetic devices, but conventional pulse stimulation activates larger nerve fibers first (with lower current intensity), which can induce rapid muscle fatigue and pain. We used well-established and validated computational models of motor nerve fibers (axons) to explore how sinusoidal low frequency alternating current (LFAC) stimulation can produce a more physiological, size-wise recruitment order. We simulated myelinated motor nerve fibers of different diameters individually inside a bipolar cuff electrode and analyzed how the membrane and ion channels changed during stimulation levels that are below thresholds for action potential firing. We found that larger fibers adapt (accommodate) more strongly during the slow depolarization of LFAC: their sodium channels become less open, while potassium activation increases, raising the current required to induce an action potential. Smaller fibers were less affected by this accommodation effect and could reach firing at lower current intensities. We also found that these effects depend on stimulation frequency; at lower frequencies, the accommodation characteristics were more defined (for all fibers), while at higher frequencies they were reduced (for large fibers) and all fiber responses became more similar. Our results suggest that the responses of intrinsic membrane dynamics to LFAC lead fiber recruitment toward a more physiological order, which facilitates the design of safer and more selective nerve stimulation strategies with LFAC.

neuroscience↗

bioRxiv2026

Microvascular pathology of proteotoxic endothelial signature characterizes Progressive Supranuclear Palsy

Cerebrovascular pathology is increasingly implicated in neurodegenerative diseases, yet its pathomechanistic contribution remains poorly defined. Building on prior evidence of dysregulated iron and oxygen homeostasis in early-affected brain regions of progressive supranuclear palsy (PSP), we hypothesized that brain microvascular alterations may play an etiological role in select neurodegenerative proteinopathies. First, we conducted a systematic neuropathological evaluation of 178 brains from the University Health Network Neurodegenerative Brain Collection, including Alzheimers disease-related neuropathologic change (ADNC; n=30), Lewy body disease with high or intermediate ADNC (n=38) and low ADNC (n=16), multiple system atrophy (MSA; n=14), PSP (n=39), frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP; n=10), and controls (n=31). Arteriolosclerosis, microinfarction, and calcification were assessed in the basal ganglia and frontal cortex. Iron burden was correlated by quantification of Perls staining in MSA and PSP, where vessel pathology was most severe. Single-nucleus RNA-sequencing (snRNA-seq) of frontal cortex tissue from control (n=5) and PSP (n=8) cases with varying arteriolosclerosis severity was performed to characterize the vascular transcriptome, with validation against an independent snRNA-seq evaluation of PSP (n= 11), Picks disease (n=9), AD (n=10), and control (n=10) brains. Histological analysis revealed disease-specific involvement of microvascular pathology in neurodegenerative diseases, identifying PSP to demonstrate most prominent and widespread vessel wall thickening across regions examined. Regression analysis using demographic, APOE and MAPT genetic risk status, and neuropathological features of cases corroborated the distinct association with PSP pathology. Elevated iron load in early affected regions of MSA and PSP brains correlated with greater vessel wall thickening, suggesting a possible pathomechanistic relationship between the two disease physiologies. snRNA-seq analysis of vascular transcriptome identified robust upregulation of heat shock proteins and hypoxia-related genes in PSP endothelial cells and pericytes across both datasets. Importantly, we found the proteotoxic signature to be strongly associated with higher vessel scores in PSP cases, linking microvascular morphology to endothelial dysfunction. Our comprehensive neuropathological evaluation coupled with correlative snRNA-seq analysis establish PSP-specific arteriolar thickening associated with endothelial proteotoxic state as a candidate pathogenic mechanism. The cerebral arteriolar unit represents a compelling therapeutic target for disease modification in PSP.

pathology↗

bioRxiv2026

Possible function of Hox2 in atrial siphon fusion of the ascidian Ciona

The hallmark of sessile adult ascidians is a vase-like shape with a single oral and atrial siphon. Ciona, however, develops two atrial siphons after metamorphosis, which subsequently fuse into one. The mechanisms underlying this fusion are unknown. This study suggests that Hox2 controls this process. Hox2-knockout animals using Transcription-Activator-Like Effector Nuclease (TALEN) retain two atrial siphons throughout their lives. During normal fusion, epidermal cells between the siphons flatten along the anterior-posterior axis. This cellular flattening does not occur in Hox2-knockout animals, suggesting that the shape change in the epidermal cells produces tension, allowing the atrial siphon openings to converge at the midline for fusion. Hox2-knockout animals lack cupular organs, which are suspected hydrodynamic sensors in the internal epithelium of the fused atrial siphon and on the sperm duct. Among several knockout attempts, atrial siphon fusion was reproduced by only one TALEN pair, suggesting that this phenotype is driven by a mutation having a broader effect than those abolishing protein function. Many ascidians, unlike Ciona, develop a single atrial siphon shortly after metamorphosis. Our findings suggest that a phylogenetically conserved gene, Hox2, establishes this group-specific atrial siphon formation mechanism in Ciona.

