Biology subjects

Kimura, K.

Publications and source records attributed to Kimura, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Genome sequence data of Ascogaster reticulata (Hymenoptera: Braconidae)

Parasitoid wasps develop in or on other arthropods and have evolved a wide range of strategies for exploiting their hosts. Ascogaster reticulata is a solitary egg to larval endoparasitoid wasp belonging to the subfamily Cheloninae of Braconidae. We constructed a genome assembly using long-read sequence data. The assembled genome was 280.5 Mbp, consisting of 209 contigs. The N50 value and GC content ratio were 13.9 Mbp and 30.43%, respectively. The percentage of transposable elements and repetitive regions was 52.43%. The predicted number of genes is 27,261. Merqury revealed that the QV value and completeness of the genome were 61.79 and 99.3917%, respectively. BUSCO analysis detected approximately 99% of the core genes. These results suggest that the genome data were of sufficient quality for use as a reference genome. Therefore, these data can be utilized as a genomic resource for comparative studies within Braconidae and for future investigations into the biology and evolution of Cheloninae parasitoid wasps.

genomics↗

bioRxiv2026

Aged-associated changes in active YAP staining during impaired skin wound healing in aged mice and delayed wound closure in ICE mice

Aging is associated with impaired skin wound healing, but the molecular mechanisms underlying this decline remain incompletely understood. In particular, how aging affects the temporal regulation of mechanosensitive signaling during tissue repair is poorly understood. In this study, we investigated age-associated changes in cutaneous wound healing, with a focus on Yes-associated protein (YAP)-associated mechanosensitive responses, and examined whether ICE mice, an inducible model of epigenetic aging, reproduce age-associated impairment in wound repair. Splinted full-thickness excisional wounds were generated in young and naturally aged mice, and wound closure, gene expression, and active YAP were evaluated over time. Aged mice exhibited delayed wound closure, particularly during the mid-to-late phases of repair. Expression of the fibrosis-related genes Tgfb1, Col1a1, and Col3a1, and the YAP-associated genes Ctgf, Cyr61, and Arhgap18 did not differ significantly between young and aged wounds at the examined time points. In contrast, Ankrd1 was selectively elevated in aged wounds at postoperative day 14. Active YAP immunofluorescence showed age-associated differences in staining patterns during wound healing, indicating altered regulation of mechanosensitive signaling with aging. Furthermore, ICE mice exhibited delayed wound closure compared with Cre control mice, reproducing a major functional feature observed in naturally aged mice. These findings suggest that age-associated impairment of wound healing is characterized not by uniform activation or suppression of YAP-related signaling, but by altered temporal regulation of mechanosensitive responses together with selective Ankrd1 elevation. ICE mice could provide a useful experimental model for investigating age-associated defects in cutaneous wound healing.

cell biology↗

bioRxiv2026

Metastable Dimeric Intermediate Drives Native-like Spidroin Fibrillogenesis, while Dehydration Promotes Cross-β Transition

The exceptional mechanical properties of spider dragline silk originate from hierarchical spidroin self-assembly, yet mimicking this process to generate native-like structures remains challenging. Here, we report a metastable dimeric intermediate as an essential molecular factor for fibrillogenesis. Small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed a compact core stabilized by solvent-excluded "dry interfaces" surrounded by extended random coil regions. Within the core, a subset of the polyalanine blocks is held by side-chain packing rather than by main-chain {beta}-sheets; the majority remain exposed in the surrounding coil regions. Upon lowering the denaturant concentration, these exposed polyalanine segments pair with their counterparts at a fibril end, forming intermolecular {beta}-sheets that act as linkers and drive the stepwise end-to-end polymerization of the dimeric particles. In solution, the resulting fibrils exhibit an ordered {beta}-structure in which the polypeptide chains run parallel to the fibril axis, in contrast to amyloid-like cross-{beta}, in which the strands lie perpendicular to it; this matches key features of native silk nanofibril architecture. However, X-ray diffraction (XRD) analysis demonstrates that these native-like nanofibrils undergo an effectively irreversible transition toward amyloid-like cross-{beta} structures upon drying. Native-like fibrils are thus achievable by self-assembly, but structural control is lost during the final dehydration step. Accordingly, we propose a general principle for decoupling self-assembly from dehydration: native-like nanofibrils are formed first, then dried under conditions that restrict molecular-chain mobility (for example, mechanical constraint or immobilization on a surface), which we expect to suppress thermodynamic relaxation into cross-{beta} structures.

