Biology subjects

Zhang, H.

Publications and source records attributed to Zhang, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Preclinical Development and Safety of Laterally Spreading Dual AAV.SPR-MYO7A Vectors for the Treatment of Usher Syndrome Type 1B

Usher syndrome type 1B (USH1B), caused by mutations in MYO7A, is the most severe form of Usher syndrome and accounts for more than 50% of USH1 cases. Affected patients are born profoundly deaf with vestibular dysfunction and develop progressive retinitis pigmentosa, for which no approved treatment exists. The MYO7A coding sequence exceeds the packaging capacity of adeno-associated virus (AAV), the gold standard vector for retinal gene therapy. To overcome this, we developed dual AAV hybrid and overlap platforms to reconstitute full-length MYO7A expression in photoreceptors and retinal pigment epithelium. Modification of cDNA split points, and reduction of non-coding sequence yielded optimized vector pairs that produced higher full-length MYO7A levels with substantially reduced truncated protein. Packaging in the laterally spreading AAV.SPR capsid enabled supraphysiological MYO7A expression in cynomolgus macaques at macular and distal retinal sites without requiring foveal detachment, an important safety consideration for USH1B patients with remaining central vision. A six-month GLP toxicology study confirmed that ATSN-301, an AAV.SPR-based dual vector reconstituting MYO7A driven by the smCBA promoter, was well tolerated at all doses, with a No Observed Adverse Effect Level established at 1.2E11 vg/eye, supporting its clinical advancement for USH1B.

genetics↗

bioRxiv2026

Transcriptomic similarity guides source selection beyond tissue lineage

Perturbational and pharmacogenomic maps remain incomplete across cellular contexts, leaving a practical source-selection question before response prediction: which measured context should inform an unmeasured target? We tested whether baseline transcriptomic similarity ranks candidate sources for held-out targets, separating source ranking from aggregation and transfer-uncertainty decisions. Similarity tracked matched-response concordance in LINCS and GSE306429; selected-source utility exceeded random expectation in GSE306429 ({Delta}U = 0.034; P = 0.002) and in exact-time-matched MIX-Seq tasks ({Delta}U = 0.046). In the full GDSC2 analysis, the drug-balanced ranking effect was 0.359 (95% CI, 0.337-0.383). In a lineage-restricted analysis of the same eligible subset, within-tissue ranking remained positive at 0.130 (95% CI, 0.119-0.141), retaining 35.8% of the same-subset global effect (0.363; 95% CI, 0.342-0.386). Global transcriptomic top-1 selection exceeded tissue-matched random selection by 0.154 (95% CI, 0.143-0.164). Thus, baseline similarity adds source-ranking information after tissue matching in the evaluated setting, while ranking alone does not resolve whether to aggregate sources or how to quantify transfer uncertainty.

bioinformatics↗

bioRxiv2026

Cancer cells hijack mitochondria from Bhlhe40+ T cells for immune evasion

Cancer cells hijack mitochondria from T cells via tunneling nanotubes (TNTs) to promote immune evasion, yet the underlying mechanisms of mitochondrial-loss remain poorly understood. Here, through targeted screening of relevant genes, we demonstrated that CD38 is a potent regulator of TNT formation through interaction with its cognate ligand, CD31. Conditional knockdown of Cd38 in murine models leads to CD8+ T cell-dependent tumor shrinkage. Single-cell RNA sequencing of tumor-infiltrating T cells revealed that cancer cells preferentially target the Bhlhe40+ effector subset. Bhlhe40 is a transcription factor that upregulates the expression of S100a4, a reported chemoattractant of nanotubes. Bhlhe40 upregulation creates mitochondrial-loss vulnerability for tumor neoantigen-specific T cells. Dual mitochondria transfer blockade with Cd38 knockdown and S100a4 inhibitor trifluoperazine markedly suppressed tumor growth. Collectively, our findings identified the Bhlhe40-S100a4 cascade as a critical regulator of cancer/T cell mitochondrial transfer, providing insights for the development of new cancer immunotherapies.

immunology↗

bioRxiv2026

NK-RCPP: a receptor-conditioned computational workflow for prioritizing neurokinin-receptor peptide candidates

