Advanced Science

Growth plate (GP) is the critical cartilaginous structure for longitudinal bone growth. Herein, employing high-resolution analytical techniques, we explore the intricate mechanisms that govern the polarized mineralization patterns within GP. The GP-epiphysis interface displays a sharp transition in tissue modulus, acting as a "protective shell" for the underlying GP, whereas the GP-metaphysis interface exhibits a gradual modulus increase, enabling efficient load redistribution to metaphysis. The unique mechanical environments at these two interfaces contribute to polarized CaP mineralization patterns, which are regulated by a complex protein-based molecular machinery. The mineralization inhibitors SPP1 and AHSG enriched at the GP-epiphysis interface could act as a line of defence against mineralization. When these two proteins coexisted with the mineralization-promoting ENPP1 and ALPL at the GP-metaphysis interface, a sequential event of precise nucleation and programmable assembly of CaP minerals occur, forming "mineralization waves" to guide bone elongation. By replicating such specific macromolecular environment at GP-metaphysis interface, a hypertable amorphous calcium phosphate (ACP) phase is well-retained in vitro, demonstrating the possibility for precise and gentle control of ACP-hydroxyapatite (HAp) transformation under physiological conditions. Our study defines a novel concept of "mineralization waves" that govern the velocity and amplitude of GP-guided mineralization process.

The pursuit of continuous, cuffless blood pressure (BP) monitoring is constrained by the superficial sensing depth of photoplethysmography (PPG). Impedance plethysmography (IPG) offers deeper tissue penetration, but its comparative value over PPG remains unquantified at scale. In this comparative study of 261 participants (130 hypertensive, 131 non-hypertensive), we utilized a custom dual-modality wearable prototype to capture simultaneous IPG and PPG signals. Over 150,000 cardiac cycles were analyzed using an unsupervised archetype discovery pipeline to quantify beat-to-beat morphological heterogeneity. IPG resolved up to three distinct morphological modes per participant, whereas co-located PPG converged into highly conserved, uniform profiles. IPG captured specific signatures of pathological arterial remodeling and physiological habitus; ventral forearm IPG pulse amplitude exhibited a significant main effect for BP status (p = 0.024), a relationship absent in the co-located PPG signal. Furthermore, increasing body mass index (BMI) significantly attenuated the prevalence of steep-upstroke archetypes in IPG (p = 0.035), quantifying a likely damping effect of adipose tissue. Deep-tissue bioimpedance captures rich, heterogeneous hemodynamic signatures including arterial-dominant morphologies that are invisible to optical sensors. Transitioning from optical pulse wave analysis to bioimpedance-based models may offer a promising pathway for accurate wearable cardiovascular monitoring.

Bacterial endocarditis is a fatal cardiovascular disease exacerbated by weakened heart contraction, yet the direct impact of cardiac contractility on bacterial adhesion remains elusive. Here, we present a novel quantitative physics model integrating finite element analysis and live-cell imaging to uncover their strong correlation. Using this model, we quantified the real-time force magnitude generated by organoid-type cardiac microtissue derived from healthy donors and dilated cardiomyopathy patients - mimicking normal and suppressed heart contractility, respectively - to the approaching bacteria in a real fluidic system. The data revealed that weakened cardiac contractility facilitated bacterial invasion of the myocardium. Verifying this finding in a mouse transverse aortic constriction model demonstrated that increasing heart contraction efficiently mitigated bacterial invasion, with a 25% increase in heart contractility reducing endocarditis risk by 80%. Our findings demonstrate that patient-derived cardiac organoids provide a physiologically relevant platform for studying bacterial infections in vitro, offering high clinical fidelity. This platform establishes a valuable tool for drug screening and the development of novel therapeutic strategies.

