Biology subjects

Qi, Y.

Publications and source records attributed to Qi, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Large-scale all-optical electrophysiology links excitability and connectivity to circuit dynamics in vivo

To understand neural computations, one would like to relate circuit dynamics to neuronal intrinsic excitability and connectivity. We developed tools for large-scale all-optical electrophysiology and applied them to approximately 300 layer 1 interneurons per mouse in vivo. Across two mice, targeted stimulation and voltage imaging identified 1,469 inhibitory and 5,752 directed electrical connections among 14,312 tested pairs. Presynaptic identity was more strongly associated with inhibitory post-synaptic potential (IPSP) amplitude, whereas postsynaptic identity was more strongly associated with IPSP decay time. Gap junctional and synaptic interactions had distinct spatiotemporal kernels, with corresponding signatures in spontaneous activity. Excitability measured under optogenetic stimulus predicted spiking responses to subthreshold voltage, depolarization rate, and spike history during spontaneous firing. Genetic subtypes also showed different behavioral-state-dependent voltage-to-spike relationships despite similar population-average subthreshold voltages. These measurements link cellular properties and pairwise interactions to spontaneous circuit dynamics, establishing an approach for functional circuit mapping in vivo.

neuroscience↗

bioRxiv2026

UniWave-2: A Hybrid Model for Nucleic Acid Waveform Feature Extraction Enhanced by Fourier and Wavelet Transforms

Motivation: Traditional methods primarily rely on statistical features such as k-mers and GC content, making it difficult to capture complex internal relationships within sequences. Deep learning models typically rely on discrete encodings, leading to issues such as information sparsity, dimensional redun-dancy, and disruption of sequence continuity. Our previous UniWave-1 framework transforms se-quences into waveform signals; however, it lacks comprehensive physicochemical integration and com-putational efficiency. Results: We propose the UniWave-2 feature extraction framework. UniWave-2 incorporates three key advancements: (i) integration of hydrophobicity into the encoding scheme; (ii) enhancement of wave-form resolution through Fourier transform, coupled with wavelet transform to precisely capture both local details and global periodic patterns; and (iii) development of a lightweight multi-scale GRU model that facilitates cross-dimensional feature interactions, captures bidirectional temporal dependencies, and integrates long- and short-range patterns with spatial positional awareness, thereby yielding rich feature representations. UniWave-2 achieved competitive performance across multiple genomic tasks, while ISM analysis demonstrated that waveform encoding enables interpretable attribution without re-lying on gradient information. These results establish UniWave-2 as a lightweight and interpretable framework for nucleic acid sequence analysis.

bioinformatics↗

bioRxiv2026

Risk-Window Palbociclib Exposure Delays ErbB2-Driven Mammary Tumorigenesis and Suppresses Mammary Epithelial Cell Stemness

ErbB2 overexpression occurs in 15-20% of invasive breast cancer (BC) and inactivation of Cyclin D1-CDK4/6 axis reduce mammary stem/progenitor cells in ErbB2-driven tumorigenesis. Here, we explored the preventive role of short-term palbociclib intervention in MMTV-ErbB2 mice. Palbociclib significantly inhibited the proliferation and stemness of ErbB2-overexpressed BC cells in vitro and in vivo. Furthermore, short-term palbociclib exposure during the early premalignant risk window significantly delayed mammary tumor development and reduced tumor multiplicity, accompanied by the suppression of epithelial proliferation and ductal/alveolar morphogenesis in premalignant tissues, as well as prolonged tumor-free survival compared with controls. Importantly, palbociclib reduced the luminal epithelial (CD24high/CD49flow), mammary reconstitution unit-enriched (CD24high/CD49fhigh) subpopulations, and luminal progenitor/TIC-enriched (CD61high/CD49fmid) subpopulations. These changes were accompanied by diminished mammary epithelial cell stemness functions, including colony-forming, mammosphere-forming, and 3D growth activities of mammary epithelial cells. Mechanistically, palbociclib-treated tissues showed inhibition of the Cyclin D1-CDK4/6-RB-E2F axis and coordinated attenuation of ER-, ErbB2- and Wnt/{beta}-catenin-associated signaling. Together, we demonstrate that short-term CDK4/6 inhibition during a premalignant risk window produces a sustained delay in ErbB2-driven mammary tumorigenesis, associated with remodeling of the mammary epithelial hierarchy and suppression of proliferative and stem/progenitor-associated activity, suggesting CDK4/6 inhibition as a strategy for ErbB2-positive BC prevention.