developmental biology↗

bioRxiv2026

Generating E. coli 0.5 controlled by a half-sized genome

Genome synthesis is a major limitation in generative biology. Here, the half-sized genome of Escherichia coli was constructed by fleshing out an imperfect minimal genome through genome-scale debugging process. Our platform consists of integrated development environment (IDE) and runtime environment (RTE). The genome IDE supported the cell-free assembly of 200-300 kb plasmids and their in vivo fusion into a single 1.7 Mb plasmid. This imperfect genome was stably maintained in E. coli as a guest genome. The RTE relies on the restriction enzyme-mediated self-digestion of the host genome in the presence and absence of the RecA recombinase. The guest genome was tested, debugged, and partially replaced by the host genome to establish E. coli controlled by a 2.3-Mb genome. This is less than half in size of the wildtype and the smallest ever reported. Enfleshing a guest genome will facilitate genome printing that transforms AI-designed genomes into physical ones.

synthetic biology↗

bioRxiv2026

A model of schema learning based on biological dimensionality reduction during sleep

Reorganizing learned knowledge into generalized representations and transferring it to future learning are essential aspects of cognition, often described as schema formation and use. However, the computational and circuit mechanisms underlying these processes remain unclear. Here, we propose a theoretical model in which schemas emerge through the formation and alignment of low-dimensional neural representations. In this model, high-dimensional input patterns are reorganized into low-dimensional manifolds through replay-driven Hebbian nonlinear dimensionality reduction. Manifold alignment simultaneously maps representations with shared task structure onto a common format, enabling downstream readout circuits to be reused across tasks. The model captures three core features of schema learning: rapid learning of similar tasks by reusing low-dimensional representations from prior experience, sleep-dependent generalization to unobserved relationships in transitive inference, and compositional recombination of schemas to solve novel tasks. Together, these results suggest a potential neural mechanism for forming, aligning, and recombining low-dimensional schemas to support future learning.

neuroscience↗

bioRxiv2026

Modulating supplementary motor area excitability enhances groove-related pleasure during music listening

Pleasurable urge to move to music is often referred to as groove. Although previous studies have shown an association between the supplementary motor area (SMA) and the groove experience, its causal role remains unclear. Here, we investigated whether the SMA is causally involved in groove experience during music listening using repetitive transcranial magnetic stimulation. Fifteen healthy participants completed three sessions on separate days: excitatory stimulation (intermittent theta burst stimulation; iTBS) over the SMA, inhibitory stimulation (continuous theta burst stimulation; cTBS) over the SMA, and sham stimulation (iTBS or cTBS) over the vertex. After each stimulation session, participants listened to five high-groove and five low-groove musical excerpts and rated urge-to-move and pleasure on a 0-100 scale. Heart rate was additionally recorded as an exploratory physiological measure during music listening. Linear mixed-effects models (LMM) showed that pleasure ratings, but not urge-to-move ratings, were higher following both iTBS and cTBS compared with sham stimulation. In exploratory LMMs, reduced log-transformed heart rate variability (HRV) significantly predicted higher pleasure ratings. These findings suggest that SMA stimulation modulates the pleasurable component of the groove experience, likely via network-level mechanisms rather than a simple linear relationship between SMA excitability and pleasure. They also raise the possibility that reduced parasympathetic activity, reflected by lower HRV, mediates the stimulation-related increase in musical pleasure. Future studies should investigate the causal roles of other brain regions as well as clarify the directionality between autonomic changes and the groove experience.