This study evaluates the feasibility of depth-sensitive bedside monitoring of cerebral blood flow (CBF) and low-frequency oscillations (LFOs) using time-domain diffuse correlation spectroscopy (TD-DCS) in healthy controls and patients with disorders of consciousness (DOC). A 1064 nm TD-DCS system equipped with superconducting nanowire single-photon detectors (SNSPDs) was used to collect 10-minute resting-state data from 25 healthy adults and 5 patients with traumatic brain injury (TBI) diagnosed with DOC, including minimally conscious state (MCS) and coma, in the subacute phase. Photon arrival times were temporally gated to distinguish superficial and cortical-weighted tissue contributions. The blood-flow index (BFI) was extracted from gated autocorrelation functions, and LFOs were quantified using power spectral density within the Slow-5 (0.01-0.027 Hz), Slow-4 (0.027-0.073 Hz), and Slow-3 (0.073-0.198 Hz) bands. Compared to healthy controls, DOC patients exhibited altered resting-state LFO amplitude and spectral distribution, suggestive of altered neurovascular dynamics in severe brain injury. An auditory "smile" command was delivered to five healthy subjects, one MCS patient, and one unresponsive wakefulness syndrome (UWS) patient to assess task-evoked hemodynamic responses. During the task, healthy participants showed clear hemodynamic responses, whereas DOC patients demonstrated attenuated and more transient responses. Overall, TD-DCS provides a noninvasive, depth-resolved approach for assessing cerebral hemodynamics and residual cortical responsiveness, supporting its potential for bedside neurocritical-care monitoring.

Biomechanical cues, including shear stress and mechanical strain, are key regulators of intestinal cellular behavior, yet their mechanostimulatory impact on fibroblasts responses during early fibrotic remodeling remains poorly understood. Using a bioengineered gut-on-a-chip model, we independently modulated flow and mechanical strain to assess fibroblast dynamics under intact or impaired epithelial barriers. Inflammation-associated fibroblasts resisted biomechanical stress, exhibiting myofibroblast-like phenotypes with hypertrophy and elevated -smooth muscle actin aligned with stress fibers. In contrast, normal fibroblasts were highly susceptible to shear stress, undergoing matrix metalloproteinase-dependent apoptotic injury, while mechanical strain alone had minimal effect. Notably, an intact epithelial barrier was both necessary and sufficient to protect fibroblasts from shear-induced damage, suggesting that "good fences make good neighbors". Under barrier dysfunction, prolonged exposure to shear stress induced the formation of stiff fibroblast aggregates composed of mechanoadaptive myofibroblast-like cells. These findings identify mechanostimulatory cues, particularly shear stress, as critical drivers of early fibrotic remodeling in inflammatory bowel disease and underscore epithelial barrier integrity as an essential biomechanical safeguard against pathological fibroblast dysregulation.

Neural decoding is useful for understanding brain functions and developing neural interface applications. However, neural interfaces based on rigid electronics often suffer from recording instability due to the foreign body responses caused by their mechanical mismatch with soft tissues, limiting the longitudinal accuracy of neural decoding methods. Herein, it is reported that flexible electronics can be integrated with machine learning algorithms to achieve long-term reliable neural decoding. Wet-spun conductive polymer microfibers showed mechanical robustness and flexibility, low impedance, and chronic biocompatibility, enabling intracerebral neural recordings in epileptic mice at a high signal-to-noise ratio eight weeks after implantation. When the signals recorded by the flexible electrodes were used in machine learning analyses with diverse complex algorithms, they consistently showed higher prediction accuracy for epileptic seizures than stiff metal electrode signals, particularly in the case of using long-term recordings for testing or small-sample datasets for training. A real-time warning system based on the flexible neural electrodes was built that predicted seizures eight minutes in advance with a low false alarm rate. Our work bridges flexible electronics and artificial intelligence for neural decoding applications such as long-term treatment of chronic neurological disorders.