cancer biology↗

bioRxiv2026

Seasonal influenza vaccine strain selection by quantifying viral fitness

The long-standing challenge of seasonal influenza vaccines providing poor protection is attributed to the rapid and continuous evolution of the virus. In theory, the recommendation of vaccine strain is to predict the dominant strain with best fitness in the upcoming season. Here, we develop FutureFlu, a biologically-grounded framework that quantifies the fitness score of influenza variants in upcoming seasons, by integrating metrics on three levels: molecular genetic divergence, individual immune escape, and population-scale transmission dynamics. The fitness scores of influenza variants present strong positive correlation with their actual observed frequencies in the next seasons. Validation across 24 seasons of three influenza subtypes shows FutureFlu recommends antigenically matched vaccine strains more frequently than annual recommendations, particularly for challenging subtypes: 83.3% versus 45.8% seasons for A/H3N2, and 75.0% versus 33.3% seasons for B/Victoria. Furthermore, FutureFlu significantly outperforms the currently used methods in vaccine strain selection whether or not there is available antigenic data from hemagglutination inhibition (HI) assay, which provides a valuable supplement for WHO vaccine recommendation. To support global public health implementation, an open online platform (futureflu.com.cn) has been established to offer real-time viral fitness predictions and vaccine recommendations.

evolutionary biology↗

bioRxiv2026

A Hymenoptera-restricted gene mediating ant castes co-opts deeply conserved machinery to control organ size

Lineage-specific genes are widespread and have been implicated as phenotypic innovation inducers, but how they acquire complex developmental functions remains poorly understood. Ant queens and workers develop dramatically different organ sizes from identical genomes under juvenile hormone (JH) control, yet the molecular effectors translating JH signalling into caste-specific organ growth remain unknown. Here we identify torch, a Hymenoptera-restricted gene, as the most consistently gyne-biased and JH-responsive gene across 68 ant species. Knockdown of torch in virgin queens of Monomorium pharaonis produces a worker-like, multi-organ growth-restricted phenotype. Mechanistically, torch harbours an E-box-like motif activated by the JH receptor Gce-Tai and acts as a GA-repeat-binding transcription factor that regulates Hippo signalling, the deeply conserved organ-size control pathway in animals. Expressing torch heterologously in mice and a growth-restricted Drosophila background shows that the gene retained its general growth-promoting activity across more than 700 million years of animal evolution in lineages that lack the gene, establishing that its function is mediated through conserved rather than ant-specific machinery. A lineage-specific gene can therefore acquire complex morphogenetic function by co-opting ancient organ-size circuitry, providing a general route by which novel genes can drive phenotypic innovation.

evolutionary biology↗

bioRxiv2026

Expanding the Ligandable Chemical Space of OTUB1 through Discovery of a Four-Membered-Ring Recruiter Chemotype