neuroscience↗

bioRxiv2026

Cytotoxicity-based High-throughput Screening System for CAR T Cell

Some chimeric antigen receptor (CAR) T cell therapies have shown strong clinical efficacy, yet systematic screening of new CAR designs remains constrained by labor-intensive, low-throughput evaluation methods. To address this limitation, we developed a cytotoxicity-centered, high-throughput screening platform that integrates single-cell pooled screening with fully automated arrayed screening enabling both large-scale library handling and quantitative functional resolution for systematic CAR design exploration. Using a mutation-based CAR design approach guided by protein fitness prediction, we generated a 4-1BB-based CAR library with approximately 10 theoretical variants while minimizing the prevalence of low-activity designs. In pooled screening, CAR T cells were evaluated at the single-cell level based on cytotoxicity and proliferation, enabling rapid enrichment of high-performing variants from a highly diverse library. Subsequent automated arrayed screening quantitatively measured cytotoxicity with high reproducibility, providing high-resolution functional data suitable for comparative ranking. Selected CAR variants demonstrated superior antitumor efficacy in a leukemia xenograft model compared with a template CAR. Furthermore, systematic analysis of mutation sites from an enhanced CAR variant identified essential mutation combinations underlying functional enhancement. Together, this study establishes a cytotoxicity-focused screening framework that provides a robust approach for optimizing CAR architectures and accelerating the development of CAR T-cell therapies.

synthetic biology↗

bioRxiv2026

PIFI Stabilizes Chloroplast NDH-PSI Supercomplex to Maintain Plastoquinone Redox Balance and PSII Efficiency

Photosynthetic electron transport is mediated by several protein supercomplexes that are spatially arranged in the thylakoid membranes of chloroplasts. The chloroplast NADH dehydrogenase-like (NDH) complex is part of the photosynthetic alternative electron transport (AET) chain, which reduces the plastoquinone (PQ) pool using reduced ferredoxin as a substrate. This NDH complex is associated with photosystem I (PSI) and mediates a portion of AET in stroma lamellae, whereas photosystem II (PSII) is concentrated in grana stacks. This study presents the findings regarding post-illumination chlorophyll fluorescence increase (PIFI), a protein crucial for regulating AET via the NDH pathway. A marked increase in NDH activity and a reduction in the PQ pool in the dark were observed in PIFI-deficient mutant strains (g-pifi) generated by genome editing. Blue native PAGE analysis indicated that PIFI was associated with the NDH-PSI supercomplex in the wild type, and the NDH complex was dissociated from PSI in the g-pifi mutants. Additionally, the g-pifi mutants exhibited a decrease in the maximum quantum yield of PSII (Fv/Fm). Notably, Fv/Fm was restored in a double mutant harboring both g-pifi and NDH-deficient pnsl1 mutations, demonstrating that deregulated NDH activity in g-pifi causes downregulation of PSII efficiency. However, the lower Fv/Fm was not observed in a mutant lacking thioredoxin m4 (trxm4), which showed deregulated NDH activity but maintained the NDH-PSI supercomplex. These data suggest that PIFI stabilizes the NDH-PSI supercomplex and maintains the spatial localization of PQ reduction via AET in thylakoid membranes, which is essential for the proper functioning of PSII.

plant biology↗

bioRxiv2026

A macrocyclic peptide-based fusion inhibitor targeting SARS-CoV-2 Spike S2 subunit

Continuous emergence of SARS-CoV-2 variants carrying mutations in Spike presents a significant challenge for durable antiviral agents. Here we screen for random 13-amino acid non-mimetic macrocyclic peptides that bind to Spike and identify PA-001 that inhibits SARS-CoV-2 infection with high potency at 0.23-2.9 nM as 50% inhibitory concentration (IC50). PA-001 bound to Spike S2 subunit and inhibited the membrane fusion during virus entry. Through drug-resistant selection, we revealed that PA-001 targeted the fusion peptide proximal region (FPPR) in S2, which has not been recognized as a drug target to date. Consistent with its highly conserved amino acid sequences beyond strains, PA-001 exhibited broad antiviral activity against all tested SARS-CoV-2 variants, in contrast to clinically-approved S1-targeting antibodies that lost activity to Omicron variants. PA-001 suppressed SARS-CoV-2 propagation and disease progression in mouse- and hamster-infection models, both by administration prophylactically and therapeutically. Combination therapy with remdesivir further enhanced antiviral profiles. In clinical phase-I trial, PA-001 was well-tolerated and showed high systemic exposure, with 4,300-10,300-fold concentration of IC50 as maximum plasma concentration by single administration to healthy volunteers. These evidence propose FPPR as an unexpected antiviral drug target accessible by macrocyclic peptides and identify PA-001 as a potent anti-SARS-CoV-2 fusion inhibitor.

microbiology↗

bioRxiv2026

Radiocesium Uptake in Two Fungal Bed-cultivated Edible Mushroom Species in Forests in Fukushima, Japan