Microenvironmental mechanics regulate morphogenesis and post-injury inflammation, however, the fragile mechanical strength and oxidative physiological environment hinder precise and consistent mechanical management after spinal cord injury (SCI). Here, we engineered self-assembling hydrogels of enantiomeric peptides with neural tissue- matching mechanical properties to persistently manipulate mechanosensing and mechanotransduction through stereo conformational recognition and consequent protein affinity difference. While hindering proliferation and morphogenesis in non-neural cells, D-hydrogel-induced intracellular tension relaxation triggered neurogenesis and ECM remolding in astrocytes, while simultaneously suppressing pro-inflammation and promoting pro-regeneration in microglia, which together enable neuroprotection from degeneration and enhance functional recovery in severe SCI rat models. These effects are mediated through neurogenic morphology changes resulting from cytoskeletal tension relaxation, leading to the opening of mechanosensitive ion channels in the cellular membrane, chromatin unfolding, and YAP nuclear translocation. This exclusive D- hydrogel-dependent neurogenesis, triggered by intracellular tension relaxation, revealed a neural-specific response to mechanical cues and provided a targeted tissue repair strategy for nerve injury. TeaserIntracellular tension relaxion activates morphogenesis specifically in neural cells through reversing neurogenic cellular morphology.

The continued emergence of antigenic drift and drug-resistant viral strains highlights the need for antiviral strategies that deliver robust efficacy, broad subtype coverage, and minimal off-target toxicity. We demonstrate a potent and broad-spectrum strategy that employs hybrid biomaterials of Urumin (a host defense peptide) and a honeycomb (HC) DNA origami through spatially organized multivalent presentation for enhanced antiviral efficacy. Molecular dynamics simulations reveal that Urumin penetrates and destabilizes the hemagglutinin (HA) trimer core, disrupting influenza A viral (IAV) entry. Arranging Urumin in trimeric clusters on the HC enables potent multivalent binding to trimeric HAs on IAV, enhancing antiviral efficacy at nanomolar concentrations, [~]1,000-fold more effective than free Urumin. In vitro assays confirm HC-Urumin outperforms free Urumin in blocking viral entry and preserving cell viability in more IAV subtypes. In vivo studies show that compared to free Urumin, HC-Urumin treatment reduces disease severity, preserves physiological behavior, and decreases mortality in infected mice, while maintaining virus-specific adaptive immune responses without altering humoral immunity. Our study offers an advanced and effective materials platform and strategy for broad-spectrum, low-dose intervention against human and animal IAVs, which can be adapted to combat other viruses by patterning corresponding host defense peptides on custom designed DNA nanostructures.

Exposure of pregnant women to toxic metals is an environmental health issue associated with various pregnancy complications. Efforts to advance our biological understanding of this problem and mitigate its adverse effects, however, have been challenged by ethical concerns of human subject research during pregnancy. Here, we present an alternative approach that leverages the design flexibility, controllability, and scalability of bioengineered human reproductive tissues to enable experimental simulation and in-depth investigation of placental exposure to environmental metals in maternal circulation. Central to this method is an in vitro analog of the maternal-fetal interface and its dynamic tissue-specific environment constructed using primary human placental cells grown in a micro-engineered device. Using cadmium as a representative toxicant, we demonstrate the proof-of-concept of emulating the human placental barrier subjected to the flow of cadmium-containing maternal blood to show how this model can be used to examine adverse biological responses and impaired tissue function on both the maternal and fetal sides. Moreover, we present a mechanistic study of maternal-to-fetal cadmium transport in this system to reveal that efflux membrane transporters expressed by trophoblasts may play an important protective role against cadmium-induced toxicity. Finally, we describe metabolomic analysis of our microphysiological system to demonstrate the feasibility of discovering metabolic biomarkers that may potentially be useful for detection and monitoring of cadmium-induced placental dysfunction.