Deubiquitinase-targeting chimeras (DUBTACs) have emerged as a promising strategy for targeted protein stabilization, but their broader application remains limited by the scarcity of ligandable deubiquitinase recruiters. Here, we report a previously unexplored four-membered-ring OTUB1 recruiter chemotype. Through systematic structure-activity relationship studies, we identified compound 21 (MS2159) as a potent and selective covalent OTUB1 ligand. Biochemical and intact protein mass spectrometric analyses demonstrated that MS2159 selectively engages the non-catalytic C23 residue of OTUB1, shows minimal reactivity toward other tested proteins, and preserves OTUB1 deubiquitinase activity. Conjugation of MS2159 with the CFTR ligand lumacaftor yielded compound 25 (MS2134), which effectively stabilized {Delta}F508-CFTR. Collectively, these findings establish a new OTUB1 recruiter scaffold, expand the ligandable chemical space of OTUB1, and provide additional opportunities for developing next-generation DUBTACs.

biochemistry↗

bioRxiv2026

Contextual Evaluation of MicroRNA Sequencing Data Harmonization: Performance in Sample Clustering

Reliable translation of microRNA sequencing data depends on effective harmonization to mitigate artifacts from variable experimental handling. Although many harmonization methods exist, prior evaluations have focused mainly on differential expression analysis, leaving the impact on subgroup discovery understudied. We present a framework for evaluating harmonization in the context of sample clustering, which integrates AI-augmented datasets, statistical evaluation pipelines, and accessible software tools, enabling systematic comparisons across diverse signal-to-artifact ratios and cluster composition settings. Using this framework, we show that harmonization can, often partially, restore clustering accuracy lost to artifacts, especially at moderate signal-to-artifact ratios, with the level of gains depending on the specific harmonization method, the paired clustering technique, and the cluster composition setting. We further confirm these findings by analyzing reconstructed cohorts from The Cancer Genome Atlas breast cancer microRNA sequencing data. Collectively, the results underscore the need for tailored harmonization to support reliable subgroup discovery and highlight the broader importance of context-specific workflows in translational genomics.

bioinformatics↗

bioRxiv2026

Programming protein shape as an explicit design layer via CAD blueprint-guided diffusion

In engineering, industrial design principles link geometric form with function. In nature, global protein shape, curvature, and chirality similarly drive critical processes like membrane remodeling and cellular motility. However, explicitly programming these global features remains a challenge for generative AI. Here, we integrate computer-aided design (CAD) blueprints with diffusion models, establishing protein shape as an explicit, programmable layer. We engineered diverse, tunable architectures, including single-chain shapes, scaffolds with unusual twist handedness, and superhelical assemblies, structurally validating 31 of 59 designs via X-ray crystallography or electron microscopy. To demonstrate shape-encoded function, we coupled fluid-flow-optimized helical propellers to an F1-ATPase rotor, yielding a prototype ATP-dependent molecular swimmer. This framework translates industrial design principles to the molecular scale, enabling the programmable engineering of biomimetic architecture with emergent functions. One-sentence summaryIntegrating CAD blueprints with diffusion-based AI translates industrial design principles to the molecular scale, enabling the explicit programming of protein shapes with emergent functions.

bioengineering↗

bioRxiv2026

MGM2 as a Unified Foundation Model for Microbiome World Exploration

Microbiomes are information-rich biological systems, yet most computational analyses still reduce communities to cohort-specific abundance tables. Here we introduce MGM2, a multimodal foundation model pretrained on 1,821,291 MicrobeAtlas samples and 225,067 OTUs clustered at 99% sequence similarity. MGM2 couples NTv3-derived microbial sequence embeddings with abundance conditioning and community-semantic alignment to learn transferable sample- and token-level representations. Frozen MGM2 representations outperformed DeepPhylo by 0.06-0.21 macro-AUROC across five temporally held-out MGnify hierarchy levels, with the largest gains for rare and fine-grained labels. In fecal microbiota transplantation, MGM2-XLarge achieved a response ROC AUC of 0.79 and reduced post-transplant Bray-Curtis distance by 15% relative to the recipient baseline. The same representation supported ASV-level trend forecasting across 24 wastewater treatment plants. Sparse autoencoder analysis resolved MGM2-XLarge token states into a 4,096-feature dictionary spanning taxonomic identity, abundance state, ecological context and technical variation. MGM2 therefore provides a sequence-aware and interpretable representation layer for microbiome classification, paired-community prediction, forecasting and feature discovery. Highlights[bullet] MGM2 integrates sequence, abundance and community semantics through pretraining on 1.82 million microbiome samples. [bullet]Frozen MGM2 improved macro-AUROC over DeepPhylo by 0.06-0.21 across five temporally held-out MGnify levels. [bullet]MGM2-XLarge reached a response ROC AUC of 0.79 and reduced post-FMT Bray-Curtis distance by 15%. [bullet]A 4,096-feature sparse autoencoder atlas resolves taxonomic, abundance, ecological and technical signals.

