Reactive oxygen species (ROS) are key mediators in the progression of atherosclerosis, making ROS imaging valuable for evaluating oxidative stress in plaques. However, most activatable fluorescent probes show limited fluorescence enhancement under physiological conditions because their fluorophores are insufficiently ionized at neutral pH, resulting in weak signal output and low sensitivity. To address this issue, we developed a pKa-engineering strategy by introducing electron-withdrawing fluorine atoms at the ortho-positions of the phenolic hydroxyl group to construct the fluorophore DCM-2F-OH. The difluoro substitution lowered the pKa to 6.0, enabling complete ionization and strong near-infrared fluorescence emission at physiological pH. Incorporation of a boronate ester trigger further afforded the activatable ROS probe DCM-2F-B. DCM-2F-B displayed a 12.8-fold fluorescence turn-on response toward ROS, significantly higher than that of the non-fluorinated analog (2.5-fold), along with a low detection limit (0.03 μM) and low cytotoxicity. The probe successfully visualized endogenous ROS in lipopolysaccharide (LPS)-stimulated macrophages and detected elevated ROS levels in aortic valve tissues from apolipoprotein E knockout (ApoE-/-) mice, consistent with histopathological analysis. This work demonstrates that pKa engineering is an effective strategy for improving activatable fluorescent probes under physiological conditions and provides a useful tool for imaging oxidative stress in cardiovascular diseases.
Layered double hydroxides (LDHs), as a typical class of two-dimensional layered nanomaterials, have attracted extensive attention in biomedical fields due to their unique layered structure, tunable chemical composition, good biocompatibility, ease of surface functionalization, and efficient drug loading and controlled release capabilities. This review systematically summarizes recent advances in the research on LDHs for biomedical applications, focusing on their potential in drug delivery, bioimaging, and other disease treatments. Studies demonstrate that LDHs function as efficient carriers for a range of bioactive molecules, including anticancer drugs, nucleic acids, and proteins. Targeted and controlled release is enabled through mechanisms like pH responsiveness and ion exchange. Their favorable biodegradability and low cytotoxicity provide a safety basis for in vivo applications. Moreover, LDHs exhibit remarkable advantages in photothermal therapy, synergistic therapy, and the construction of multifunctional theranostic platforms. Despite significant progresses, challenges remain regarding long-term biosafety, scalable fabrication, and clinical translation. In the future, through rational design and multifunctional integration, LDHs are expected to become key components in next-generation intelligent nanotheranostic platforms.
Drug-induced liver injury (DILI) is a complex and intractable disease because existing anti-oxidate therapies in clinic fail to modulate multiple pathological pathways concurrently. Here, we present a direction-aware framework that integrates disease-network analysis, AI-guided molecular screening, and self-assembled nanomedicine design for precise protection of DILI. Time-resolved transcriptomic profiling of DILI identifies two complementary repair axes: the suppression of cytokine-cytokine receptor signaling for inflammation control together with the activation of glutathione biosynthesis for antioxidant defense. Guided by these DILI-driven mechanisms, we develop a dual-constraint deep-learning model that jointly evaluates the interaction between therapeutic molecules and disease targets, enabling the identification of candidate molecules whose biological effects match the desired intervention. Through independent screening from FDA-approved active pharmaceutical ingredients pool, we explore hesperidin (HES) and ursodeoxycholic acid (UDCA) as combination molecules capable of self-assembling into uniform nanomedicines (HUNMs) with predicted biological activities. Flash nanocomplexation-based engineering of HES and UDCA produces stable carrier-free nanocrystals with improved aqueous dispersibility. In an acetaminophen-challenged DILI mice, HUNMs alleviate hepatic injury, suppress inflammatory responses, restore glutathione homeostasis, and accelerate liver recovery. Together, our insights highlight an AI-native strategy that harnesses smart molecules to develop a precise and translatable nanomedicine for efficient management of DILI and other complex diseases.