The anatomical complexity and distinctive tissue environment of the human oral cavity pose major challenges to modeling oral infection and host-microbe interactions in preclinical laboratory settings. Here we present a bioengineered oral microphysiological system comprising vascularized human gingival tissue integrated with tooth analogs that together recreate a functional unit of the human oral cavity. We incorporated Streptococcus mutans and Candida albicans into this system to model cross-kingdom biofilm formation, microbial dissemination, and host-microbial interactions at the gingival-tooth interface. Single-cell RNA sequencing and global metabolomics analysis revealed that fungal colonization induces epithelial-to-mesenchymal transition associated with distinct transcriptional and metabolic signatures. Our platform also allowed us to simulate SARS-CoV-2 infection and examine gingival responses to live-virus challenge. Finally, we integrated the engineered gingival tissue with controlled human saliva flow to show that hyposalivation potentiates the pathogenic capacity of fungal infection. This work demonstrates the potential of oral microphysiological systems as an experimental platform for in vitro modeling and mechanistic investigation of host-microbe interactions under controlled, human-relevant conditions.

Aging manifests as progressive deterioration in cellular and systemic homeostasis, requiring systems-level perspectives to understand the gradual molecular dysregulation of underlying biological processes. Here, we report systems-level changes in the molecular regulation of biological processes under multiple lifespan-extending interventions in mice and across age in humans. In mouse cohorts, Differential Rank Conservation (DIRAC) analyses of liver proteomics and transcriptomics show that mechanistically distinct prolongevity interventions tighten the regulation of aging-related biological modules, including fatty acid metabolism and inflammation processes. An integrated analysis of liver transcriptomics with mouse genome-scale metabolic model supports the shifts in fatty acid metabolism. Additionally, the difference in DIRAC patterns between proteins and transcripts suggests biological modules which may be tightly regulated via cap-independent translation. In a human cohort spanning the majority of the adult lifespan, DIRAC analyses of blood proteomics and metabolomics demonstrate that regulation of biological modules does not monotonically loosen with age; instead, the regulatory patterns shift according to both chronological and biological ages. Our findings highlight the power of systems-level approaches to identifying and characterizing the biological processes involved in aging and longevity.

Plastic-derived micro- and nanoplastics are pervasive, but how environmentally aged particles affect vascular barriers is poorly understood. We hypothesized that photoaged plastics impair endothelial force-sensing, triggering gut-brain-heart barrier failure. Ultraviolet (UV) exposure converted pristine nanoplastics into oxidized, irregular photoaged microplastic aggregates (> 1.2 {micro}m). In human aortic endothelial cells, photoaged particles increased membrane stiffness and activated transcriptional programs linked to permeability, junction disruption, inflammation, and cytoskeletal remodeling. Mechanistically, photoaged particles selectively inhibited Piezo1-mediated Ca2+ signaling and downstream Notch activity without changing PIEZO1 expression, and endothelial CRISPR inhibition of PIEZO1 recapitulated these effects. In zebrafish, photoaged plastic exposure increased gut-vascular permeability and systemic spread with brain and heart accumulation, accompanied by reduced neurovascular and myocardial Ca2+ signals, depressed cardiac contractility, and abnormal locomotor behavior. Thus, photoaged plastics compromise vascular barriers through disrupted endothelial Piezo1-Notch mechanotransduction.

End-stage liver disease (ESLD) is one of the leading causes of death worldwide. Currently, the only curative option for patients with ESLD is liver transplantation. However, the demand for donor livers far exceeds the available supply, partly because many potentially viable livers are discarded following biopsy evaluation. While biopsy is the gold standard for assessing liver histological features related to graft quality and transplant suitability, it often leads to high discard rates due to its susceptibility to sampling errors and limited spatial coverage. Besides, biopsy is invasive, time-consuming, and unavailable in clinical facilities with limited resources. Here, we present an AI-assisted photoacoustic/ultrasound (PA/US) imaging framework for quantitative assessment of human donor liver graft quality and transplant suitablity at the whole-organ scale. With multimodal volumetric PA/US images as the input, our deep-learning (DL) model accurately predicted the risk level of fibrosis and steatosis, which indicate the graft quality and transplant suitability, when comparing with true pathological scores. DL also identified the imaging modes (PAI wavelength and B-mode USI) that correlated the most with prediction accuracy, without relying on ill-posed spectral unmixing. Our method was evaluated in six discarded human donor livers comprising sixty spatially matched regions of interest. Our study will pave the way for a new standard of care in organ graft quality and transplant suitability that is fast, noninvasive, and spatially thorough to prevent unnecessary organ discards in liver transplantation.