Prioritizing peptides for closely related G protein-coupled receptors requires comparing target-directed scores with evidence from other structural contexts. We applied the Neurokinin Receptor-Conditioned Peptide Prioritization workflow (NK-RCPP) to 96 generated candidates for neurokinin receptors NK1R, NK2R and NK3R. The retrospective analysis combined receptor-matched on-target REF15 scores with 4,518 paired structural-neighbour comparisons covering 82 candidates. Lower on-target scores than the matched controls were observed for 14/32 NK1R, 23/32 NK2R and 6/32 NK3R candidates. Adding the neighbour-score criterion retained 16 of the 33 evaluable on-target-favoured candidates, reducing that pool by 51.5%. At matched receptor-specific shortlist sizes, half of the candidate identities differed from on-target-only selection. NK2R-04 and NK2R-19 remained selected at the higher neighbour-fraction threshold of 0.75. Fourteen NK2R candidates lacked neighbour summaries, leaving their joint-rule status unresolved. Sequence-frequency and embedding analyses described the candidates within a 1,048-peptide comparison space, and an exploratory 10-peptide NK2R comparison showed differences in standardized scores with uncertain score-potency agreement. These findings show how paired structural-neighbour scoring and threshold choice shape candidate selection within a receptor family. The resulting shortlist provides a defined set for testing whether joint prioritization enriches for active, receptor-preferred peptides.

bioinformatics↗

bioRxiv2026

Tryptophan Restores Recovery Competence Following Starvation in the Absence of Lysosomal Amino Acid Sensing

How organisms recover from starvation to nutrient-replete physiology remains poorly understood. In C. elegans, we previously observed that transcription factor hlh-30 or a combination of glucose and linoleic acid in its absence are essential for survival with refeeding following starvation, i.e. recovery competence. A forward genetic screen to evaluate mechanisms identified depd-5, which encodes a GATOR1 subunit, loss of which is expected to impair amino acid sensing with unrestrained raga-1 and mTOR activation. Surprisingly, glucose refeeding reduced recovery competence in depd-5(lf, loss-of-function) animals, which was rescued by amino acids, specifically tryptophan. Increasing tryptophan levels in depd-5(lf) worms through supplementation, reduced tryptophan catabolism with tdo-2 ablation and prolonged starvation restored recovery competence. raga-1(gf, gain-of-function) recapitulated whereas raga-1(lf) rescued the impaired recovery competence in starved depd-5(lf) worms. An analogous DEPDC5 mutation in mammalian cells impaired starvation-induced mTOR inactivation. We conclude that tryptophan counters unrestrained mTOR activity to restore recovery competence following starvation.

cell biology↗

bioRxiv2026

MPFIL: Multi-Level Protein Feature Integration for Predicting Mutation-Induced Protein-Protein Binding Affinity Changes

Predicting mutation-induced changes in protein protein binding affinity ({triangleup}{triangleup}G) is challenging because mutations can affect both local residue environments and the structural organization of protein complexes. Existing approaches often rely on handcrafted physicochemical, energetic, or evolutionary features, which introduce additional computational costs and require domain-specific knowledge. In this work, we propose MPFIL, a multi-level protein feature integration framework for predicting mutation-induced protein-protein binding affin ity changes. MPFIL integrates structural and sequential infor mation to characterize mutation effects from complementary perspectives. Its structural feature extraction module captures mutation-induced structural changes and differential information between wild-type and mutant complexes, while sequence feature extraction modules model local residue context and long-range dependencies. The resulting features are integrated for {triangleup}{triangleup}G prediction. We evaluate MPFIL on three benchmark datasets. Experimental results demonstrate competitive and consistent performance across different protein complex settings, while ablation studies verify the contribution of the structural and sequential features. These results support the effectiveness of multi-level protein feature integration for modeling mutation induced changes in protein-protein binding affinity.

bioinformatics↗

bioRxiv2026

METTL3-mediated m6A modification alleviates skeletal muscle atrophy in ALS by regulating ACSL1-dependent triglyceride metabolism

Skeletal muscle-intrinsic defects are increasingly recognized as critical contributors to the pathogenesis of amyotrophic lateral sclerosis (ALS), yet the underlying molecular mechanisms remain poorly understood. N6-methyladenosine (m6A) is the most abundant post-transcriptional RNA modification in mammalian mRNAs that plays important regulatory roles in diverse physiological and pathological processes, however its role in ALS-associated skeletal muscle atrophy remains unknown. Here we found that global m6A methylation and the expression of its methyltransferase METTL3 are reduced in the skeletal muscle of hSOD1G93A transgenic ALS mice. Multi-omics analyses reveal that m6A dysregulation is closely associated with impaired triglyceride metabolism. Functionally, METTL3 promotes triglyceride accumulation in ALS skeletal muscle in an m6A-dependent manner; overexpression of METTL3 in ALS myotubes improves energy supply and alleviates muscle atrophy by promoting triglyceride accumulation. In vivo overexpression of Mettl3 in skeletal muscle attenuates muscle atrophy, improves motor performance, and prolongs survival of ALS mice. Mechanistically, we identify the fatty acid activation enzyme ACSL1 as a key downstream effector of METTL3-mediated regulation of triglyceride metabolism. METTL3-mediated m6A modification promotes the expression of Acsl1 by enhancing its mRNA stability. Further investigation revealed that the regulatory effect of m6A on Acsl1 expression depends on the m6A reader protein IGF2BP2. Collectively, our findings establish m6A modification as a critical regulator of skeletal muscle atrophy in ALS by maintaining triglyceride metabolic homeostasis, highlighting a promising therapeutic target for ALS.