biophysics↗

bioRxiv2026

Immunomodulatory mechanisms of submicron phosphatidylserine-exposing polymeric particles (PSPs)

Macrophages are highly dynamic cells that maintain tissue homeostasis by regulating both initiation and resolution of inflammation. During efferocytosis, macrophages recognize the eat me signal, phosphatidylserine (PS), exposed at the surface of apoptotic cells, leading to the resolution of inflammation and acquisition of a pro-resolving phenotype. Inspired by this endogenous mechanism, PS-based biomaterials have demonstrated immunomodulatory potential. However, the molecular mechanisms underlying PS-mediated macrophage reprogramming remain poorly understood. Here, submicron PS-exposing polymeric particles (PSPs; [~]300 nm) were developed to improve the suitability of PSP formulations for future systemic administration while preserving their immunomodulatory activity. PSPs were efficiently internalized by macrophages through both actin- and dynamin-dependent pathways. PSP treatment significantly reduced IL-6 and IL-12p70 production in LPS-stimulated macrophages, whereas induction of the classical anti-inflammatory M2 marker CD206 was limited. Transcriptomic analysis revealed coordinated attenuation of inflammatory signaling pathways, including downregulation of Myd88, Nfkb1, Rel, and Irf8, together with activation of NRF2-associated antioxidant pathways characterized by increased expression of Nfe2l2, Hmox1, Prdx1, Gclm, and Gclc. Activation of antioxidant-associated genes together with reduced Irf8 expression suggests that PSP promotes inflammatory resolution through coordinated redox adaptation and selective attenuation of inflammatory signaling. Collectively, these findings provide mechanistic insight into PS-mediated macrophage reprogramming and support the future development of systemically administered therapies for chronic inflammatory diseases, including vascular inflammatory disorders. HighlightsO_LISubmicron PSPs retain immunomodulatory activity of apoptotic cell-mimicking biomaterials. C_LIO_LIPSPs are rapidly internalized through actin- and dynamin-dependent pathways. C_LIO_LIPSPs attenuate inflammatory signaling and selectively suppress IL-6 and IL-12p70 production. C_LIO_LIPSPs induce NRF2-associated antioxidant and glutathione responses. C_LIO_LITranscriptomics reveals an early redox-adaptive macrophage program. C_LI

bioengineering↗

bioRxiv2026

Identification of the Down syndrome critical region 3 gene as a mammalian cell size regulator

Using a genetic screening approach based on an inducible gene-activating system and cell sorting, Down syndrome critical region 3 (DSCR3) was isolated as a gene whose overexpression increased cell size. Fibroblasts derived from individuals with Down syndrome (DS) exhibit elevated DSCR3 expression at both the mRNA and protein levels, correlating with increased cell volume compared to fibroblasts from healthy donors. Despite a slower proliferation rate, DS fibroblasts demonstrate higher basal and maximal mitochondrial respiration, suggesting enhanced metabolic activity associated with increased cell size. siRNA-mediated knockdown of DSCR3 reduces cell size in both DS and normal fibroblasts, indicating its general role in cell size regulation. As DSCR3 is a component of the retriever complex involved in endosomal cargo recycling, these findings position membrane protein trafficking as a novel module for cell size control.

cell biology↗

bioRxiv2026

Akkermansia muciniphila-derived LPS links gut dysbiosis to pathogenic miR-21 signaling in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic T cell-mediated autoimmune disease characterized by blood-brain barrier (BBB) disruption, neuroinflammation, and demyelination of the central nervous system (CNS). Emerging evidence links gut microbiota to disease pathogenesis, but the microbial factors that regulate pathogenic microRNA (miRNA) programs are largely unknown. Here, using experimental autoimmune encephalomyelitis (EAE, a MS mouse model), we investigated whether gut microbiota exacerbate EAE pathogenesis by modulating host miRNA expression. Antibiotic-induced depletion of the gut microbiota markedly attenuated EAE scores and reduced circulating inflammatory miRNAs, with miR-21 emerging as the dominant pathogenic candidate. Functional inhibition of miR-21 significantly ameliorated disease severity and reduced CNS T-cell infiltration. Mechanistically, miR-21 enhanced IL-17 and GM-CSF production by CD4 T cells and promoted immune-cell entry into the CNS through endothelial activation and blood-brain barrier dysfunction. We identified a transient expansion of Akkermansia muciniphila during the prodromal phase of EAE that positively correlated with circulating miR-21 levels. Colonization of antibiotic-treated mice with A. muciniphila exacerbated EAE and increased serum miR-21, whereas monocolonization of germ-free mice was insufficient to induce systemic miR-21, indicating a requirement for an inflammatory host environment. Further analyses revealed that atypical lipopolysaccharides (LPS) derived from A. muciniphila induce epithelial miR-21 production through coordinated TLR2/TLR4 signaling. Circulating miR-21 subsequently promoted endothelial dysfunction through the TIMP3-ADAM17 pathway, facilitating pathogenic T-cell migration into the CNS. Importantly, circulating miR-21 was also elevated in patients with MS. Collectively, these findings identify a previously unrecognized A. muciniphila-LPS-miR-21 axis linking gut dysbiosis to neuroinflammation and suggest that host-derived miRNAs function as systemic mediators through which microbial signals influence CNS autoimmunity.

