Advanced Science

Cervical spinal cord injury (SCI) causes profound and persistent loss of hand function, and effective neuromodulation strategies remain limited. We report the first-in-human implantation of a 32-contact cervical epidural paddle array in two individuals with severe chronic SCI. Individualized motor pool recruitment maps, derived from systematic bipolar and multipolar configurations, enabled person-specific stimulation parameters. Optimized stimulation restored volitional hand opening, closing and coordinated upper-limb movements that were previously unattainable. This approach achieved a >91% success rate in complex reach-grasp-lift-release sequences, supported by substantial gains in range of motion, grip, and pinch strength. Electrophysiological and kinematic analyses demonstrated parameter-dependent, selective recruitment of flexor and extensor motor pools. Personalized stimulation programs integrated with goal-directed activities enabled functional hand use in home and community settings, sustained over several months of continued autonomous use. These findings establish a mechanistically grounded and translational framework for restoring upper-limb function after chronic severe SCI.

Glaucoma is characterized by progressive stiffening of the trabecular meshwork (TM), which elevates intraocular pressure and contributes to tissue dysfunction. Although substrate stiffness and mechanical stimulation both regulate TM homeostasis, their combined effects remain poorly understood. Here, a hydrogel-integrated microfluidic platform is presented that enables simultaneous control of substrate stiffness via tunable gelatin methacryloyl (GelMA) hydrogels and equi-biaxial quasi-static stretch via hydraulic actuation. Finite element analysis validates the applied strain field, and optimized crosslinking ensures structural stability. Primary normal TM (nTM) and glaucomatous TM (gTM) cells cultured under coupled conditions exhibit selective mechanotransduction dysregulation rather than global mechanosensory impairment. While nTM cells dynamically regulate -smooth muscle actin (-SMA), myocilin (MYOC), matrix metalloproteinase-2 (MMP2), and collagen type I (COL1), gTM cells display constitutively elevated -SMA, loss of mechanical regulation of MMP2, and impaired stretch-mediated COL1 suppression, while retaining stiffness-dependent focal adhesion kinase and MYOC sensitivity. Key differences between normal and glaucomatous cells emerge only under combined stiff and stretched conditions, underscoring the importance of coupled mechanical cues in revealing disease-relevant phenotypes. These findings implicate tissue stiffening in selective pathway dysregulation and highlight mechanotransduction-targeted therapeutic strategies.

Persistent hyperglycemia impairs wound healing in diabetic patients, and severe cases may even lead to disability or death. Glycemic control alone cannot effectively prevent the occurrence of diabetic foot ulcers, a serious complication of diabetes. However, safe, efficient, and cost-effective therapies remain unavailable and are urgently needed. Using a novel sports medicine paradigm, we predicted, based on reverse-transcriptomics, that exercise-induced sweat has the potential to promote would healing in diabetic foot ulcers. Subsequent animal experiments demonstrated that sweat can indeed promote re-epithelialization and collagen deposition, upregulate the expression of the proliferation marker Ki-67, the angiogenesis marker CD31, and -SMA, and significantly accelerate wound healing in a mouse model of diabetic foot ulcers. This study provides a new direction for sports medicine and offers a novel therapeutic strategy for patients with diabetic foot ulcers.

The gut-brain axis has emerged as a crucial factor in neurodegeneration, with growing evidence linking gut dysbiosis and metabolic dysfunction to Alzheimers disease (AD) progression. Unfortunately, the lack of human-relevant in vitro models limits our ability to effectively explore the mechanism of this axis. To address this gap, we have developed a human induced pluripotent stem cell (iPSC)-derived gut-blood-brain barrier (BBB)-brain microphysiological system that enables systematic investigation of gut-brain interaction in the context of AD under controlled conditions. Our findings reveal that the interaction between gut and brain organoids can promote the maturation of brain organoids, making them more similar to their physiological characteristics in vivo. Additionally, co-culture gut and brain organoids better recapitulates the pathological features of AD. We also discovered that gut organoids of AD can trigger neurodegenerative disease manifestations in healthy brain organoids. In summary, our microphysiological system provides a novel and versatile in vitro platform for studying the interaction between the gut and brain in neurodegenerative diseases.