Cellular senescence involves progressive acidification, but how cells sense and adapt to this pH shift remains unclear. Here we report that metabolic enzyme GOT1 functions as a pH sensor that undergoes liquid-liquid phase separation (LLPS) to combat senescence. Proteomic analysis identified GOT1 upregulation in aged human lung cells. Acidic conditions mimicking senescence directly induce GOT1 LLPS via its N-terminal intrinsically disordered region (IDR1), recruiting ME1 to form dynamic enzymatic co-condensates that scavenge reactive oxygen species and alleviate oxidative stress. To quantitatively interrogate GOT1's pH microenvironment during senescence, we engineered BDP-PLP, a first-in-class fluorescent probe conjugating the native GOT1 cofactor pyridoxal phosphate to a BODIPY fluorophore. Operating via a binding-inhibited PET mechanism, this probe enables high-specificity GOT1 targeting and pH-dependent fluorescence lifetime imaging (FLIM). Using FLIM, we achieved quantitative real-time visualization of pH dynamics within GOT1 condensates in living cells, revealing that phase separation generates a highly acidic local microenvironment critical for its anti-senescence function. This study uncovers a pH-triggered phase separation mechanism that bolsters antioxidant defense via metabolic enzyme co-condensation, offering new perspectives on metabolic adaptation in aging and establishing a chemical tool for probing microenvironmental dynamics.
Nitric oxide (NO)-releasing materials have attracted considerable interest for their potential in diverse biomedical applications. However, a persistent challenge is the leaching of NO donors from these materials, which leads to significantly reduced NO release durations and undesirable burst release effects. Herein, we demonstrate a novel hydrogen-bonding strategy to address NO donor leaching and the burst release of NO. A nitrosothiol-based NO hydrogel was developed by incorporating a SNAP analog bearing a 4-hydroxyphenethyl sidechain into polyvinyl alcohol (PVA)/PEG hydrogels. The hydrogel containing the structurally modified SNAP analog exhibited an elongation of over 300% and sustained NO release under physiological conditions. Hydrogen-bonding interactions contributed to the enhanced retention of the SNAP analog within the hydrogel network, extending donor retention to 144 h compared with the hydrogel containing unmodified SNAP (10 h). These NO-releasing hydrogels exhibited potent antibacterial activity against both S. aureus and E. coli, with bacterial reduction exceeding 99%, and caused pronounced disruption of surface-associated bacterial structures. This novel hydrogen-bonded NO donor-hydrogel system offers a promising strategy for durable and controlled NO release, with potential applications in wound dressings, tissue scaffolds, and flexible sensors.
Viscosity and polarity are critical physiological parameters implicated in the onset and progression of nonalcoholic fatty liver disease (NAFLD). Real-time monitoring of their dynamic variations is of significant importance for the early diagnosis of NAFLD. In this study, BODIPY was utilized as the fluorophore core to successfully develop a dual-responsive fluorescent probe, Mp BDP-CF 3 , through the incorporation of a trifluoromethyl (CF3) group at the meso position and methoxyphenyl groups at the 3,5-positions. The probe demonstrated a high fluorescence quantum yield (approximately 47.86% in 1,4-dioxane) and excellent photostability. It exhibited remarkable fluorescence sensitivity to variations in both viscosity and polarity within solution and cellular environments, achieving a detection limit as low as 0.41 cP for viscosity. Furthermore, Mp BDP-CF 3 demonstrated effective targeting of lipid droplets during NAFLD progression. Subsequent in vivo investigations revealed that Mp BDP-CF 3 facilitated high-contrast fluorescence imaging and precise discrimination of NAFLD in BALB/c nude mouse models, underscoring its substantial potential for early NAFLD diagnosis.
Ultrasound (US) was once predominantly used for diagnosis but not anymore. It has the capability to noninvasively deliver energy to the deep tissues and also enable spatiotemporal control. Unlike high-intensity focused ultrasound, which primarily ablates tumors through localized heat, nonthermal US converts acoustic energy into mechanical, cavitation, and physicochemical effects. The bioeffects that are induced under nonthermal US provide a mechanistic basis for cancer therapies. Here, we present a mechanism-guided framework that connects acoustic parameters and nonthermal bioeffects with therapeutic modalities. Nonthermal US-based cancer treatments are classified into six representative modalities: histotripsy, ultrasound-targeted microbubble destruction, sonogenetics, sonomechanical therapy (SMT), sonodynamic therapy (SDT), and sonopiezoelectric therapy (SPT). Through a review of recent advances in these modalities, we highlight emerging opportunities and future directions with a focus on standardizing acoustic dosimetry, biosafety assessment, and material optimization. These insights collectively establish a foundation for creating more reliable and clinically relevant nonthermal US-based therapies for precision oncology.
