Abstract:Objective Conventional pulmonary function testing remains the primary clinical approach for evaluating respiratory dysfunction, but its reliability is strongly dependent on subject cooperation and test execution. In addition, global spirometric indices provide limited information on regional ventilation heterogeneity, which is important for understanding the spatial characteristics of pulmonary functional abnormalities. To address these limitations, this study proposed a dual-branch fusion method based on electrical impedance tomography (EIT) for the identification of pulmonary function abnormalities and evaluated its feasibility in screening and auxiliary classification tasks.Methods A total of 507 valid EIT-pulmonary function examination samples were included, comprising 244 samples with normal pulmonary function and 263 samples with abnormal pulmonary function. During forced expiration, thoracic EIT boundary voltage signals were collected, and corresponding conductivity image sequences were reconstructed to represent both respiratory dynamic changes and intrapulmonary ventilation distribution. For each valid examination sample, 64 consecutive frames from the expiratory phase were selected as the model input. A dual-branch architecture was then constructed, in which one branch used boundary voltage signals to characterize temporal respiratory dynamics, while the other branch used reconstructed conductivity images to describe spatial ventilation patterns. Residual network (ResNet) models with different depths, including ResNet18, ResNet50, and ResNet101, were employed for feature extraction. The extracted voltage and image features were concatenated to generate a unified spatiotemporal representation. Several machine learning classifiers, including support vector machine, k-nearest neighbor, decision tree, linear discriminant analysis, and Stacking ensemble learning, were further applied for classification. Five-fold cross-validation was used to evaluate model performance in binary classification between normal and abnormal pulmonary function, three-class classification among normal, obstructive, and non-obstructive pulmonary function patterns, and four-class classification involving multiple ventilatory dysfunction types. Accuracy and macro-averaged F1-score were used as the main evaluation metrics. Feature extraction, feature standardization, classifier training, and model evaluation were independently completed within each fold to reduce the risk of information leakage.Results On average, the fused voltage-conductivity channel outperformed the single voltage and single conductivity-image channels. The average accuracy and F1-score of the fusion channel reached 0.835 ± 0.053 and 0.765 ± 0.073, respectively, representing improvements of 6.5% and 9.7% compared with the voltage-only channel. Among the different network depths, ResNet18 achieved the best overall performance, suggesting that a relatively shallow residual network may be more suitable for learning EIT-based spatiotemporal features, probably because the key pathological information in EIT is mainly reflected by low-frequency temporal changes and large-scale regional ventilation differences rather than fine image textures. Among the classifiers, the Stacking strategy achieved the highest comprehensive performance, with an accuracy of 0.830 8 ± 0.059 9 and an F1-score of 0.821 9 ± 0.060 7, indicating that ensemble learning can further improve the robustness of multi-class recognition. The overall advantage of the fusion channel also supports the complementary value of temporal boundary-voltage dynamics and spatial conductivity-distribution information in characterizing pulmonary function abnormalities.Conclusion These findings demonstrate that dual-branch fusion of EIT voltage signals and conductivity images can effectively characterize spatiotemporal features associated with abnormal pulmonary ventilation. By integrating temporal respiratory dynamics with regional ventilation distribution, the proposed framework reduces dependence on a single predefined EIT index and provides a more comprehensive representation of pulmonary function abnormalities. The proposed method offers a non-invasive and radiation-free approach for pulmonary function abnormality screening, auxiliary classification, and bedside dynamic assessment, and may support the future development of wearable or portable EIT-based respiratory monitoring systems.