bioinformatics↗

bioRxiv2026

Human 28S rRNA analysed by state-of-the-art oligonucleotide mass spectrometry: benchmarking current capabilities and a call to action for MS-Seq

Oligonucleotide mass spectrometry (MS-Seq) is emerging as a powerful approach for sequence-resolved RNA modification analysis, yet the field lacks standards for experimental workflows, data analysis and reporting. To assess current capabilities, the Human RNome Project Consortium conducted a cross-platform benchmarking study using a common RNA sample. A partial RNase T1 digest of human 28S rRNA was distributed to participating laboratories and analysed using existing LC-MS/MS workflows spanning different chromatographic strategies and mass spectrometers. To enable direct comparison, datasets were analysed using a harmonized NucleicAcidSearchEngine (NASE) workflow. Despite substantial methodological differences, laboratories recovered highly overlapping oligonucleotide sets and generated similar sequence coverage maps with a global coverage of 54.16%, demonstrating reproducible sequence information across platforms under standardized sample and analysis conditions. The benchmark further revealed incomplete sequence coverage, platform-specific differences in data architecture and increased assignment ambiguity during dynamic modification searches. Together with the community consensus developed during the HRPC workshop, these findings define priorities for the field, including improved sensitivity, standardized data analysis and reporting, community repositories, and robust bioinformatic workflows for confident de novo RNA modification discovery. This study provides an experimental benchmark and roadmap toward routine MS-based mapping of the human RNome. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/739151v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1fb1b9borg.highwire.dtl.DTLVardef@d172a5org.highwire.dtl.DTLVardef@bdc185org.highwire.dtl.DTLVardef@1ec1cc9_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2026

Rapamycin Mitigates a Sex-biased Convergent Aging Trajectory

Rapamycin extends lifespan across species, yet its cell-type-specific benefits and vulnerabilities remain unclear at whole-organism scale. Here, we present the Rapamycin Fly Cell Atlas (Rapa-FCA), a whole-organism single-nucleus transcriptomic atlas of Drosophila spanning both sexes, multiple ages, 18 cell classes, and 181 cell types. Rapamycin elicited a highly heterogeneous response, with prominent effects in reproductive, digestive, and neuromuscular systems and modest responses in most neuronal populations. Across diverse tissues, we identified a rapamycin-sensitive Convergent Aging Trajectory (CAT), marked by Fkbp12 enrichment and mTORC1-linked metabolic programs, including glycolysis and lipid synthesis. CAT-high nuclei accumulated with age and were preferentially reduced by rapamycin, especially in females, consistent with stronger female lifespan extension. By integrating CAT abundance, aging-clock predictions, and nucleus-ratio changes, we mapped sex- and cell-type-specific geroprotection effects of rapamycin. Together, the Rapa-FCA provides an organism-wide framework for resolving how rapamycin reshapes cellular aging across sex, tissue, and cellular state.

developmental biology↗

bioRxiv2026

Identification of Heterogeneous Cortical Thickness Patterns Associated with Prenatal Gestational Diabetes Exposure: A SuStaIn-Based Subtyping Study