In Japan, mushrooms have long been valued as food resources, but the Fukushima nuclear accident disrupted production and shipment due to radiocesium contamination. Limited knowledge has particularly hindered outdoor fungal bed cultivation. To address this, we conducted cultivation experiments with Lyophyllum decastes and Lepista nuda across 14 broad-leaved deciduous forest sites in Fukushima Prefecture. Air dose rates at 1 m height ranged from 0.04 to 0.89 {micro}Sv/h. Radiocesium concentrations (combined 137Cs and 134Cs, expressed as Bq/kg at 90% water content) in fruit bodies were 0.4-12 (mean 2.3) for Ly. decastes and 0.2-43 (mean 8.1) for Le. nuda. Both species remained below Japans food safety threshold of 100 Bq/kg, indicating that safe cultivation is feasible across broad areas. Uptake patterns differed: concentrations in Ly. decastes correlated with contamination in litter and multiple soil layers, whereas in Le. nuda it correlated only with litter contamination. These findings suggest that clean soil fills for Ly. decastes and clean litter covers for Le. nuda could serve as mitigation strategies tailored to each species. Aggregated transfer factors (2.76 x 10-5 m2/kg for Ly. decastes and 6.60 x 10-5 m2/kg for Le. nuda) were lower than those reported for wild mushrooms. Overall, this study provides new insights into reducing radiocesium assimilation by cultivated mushrooms and supports the revival of outdoor fungal bed cultivation in contaminated landscapes.

ecology↗

bioRxiv2025

Intensity-dependent corticospinal facilitation by repetitive peripheral magnetic stimulation: Evidence for a major contribution of group I afferents

BackgroundRepetitive peripheral magnetic stimulation (PMS) is increasingly used in neurorehabilitation, yet the optimal stimulation intensity for inducing corticospinal facilitation and the underlying afferent mechanisms remain unclear. We investigated the intensity-dependent effects of repetitive PMS on corticospinal excitability and single motor unit responses, and tested group I afferent contribution. MethodsHealthy participants received repetitive PMS (25 Hz; 2-s ON/2-s OFF) over the extensor carpi radialis (ECR) in a crossover design at 0.9x motor threshold (MT), 1.2xMT, and high intensity sufficient to induce maximal wrist dorsiflexion (mean 1.8xMT). Motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation were recorded from the ECR and flexor carpi radialis (FCR) before and during the intervention (total 15 min). The lasting effects were assessed after 9 min of high-intensity PMS for 50 min. To examine group I afferent contribution, the same high-intensity protocol was applied during upper-arm ischemia after reducing the ECR H-reflex to <10% of baseline. Sensory-motor input characteristics across stimulation intensities were compared using post-stimulus time histograms of ECR single motor unit firings during weak voluntary contraction. ResultsHigh-intensity PMS significantly increased ECR MEPs after 9 min of intervention, whereas 1.2xMT of PMS required 15 min to induce a marked effect. PMS at 0.9xMT did not induce significant MEP changes. Across all intensities, the FCR MEPs remained unaltered. ECR MEPs remained markedly elevated for up to 30 min after 9 min of high-intensity PMS. In contrast, PMS delivered during ischemia produced no MEP enhancement. The motor unit analysis revealed that suprathreshold PMS elicited an early peak in firing probability--consistent with monosynaptic Ia excitation--whose amplitude increased with stimulation intensity, whereas PMS at 0.9xMT produced no discernible peak. ConclusionsRepetitive PMS above MT facilitates corticospinal excitability in an intensity-dependent manner. Facilitation was abolished during ischemia. Together with the presence of a short-latency peak in motor unit firing via a monosynaptic pathway, this finding supports a major contribution of large-diameter muscle afferents, with a substantial Ia component, to PMS-induced corticospinal facilitation.

neuroscience↗

bioRxiv2025

Comprehensive and accelerated mapping of driver mutations through single-nucleotide random mutagenesis of target genes

Identification of novel driver mutations is crucial for personalized medicine and drug discovery. However, genome-wide studies based solely on patient-derived data cannot confirm the oncogenic potential of detected mutations. Here, we present a genome editing-based method for comprehensive random mutagenesis of target genes. This approach generates cultured cells harboring random single-nucleotide variants (SNVs) within 10 days of DNA amplicon preparation. To validate its utility, random mutations were introduced into endogenous exons of lung cancer-related oncogenes in HEK293 cells. Spheroid formation assays and xenograft models demonstrated enhanced tumorigenicity, indicating the acquisition of oncogenic potential. Amplicon-sequencing of spheroid transcripts revealed significant enrichment of variants annotated as pathogenic in clinical databases and computational predictions. Furthermore, osimertinib treatment of lung cancer cell lines with random SNVs on EGFR identified 58 novel resistance mutations. These results highlight the effectiveness of our mutagenesis platform, offering a powerful tool to advance cancer research and precision medicine.

bioengineering↗