Senescence-associated β-galactosidase (SA-β-gal) is a key biomarker of cellular senescence and has been demonstrated to be a major driver of various age-related diseases and tumor resistance. However, noninvasive in vivo imaging of SA-β-gal remains challenging due to the poor membrane permeability of existing probes, their reliance on intratumoral injection, and limited blood-brain barrier (BBB) permeability. This study reports a novel, rationally designed near-infrared (NIR) fluorescent probe, DCIP-AcGal, which involves a synergistic lipophilicity-oriented design strategy by leveraging a BBB-penetrating fluorophore with acetylated β-galactose. Its SA-β-gal-sensitive NIR emission property enables deep-tissue imaging of senescent cells with low background and high signal-to-noise ratio. The probe's favorable lipophilicity facilitates passive diffusion through the cell membranes, ensuring efficient activation by SA-β-gal within the lysosomes. Its specificity for imaging senescent cells has been validated through GLB1 knockdown, and its fluorescence intensity and distribution correlate positively with p21 expression. Following intravenous injection, DCIP-AcGal enables noninvasive imaging of palbociclib-induced tumor senescence in living mice. Furthermore, due to its favorable lipophilicity, the probe crosses the BBB, allowing for the assessment of senescence accumulation in the brains of aged mice. These findings demonstrate that probe DCIP-AcGal provides a valuable tool for investigating senescence biology and monitoring the efficacy of senotherapeutic interventions.
In this work, we replaced hydrogen with halogen atoms in the BC7 molecule to enhance the parent molecule's photostability and improve the photothermal therapy (PTT) and photodynamic therapy (PDT) effects. We further improve and enhance the properties of BC7 while maintaining its optical properties, tumor targeting, and optical therapeutic effects. Nuclear magnetic resonance (NMR) spectroscopy of the four synthesized compounds confirms the success of the target products. We briefly screen BC7-Cl, BC7-Br, and BC7-I by in vitro photothermal experiments, in-solution reactive oxygen species detection, and stability tests. We intravenously inject three molecules into a tumor-bearing mouse model for in vivo tumor imaging and observe that their tumor-targeting ability has been markedly improved. In further optical treatment experiments, BC7-Br showed the best therapeutic effect and a significant improvement compared to BC7. This study provides innovative strategies and theoretical support for the construction and development of new materials with dual functions of efficient PTT and PDT.
Targeted protein degradation (TPD) has emerged as a transformative therapeutic strategy that offers unprecedented opportunities to eliminate traditionally "undruggable" proteins that have posed significant challenges in traditional drug development. Current TPD approaches, including proteolysis-targeting chimeras (PROTACs), molecular glues, and lysosome-targeting chimeras (LYTACs), encounter several limitations. These include the complexity of forming stable ternary complexes, suboptimal design of linkers, a limited repertoire of E3 ligases, and inadequate pharmacokinetic properties. Artificial intelligence (AI) has rapidly become essential in addressing these challenges, revolutionizing the TPD drug discovery process through data-driven insights and predictive modeling. This review systematically explores AI applications in TPD development, covering the prediction and design of stable ternary complexes, rational optimization of linkers, high-throughput screening for E3 ligase ligands, and accurate predictions of degradation efficiency and ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) properties. Additionally, this review underscores AI's pioneering role in discovering molecular glues, from target identification to activity prediction, and discusses the AI-driven optimization of emerging TPD modalities, such as LYTACs and PROTAC/IMiD bifunctional molecules. Despite significant progress, several critical challenges remain, such as the absence of standardized datasets, the static modeling of dynamic biological systems, and the opaque nature of advanced AI architectures. Future research should concentrate on integrating multi-omics data to improve model training, developing dynamic and mechanistic AI frameworks, advancing explainable AI (XAI) to enhance mechanistic interpretability, and encouraging transdisciplinary collaboration to expedite clinical translation. By integrating AI with structural biology, pharmacology, and experimental validation, TPD technologies hold the potential to expand the druggable proteome and provide novel therapeutic solutions for cancer, neurological disorders, and other persistent diseases.