The spleen, as the largest immune organ, plays a pivotal role in modulating immune responses, particularly in the context of carcinogenesis and tumor progression. Non-pharmacological manipulation, particularly splenic ultrasound stimulation (SUS), has demonstrated significant immunomodulatory efficacy in alleviating chronic inflammatory diseases, suggesting its potential to revitalize splenic immunocompetence suppressing tumor proliferation, yet remains underexplored. This study applied low-frequency pulsed focused ultrasound (FUS) noninvasively stimulating the spleen (FUS sti. spleen) to investigate the efficacy in enhancing antitumor immunity and suppressing hepatocellular carcinoma (HCC). The results showed that FUS sti. spleen significantly suppressed tumor proliferation, achieving a suppression rate of >70% for H22-HCC and >83% for Hepa1-6-HCC, along with significantly prolonged survival. Comprehensive flow cytometry, single-cell RNA sequencing (scRNA-seq) and cytokine analyses demonstrated that SUS profoundly reshaped the splenic and intratumoral immune landscape, specifically activating cytotoxic CD8+ T cells and NK cells while suppressing immunosuppressive cell populations. Mechanistically, FUS facilitated calcium influx in splenic immunocytes, activating multiple signaling pathways, such as TNF, NF{kappa}B, MAPK, HIF-1, and ErbB, thereby counteracting tumor-driven immunosuppressive polarization while potentiating robust immune activation that impedes malignant progression and neoplastic proliferation. Leveraging above insights, we developed spleen-targeted nanodroplets encapsulating bioavailable calcium ions (STNDs@Ca{superscript 2}), which, upon FUS stimulation, undergo cavitation-mediated controlled release of Ca{superscript 2}, further amplifying immunocyte activation and tumor suppression, achieving a remarkable H22-HCC suppression rate of over 90%. This study highlights the therapeutic potential of ultrasound-mediated splenic immunomodulation, both as a standalone intervention and in synergy with STNDs@Ca{superscript 2}, as a novel and noninvasive strategy for cancer immunotherapy.

Ensuring the identity and optimal aging state of cell products is critical for the efficacy and safety of cell therapies. Despite rapid iterations, there remains an urgent need for robust and easy-to-implement tests to characterize cell products. Here, we present CIAdex (Cell Identification and Aging Index), an analytical framework that utilizes single-cell Fourier-transform infrared (FTIR) spectral fingerprints and machine learning to achieve precise label-free cell identity assessment and aging quantification. CIAdex employs a Feature Extraction Processor to automatically extract FTIR spectral variables corresponding to distinct cellular biomolecular features, enabling reliable distinction of lineage-, donor-, and batch-specific cell populations using linear discriminant analysis. Through application of the XGBoost algorithm, a quantitative aging index (trPDL/trPN) was further generated for tracking cellular aging dynamics along culture expansion. Notably, trPDL/trPN quantitatively represent subtle age-related shifts among different batches and drug-induced senescence or rejuvenation effects which are unmeasurable by existing methods. Together, our work demonstrates that CIAdex, by simultaneous label-free identity verification and aging quantification of cell populations, offers a transformative approach to interpret single-cell FTIR spectral fingerprints and provides novel metrics for quality control in cell manufacturing with significant potential for optimization and assurance of cell therapies safety and efficacy.