immunology↗

bioRxiv2026

An ancient polymorphism in myosin I a/b determines the left-right asymmetry of Japanese snails

Left-right (LR) asymmetry is a fundamental feature of animal development, yet progress in understanding its establishment has been limited by the fact that almost all animals exhibit invariant chirality. Snails are the exception because some wild-living species show abundant chiral variation, yet the existing knowledge is from snails in which the chirality mutation is rare and causes pathology. Here, we show that a chiral polymorphism in Japanese Euhadra snails is due to functional variation in an unconventional myosin I a/b, an isoform not previously implicated in LR specification in any animal. Both the sinistral and dextral gene variants are ancient, impart minimal transcriptional differences in the single-cell embryo and are not pathological. Phylogenetic and structural modelling suggests that dominant-acting amino acid substitutions in the myosin actin-binding domain and/or motor-level junction were enabled by relaxed selection. These results broaden the known molecular repertoire underlying LR asymmetry, suggest key mutations and positions that should be explored in other model animals, and highlight snails as a powerful model for understanding the origins of animal LR asymmetry.

evolutionary biology↗

bioRxiv2026

Osteopontin Upregulation Defines a Pre-Rupture State in Thoracic Aortic Aneurysms in Mice and Humans

BackgroundThoracic aortic aneurysm (TAA) is a life-threatening condition with an unpredictable lisk of rupture. Current clinical parameters have limited ability to accurately predict imminent rupture. Osteopontin (OPN) has been implicated in aortic aneurysm pathology, however, it role as a marker of imminent rupture remains. unclear. We investigated the dynamics of OPN expression dynamics in a mouse model with predictable rupture timing and validated our findings in human TAA. MethodsOne-month-old fibrillin-1 hypomorphic (Fbn1mgR/mgR) mice were used as a TAA model; with wild-type (WT) mice served as controls. Angiotensin II (AngII) was administered to Fbn1mgR/mgR to induce acute aortic rupture. Single-section transcriptome analysis and immunofluorescence staining were performed on ascending aortic tissue at 24 and 72 hours after AngII infusion, with pre-treatment Fbn1mgR/mgR and WT mice serving as controls. To determine conservation in human disease, we reanalyzed publicly available single-cell RNA sequencing data from ascending thoracic aortic aneurysm (ATAA) patients. ResultsAngII infusion induced progressive mortality beginning at 24 hours, with approximately 60% survival at 72 hours and nearly no survival by 8 days in Fbn1mgR/mgR mice. At this pre-rupture time point, OPN showed prominent upregulation at both mRNA and protein levels in ascending aortic tissues compared to controls. Immunofluorescence staining revealed increased OPN expression in the aortic wall, particularly in regions exhibiting structural deterioration. Reanalysis of human ATAA single-cell data showed elevated OPN expression compared to controls, with enrichment in immune cell populations, especially macrophages. Within the macrophage compartment, subcluster analysis identified a stress-responsive subpopulation (MC1) that was markedly expanded and almost exclusively composed of ATAA-derived cells, representing the primary source of OPN upregulation. ConclusionsOPN upregulation represents a conserved molecular signature of the pre-rupture state in TAA across mice and humans. Our mode, which enables predictable rupture timing, allowed the capture of acute pre-rupture molecular changes, suggesting OPN as a potential biomarker for predicting imminent aortic rupture.

molecular biology↗

bioRxiv2026

The epidermal stem cell-supporting matricellular protein fibulin 7 modulates skin inflammatory response in a psoriasis model