Efficient optimization of chemical reaction conditions is crucial for enhancing reaction yield and selectivity, yet traditional methods face inherent limitations including experimental inefficiency, low predictive accuracy, and poor interpretability. This study proposes a novel framework for reaction condition optimization by integrating the Kolmogorov-Arnold network (KAN) model and Bayesian optimization (BO) algorithm. The KAN model establishes accurate and explicit mappings between reaction conditions and outcomes like yield, while BO iteratively optimizes reaction outcomes to identify optimal conditions based on the KAN model, demonstrating efficacy even with sparse data. This framework is implemented as the BayesianKAN software and validated in two reaction systems: hydrogen peroxide (H2O2) synthesis and photocatalytic acceptorless dehydrogenation to flavones. KAN exhibits superior fitting accuracy and generalization ability compared to the traditional response surface methodology and other seven common machine learning approaches. Experiments also verify the feasibility and effectiveness of BayesianKAN, achieving 60.7% and 5.2% increases in H2O2 production and flavone yield, respectively.
MYC-amplified osteosarcoma, a poor-prognosis molecular subtype, presents formidable therapeutic challenges due to its aggressive phenotype, chemoresistance, and the "undruggable" oncogenic driver MYC. A single-molecule prodrug, DHU-NO3, was developed to achieve precise synergy between photodynamic therapy (PDT) and nitric oxide (NO) gas therapy for the treatment of MYC-amplified osteosarcoma. This prodrug covalently links the clinically approved photosensitizer methylene blue (MB) to an NO donor and undergoes a sequential activation cascade: reactive oxygen species-triggered MB release, 405 nm light-controlled NO generation, and PDT initiation under 650 nm laser irradiation. In 143B osteosarcoma cells, this temporally coordinated regimen efficiently induces apoptosis and potently suppresses the MYC signaling network. In the 143B osteosarcoma subcutaneous xenograft model, DHU-NO3-mediated sequential phototherapy demonstrated robust tumor growth inhibition with favorable biosafety. This work establishes a spatiotemporally programmable prodrug platform and provides a potent strategy to combat MYC-amplified osteosarcoma by indirect pathway suppression.
Porous two-dimensional (2D) materials have emerged as promising electrocatalysts for the urea oxidation reaction (UOR) due to their high surface area, tunable electronic properties, and enhanced charge and mass transport. These structural advantages improve catalytic activity, selectivity, and reaction kinetics. However, challenges such as catalyst deactivation, structural instability, and limited mechanistic understanding still restrict their broader application. While previous studies have explored compositional tuning and catalytic performance, a systematic understanding of how porosity, defect engineering, and electronic structure modulation govern UOR efficiency and durability remains limited. This review comprehensively evaluates porous 2D materials for UOR, focusing on structure-activity relationships and fundamental catalytic mechanisms. Special attention is given to how pore architecture, surface defects, and electronic configurations regulate reaction pathways. Porous 2D materials are categorized into six major classes: oxides and their derivatives, hydroxides, sulfides, phosphides, carbides and nitrides, and other emerging materials. Unlike conventional reviews that primarily classify materials based on composition and performance, this work highlights the role of microstructural design in optimizing UOR electrocatalysis. By addressing existing knowledge gaps and identifying key structural parameters for catalyst optimization, this review provides a systematic framework for guiding the rational design of porous 2D materials in sustainable electrochemical energy conversion.
The development of safe and effective vaccines remains a critical priority in modern medicine. Traditional adjuvants, while effective in enhancing immune responses, often suffer from limitations including local reactogenicity, limited mucosal immunity, and instability of antigens. Chitosan, a natural polysaccharide derived from chitin, and its derivatives have emerged as promising candidates for nanoparticle-based vaccine delivery due to their biocompatibility, biodegradability, mucoadhesive properties, and intrinsic immunostimulatory activity. Chemical modifications of chitosan, such as trimethylation, quaternization, and thiolation, enhance its solubility, stability, and immune modulatory functions. Chitosan derivative nanoparticles (CDNPs) facilitate efficient antigen encapsulation, protect labile biomolecules, and promote uptake by antigen-presenting cells. They can trigger both humoral and cellular immune responses, particularly through mucosal administration routes. This review comprehensively discusses the physicochemical properties of chitosan derivatives, mechanisms of immune enhancement, methods for nanoparticle preparation, applications in preclinical and clinical vaccine studies, and challenges for translation to human use. Insights from recent literature suggest that CDNPs hold significant potential to revolutionize vaccine delivery platforms, especially for mucosal vaccines and next-generation immunotherapeutics.