neuroscience↗

bioRxiv2026

Engineering Upd-like to broadly restrict arbovirus infection and vector competence in mosquitoes

The global expansion of mosquito-borne viruses poses a growing threat to public health, yet the endogenous signals that activate antiviral immunity in mosquito vectors remain incompletely understood. Here, we identify Aedes aegypti Upd as an activator of JAK/STAT signaling that restricts arbovirus infection. Loss of Upd increased infection and dissemination of the flaviviruses ZIKV and DENV, as well as the alphavirus SINV, whereas blood meal inducible midgut overexpression reduced ZIKV and SINV infection. Mechanistically, Upd associated with the extracellular region of the Domeless (Dome) receptor, and Upd deficiency markedly reduced STAT phosphorylation and nuclear accumulation in the midgut. Upd expression was transiently induced in the midgut by an uninfected blood meal but remained elevated following ZIKV challenge, linking physiological responses to blood feeding with antiviral defense. In mammal mosquito transmission models, Upd overexpression reduced viral acquisition from infected hosts, whereas Upd deficiency enhanced viral dissemination and increased transmission following systemic infection. Together, these findings identify Upd as an endogenous regulator of mosquito JAK/STAT signaling and provide proof of principle that temporally targeted Upd expression can reduce arbovirus susceptibility and vector competence.

microbiology↗

bioRxiv2026

Early hematopoietic fate decisions are independent of bone marrow niche

Hematopoietic stem cells (HSCs) reside in specialized bone marrow microenvironments, termed niches, which regulate self-renewal and differentiation through local support programs. Although multiple anatomically distinct niches with unique cellular compositions have been described, it remains unclear to what degree these niches differentially shape HSC fate. In parallel, hematopoietic output is modulated with age, yet it remains unclear if this shift in HSC fate is driven by niche-specific modifications. To address these questions, we applied Multiplexed Error Robust Fluorescence in situ Hybridization to jointly define the cellular and spatial organization of the femoral marrow in young and aged mice. We find that while HSC niches are anatomically and compositionally distinct, stem cell lineage priming is not strongly associated with niche identity consistent with our demonstration that fate-shaping signals are provided redundantly across niches. Moreover, we find that while aging remodels the cellular composition of the marrow, reshapes cellular expression, and alters hematopoietic differentiation, HSC niches are preserved, and HSC fate remains niche agnostic. Together these findings separate anatomical niche identity from early HSC fate decisions and are consistent with a model in which hematopoietic support is distributed across different niche locations rather than partitioned among discrete sites.

immunology↗

bioRxiv2026

Vascularized Organoids Emerge from a Heart-Foregut-like Intermediate Recapitulating Early Human Development

During early cardiogenesis, the heart regions lie in close contact with foregut endoderm, which supplies essential nutrients and signaling cues to orchestrate primitive heart tube assembly. Here, we show that self-organized heart/foregut like-structures emerge from human pluripotent stem cells (hPSCs). The initial heart/foregut-like structures, termed the Seed, are encased by surrounding supportive cells termed Soil, which provide a matrix microenvironment; the Seed progressively develops into chambered human cardiac organoids (hCOs) containing cardiomyocytes (CMs), epicardial cells, cardiac fibroblasts, and endothelial cells. Optimization of the induction system further enhanced hCO maturation and vascularization. Therefore, this Soil-Seed system provides a model to dissect human early heart/foregut self-organization.

developmental biology↗

bioRxiv2026

N-Acetylglucosamine ameliorates knee osteoarthritis by enhancing UDP-GlcNAc conversion and chondrocyte DNA repair capacity