ECM composition and organization are greatly altered during inflammation but it is still elusive if ECM dynamics may protect tissue stem cells against aberrant inflammation. Fibulin 7 (encoded by Fbln7) is part of the basement membrane ECM where epidermal stem cells (EpSCs) reside. It supports the long-term potential of fast-cycling EpSCs and moderates aging-related inflammatory markers in keratinocytes. Here, we assessed fibulin 7s role during imiquimod (IMQ)-induced inflammation in 1-year-old mouse dorsal skin. We found that loss of Fbln7 aggravates epidermal inflammation, marked by increased epidermal thickness, proliferation, and phosphorylation of JNK (c-Jun N-terminal kinase). Fast-cycling EpSCs labeled with Slc1a3-creER-TdTomato demonstrated that IMQ-induced proliferation in Fbln7 KO mice is contributed by cell divisions in the suprabasal layers, a hallmark of inflammatory epidermal responses. EpSC transcriptomes further reveal IMQ-modulated genes that are more substantially affected in Fbln7 KO mice, including IL-17 pathway-related genes known in psoriasis pathogenesis. Mechanistically, fibulin 7 directly binds to IL-17A and decreases IL-17A-mediated p38 MAPK activation. In public human psoriasis datasets, FBLN7 is reduced in lesional skin compared with non-lesional or normal skin, and it is significantly correlated with common psoriasis-associated genes. Altogether, fibulin 7 is potentially beneficial to protect against skin inflammation.

cell biology↗

bioRxiv2026

Sowing date effects on anther dehiscence, pollen germination on the stigma, and fertility under heat in Japanese rice

Heat-tolerant rice cultivars are essential for mitigating global warming impacts. Basal anther dehiscence length (BDL) is a promising visible morphological marker for heat tolerance through stable pollination. We investigated the effects of sowing date on anther morphology, pollination, and fertility under controlled high-temperature conditions (35, 37, or 39 {degrees}C at flowering). Three japonica cultivars-- Akitakomachi (early heading), Koshihikari (medium), and Hatsushimo (late)--were sown monthly over 3 months and grown in pots. At heading, the plants were exposed to the temperature treatments for 3 days, and the proportion of florets with [≥]10 germinated pollen grains on the stigma (GP10) and seed set were assessed. Among anther traits, BDL showed the greatest variation, with all cultivars from the second sowing exhibiting the shortest BDL. Analysis of variance revealed significant effects of genotype, sowing date, and their interaction on anther traits and fertility. Regression analysis indicated that fertility was associated with GP10, with BDL contributing significantly to GP10 in the late-heading Hatsushimo, together with maximum temperature at flowering. Thus, both genotype and environment shape anther morphology, pollination, and fertility, indicating that BDL plasticity and genotype-specific environmental responses must be carefully considered when using BDL as a breeding marker for heat tolerance. HighlightVariation in sowing date significantly affects anther morphology and heat tolerance in rice. Genotype-specific responses to the growing environment require careful consideration for reliable breeding assessments.

plant biology↗

bioRxiv2026

Anterior cingulate cortex projections to the amygdala in primates: topographic and layer-specific organization underlying emotion and mood regulation

Emotion and mood regulation critically depends on interactions between the anterior cingulate cortex (ACC) and the amygdala. However, the detailed architecture of ACC projections to their major targets, the basal (BA) and accessory (AcBA) basal nuclei of the amygdala, remains unclear. To address this issue, a combined retrograde and anterograde tracing with viral vectors were performed in macaques to map the projection patterns from pregenual (pgACC), subgenual (sgACC), and dorsal (dACC) subareas. Data revealed that ACC neurons projecting to the BA arose predominantly from the superficial layers (II/III) of all subareas and the deep layers (V/VI) of the sgACC, whereas ACC neurons projecting to the AcBA originated mainly in the deep layers of the sgACC and dACC. The present study defines the topographic and layer-specific organization of ACC-amygdala connectivity in primates and subserves to provide an anatomical basis for future causal and translational approaches, such as targeted interventions against ACC-related mood disorders. TeaserPrimate anterior cingulate cortex has topographic and layer-specific projections to amygdala that are involved in emotion and mood regulation.

neuroscience↗

bioRxiv2025

Tetraploid Caenorhabditis elegans embryos exhibit enhanced tolerance to osmotic stress