Room temperature phosphorescent (RTP) molecules, owing to their unique afterglow characteristics, can translate microscopic electronic transitions into macroscopic smart responses, and have gradually emerged as a research hotspot in the field of smart molecules. Benefiting from the renewability, biocompatibility, and structural tunability of cellulose, these materials provide an ideal and sustainable platform for constructing RTP systems. Significant progress has been made in this field, leading to the emergence of numerous cellulose-based RTP material systems with novel structures and excellent performance. This review summarizes recent advances in RTP systems cellulose derivatives. First, it elucidates the underlying mechanisms of cluster-triggered emission, followed by a detailed discussion of four key construction strategies: regulation of aggregation structures, reconstruction of clustered emission centers, hydroxyl functionalization, and host-guest doping. In addition, innovative applications in information security and environmental monitoring are highlighted. Finally, current challenges are discussed, and perspectives on the rational design of future biomass-based RTP materials are provided.
A fundamental constraint of conventional messenger RNA (mRNA) delivery systems is their obligatory trafficking through endosomal-lysosomal compartments, wherein cargo degradation and activation of endosomal Toll-like receptors precipitate substantial translational attrition and deleterious inflammatory cascades. We herein report a chemically engineered platform that circumvents these limitations ab initio. Through strategic perfluoro-acylation of branched polyethyleneimine (PEI, 25 kDa) with pentafluoropropionic anhydride, we install approximately 26 fluoro-amide "zipper" moieties per polymer chain that orchestrate direct, energy-independent trans-bilayer translocation without recruitment of clathrin, caveolae, or lipid raft microdomains-thereby precluding lysosomal entrapment and catabolism. Bio-orthogonal copper-free click chemistry between azide- and dibenzocyclooctyne (DBCO)-terminated PEI-F derivatives, coupled with redox-labile disulfide crosslinkers, engenders polyplexes of exceptional extracellular stability that undergo quantitative glutathione-triggered disassembly within the cytosolic milieu. This endosome-evasive entry mechanism effectively sequesters single-stranded mRNA from Toll-like receptor 3, TLR7, and TLR8 surveillance, establishing a "TLR-attenuated" delivery paradigm characterized by undetectable interferon-α, interferon-β, TNF-α, and IL-6 induction. In human umbilical vein endothelial cells, GFP-mRNA transfection exceeds 90% fluorescent positivity with 4.8-fold superior luciferase expression relative to Lipofectamine™ 3000, whilst maintaining >95% viability. Therapeutic translatability is demonstrated in a murine hindlimb ischemia model, wherein a single 10 μg intramuscular dose of mVEGF-A polyplexes restores blood perfusion to 118% of baseline within 28 days-representing marked superiority over the commercial gold standard and effectuating complete tissue salvage without necrosis. Comprehensive hematological and immunological profiling corroborates the absence of hematotoxicity, systemic inflammation, or innate immune activation. This modular, purely synthetic platform resolves the classical stability-availability paradox whilst eliminating the immunogenic liabilities inherent to endocytic delivery, furnishing a readily translatable scaffold for precision regenerative medicine.
The reversible transformation between organic molecules and radicals is an effective method for realizing dual emissions in a single material. Herein, in this work, we report a high proportion n-electron [2,2'-biisoindoline]-1,1',3,3'-tetraone (4A2B) crystal with the reversible homolysis. The crystal exhibits dual room-temperature phosphorescence (RTP) emissions at 400 nm (τ = 114.28 μs) and 575 nm (τ = 35.78 μs), originating from the 4A2B molecule and the corresponding radical ion pair generated by reversible homolysis of the weak N-N bond, respectively. Thanks to that, the radical ion pair performed a great photostability (t 1/2 = 1.64 × 105 s) and an outstanding repeatability under 100 times on-off excitation cycles. The RTP emission of the 4A2B molecule has a strong response on high energy excitation due to the ultrafast intersystem crossing process (1.22 × 1013 s-1) within the high-lying excited states. Furthermore, the 4A2B crystal with these two long-lived RTP emissions is able to be applied in optical anti-counterfeiting, optical encryption and high-resolution bioimaging. This research elucidates that the incorporation of organic molecules and radical ion pairs may provide a new method to achieve dual emissions containing RTP emissions, fluorescence or thermally activated delayed fluorescence.