Background: Knee osteoarthritis (KOA) is a degenerative joint disease characterized by progressive cartilage degradation and synovial inflammation, while current therapies offer only symptomatic relief. This study aimed to determine whether N-acetylglucosamine (GlcNAc) is superior to glucosamine hydrochloride (GlcN{middle dot}HCl) in slowing the progression of KOA and to elucidate the underlying mechanisms involved. Methods: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to assess serum concentrations of UDP-GlcNAc to conduct a comprehensive evaluation of the pharmacokinetics and pharmacodynamics associated with both GlcNAc and GlcN{middle dot}HCl. Using a destabilization of the medial meniscus (DMM) rat model, we performed micro-computed tomography and histopathological assessments to evaluate structural changes in the joint tissues. Additionally, we conducted bulk RNA sequencing, flow cytometry and ELISA to validate the efficacy of GlcNAc in anti-inflammatory immunoregulation and DNA repair in knee cartilage. Results: GlcNAc achieved higher peak serum concentrations and more efficient conversion to UDP-GlcNAc than GlcN{middle dot}HCl, with UDP-GlcNAc levels 4.57 times higher at 9 hours after administration. GlcNAc significantly attenuated KOA-like pathological changes, downregulated catabolic markers Mmp3 and Adamts4, decreased pain-related neural markers Pgp9.5 and Ngf, and alleviated pain sensitivity. RNA-seq identified 4,395 downregulated DEGs in DMM rats, of which GlcNAc upregulated 2,808 compared to only 391 by GlcN{middle dot}HCl. GlcNAc promoted DNA repair and adaptive immune cell differentiation pathways, and decreased serum IL-1{beta}, IL-6, and IL-17. Four candidate DNA repair genes, Hist1h2an, Poli, Ube2t, and Hells, were identified as significantly upregulated by GlcNAc, with GSEA confirming homologous recombination repair pathway enrichment. Conclusion: This study shows GlcNAc outperforms GlcN{middle dot}HCl in rat DMM knee osteoarthritis via multiple mechanisms, including 4.57-fold higher UDP-GlcNAc conversion, reduced cartilage degeneration, immune rebalancing, and DNA repair activation, supporting GlcNAc as a promising KOA candidate warranting further clinical study.

pharmacology and toxicology↗

bioRxiv2026

The limbic navigation system as a hierarchical RNN

Path-integration-trained recurrent neural networks (RNNs) can reproduce grid-like and head-direction-like representations, but most existing models rely on low-dimensional velocity inputs and do not capture the hierarchical organisation of the brain's self-localisation circuit. We introduce a hierarchical recurrent neural network (HRNN) that separately processes angular velocity and linear speed to perform simultaneous angular and positional path integration. Trained on these tasks, the HRNN generalises to longer timescales than seen during training and develops neural response properties resembling all the key cell types: angular velocity cells, head direction cells, speed cells, conjunctive and pure grid cells. The learned circuit also recovers key motifs of the navigation system, including attractor dynamics and asymmetric connectivity patterns. Perturbation analyses reveal functional information flow across the hierarchy, consistent with experimental observations. Finally, introducing theta-paced mutual inhibition between two learned angular-velocity subpopulations enables the HRNN to reproduce the bidirectional theta sweeps observed in empirical data. These results establish hierarchical task-driven RNNs as a biologically interpretable framework for linking neural representations, circuit motifs, and population dynamics in spatial navigation, while enabling targeted in silico perturbations and the generation of experimentally testable predictions.

neuroscience↗

bioRxiv2026

Inflammation is an essential trigger for POLE-driven ultramutated colorectal cancer

Pathogenic variants in the exonuclease (proofreading) domain of DNA polymerase {varepsilon} (POLE) are frequently detected in colorectal cancers (CRC) with high tumor mutational burden (TMB), yet their functional role in intestinal tumorigenesis remains unknown. Here, we investigated the impact of mutation in POLE on intestinal homeostasis using a conditional mouse model expressing the PoleP286R pathogenic driver in intestinal epithelial cells. PoleP286R mutant mice developed invasive adenocarcinomas in the small intestine with incomplete penetrance and extremely long latency but did not spontaneously develop colorectal tumors. However, induction of colitis with dextran sodium sulfate (DSS) resulted in a striking phenotypic shift to colorectal adenocarcinomas at high frequency. Molecular and histological analyses revealed hyperactivation of Wnt/{beta}-catenin, NF-{kappa}B, ERK and STAT3 pathways, accompanied by markedly higher proliferation of intestinal epithelial cells, in DSS-treated PoleP286R mutant mouse colons. Whole genome sequencing of the tumor demonstrated exceptionally high TMB (>100 mutations/megabase) and characteristic trinucleotide signatures, consistent with human POLE tumors. Recurrent mutations in Apc and Ctnnb1 underscored the central role of Wnt signaling in POLE-driven hypermutated CRC. In addition, intestinal organoids derived from PoleP286R mutant mice exhibited accelerated growth and increased expression of pro-proliferative genes. Together, these findings highlight POLE as a gatekeeper of intestinal genomic integrity and identify inflammation as the key driver that unleashes POLE-mutant CRC. Furthermore, this work establishes a mechanistically faithful preclinical platform for studying the molecular pathogenesis of CRC and advancing therapeutic drug discovery.