The kidney epitheliums pivotal role in molecular filtration, metabolism, and excretion highlights the crucial importance of understanding kidney physiology in drug development. However, our knowledge is largely derived from non-human or non-physiological models, potentially limiting its applicability to humans. To address this significant gap, we have pioneered a human kidney epithelial microphysiological analysis platform (Epi-MAP) designed to establish, mature, and monitor renal functions of the human collecting epithelium within a physiologically relevant microenvironment. We first demonstrate the highly mature collecting duct physiology derived from human stem cells, enabled by the Epi-MAPs microenvironments that recapitulate in vivo asymmetries in fluidic and biochemical conditions. The integrated biosensors of the Epi-MAP provide long-term, time-resolved epithelial maturation trajectories, revealing advanced integrity and functional maturity with transepithelial metrics. Furthermore, Epi-MAPs electrophysiological analytics for measuring water flux, in conjunction with transepithelial potential and resistance, allow for real-time decoding of intricate epithelial responses to substance stimulation, showcasing its effectiveness as a robust pharmacological test model. This human cell-derived, physiologically advanced model on a chip stands as a robust in vitro tool, offering comprehensive insights into human kidney biology and significantly enhancing drug discovery process based on human physiology.

Peripheral neuropathy (PN) is the most common complication of diabetes. It is characterized by sensory loss which often causes major health consequences including foot ulceration, chronic pain, poor mobility and increased risk of falls. However, present treatments do not counteract the cause of the disease, namely lack of sensory feedback, but rather aim at partial and temporal symptoms relief (e.g. analgesics for pain or creams for ulcers healing). Electrical stimulation is a promising solution for sensory restoration, but it is yet unknown if it can elicit perceivable sensations in PN damaged nerves and whether it could lead to any health or functional benefits. To this aim, we designed a wearable sensory neuroprosthesis providing targeted neurostimulation at the ankle level (NeuroStep) restoring feet lost sensations. We tested it in 14 participants with PN, evaluating its effects on functional outcomes and pain, and the cortical activation related to the restored sensations. Our system was able to restore lost sensations in all participants. The nerves of PN participants resulted significantly less excitable and sensitive than healthy individuals (N=22). Thanks to the neurostimulation, participants improved cadence and functional gait, with even stronger improvements in individuals with higher risk of falls. A full day of NeuroStep use led to a clinically significant reduction of 30.4% {+/-} 9.2% in neuropathic pain. Restored sensations activated cortical patterns, as measured via fMRI, similar to the naturally located foot sensations, thus not requiring training by the user. NeuroStep restores intuitive sensations in PN participants, improving mobility and decreasing pain, possibly replacing multiple inefficient treatments. It holds potential to drastically improve patients quality of life thanks to functional and health benefits, while paving the way to new effective neuromodulation treatments.

BackgroundCalcium ions (Ca{superscript 2}) serve as universal intracellular messengers regulating diverse physiological processes, while dysregulated Ca{superscript 2} homeostasis triggers cytotoxicity. Molecular hydrogen (H2) exhibits protective effects against oxidative stress-related pathologies, but its mechanism of action remains incompletely understood. Transient receptor potential canonical 4 (TRPC4) channels and their associated protein TRPC4AP are critical mediators of Ca{superscript 2} influx ( [Ca{superscript 2}]i), yet their role in H2-mediated calcium signaling is unexplored. This study investigates the molecular mechanism by which H2 modulates Ca{superscript 2} dynamics through the TRPC4-TRPC4AP axis, aiming to establish its therapeutic potential for calcium-related disorders. MethodsThe study employed heterogeneous cellular models (e.g., mesenchymal stem cells, neurons, fibroblasts) and in vivo two-photon calcium imaging in C57BL/6J mice. Techniques included CRISPR-Cas9 knockout, siRNA-mediated gene silencing, molecular docking (AlphaFold 3), and protein-protein interaction analysis. Calcium flux was quantified via fluorescence imaging, while mitochondrial integrity and cytoskeletal dynamics were assessed using JC-1 staining, ATPase activity assays, and live-cell imaging. Structural validation of TRPC4-TRPC4AP binding sites utilized mutagenesis and complementation experiments. ResultsH2 selectively enhanced extracellular Ca{superscript 2} influx via TRPC4-TRPC4AP, with no cytotoxicity or mitochondrial dysfunction observed. Key arginine residues (730Arg-731Arg) in the TRPC4 CIRB domain formed hydrogen-bond networks essential for channel activation. In vivo, H2 increased neuronal Ca{superscript 2} transient frequency and amplitude in the primary motor cortex. TRPC4AP knockout abolished H2-induced Ca{superscript 2} influx, while mutagenesis of 730Arg/731Arg disrupted channel activity. H2 also promoted cytoskeletal remodeling and cell motility, dependent on TRPC4AP-mediated Ca{superscript 2} signaling. ConclusionsThis study identifies H2 as a novel calcium agonist that activates the TRPC4-TRPC4AP axis to regulate extracellular Ca{superscript 2} influx. The 730Arg-731Arg motif in TRPC4 serves as a critical H2-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic basis for H2-based therapies targeting calcium dysregulation in neurodegenerative, inflammatory, and metabolic diseases, while highlighting TRPC4AP as a pivotal molecular switch for gasotransmitter signaling.