ImportancePrenatal exposure to gestational diabetes mellitus (GDM) has been associated with adverse metabolic, neurodevelopmental, and psychiatric outcomes in offspring. However, whether GDM-exposed youth exhibit heterogeneous neuroanatomical patterns remains unclear. ObjectiveTo identify distinct cortical thickness subtypes among GDM-exposed youth and examine their associations with anthropometric, neurocognitive, psychiatric/behavioral and neuroimaging measures both cross-sectionally and longitudinally. Design, Setting, and ParticipantsThis cohort study used the Adolescent Brain Cognitive Development (ABCD)(R)data, a multisite longitudinal population study. Subtype and Stage Inference (SuStaIn), an unsupervised machine learning framework, was applied to cross-sectional structural MRI data to identify cortical thickness patterns in 573 GDM-exposed youth and 2854 healthy controls. Posthoc longitudinal analyses included 1,853 observations from a subset of GDM-exposed youth with 1-, 2-, and 4-year follow-up visits to examine subtype differences in developmental trajectories over time. Exposure(s)Prenatal exposure to GDM. Main Outcome(s) and Measure(s)The primary outcomes included identification of cortical thickness subtypes and their inferred regional ordering patterns. Secondary outcomes included subtype-specific anthropometric, neurocognitive, psychiatric/behavioral and neuroimaging measures. ResultsThe GDM-exposed sample had a mean age of 119.02 {+/-} 7.34 months and was 47.5% female. Two cortical thickness subtypes were identified. Between subtypes, Subtype 1 (63.2%) was characterized by earlier inferred insula involvement and was associated with greater height (d = 0.36, pFDR < 0.001) and weight (d = 0.26, pFDR = 0.007), whereas Subtype 2 exhibited earlier inferred frontal involvement and nominally higher Attention-Deficit/Hyperactivity Disorder (ADHD) prevalence (d = 0.08, p = 0.036), steeper longitudinal cortical thinning across all six cortical regions of interest ({beta} range: -0.05 to -0.13, all pFDR < 0.05), and a smaller decline in Obsessive-Compulsive Disorder (OCD) prevalence over time ({beta} = -1.02, pFDR = 0.049). Conclusions and RelevanceGDM exposure was associated with two distinct offspring cortical thickness subtypes, each showing different inferred regional ordering patterns and clinical associations. One subtype showed an insula-cingulate-predominant pattern associated with anthropometric measures, whereas the other showed a frontal-predominant pattern associated with nominally higher psychiatric measures and faster cortical thinning over time.

neuroscience↗

bioRxiv2026

Intrinsic regulation of intestinal stem cell fate and homeostasis by Tet

How developmental progenitors navigate divergent trajectories to either establish adult stem cell pools or undergo terminal differentiation remains a fundamental gap in stem cell biology. Here, we identify the well conserved Tet protein as an essential transcriptional regulator of intestinal stem cell (ISC) establishment. Developmental Tet depletion causes region-specific ISC loss and compromises adult lifespan, while adult-specific loss drives progressive stem cell exhaustion. Overexpression of Tet leads to ISC expansion in both developing and adult guts. Utilizing a comprehensive single-nucleus transcriptomic atlas spanning gut development, we demonstrate that Tet stabilizes progenitor identity by maintaining epithelial integrity, niche signaling, and fate maintenance. By defining this developmental trajectory, we reveal Tet as a critical factor that drives proper ISC maturation and maintains long-term adult epithelial homeostasis.

developmental biology↗

bioRxiv2026

SSPSPredictor: A Sequence and Structure based Deep Learning Model for Predicting Phase-Separating Proteins