Food spoilage driven by microbial growth severely endangers public health and global food security. Traditional methods for food freshness evaluation suffer from cumbersome operation, time-consuming procedures, and poor portability. In contrast, fluorescent probe-based detection offers distinct advantages including high sensitivity, fast response, and visual detectability. This review summarizes recent advances in organic small-molecule fluorescent probes for food freshness assessment over the past 3 years. We focus on representative sensing mechanisms of these probes, including nucleophilic substitution, protonation/deprotonation, hydrogen-bonding interactions, Lewis acid-base reactions, and Michael addition as well as typical target analytes such as biogenic amines, hydrogen sulfide, sulfur dioxide derivatives, pH, ATP, and hydrazine. The representative applications of fluorescent probes in portable test strips, smartphone-assisted sensing platforms, and intelligent detection systems are emphatically introduced. Although these probes have achieved remarkable results in sensitivity, response speed and practicality, challenges still exist such as single detection target, insufficient anti-interference ability, and lack of prospective prediction function. Future research will focus on expanding detection scopes, developing multi-target recognition probes, combining artificial intelligence to construct spoilage prediction models, and promoting the industrial application of portable and intelligent food freshness detection technology.
In response to the increasing demands in advanced lithography for high-resolution and highly stable photoresists, the development of high-performance resist materials has become critically important. In this study, a series of poly (tert-butyl methacrylate-co-adamantyl methacrylate-co-[trifluoroethyl methacrylate/1,1,1,3,3,3-hexafluoroisopropyl isobutyrate methacrylate]) copolymers (PTAF) were rationally designed and synthesized, and their preparation conditions were systematically optimized via a single-factor experimental design. The molecular weight distribution, thermal properties, hydrophobicity, and electron-beam lithography (EBL) performance of the resulting polymers were comprehensively investigated. The results show that PTAF exhibits a narrow molecular weight distribution (polydispersity index = 1.2-1.3) and a number-average molecular weight (M n ) of approximately 4271 g mol-1. The polymer demonstrates good thermal stability, with an initial decomposition temperature (T d) of 212°C and a glass transition temperature (T g) of 105°C, along with pronounced hydrophobicity. EBL results reveal that PTAF displays intrinsic negative-tone behavior in the absence of externally added photoacid generators, enabling fabrication of high-resolution patterns with a minimum linewidth of 16.5 nm. Mechanistic studies indicate that fluorinated monomers undergo ionization upon irradiation, initiating in situ acid generation, which subsequently triggers the deprotection of tert-butyl ester groups during post-exposure baking. This process leads to a polarity reversal and a corresponding solubility switch in the resist system. Overall, this work establishes a high-performance ternary copolymer photoresist platform and elucidates its negative-tone imaging mechanism, providing valuable insights for the design of polymer-based resists in advanced lithography, particularly for extreme ultraviolet applications.
The dysregulation of microRNA (miRNA) expression is closely linked to the pathogenesis of lung cancer, rendering the quantification of trace miRNAs clinically indispensable. However, achieving ultrasensitive detection of miRNAs in complex biological matrices, including living cells, tissues, and blood serum, remains significant challenges. To address this, we developed a fluorogenic detection platform for miR-21 based on tetrazine-mediated transfer (TMT) reactions. While TMT strategies have been previously explored, we report a refined probe design optimized through computational screening. By minimizing the energy gap (ΔE) between the fluorophore's emissive state and the tetrazine's dark state, we achieved an efficient quenching mechanism with a low background (fluorescence quantum yield <0.01). By integrating hybridization-mediated target recycling with the inverse electron demand Diels-Alder reaction, the Inverse Diels-Alder Cycloaddition Reaction (IDCR) probe achieves signal amplification. Although the theoretical catalytic turnover is significantly enhanced at low target concentrations, the practical detection limit is primarily governed by the high signal-to-background ratio afforded by our optimized probe. This mechanism yields an ultralow limit of detection (3.58 × 10-18 M) with a broad linear range (100 aM to 100 nM). Building upon foundational bioorthogonal chemistry, the IDCR probe enables high-contrast imaging in living cells and tissues and distinguishes lung cancer patients from healthy individuals.