cancer biology↗

bioRxiv2026

Establishing toxicologically relevant primary alveolar macrophage models to enable a realistic assessment of nanomaterials safety

Alveolar macrophages (AMs) are key sentinels of the distal lung, maintaining surfactant homeostasis and clearing inhaled particles through continuous surveillance and phagocytosis. Mechanistic studies of particle-cell interactions, important to assess the respiratory safety or toxicity of particles, however, are limited by the lack of suitable in vitro models that preserve the core features of resident AMs. Here, we benchmarked six murine AM models, including short-term cultured primary AMs (AMs 3 h, AMs 24 h), primary AMs maintained with GM-CSF, TGF-{beta}, and the PPAR-{gamma} agonist rosiglitazone (AMs GTR), bone marrow-derived AM-like cells (BM-AMs), the immortalized AM cell line MH-S, and the non-transformed fetal liver-derived AM-like cell line MPI, against in vivo AMs based on surface phenotype, transcriptomic identity, and their responses to three carbon-based nanomaterials (CBNs). Both cell lines showed limited AM identity, characterized by low CD11c and SiglecF but high CD11b surface expression. In contrast, AMs GTR and BM-AMs more closely preserved resident AM-like surface marker profiles. Transcriptomic features, although differing for alterations in metabolism-related pathways, particularly oxidative phosphorylation, revealed most in vivo-like for the primary AM models. Functional, toxicological assessment using soot-like carbon nanoparticles (CNP), double-walled carbon nanotubes (DWCNT) and multi-walled carbon nanotubes (MWCNT) demonstrated strong model-dependent responses. Consistent with MWCNT-induced reduction in AM numbers observed in vivo, AMs GTR and BM-AMs exhibited the strongest in vitro responses, including cytotoxicity and inflammatory cytokine release. MH-S cells in contrast stood out for their cytotoxic sensitivity supporting their use for high throughput cytotoxicity tests. Overall, these findings identify AMs GTR and BM-AMs as the closest models for studying AMs biology and AM-intrinsic responses to poorly soluble nanoparticles like CBNs, and with regard to availability of higher cell numbers, BM-AMs might provide a scalable platform for mechanistic and higher-throughput nanotoxicology studies.

pharmacology and toxicology↗

bioRxiv2026

DCAF: Unifying Data Validity, Evaluation Validity and Predictive Modeling for Single-cell Perturbation Simulation

Single-cell perturbation profiling is limited by cost and throughput, while computational methods remain constrained by inconsistent evaluation and underperformance relative to simple baselines. Here we present DCAF, a unified framework for single-cell perturbation simulation that that jointly addresses data validity, evaluation validity and predictive modeling. Among its three components, DCAF-data addresses the single-measurement limitation by reconstructing pre- and post-perturbation status using entropy-regularized optimal transport, and is demonstrated on both cell line and multi-cell type datasets. DCAF-criterion evaluates log fold changes in non-silenced genes to improve sensitivity to model error and robustness to zero inflation noise, validated on synthetic datasets. DCAF-model combines JEPA with explicit modeling for regulatory-dynamics modeling for interpretable prediction. Across benchmarks, DCAF outperforms state-of-the-art deep-learning methods, as well as mean and linear baselines in cross-gene, cross-cell-type and cross-region generalization. In adult Mouse brain, DCAF recapitulates known cell-type-specific regulatory heterogeneity of Dgcr8 knockdown at both cell type and gene levels. DCAF thus provides a rigorous and biologically grounded foundation for reliable perturbation simulation across development, disease and therapeutic discovery.