Liquid-liquid phase separation (LLPS) underlies the formation of biomolecular liquid condensates (also referred to membraneless organelles, MLOs), which are essential for spatially organizing various biochemical processes within cells. Proteins that play a key role in driving condensates formation are termed phase-separating proteins (PSPs). Given experimental identification of PSPs remains labor-intensive and time-consuming, multiple computational tools have been developed based on empirical features or deep learning. In this study, we propose SSPSPredictor, a novel multimodal predictive model for PSPs with folded or intrinsically disordered structures, leveraging the fusion of sequence information from a protein language model ESM-2 and structural insights from a graph neural network GVP. Compared with existing tools, SSPSPredictor achieves balanced performance in identifying endogenous PSPs, predicting relative LLPS propensities, and recognizing key regions that drive LLPS. Moreover, SSPSPredictor exhibits good interpretability in identifying driving regions along protein sequences, although no relevant supervision was provided during training. Further predictive analysis of the human proteome using SSPSPredictor reveals that the proportion of intrinsically disordered proteins (IDPs) undergoing LLPS is significantly higher than that of folded proteins. In addition, pathogenic variants, especially those located in disordered regions, exhibit higher LLPS propensity than other mutations, uncovering a link between LLPS and diseases at the amino acid level.

bioinformatics↗

bioRxiv2026

An oxytocin-gated circuit from the hypothalamus silences olfactory tubercle neurons to drive prosocial grooming

Spontaneous helping behaviors such as allogrooming are vital for survival in social species, yet their underlying neural mechanisms remain largely unknown. Although oxytocin (OXT) is known to modulate allogrooming, the precise neural circuits, molecular pathways, and sensory drivers remain unclear. Here, using a social emergency paradigm in mice, we show that observer mice selectively allogroom distressed, anesthetized conspecifics, a behavior that is guided by main olfactory system cues and mitigates the demonstrators anxiety. We identify an oxytocinergic circuit from the paraventricular hypothalamus to the medial olfactory tubercle (PVNOXT[->]mOT) that is necessary and sufficient for this helping behavior. In vivo fiber photometry reveals that PVNOXT[->]mOT activity and OXT release are temporally locked to allogrooming initiation. This behavioral effect requires oxytocin receptor (OXTR) signaling specifically on mOT dopamine D1 receptor-expressing neurons, where OXTRs suppress neuronal excitability via G protein-gated inwardly rectifying K+ (GIRK) channels. Disruption of this inhibitory mechanism induces neuronal hyperexcitability and impairs allogrooming, a deficit rescued by restoring GIRK function. These findings define a PVNOXT-mOT circuit for prosocial helping, revealing an oxytocinergic pathway with potential therapeutic relevance for neuropsychiatric disorders characterized by social deficits.

neuroscience↗

bioRxiv2026

Neuropeptide Y deficiency in the bone marrow drives hematopoietic stem and progenitor cell aging

Aging-related blood disorders are linked to defects in the regenerative and multilineage differentiation ability of hematopoietic stem and progenitor cells (HSPCs). While remodeling of the bone marrow (BM) microenvironment where HSPCs reside is known to contribute to these age-associated defects, the underlying factors and mechanisms remain poorly defined. Here, we discovered that the age-related decline of the neurotransmitter neuropeptide Y (NPY) in the BM is a critical driver of HSPC dysfunction. Using mouse models, we demonstrated that NPY levels decrease in the BM with age, and that genetic NPY overexpression or exogenous NPY administration in old mice substantially reverses aging-associated phenotypic and functional defects in HSPCs. Transcriptome analysis revealed that NPY supplementation in old mice restores aging-disrupted molecular pathways in their HSCs, including oxidative stress responses, myeloid differentiation, stemness, mitochondrial activity, and RhoA signaling. However, NPY genetic loss in young mice led to a decline in HSCs regenerative capacity and increased oxidative stress. Importantly, NPY levels also decline in elderly humans, and ex vivo treatment of human BM-derived HSPCs with NPY enhances their in vivo repopulating capacity. These results suggest that NPY supplementation or preservation of NPY-producing nerve fibers could be a therapeutic strategy to rejuvenate aged HSC function.