Polyploidy is a widespread phenomenon in development and evolution and is often associated with altered cellular physiology and developmental robustness. However, the influence of genome doubling on embryonic robustness to environmental stress remains poorly understood. Here, we investigated developmental traits and osmotic responses in tetraploid Caenorhabditis elegans embryos. Tetraploid animals exhibited enlarged body and tissue sizes and produced embryos with approximately 1.6-fold greater volume than diploids, accompanied by moderately delayed development and partial embryonic arrest. When exposed to a range of osmotic environments, both diploid and tetraploid embryos swelled or shrank in response to external osmolarity. Strikingly, tetraploid embryos at the early stage maintained normal cell division across a broader range of osmotic conditions than diploids. Quantitative analyses further revealed that tetraploid embryos exhibit reduced cytoplasmic mass density, largely reflecting lower protein concentration, while lipid and RNA levels remain unaffected. These compositional differences likely buffer volume fluctuations and underlie the enhanced osmotic tolerance of tetraploid embryos. Our findings demonstrate that genome doubling reshapes embryonic cellular physiology in a non-proportional manner to ploidy, thereby enhancing robustness during early development.

developmental biology↗

bioRxiv2025

Decoding the emergent properties of microbial community functions through sub-community observations and interpretable machine learning

The functions of microbial communities, including substrate conversion and pathogen suppression, arise not as a simple sum of individual species capabilities but through complex interspecies interactions. Understanding how such functions arise from individual species and their interactions remains a major challenge, limiting efforts to rationally understand microbial roles in both natural and engineered ecosystems. Because current holistic (meta-omics) and reductionist (isolation- or single-cell-based) approaches struggle to capture these emergent microbial community functions, this study explores an intermediate strategy: analyzing simple sub-community combinations to enable a bottom-up understanding of community-level functions. To examine the validity of this approach, we used a nine-member synthetic microbial community capable of degrading the environmental pollutant aniline, and systematically generated a dataset of 256 sub-community combinations and their associated functions. Analyses using random forest models revealed that the sub-community combinations of just three to four species enabled the quantitative prediction of functions in larger communities (5-9-member; Pearsons r = 0.78-0.80). Prediction performance remained robust even with limited sub-community data, suggesting applicability to more diverse microbial communities where exhaustive sub-community observation is infeasible. Moreover, interpreting models trained on these simple sub-community combinations enabled the identification of key species and interspecies interactions that strongly influence the overall community function. These findings provide a methodological framework for mechanistically dissecting complex microbial community functions through sub-community-based analysis.

microbiology↗

bioRxiv2025

Behavioural and physiological evidence for the development of cardiac-exteroceptive integration during the first year of life

Continuous integration of environmental exteroceptive and internal interoceptive signals is fundamental for perception and adaptive behaviours, yet its developmental trajectory remains poorly understood. Here, we introduced a modified iBEATs paradigm, based on prior work, to investigate cardiac-exteroceptive integration in 3-8-month-old infants. Using behavioural measures of looking time and physiological measures of pupillometry, we found that the ability to detect cardiac-exteroceptive synchrony emerges during the first year of life. Critically, this was evident only when stimuli coincided with systole, the baroreceptor-active phase of the cardiac cycle, supporting central multimodal integration interpretable within a predictive coding framework. Furthermore, individual differences in behavioural sensitivity were accounted for by the degree of autonomic maturation. These findings provide the first evidence for the developmental emergence and mechanisms of cardiac-exteroceptive integration, and establish the modified iBEATs paradigm as a promising tool for assessing interoceptive development in early life.

neuroscience↗

bioRxiv2025

Rare k-mers reveal centromere haplogroups underlying human diversity and cancer translocations