bioinformatics↗

bioRxiv2026

Microbial Reductive Dechlorination of Hexachlorobutadiene by Dehalococcoides

Hexachlorobutadiene (HCBD), a persistent organic pollutant (POP), poses a significant threat to ecological environments and human health. While anaerobic microbial transformation is a promising remediation strategy, the specific microorganisms and metabolic mechanisms involved have remained poorly understood. This study demonstrates the successful enrichment of an anaerobic microbial consortium from river sediment, with lactate as an electron donor, capable of transforming HCBD. Within 240 days, 62.5 mol of HCBD was sequentially dechlorinated through pentachlorobutadiene (PCBD), tetrachlorobutadiene (TeCBD), trichlorobutadiene (TCBD), and dichlorobutadiene (DCBD) to chlorobutadiene (CBD). Co-amendment with HCBD and trichloroethene (TCE) resulted in complete removal of both compounds. 16S rRNA gene sequencing and quantitative PCR showed enrichment of Dehalococcoides, linking its population growth to HCBD and TCE dechlorination. This study demonstrates that Dehalococcoides growth was associated with HCBD transformation in the enrichment cultures, broadens our understanding of the metabolic capabilities of Dehalococcoides, and identifies a candidate gene for this important environmental process, offering valuable insights for the bioremediation of sites contaminated with HCBD.

microbiology↗

bioRxiv2026

A Compact Regulatory Logic Governs Fibroblast Cell State Plasticity in Fibrosis

Fibroblast activation drives organ failure across human diseases, yet its regulatory basis remains largely undefined. Here, we repressed 280 conserved disease-activated enhancers using CRISPR droplet sequencing (CROP-seq) in ~550,000 fibroblasts, constructing a regulome of 15 gene programs governing the profibrotic state. Systematic TF perturbation converged on MEOX1 as a central regulator, and fibroblast-specific Meox1 deletion in mice improved cardiac function, reduced fibrosis, and rewired profibrotic fibroblasts toward quiescence in heart failure. Effector gene perturbation in human cardiac fibroblasts revealed distinct regulators of contractility, migration, and inflammatory signaling, and these programs were conserved across profibrotic states in human lung, liver, and skin. Simultaneous repression of just six enhancers using a multiplexed dCas12a-KRAB system collapsed the profibrotic network, suppressed migration and contraction, and rewired fibroblast paracrine signaling to attenuate macrophage inflammation. These findings define the regulatory logic of fibroblast plasticity and establish a new framework for reprogramming profibrotic cell states in disease.

genomics↗

bioRxiv2026

Modulating Sleep Oscillations Through Targeted Memory Reactivation: Linking Functional Dynamics to Brain Microstructure

Background: Sleep-dependent memory consolidation reputedly rely on the coordinated activity of sleep oscillations, which may facilitate the reactivation and redistribution of newly acquired memories. Targeted memory reactivation (TMR) during sleep can experimentally modulate these processes, although effects remain variable. Moreover, how learning-related microstructural plasticity relates to sleep oscillatory activity and spindle-related network dynamics remains poorly understood. We investigated whether auditory TMR modulates sleep spindles, slow oscillations (SOs) and their co-occurrence, whether these changes relate to motor memory consolidation and diffusion MRI (dMRI) measures of brain microstructure, and whether learning and TMR alter the evolution of cortico-cortical connectivity during spindle development. Methods: Sixty healthy young adults underwent five dMRI sessions before and after motor sequence learning (Day 1), after a post-training night of regular sleep (RS) or TMR (Day 2), and before and after relearning (Day 5). Baseline and experimental nights were recorded with 256-channel high-density EEG. Oscillation densities were analyzed using mixed-design ANOVAs and related to behavioral and dMRI changes using Pearson or Spearman correlations. Intra-spindle connectivity between 148 source-reconstructed cortical regions was compared between spindle onset and end, across nights and between groups using Network-Based Statistics. Results: Spindle density, SO density and SO-spindle co-occurrence remained stable across nights in the RS group but decreased after TMR. Higher spindle density and co-occurrence were associated with greater overnight performance gains, and all oscillatory measures were associated with learning-related dMRI changes in subcortical regions, notably striatum and thalamus. Connectivity increased from the beginning to the end of the spindle duration during the baseline night in both groups, but during the experimental night only in the RS group. Conclusions: Motor learning is associated with coordinated changes in sleep oscillatory activity, within-spindle cortical connectivity and brain microstructure, supporting a dynamic interaction between learning-induced plasticity and sleep-dependent network reorganization. Under the present stimulation conditions, TMR did not enhance these processes but instead reduced sleep oscillatory activity and attenuated spindle-related connectivity dynamics, underscoring the importance of precisely targeting endogenous sleep rhythms when using TMR.

neuroscience↗