Clostridioides difficile infection (CDI) is a leading healthcare-associated diarrhea with high recurrence rates, partially due to antibiotic-induced dysbiosis and dysregulated host inflammation. Specialized pro-resolving mediators (SPMs), such as Lipoxin A4 (LXA4), offer promise in controlling excessive inflammation and promoting tissue repair, yet their role in CDI remains unexplored. Here, we developed a compact, gas-tight gut-on-a-chip (GOC) system that reconciles the anaerobic requirements of C. difficile with the oxic needs of human intestinal epithelium, enabling physiologically relevant co-culture within a standard incubator. A CDI in vitro model was established based on this GOC system. Using the model, we demonstrated that prophylactic administration of LXA4 significantly preserved epithelial barrier integrity, attenuated pro-inflammatory cytokine secretion (IL-8 and IFN-{gamma}), and reduced bacterial colonization. Transcriptomic analysis revealed that LXA4 pretreatment upregulated genes involved in cell junction organization while downregulated immune activation pathways. These protective effects were validated in a murine CDI model, where LXA4 pretreatment reduced weight loss, pathological damage, and fecal bacterial burden. Furthermore, prophylactic administration of LXA4 synergized with vancomycin treatment further enhanced antibiotic efficacy while allowing a 50% dose reduction without compromising therapeutic outcomes. Our study establishes a robust approach for CDI research and highlights the prophylactic and adjuvant potential of inflammation-resolving strategies, offering a novel approach to mitigate CDI incidence and improve treatment outcomes.

The lymphatic system plays various crucial but underappreciated roles in fluid transport and immune response in numerous organs and tissue types. Consequently, generation of lymphatic vessels has been postulated as an innovative therapeutic strategy for various diseases. However, there is a lack of efficient and reliable method to differentiate human pluripotent stem cells into lymphatic endothelial cells (LEC) for lymphatic regeneration. Current differentiation methods suffer from poor yield and low lymphatic marker expression, while also having limited clinical applicability due to its reliance on either the embryoid body intermediates or xenogenic supporting cells. Given that LECs exclusively rely on anaerobic and fatty acid metabolism due to the hypoxic environment of the lymph, here we report that the unique lymphatic-specific metabolic pathways can be exploited to promote lymphatic identity in differentiated LECs (dLECs). We show that dLECs express elevated levels of lymphatic markers compared to native endothelial cells, which is up to 15 times higher than the current leading standard of dLECs. Moreover, dLECs can form lymphatic vascular networks in both 2D and 3D, as well as secrete important lymphangiocrine for organ maturation. Upon implantation into double-ligation tail lymphedema and mammary fat pad models, dLECs were able to integrate with the host lymphatic vessels, restore fluid flow, and reduce swelling. Collectively, we show that metabolite supplementation can drive stem cell differentiation into dLECs, which can be incorporated into new alternative methods for personalized therapies and disease modeling, as well as provide a direct therapeutic option for lymphedema and lymphatic disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/686405v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1c4caa5org.highwire.dtl.DTLVardef@d42b08org.highwire.dtl.DTLVardef@1554ffcorg.highwire.dtl.DTLVardef@1f64ae6_HPS_FORMAT_FIGEXP M_FIG C_FIG A xeno-free and stepwise differentiation protocol with VEGF-C and sodium acetate induces lymphatic identity in iPSC-derived endothelial cells. Differentiated LECs (dLECs) express key lymphatic markers as verified using bulk RNA-sequencing, qPCR, FACS, and immunostaining. These dLECs secrete important cytokines for lympangiocrine signaling, able to form robust 2D and 3D lymphatic networks, as well as demonstrate in vivo functionality and host integration in murine models.

