Biology subjects

Iwama, S.

Publications and source records attributed to Iwama, S..

12 recordsLinked to original sources

bioRxiv2026

STEER-seq: a recombinase based platform for pooled multimodal single-cell perturbation screening

Pooled gain-of-function screening is a powerful method for screening for transcription factors whose overexpression leads to changes in cellular identity. However, variable transgene copy number, integration site and genomic context from random viral integration can confound quantitative comparisons between perturbations. We present STEER-seq (Single-cell Transgene Expression via Exchange Recombination sequencing), a virus-free platform for pooled ORF gain-of-function screening in human induced pluripotent stem cells (hiPSCs). STEER-seq combines irreversible Bxb1-mediated integration at a defined genomic safe-harbour locus with promoterless donor libraries and barcode-enabled perturbation tracking, enabling single-copy expression from a common promoter and genomic context and direct recovery of perturbation identity by single-cell sequencing. In a focused haematopoietic screen, SPI1 expression alone was sufficient to drive differentiation towards HSC/MPP-like progenitors in the absence of exogenous cytokines, with transient induction generating progenitors that retained multilineage haematopoietic differentiation potential. Joint RNA-chromatin accessibility profiling linked transcription factor-induced transcriptional responses to chromatin remodelling and enabled reconstruction of perturbation-induced gene regulatory networks, including a RUNX2-driven osteoblast programme. Scaling STEER-seq to around 2,000 human coding sequences enabled systematic discovery of transcription factors capable of inducing distinct cell-state programmes, including neural progenitor-, melanocyte- and vascular smooth muscle-like states, as well as maintenance of pluripotency. STEER-seq provides a scalable, quantitative framework for single-cell and multimodal gain-of-function screening, enabling functional discovery and engineering of human cell identity.

genomics↗

bioRxiv2026

SELECT-seq allows Pre-Sequencing Enrichment of SNP Edits in One-Pot Single-Cell Whole-Transcriptome Sequencing

Advances in high-throughput sequencing have associated millions of putative genetic variants with disease. However, scalable experimental methods to establish causal relationships between genetic variants and downstream transcriptional outcomes remain a major challenge. Single-cell methods that integrate genotyping with transcriptomic profiling provide a way to address this, but do not enable pre-sequencing enrichment of correctly edited cells, limiting scale. We present SELECT-seq (SNP Enrichment Leveraging Cas12a Targeting), a rapid method that allows SNP-specific PCR amplification and Cas12a-mediated fluorescence detection simultaneously with whole-transcriptome amplification. This one-pot workflow enables identification and enrichment of SNP-bearing single cells, making a rapid and scalable methodology for analysis of genotype-phenotype linkage avoiding laborious single cell cloning steps. As a proof of principle we show that SELECT-seq distinguishes U-2 OS and T-47D cell lines based on a PIK3CA (NM_006218.4:c.3463A>G) mutation while preserving transcriptome integrity. It physically enriches a rare NRF2 T80K (NM_006164.5:c.390C>A) mutant cells (6.7%) from a prime-edited pool, achieving 86% genotype accuracy, and shows 87.5% directional concordance in the transcriptomic effects compared with a clonal NRF2 T80K cell line. SELECT-seq thus provides a rapid, scalable and widely accessible approach for mapping genotype-phenotype relationships at single-cell resolution.

molecular biology↗

bioRxiv2026

Design and validation of a novel portable electrode holder for motor-related EEG measurement