cell biology↗

bioRxiv2026

Looking backward for the future: long-term population recovery, habitat associations, and future climatic vulnerability of the critically endangered Sino-Mongolian beaver (Castor fiber birulai) in China

Despite the successful population recovery of the Eurasian beaver (Castor fiber) across much of Eurasia, its subspecies, the Sino-Mongolian beaver (C. f. birulai), remains critically endangered, with an estimated population of approximately 1,500 individuals confined to a small number of isolated and fragmented refugia along the China-Mongolia border. Effective conservation of this highly threatened subspecies requires a holistic perspective that integrates constraints on population dynamics, habitat associations, and future climatic vulnerability. Here, we combined systematic annual field surveys conducted between 2003 and 2023 with historical survey records from 1975 to 1989 in northern Xinjiang, China, to synthesize long-term spatiotemporal population dynamics, evaluate habitat preferences based on nine local environmental variables, and assess future climatic vulnerability using ensemble species distribution models (SDMs) under projected climate change scenarios. We detected a significant and phased population recovery, with beaver colony numbers increasing from 27 (approximately 100 individuals) in 1975 to 227 (681-908 individuals) in 2023. This recovery closely corresponded with major conservation milestones, including the establishment and upgrading of nature reserves, strengthened legislative protection, and enhanced multi-stakeholder collaboration. Habitat analyses further indicated that the Sino-Mongolian beaver preferentially occupied areas characterized by minimal anthropogenic disturbance and stable hydro-geomorphic conditions. Critically, SDM projections revealed that only 14% of the current study area presently exhibits high climatic suitability, and these highly suitable habitats are expected to disappear entirely by the 2050s. Together, our findings provide a comprehensive overview of the historical population recovery and conservation trajectory of the Sino-Mongolian beaver in China, and offer robust scientific support for developing adaptive management strategies in the face of ongoing climate change and increasing human pressures.

ecology↗

bioRxiv2026

Accessible and reproducible mesoscale fMRI at 5.0 T: A Pulseq-based open framework for human laminar mapping

Mesoscale, layer-specific functional MRI (fMRI) enables noninvasive access to cortical microcircuitry, yet widespread adoption has been constrained by a reliance on ultra-high field ([≥]7.0 T) systems and proprietary pulse sequences. To bridge this gap and enhance accessibility, we developed an open-source framework at 5.0 T for mapping laminar brain activity. This framework integrates a Pulseq-based 3D vascular space occupancy (VASO) sequence with an end-to-end data acquisition and analysis pipeline. At matched sub-millimeter resolution (0.8 mm in-plane), the Pulseq-based 3D implementation increased slab coverage by [~]1.82-fold and improved temporal signal-to-noise ratio by [~]1.50-fold relative to a vendor-provided 2D-VASO sequence. Validated using a finger-tapping paradigm, individual cerebral blood volume-weighted (VASO) laminar activation profiles consistently revealed the canonical "double-peak" pattern, with distinct superficial and deep peaks in the primary motor cortex. These profiles exhibited excellent cross-visit reliability (r = 0.80), and peak depths showed good spatial reliability (ICC = 0.69 for deep layers; ICC = 0.58 for superficial layers). Between-subject reproducibility was high (r = 0.86). Deploying the identical Pulseq protocol at an independent imaging site reproduced the characteristic double-peak laminar profiles (r = 0.63). At the group level, 5.0 T laminar profiles closely matched established 7.0 T findings, robustly resolving both deep and superficial peaks despite the lower field strength. Notably, for each participant, a single 13-minute VASO run was sufficient to resolve reliable laminar activation patterns that exhibited high consistency with multi-run averages (r = 0.78), highlighting the potential for high-throughput population studies or clinical research settings. The Pulseq-based 3D VASO sequence file, image reconstruction pipeline, and data analysis scripts are openly available to facilitate the adoption of this framework. This work establishes a practical route towards more accessible and reproducible mesoscale fMRI for studying human laminar functional architecture.

neuroscience↗