The thymus plays a pivotal role in the generation of immunocompetent T cells. Although its function is dependent on its complex extracellular matrix, its 3D architecture and mechanical properties remain poorly characterised This knowledge gap limits efforts to model and engineer the organ, which is a critical step towards the development of strategies for the treatment of many haematological and autoimmune diseases. Here, we provide the first comprehensive multiscale dataset of bovine thymic extracellular matrix architecture and viscoelastic behaviour, including quantitiative descriptors such as relaxation times, instantaneous and equilibrium elastic moduli, storage and loss moduli, and spatial mechanical heterogeneity. Taken together, our data define the thymus as a compliant, highly dissipative viscoelastic organ with a fibrillar architecture. They also represent a unique database, which, for the first time, paves the way for quantitative thymus tissue engineering.

Stem cell aging significantly impairs therapeutic efficacy, requiring innovative strategies to restore potency. We present a microfluidic cell-compressing platform for reactivation (-CPR) designed to apply controlled hydrodynamic deformation to late-passage stem cells. This mechanical stimulation facilitates functional reactivation without external chemical cues. Within a defined window, -CPR effectively reduces oxidative stress, SA-{beta}-Gal activity, and {gamma}H2AX foci, while simultaneously restoring proliferation and canonical stemness markers (OCT4, SOX2, and KLF4). Mechanical stimulation via -CPR induces coordinated structural remodeling: nuclei become more compact, actin cortex organization is restored, -actinin redistributes to focal adhesions, and microtubule networks are restructured, suggesting a rebalanced intracellular tension. Transcriptomic and proteomic analyses reveal that this process reprograms extracellular matrix remodeling and DNA repair pathways while attenuating pro-fibrotic and senescence-associated secretory phenotype (SASP)-associated pathways. Crucially, this reactivation occurs without compromising fundamental MSC hallmarks, preserving intrinsic immunophenotypes and multilineage differentiation potential. Functionally, -CPR-processed stem cells demonstrate restored in vitro wound closure and enhanced tissue repair in vivo, with efficacy appearing dependent on mechanical dosage. This platform establishes a non-genetic, mechanobiological approach to restoring stem cell function, offering a scalable strategy for functional reactivation and potentially paving the way toward comprehensive cellular rejuvenation.

Urinary tract infections (UTIs) remain a major health burden, yet mechanistic studies are limited by the lack of experimental models that enable high spatiotemporal resolution tracking of infection dynamics, while recapitulating the stratified architecture of the bladder epithelium, urine tolerance and fluid dynamics. Here, we present a modular microphysiological platform integrating a fully stratified, urine-tolerant human urothelium cultured on standard transwell inserts within a custom-designed perfusion device compatible with live imaging. Urine flow enables real-time, high-resolution imaging of uropathogenic Escherichia coli (UPEC) infections under physiologically relevant conditions, including clearance of planktonic bacteria and nutrients replenishment, while retaining tissue-associated populations. This system revealed UPEC attachment via the type 1 fimbrial adhesin FimH and its inhibition by D-mannose treatment. Moreover, the platform captured L-form formation upon treatment with the frontline antibiotic fosfomycin and regrowth of walled bacteria following drug withdrawal. The platform further uncovered strain-specific lysis through bacteriophages in contrast to the activity of broad-spectrum antibiotics. In summary, this system constitutes a scalable platform with high predictive power for studying UTI pathogenesis and preclinical therapeutic testing.

Demyelinating diseases are a group of complex neurodegenerative disorders characterized by damage to the myelin, the protective sheath that insulates and supports efficient nerve signal conduction. Such a loss of myelin causes the formation of lesions not only in the brain but also often in the spinal cord (SC). Despite the high prevalence of SC lesions among patients, existing models mostly focus on those in the brain, inadequately capture the unique anatomical and physiological features of SC pathology. In this study, we developed a robust, reproducible in vitro model of SC demyelination by combining microwell technology and piezoelectric scaffolds to engineer human neural stem cell (hNSC)-derived nerve tissues featuring aligned, myelinated, extended axons up to 2000 {micro}m in length. We utilized distinct chemical treatments to induce demyelination with or without axonal degeneration: a cuprizone cocktail, a copper chelator combined with inflammatory cytokines, and lysophosphatidylcholine (LPC). Electrophysiological assessments validated the physiological relevance of our model, demonstrating impaired signal transmission and neural connectivity akin to in vivo demyelination pathology. Our versatile platform thus provides a valuable tool for elucidating SC demyelination pathophysiology and exploring potential therapeutic interventions.