ObjectiveA simplified headset to measure electroencephalography (EEG) from sensorimotor areas is necessary to monitor motor-related neural responses accurately in real-world environments. The aim of the present study was to design and validate a novel, easy-to-use, and reliable EEG electrode holder that enables positioning of the EEG electrodes directly over the sensorimotor cortex, while enabling flexibility in relation to varying head size. ApproachThe spatial distribution of motor-related EEG activity was estimated using a dataset of high-density EEG (HD-EEG) from 82 participants. International databases of head shape were analyzed to determine dimensional requirements for a headphone-shaped holder. The proposed headset was validated by comparing its recordings with those obtained with a commercially available HD-EEG (Experiment 1) and by computing the actual position and recorded EEG motor-related activity obtained using the proposed holder (Experiment 2). Main resultsThe estimated distance between the measurement electrode and the top of the head, used as a design requirement for the proposed headset, ranged from 56.2 to 80.0 mm. Experiment 1 showed that the center frequencies of alpha-band recorded by the proposed and by the HD-EEG headsets were highly correlated (r=0.97). Experiment 2 showed that, by using the proposed headset, the actual placement of electrodes was within 8 mm from the ideal positions. Moreover, the experiment showed consistent results in terms of task-, location-, and frequency-specific modulation of sensorimotor activities in the alpha-band. For example, significant contralateral motor-related event-related desynchronization in the alpha-band, and significant alpha-band power increase during the eyes closed condition, namely event-related synchronization. SignificanceThe proposed electrode holder is easy to use and adjustable to compensate for varying head size and it may enable reliable measurement of motor-related EEG. It could support practical application of motor-related EEG acquisition in real-world contexts in several applications including sports, rehabilitation, and artistic performance.

neuroscience↗

bioRxiv2025

Pre-movement neural population activity in human motor cortex reflects the subsequent outcome of futsal free kicks

Brain states immediately before movement may shape skilled athletic performance, but translating electroencephalography (EEG)-based monitoring to sport has been limited by dense montages and laboratory-constrained tasks. We asked whether a portable three-channel EEG system can detect outcome-related preparatory activity during a realistic futsal free-kick task. EEG from C3, Cz, and C4 during the 2 s before each kick was used to decode successful versus unsuccessful trials with channel-wise linear support vector machines (SVMs), and the same approach was tested for transfer across recording days. To relate decoding to interpretable physiology, alpha- and beta-band power were also compared between outcomes. Successful and failed kicks were decoded significantly above label-shuffled null distributions within recording days across all channels, showing that low-density EEG can capture sensor-level information linked to subsequent performance. However, decoding did not generalize reliably across days, indicating that these neural signatures were session specific. Spectral analysis showed selectively increased C3 beta-band power before successful kicks, whereas alpha-band power showed no significant outcome-related differences. These findings support portable EEG as a practical route toward monitoring preparatory brain states in sport, while suggesting that real-world use will require short day-specific calibration or adaptive decoding.

bioengineering↗

bioRxiv2025

Chronotype-specific impacts of sleep-wake schedule on behavioral, physiological and psychological parameters

Diurnal variations in human performance are known to be associated with chronotype, which reflects individual differences in preferred sleep-wake cycles. Early chronotypes (ECs) exhibit superior motor and cognitive functions in the morning, whereas late chronotypes (LCs) tend to perform better in the evening. However, previous studies have examined these patterns under socially imposed nighttime sleep schedules, without considering individuals preferred sleep timing. Thus, it remains unclear whether these chronotype-dependent performance peaks persist when individuals follow their preferred sleep schedules. To address this question, we measured subjective states, sleep pressure (using electroencephalography), and motor and cognitive functions at 1.5 and 12.5 hours after awakening under two conditions: the Clock-Time condition (10:30 PM-6:30 AM sleep aligned with social rhythms) and the Self-Time condition (8 hours of sleep aligned with individual preferences). The results revealed distinct chronotype-specific patterns. ECs exhibited increases in fatigue and sleepiness dependent on awake duration, showing typical sleep pressure accumulation. LCs were more affected by sleep timing and showed atypical patterns of sleep pressure accumulation compared with ECs. Motor ability tended to improve in the afternoon in both chronotypes, irrespective of sleep-wake schedule, whereas cognitive performance peaked at different stages of wakefulness - earlier for ECs and later for LCs - independent of the time of day. These findings suggest that chronotype-aligned sleep-wake schedules could help optimize fatigue management, cognitive performance, and motor control. Diurnal variations in performance cannot be attributed to clock time alone but emerge from the combined effects of chronotype, awake duration, and sleep-wake regulation. Statement of SignificanceThis study provides new insights into the ways in which chronotype and sleep-wake schedules jointly shape behavioral, physiological, and psychological dynamics across the day. Whereas previous research has largely emphasized circadian rhythms, our findings underscore the critical role of actual sleep timing in modulating daily function ing. This integrative perspective extends current understanding of the ways in which individual differences in biological timing and social constraints interact to influence human health and performance. Importantly, the results highlight the need for future studies to disentangle circadian and sleep-wake schedule effects, which could inform personalized strategies for optimizing well-being, productivity, and clinical interventions in disorders related to sleep and circadian misalignment.