Centromeres are among the most diverse and dynamically evolving regions of the human genome and are commonly affected in various human cancers. However, organized into highly repetitive -satellite higher-order repeats (HORs), human centromere sequences have long resisted detailed genomic analysis. Although the development of long-read sequencing platforms has enabled the analysis of complete centromere sequences, their application to a large set of samples is still largely limited, preventing our understanding of centromere variation and haplotype structures across large human populations and the structural basis of centromere-involving translocations in cancer. Here we show that rare k-mers present in centromeric regions can serve as effective markers for dissecting the complexity of centromere structure, particularly that of active -satellite HOR arrays (aHOR arrays), across human populations and for understanding centromere-involving abnormalities in cancer. Based on rare k-mer-based clustering, centromere aHOR arrays are clustered into discrete haplogroups (aHOR-HGs) with distinct structural features. These k-mers were also used to develop a framework that enables the inference of haplogroups in a given sample based on short-read whole genome sequencing (WGS) data (ascairn). By applying ascairn to large-scale human population datasets (n > 3,300), we revealed the diversity of aHOR-HGs and their geographic histories across populations. The rare k-mer-based approach was also applied to investigate the structure of 1p/19q co-deletion, a highly recurrent centromere-involving translocation in IDH-mutated oligodendrogliomas. Analyzing short-read WGS data from 142 cases with 1p/19q co-deletion using rare k-mers, we showed that breakpoints of 1p/19q co-deletion were mapped to aHOR arrays in chromosomes 1 (D1Z7) and 19 (D19Z3), which was validated by long-read sequencing of two 1p/19q co-deletion-positive cases. Notably, the translocation preferentially involved haplogroups composed of haplotypes containing larger regions susceptible to rearrangement. These results highlight the role of rare k-mers in dissecting the complexity of centromere sequences and their evolutionary history as well as understanding centromere-involving abnormalities associated with human diseases.

genomics↗

bioRxiv2025

Xrp1 drives damage-induced cellular plasticity of enteroendocrine cells in the adult Drosophila midgut

Cellular plasticity, the ability of a differentiated cell to adopt another phenotypic identity, is restricted under basal conditions, but can be elicited upon damage to facilitate regeneration. Such damage-induced cellular plasticity restores homeostasis and prevents pathology, yet its underlying molecular basis remains largely unexplored. Here, we reported damage-induced cellular plasticity of secretory enteroendocrine cells (EEs) in the adult Drosophila midgut. We found that ionizing radiation enhanced EE plasticity such that it promoted EEs to dedifferentiate into ISCs and subsequently re-differentiate towards ECs. We identified that radiation induced the production of a stress-inducible transcription factor Xrp1 in EE lineages, and its upregulation was necessary for EE plasticity. Single-cell RNA sequencing of guts with EE-specific Xrp1 overexpression revealed ectopic expression of progenitor-specific genes in EEs, which was necessary for Xrp1 to drive EE plasticity. Our work provides a mechanistic framework for understanding cellular plasticity and suggests its potential role in damage-induced responses.

cell biology↗

bioRxiv2025

Spatiotemporal dynamics of CD73 in mouse retina under physiological conditions

Adenosine is essential to energy metabolism and neuromodulation in the central nervous system. The retina is a highly energy-demanding neural tissue, and dysregulation of adenosine signaling causes retinal diseases. However, the dynamics of ecto-5-nucleotidase (CD73), a key enzyme for extracellular adenosine generation, remain elusive. Here, we investigate its spatiotemporal profile from development to adulthood using two transgenic mouse lines. We found that CD73 is transiently expressed in the early astrocyte lineage (Embryonic day 16.5 to Postnatal day [P] 3), becomes prominent in the rod-photoreceptor lineage by P3, and appears in the inner nuclear layer from P7 onward. CD73 deletion delays rod-response recovery and shortens the implicit time in scotopic ERG under dim light. These findings indicate that light and other physiological cues influence CD73 expression, which has significant consequences for retinal function, thereby providing a foundation for exploring disease-related alterations in CD73 and designing therapies that restore adenosine homeostasis.

developmental biology↗

bioRxiv2025

Perturbing postural stability during treadmill walking with dysfunctional electrical stimulation

Functional electrical stimulation is commonly used to enhance human movement through low-level electrical activation of muscles. More recently, dysfunctional electrical stimulation (DFES) has been proposed as a method to perturb gait by artificially inducing discomfort and mimicking inadequate muscle activity. Here we investigated strategies to induce internal perturbations during treadmill walking using DFES by systematically varying the timing and target muscle. Eleven healthy participants walked at three different speeds while DFES was applied to the tibialis anterior (TA), soleus (SOL), rectus femoris (RF), and biceps femoris (BF) muscles at 25%, 50%, 75%, and 100% of the gait cycle, each for a duration of 0.2 seconds. The gait cycle was time-locked to heel contact (0%). Results showed a significant reduction in the anterior-posterior margin of stability compared to baseline, particularly when DFES was applied to the SOL at 75%, the RF at 50%, and the BF at 75% of the gait cycle. Under these conditions, increased knee flexion and shorter stride intervals were observed relative to baseline. In conclusion, we identified effective DFES conditions to induce postural instability during walking. By mimicking inadequate muscle activity, DFES provides a promising method to study dynamic balance control and mechanisms underlying falls in neurological populations.

bioengineering↗