Cryopreservation depends critically on suppression of ice formation by cryoprotective agents (CPAs), but limited data is available on the CPA concentration required for vitrification (Cv). Here, we introduce a high-throughput 384-well platform that integrates automated liquid handling, randomized plate layouts, and a binary-search strategy to rapidly determine Cv across hundreds of formulations. Relative to conventional methods, this approach increases throughput by [~]50-fold, compressing a year of measurements into one week, while markedly reducing manual labor. Across [~]200 CPA compositions, we demonstrate that environmental boundary conditions strongly influence vitrification behavior: plates sealed with silicone mats exhibited lower Cv than open plates, indicating that sealed configurations promote vitrification. Further, the data reveal a decrease in Cv with increasing CPA molecular weight, consistent with enhanced ice suppression by larger molecules. We also present a simple mixture model that accurately predicts Cv for a broad range of CPA formulations, including mixtures containing up to seven CPAs (R{superscript 2} > 0.94), and use this model to evaluate published CPA toxicity data to identify formulations that operate near their vitrification threshold while maintaining relatively low toxicity. Together, these results establish a framework for rapid Cv determination, predictive modeling of vitrification behavior, and rational design of CPA formulations.

Meningeal lymphatic vessels (mLVs) are vital for brain waste clearance, making them a promising therapeutic target. However, effective modulation strategies for mLVs with translational potential remain underdeveloped. Here, we develop a low-intensity focused ultrasound (LIFU) strategy that precisely targets the vault cranial meninges to non-invasively facilitate mLVs drainage. Using models of Alzheimers disease (AD) and aging, we demonstrate that this approach promotes CSF drainage, prevents cognitive decline, and reduces pathological biomarkers. Mechanistically, RNA sequencing combined with calcium imaging in vitro reveals that LIFU activates the Piezo1 ion channel in lymphatic endothelial cells, whereas pharmacological inhibition of Piezo1 abolishes LIFUs therapeutic effects. Compliant with FDA safety guidelines, this LIFU protocol demonstrates strong clinical translatability. If its efficacy is clinically confirmed, LIFU offers a promising therapy for neurodegenerative diseases triggered by waste accumulation.

The development of neural probes has enabled a deeper understanding and improved treatment for neurological disorders. Microneurography is currently the gold standard for assessing the electrophysiological signature of pain mechanisms in the human peripheral nervous system. However, its clinical utility is limited by the low recording yield and signal-to-noise ratio of single-electrode probes. To overcome these limitations, we developed mechanically robust, multi-electrode probes designed for acute percutaneous insertion and recording in peripheral nerves. The electrical and mechanical stability of these probes was confirmed through repeated insertions in artificial human skin and rat peripheral nerves. In addition, ex vivo and in vivo experiments demonstrated enhanced functional performance, with multi-site recordings enabling the isolation of single-fiber activity. Importantly, our probes can be operated analogously to conventional microneurography needles while substantially increasing the information yield, providing enhanced capabilities for minimally invasive peripheral nerve assessment.

Neurodegenerative diseases involve structural and morphological alterations in tissue architecture that are difficult to capture in single thin sections. Three-dimensional multiplexed pathology, however, remains limited by the lack of clearing methods applicable to formalin-fixed paraffin-embedded (FFPE) clinical specimens. As the development of tissue-clearing methods requires the optimization of multiple parameters, we employed a neural network-based Complex System Response (CSR) approach to guide the design of FIDELITY, an epoxy-free delipidation and epitope-retrieval pipeline for whole FFPE specimens. FIDELITY preserves tissue rigidity, enhances immunostaining efficiency, and supports at least five rounds of multiplex labeling without deformation. It enables whole-brain atlas registration, quantitative neuronal profiling, and volumetric pathology of archived human Alzheimers and glioma specimens while remaining compatible with routine histology. Altogether, FIDELITY provides accurate 3D metrics and offers translational potential to bridge spatial mapping and conventional pathology.