neuroscience↗

bioRxiv2025

Generating long deletions across the genome with pooled paired prime editing screens

Engineered deletions are a powerful probe for studying genome architecture, function, and regulation. Yet, the lack of effective methods to create them in large numbers and at multi-kilobase scale has rendered interrogating all the gigabases of human DNA elusive. Prime editing based approaches are promising contenders to fill this gap, but have not been adopted for large-scale pooled screening so far. Here, we performed the first high-throughput paired prime editing deletion screens. To establish the technology, we generated 3,802 deletions of up to 1.2 Mb in a reporter locus in two human cell lines, and identified key determinants of paired prime editing efficiency to be deletion length, predicted pegRNA efficacy, and contact frequency. We used these learnings to generate therapeutically important deletions at rates that are expected to give clinical benefits for retinitis pigmentosa and Duchenne muscular dystrophy. Combining several technological advances, we then performed the first pooled paired prime editing screen to delete over 10,471 sequences of median length 1.1 kb that target 149 genes across the genome. Our screens were high quality, recovering 75% of positive controls. We discovered 462 non-coding regions with fitness effects, and validated deletions with impact as well as tolerated ones in regions with functional annotations. Our screens are the largest pooled characterization of precise deletions to date, demonstrating that prime deletion screens can perturb the genome at scale, and highlighting that most non-coding DNA around essential genes is dispensable for cell growth in one condition.

genomics↗

bioRxiv2025

Human intention inference with a large language model can enhance brain-computer interface control: A proof-of-concept study

Brain-computer interface (BCI) control enables direct communication between the brain and external devices. However, the accuracy of BCI control based on intentions inferred from unstable neural signals remains limited, even with data-driven approaches to tailor neural decoders. Here we propose a knowledge-driven framework for inferring human intentions, leveraging large language models (LLMs) that incorporate prior knowledge about human behavior and neural activity. We developed a neural decoder that integrates neural and oculomotor signals with contextual information using an LLM agent. Its feasibility was tested in a real-world BCI task involving interaction with a computer application. The LLM-based decoder achieved an average accuracy of 79% among participants whose neural signals were responsive (11 out of 20) in inferring the intention to select arbitrary posts in a social networking service. Ablation analyses revealed that the integration of contextual information, multimodal signals, and empirical knowledge is critical for decoding accuracy. This study demonstrates the feasibility of a neural decoding framework using an LLM, paving the way for improved performance in BCI-driven external device operation for individuals with disabilities. HighlightsO_LILarge language models can infer human intent by integrating neural and oculomotor signals with screen context. C_LIO_LIThe proposed model outperforms conventional data-driven approaches in decoding accuracy. C_LIO_LIAblation analyses reveal that integrating contextual information, multimodal signals, and empirical knowledge is critical for accurate decoding. C_LI

neuroscience↗

bioRxiv2025

Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

The roles of circulating complement C3, produced in hepatocytes, in immune defense are well defined in the literature. Interestingly, the C3 gene evolved before the circulatory system, suggesting an essential but yet unknown role of locally produced C3. Here, we investigated the effect of C3 on cancer immunotherapy and found that cancer-associated fibroblast (CAF)-derived C3, not hepatocyte-derived C3, determines the efficacy of immune checkpoint blockade (ICB). Tumors developed in CAF-specific C3 knockout mice exhibited resistance to anti-PD-1 therapy, with increased immunosuppressive M2-like macrophage infiltration. Mechanistically, the C3 degradation product iC3b suppressed myeloid cell infiltration via complement receptor 3 signaling, and the therapeutic targeting of this pathway restored ICB sensitivity of immunotherapy-resistant tumors. In human cancers, stromal C3 expression correlates with reduced M2-like macrophage infiltration and improved immunotherapeutic outcomes. These data demonstrate the role of locally produced C3 in the innate immune response, which may have been conserved across many organisms.

cancer biology↗

bioRxiv2024

Enhanced human sensorimotor integration via self-modulation of the somatosensory activity