Plant-derived extracellular vesicles (PEVs) are promising candidates for oral drug delivery, yet their clinical translation is hindered by limited targeting precision and inconsistent systemic absorption. While surface engineering can enhance tissue accumulation, strategies that preserve biocompatibility and enable scalable production remain limited. Here, we introduce a simple thermal processing approach, boiling, to structurally reconfigure ginger EVs into functionally enhanced, thermally reassembled nanoparticles (B-GEVs). The surface architecture of B-GEVs is enriched with key vesicle trafficking regulators, including V-type proton ATPase subunit G, ARF1, and {beta}-adaptin-like protein. This specific composition drives their tissue-specific accumulation in the intestine and liver and potentiates clathrin-dependent cellular uptake in intestinal cells by 8.57-fold. Beyond superior intrinsic anti-inflammatory activity through NLRP3 inflammasome suppression, B-GEVs function as an efficient oral delivery platform. When loaded with TNF- siRNA, they enable a synergistic therapy that simultaneously modulates upstream inflammation and silences key downstream mediators, showing potent efficacy in colitis. Our findings position boiling as a natural strategy for enhancing the bioactivity and targeted oral delivery potential of ginger-derived EVs.

Nitrogen mustard (NM)-caused severe cutaneous damage lacks effective targeted therapies. Vitamin D3 (VD3) shows promise as a therapy for NM-induced dermal toxicity; however, the underlying mechanisms remain elusive. Herein, we initially confirmed that NM induced gut flora dysbiosis, characterized by a decrease of Akkermansia muciniphila (AKK) abundance, thereby leading to butyrate reduction. Antibiotics (ABX) significantly promoted NM-induced skin injury, whereas fecal microbiota transplantation of the controls feces (HC-FMT) or AKK administration attenuated NM-induced dermal toxicity. HC-FMT or AKK significantly increased butyrate levels in feces and serum of NM-treated mice. Butyrate notably attenuated ABX-caused acceleration of NM-induced skin injury. Meanwhile, NM markedly decreased the expression of -defensins, MMP7, and VDR. NM failed to further decrease AKK abundance and BA contents in intestinal MMP7-deficient mice, which was abolished by human alpha defensin 5 (HD5) overexpression. And intestinal MMP7 deficiency enhanced NM-caused skin injury, which was markedly attenuated by HD5 overexpression, AKK transplantation, or BA supplementation. Moreover, NM also failed to further reduce MMP7 and -defensin expression, AKK abundance, and butyrate levels in intestinal VDR-silenced mice. Finally, VD3 remodeled the gut microbiome particularly enriching AKK, increased butyrate contents and promoted the expression of -defensins, MMP7, and VDR, thereby attenuating NM-induced skin damage. The protective effect of VD3 against NM-caused dermal toxicity was abolished by either ABX or intestinal-specific knockdown of MMP7 or VDR in mice; however, this impairment was reversed by butyrate or AKK. In conclusion, VD3 attenuated NM-caused dermal toxicity by promoting BA production via remodeling the gut microbiota, and this effect was partially mediated by the intestinal VDR--defensin signaling pathway. These highlight that targeting the gut flora or supplementing with BA could be potential therapies for NM-induced dermal toxicity.