Human induced pluripotent stem cells (hiPSCs) offer a powerful platform to model chondrogenesis and enable regenerative strategies, yet regulation of cell-fate commitment remains elusive. Here, we systematically mapped cell-fate trajectories from 7 time points during a 49-day chondrogenic hiPSC differentiation protocol using single-nucleus multimodal transcriptomic and chromatin accessibility profiling (scRNA-seq and scATAC-seq). Integrative analysis of dynamics revealed branching differentiation trajectories with clear bifurcation points and distinct cell-fates. Notably, the chondrogenic trajectory originated at day 6 as a neurogenic development and branched off at day 21 to a chondrogenic cell-fate. Through transcription factor activity analysis (TFAA) and cis-co-accessibility networks, we found that NFIA and NFIB drove chondrogenic distinction, exhibited in both modalities as directly targeting chondrogenic genes such as COMP, FIBIN, VIM. This was then confirmed by experimental validation as modulation of NFIA expression at this point further enhanced chondrocyte formation. Together, our study provides a high-resolution multimodal atlas of chondrogenic differentiation and identified critical transcriptional regulators that can now be leveraged to implement regenerative cartilage therapies from hiPSCs.

The superior stealth properties and high information density make DNA a sought-after candidate in the field of molecular steganography. Here, we developed the InfinMark end-to-end DNA steganography framework for anti-counterfeiting applications by combining the characteristics of both the Internet of Things (IoT) and DNA-of-Things (DoT). InfinMark includes five modules: Information Transcoding, Fingerprint Writing, Nano-encapsulation, Invisible Marking, and Multi-level Rapid Authentication. It ensures precise anti-counterfeiting information reading and writing through a dynamic DNA-compatible transcoding algorithm, achieves seamless embedding by developing scalable nanoparticle manufacture methods, and supports cross-scenario on-site verification, ultimately granting it comprehensive anti-counterfeiting capabilities spanning from source labeling to terminal tracing. By addressing the bottlenecks in IoT and DoT integration, lifecycle tracking, as well on-site product authentication, this research constructs a full-chain bimodal anti-counterfeiting system, thereby showcasing the practical application of informational DNA nanoparticles in various aspects of production and daily life.

Chronic periodontitis represents one of the most prevalent inflammatory tissue-destructive conditions in humans, yet the molecular thresholds separating reversible inflammation from permanent structural collapse remain undefined. Using single-cell RNA sequencing data from 12,104 cells (GSE152042) spanning three disease states -- healthy gingival tissue, mild periodontitis, and severe periodontitis -- we constructed a variational autoencoder (VAE)-derived 20-dimensional latent disease manifold and applied formal hysteresis quantification to measure transcriptional irreversibility. Chi-square analysis across 9,163 cells occupying transitional pseudotime bins yielded {chi}{superscript 2} = 11,971 (p < 10-300, df = 4), with Cramers V = 0.81, confirming strong state-memory effects inconsistent with freely reversible disease dynamics. Non-negative matrix factorisation (NMF; k = 15) identified biologically coherent gene programs whose co-activation topology was encoded as a hypergraph constraint network; in severe disease, 16 of 76 healthy constraints collapsed by more than 60%, and the Fibroblast-Epithelial coupling (Programs 1-4) was reduced by 84%. A six-agent agentic AI simulation faithfully recapitulated observed shifts in cellular composition and established a temporal threshold beyond which tissue damage trajectories diverge irreversibly. We introduce the Regenerative Permission Index (RPI), a composite single-cell metric (range: 0.060-0.644), whose mean in severe periodontitis (0.323) falls well below the 0.50 permissibility threshold, indicating that all tested biomaterial interventions will fail. Five-fold cross-validated classification achieved 88% accuracy (Random Forest, AUC = 0.992), and permutation testing confirmed that constraint network patterns are biologically specific rather than artefactual (p < 0.01). Together, these findings provide a quantitative basis for understanding periodontal irreversibility and position RPI-guided decision-making as a framework for precision regenerative medicine.