Kinesthetic motor imagery, the mental rehearsal of movement and its associated sensations1,2, activates somatosensory and motor cortices3,4. Motor imagery improves performance in neurorehabilitation5, sports6, and instrument play7. However, the mechanism that enhances performance through repetitive motor imagery remains unclear. Here, we combined individually tailored motor imagery training combined with closed-loop neurofeedback training, neurophysiology, and behavioral assessment to characterize how the training can modulate the somatosensory system and improve performance. The closed-loop training using real-time feedback of human electroencephalogram (EEG) signals enhanced participants self-modulation ability of intrinsic neural oscillations in the primary somatosensory cortex (S1) within 30 minutes. Further, the short-term reorganization in S1 was corroborated by the post-training reactivity enhancement revealed by changes in somatosensory evoked potential (SEP) amplitude of early component originating from S18. Meanwhile those derived from peripheral sensory fibers were maintained, suggesting that the closed-loop training manipulated cortical activities. Behavioral evaluation demonstrated improved performance during keyboard touch-typing indexed by resolved the speed-accuracy trade-off9. Collectively, our results provide evidence that mentally rehearsed movement during kinesthetic motor imagery induces functional reorganization of S1 activities that can contribute to performance improvement.

neuroscience↗

bioRxiv2023

An Alzheimer's disease-associated common regulatory variant in PTK2B has causal effects on microglial function

Genome-wide association studies (GWAS) are revealing an ever-growing number of genetic associations with disease, but identifying and functionally validating the causal variants underlying these associations is very challenging and has only been done for a vanishingly small number of variants. Here we validate a single nucleotide polymorphism (SNP) associated with an increased risk of Alzheimers disease (AD) in an intronic enhancer of the PTK2B gene, by engineering it into human induced pluripotent stem cells (hiPSCs). Upon differentiation to macrophages and microglia, this variant shows effects on chromatin accessibility of the enhancer and increased binding of the transcription factor CEBPB but only subtle effects on PTK2B or CLU expression. Nevertheless, this variant results in global changes to the transcriptome and phenotype of these cells. Expression of interferon gamma responsive genes including chemokine transcripts and their protein products are altered, and chemotaxis of the resulting microglial cells is affected. This variant thus causes disease-relevant transcriptomic and phenotypic changes, and we propose that it acts by altering microglia reactivity, consistent with the role of these cells in progression of AD.

neuroscience↗

bioRxiv2022

Two common issues in synchronized multimodal recordings with EEG: Jitter and Latency

Multimodal recording using electroencephalogram (EEG) and other biological signals (e.g., electromyograms, eye movement, pupil information, or limb kinematics) is ubiquitous in human neuroscience research. However, the precise time alignment of data from heterogeneous sources is limited due to variable recording parameters of commercially available research devices and experimental setups. Here, we introduced the versatility of a Lab Streaming Layer (LSL)-based application for multimodal recordings of high-density EEG and other devices such as eye trackers or hand kinematics. To introduce the benefit of recording multiple devices in a time-synchronized manner, we discuss two common issues in measuring multimodal data: jitter and latency. The LSL-based system can be used for research on precise time-alignment of datasets, such as detecting stimulus-induced transient neural responses and testing hypotheses well-formulated in time by leveraging the millisecond time resolution of the system.

neuroscience↗

bioRxiv2021

De novo brain-computer interfacing deforms manifold of populational neural activity patterns in human cerebral cortex

Human brains are capable of modulating innate activities to adapt to novel environmental stimuli; for sensorimotor cortices (SM1) this means acquisition of a rich repertoire of motor behaviors. We investigated the adaptability of human SM1 motor control by analyzing net neural population activity during the learning of brain-computer interface (BCI) operations. We found systematic interactions between the neural manifold of cortical population activities and BCI classifiers; the neural manifold was stretched by rescaling motor-related features of electroencephalograms with model-based fixed classifiers, but not with adaptive classifiers that were constantly recalibrated to user activity. Moreover, operation of a BCI based on a de novo classifier with a fixed decision boundary based on biologically unnatural features, deformed the neural manifold to be orthogonal to the boundary. These principles of neural adaptation at a macroscopic level may underlie the ability of humans to learn wide-ranging behavioral repertoires and adapt to novel environments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/450263v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@12a66c3org.highwire.dtl.DTLVardef@c090e8org.highwire.dtl.DTLVardef@698f87org.highwire.dtl.DTLVardef@4e135c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