The global crisis of multidrug-resistant pathogens necessitates innovative antimicrobial peptide (AMP) discovery. Deep-sea microbiomes represent an underexplored resource for novel AMPs, but their mining is hindered by biases in current prediction methods, including sequence length imbalance, N-terminal methionine artifacts, and lack of microbial optimization. To overcome these, we developed XAMP, a dual-engine predictor integrating XAMP-E (based on ESM-2 for high-accuracy feature representation) and XAMP-T (a one-layer Transformer for accelerated screening). By training on debiased datasets, XAMP achieved a median AUC of 0.972, an approximately 10% improvement over state-of-the-art tools, with XAMP-T operating 5 to 40 times faster. Applying this pipeline to deep-sea metagenomes, we identified 2,355 promising AMP candidates. Experimental validation of six synthesized peptides against ESKAPE pathogens demonstrated potent, broad-spectrum activity, particularly against Gram-negative bacteria, which dominate deep-sea ecosystems and represent a major challenge in nosocomial infections. This study establishes a robust computational-experimental framework for discovering therapeutic candidates from extreme environments to combat antibiotic resistance crises.

Cells dynamically integrate biochemical and mechanical signals arising from their surrounding microenvironment to regulate morphology and behavior. Mechanical cues like matrix stiffness, surface topography, and other physical perturbations modify biophysical signals. Surface topography, particularly curvature regime acts as any important mediator of mechanotransduction by coordinating cytoskeletal organization, focal adhesion dynamics, and nuclear architecture. Curvature response has been demonstrated at broader length scales and influences nucleus shape change, chromatin organization, and gene regulation, positioning the nucleus as an active mechanosensitive hub. Bone tissue consists of a curvature-rich microenvironment defined by a trabecular architecture at tissue scale and by resorption cavities such as Howships lacunae at cellular scale. While these geometries are essential for homeostasis, their role in pathological context remains poorly understood. Osteosarcoma develops within this mechanically complex multiscale architecture, but how bone-inspired curvature regulates nuclear behavior and signaling in osteosarcoma cells remains unclear. Here, we engineered three-dimensional (3D) concave hemispherical substrates that recapitulate nucleus-scale bone micro-curvature and assessed their effects on human SaOS-2 osteosarcoma cells. In comparison with flat surfaces, concave confinement resulted in pronounced nuclear rounding and softening, accompanied by Lamin A/C reorganization and increased heterochromatin compaction marked by H3K9me3. Curvature-driven nuclear remodeling selectively modulated Hippo pathway main effectors YAP/TAZ without activating NF-{kappa}B mediated canonical inflammatory responses. Furthermore, cells maintained overall viability without elevated pathological DNA damage or apoptotic signaling, suggesting an adaptive, damage-tolerant nuclear response. Overall, these findings indicate nucleus-scale curvature as a critical regulator within the bone microenvironment that governs nuclear modelling and mechanosensitive signaling in osteosarcoma cells. Incorporating physiologically relevant geometry into in vitro models establishes new insight into cancer microenvironment crosstalk and highlights nuclear interior and outer architecture as a key regulator of tumor cell behavior.

Cortisol is a primary biomarker of stress, released in sweat at concentrations that directly correlate with physiological stress levels. Detecting cortisol non-invasively offers significant potential for real-time stress monitoring and healthcare applications. Biosensors capable of binding cortisol can thus enable the development of novel diagnostic platforms for personalised health management. In our earlier work, a 38-mer peptide fragment derived from the protein 2V95 was identified as a functional binder to cortisol. In the present study, we applied generative artificial intelligence (AI) approaches to expand the sequence space and identify superior candidate peptides with improved binding affinity. By integrating sequence-based and structure-based AI models, we generated and screened a peptide library of nearly 10,000 sequences against cortisol, leading to the identification of high-affinity candidates for further evaluation.