Abstract:Steroid-associated osteonecrosis of the femoral head (SANFH) is a progressive osteoarticular disorder associated with prolonged or high-dose glucocorticoid exposure. Its development involves local ischemia, oxidative stress, dysregulated bone metabolism, and disruption of the osteoimmune microenvironment. Macrophages exhibit marked phenotypic plasticity, and their polarization is essential for maintaining the dynamic balance among inflammation, angiogenesis, and bone remodeling. Persistent glucocorticoid stimulation, hypoxia, excessive reactive oxygen species, and damage-associated signals released from necrotic tissues can shift macrophages toward a pro-inflammatory phenotype. Classically activated macrophages (M1 macrophages) predominantly mediate inflammatory responses. By releasing tumor necrosis factor-α, interleukin-1β, interleukin-6, and other mediators, promoting osteoclast activation, suppressing the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and impairing vascular endothelial function, M1 macrophages accelerate trabecular destruction and expansion of the necrotic lesion. In contrast, alternatively activated macrophages (M2 macrophages) contribute to inflammation resolution, neovascularization, osteogenic repair, and tissue remodeling, thereby supporting regeneration within the necrotic region. Macrophage-derived exosomes further influence disease progression through intercellular communication. Exosomes from different macrophage phenotypes can differentially regulate adipogenic differentiation, neutrophil extracellular trap formation, and endothelial phenotypic transition, thereby affecting the repair capacity of the necrotic area. These findings indicate that macrophage polarization shapes the local microenvironment not only through soluble mediators but also through vesicle-mediated communication with BMSCs, neutrophils, endothelial cells, and other cell populations. At the molecular level, the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, nuclear factor kappa B (NF-κB), Janus kinase/signal transducer and activator of transcription (JAK/STAT), and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) pathways jointly regulate macrophage polarization and its downstream osteoimmune effects. Dysregulation of these pathways may sustain inflammatory activation, aggravate oxidative and vascular injury, inhibit osteogenesis, and impair tissue repair. NLRP3 inflammasome activation links danger signals and oxidative stress to inflammatory cytokine maturation and pyroptotic injury; NF-κB signaling promotes pro-inflammatory gene transcription and M1 polarization; JAK/STAT signaling participates in the balance between inflammatory and reparative macrophage programs; and PI3K/Akt/mTOR signaling regulates cellular metabolism, survival, autophagy, and regeneration. Notably, the biological effects of PI3K/Akt/mTOR signaling are cell-type dependent, and Akt-mediated repair signaling should be distinguished from mTOR-related autophagy regulation. The pathological significance of macrophage polarization in SANFH is not determined simply by an increase or decrease in a single phenotype. Rather, disease progression appears to result from an imbalance between persistent pro-inflammatory activity and insufficient reparative responses at different stages of the disease. Such an imbalance disrupts the coordinated coupling of inflammation resolution, vascular regeneration, and bone remodeling, ultimately contributing to structural deterioration of the femoral head. This review summarizes the pathological roles, signaling regulation, and exosome-mediated intercellular communication associated with macrophage polarization imbalance in SANFH. A more precise understanding of these mechanisms may clarify the osteoimmune basis of SANFH and support the development of macrophage-targeted interventions. However, most available evidence is derived from cellular and animal studies, and the temporal evolution of macrophage phenotypes in patients remains insufficiently characterized. Therapeutic strategies should therefore move beyond the simple suppression of M1 macrophages or enhancement of M2 macrophages and instead aim to restore a stage-appropriate balance between inflammatory control and tissue repair while promoting angiogenesis and bone reconstruction.

Abstract:Objective Community-acquired pneumonia (CAP) is a common infectious disease encountered in emergency departments, and timely severity stratification is essential for early risk identification and individualized treatment. Chest CT can accurately evaluate the extent of pulmonary involvement; however, its use for repeated bedside assessment is limited by radiation exposure, cost, limited portability, and the need for patient transport. In contrast, EIT is a radiation-free, noninvasive, and bedside functional imaging modality that can continuously monitor regional ventilation. Nevertheless, its relatively low spatial resolution limits its ability to directly characterize the anatomical extent of pulmonary lesions. Therefore, this study constructed a CT shape-prior-guided 3D CT-EIT composite image and explored its value for CAP severity assessment.Methods A total of 60 adult patients with CAP who were admitted to the Emergency Department of the Third Hospital of Hebei Medical University between October 2023 and October 2024 were retrospectively included. According to a five-lobe CT score ranging from 0 to 25, the patients were divided into a light pneumonia (LP) group and a moderate-to-severe pneumonia (MSP) group. Patients with CT scores of 0–8 were classified into the LP group (n=29), whereas those with CT scores of 9–25 were classified into the MSP group (n=31). Clinical indices, including oxygenation index (PaO₂/FiO₂), fibrinogen, and pneumonia severity index (PSI), were collected. The 3D EIT composite image based on the individual CT images of each patient was constructed, and the percentage of ventilation blocked regions (PVBR), ventilation distribution center error (VDCE), and global ventilation delay index (GVDI) were established. Receiver operating characteristic (ROC) curves were plotted to evaluate the discriminative performance, and Pearson correlation coefficient (r) was used to assess the correlation between 3D EIT ventilation indices and CT score.Results There were no statistically significant differences in baseline characteristics between the two groups (P>0.05). Compared with the LP group, the MSP group showed lower PaO₂/FiO₂ (166 mmHg vs. 298 mmHg, P<0.01), higher fibrinogen (4.59 g/L vs. 3.90 g/L, P<0.01), higher PSI (114.23 vs. 94.52, P<0.01), higher PVBR (39.33 vs. 26.33, P<0.001), higher VDCE (42.21 vs. 35.33, P<0.05), and higher GVDI (0.48 vs. 0.38, P<0.001). Among the conventional clinical indices, PaO₂/FiO₂ showed the highest area under the ROC curve (AUC=0.723). Among the 3D EIT ventilation indices, PVBR showed the best performance, with an AUC of 0.775 and a specificity of 96.6%. CT score was negatively correlated with PaO₂/FiO₂, but positively correlated with fibrinogen, PSI, and 3D EIT ventilation indices. Among these, CT score showed the strongest correlation with PVBR (r=0.696).Conclusion 3D CT-EIT composite imaging is feasible for rapid bedside functional assessment of CAP severity. PVBR, VDCE, and GVDI provide complementary information on the extent of ventilation blockage, the spatial redistribution of ventilation, and the temporal delay in ventilation, respectively. Among these indices, PVBR showed the best discriminatory performance and the strongest correlation with CT-defined lesion severity. Although 3D EIT cannot replace CT for structural diagnosis, it may serve as a practical radiation-free functional complement for bedside severity stratification, serial ventilation monitoring, and early clinical decision-making in patients with CAP.

Abstract:Kynurenine pathway (KP) is a major catabolic route of tryptophan, generating a series of bioactive metabolites including kynurenine, kynurenic acid, quinolinic acid, and 3-hydroxykynurenine. Beyond its fundamental role in amino acid metabolism, KP exerts critical regulatory functions in neurodevelopment, synaptic plasticity, and immune modulation. Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition with onset in early childhood, characterized by persistent deficits in social communication and restricted, repetitive behaviors. Despite extensive research, the etiological mechanisms underlying ASD remain highly complex and incompletely understood, involving genetic, environmental, and immunological factors. Emerging clinical evidence has consistently revealed a significant imbalance in KP metabolites in individuals with ASD, often accompanied by altered ratios of neuroprotective versus neurotoxic byproducts. However, the primary drivers of this metabolic dysregulation and its causative contribution to ASD pathogenesis are not yet fully elucidated. In this review, we systematically examine the enzymatic steps of KP and the principal physiological functions of its major metabolites, with particular emphasis on their dual roles in neuroprotection and neurotoxicity. We then analyze the potential pathogenic mechanisms through which KP dysfunction may contribute to ASD, focusing on two interconnected etiological dimensions. First, KP imbalance can promote oxidative stress, which in turn triggers chronic inflammatory responses via microglial activation and release of pro-inflammatory cytokines, thereby disrupting neuronal homeostasis. Second, aberrant KP metabolism affects neurotransmitter systems, particularly glutamatergic and dopaminergic signaling, leading to impaired neural circuit development and synaptic pruning. By integrating current findings on the KP–ASD association, this review offers a comprehensive etiological framework and a clinically relevant paradigm. Furthermore, we highlight that specific KP metabolites, such as the kynurenic acid/quinolinic acid ratio, hold promise as peripheral biomarkers for early diagnosis and disease stratification. Finally, we discuss the therapeutic potential of targeting KP enzymes or receptors for personalized intervention strategies, while acknowledging the challenges in translating these findings into clinical practice.

Abstract:Polyamine homeostasis is tightly coupled to tumor-cell proliferation, metabolic reprogramming, stress adaptation, and immune escape. Ornithine decarboxylase antizyme 1 (OAZ1) is a central negative regulator of this network. When intracellular polyamine levels increase, the ribosome shifts one nucleotide downstream and switches to a new reading frame during translation of OAZ1 mRNA, thereby enabling the production of the full-length active OAZ1 protein. OAZ1 then restrains polyamine accumulation through two complementary mechanisms. It binds ornithine decarboxylase (ODC), inhibits enzyme activity, promotes dissociation of the active ODC homodimer, and facilitates ubiquitin-independent proteasomal degradation of ODC, thereby reducing endogenous putrescine synthesis. In parallel, OAZ1 suppresses the polyamine transport system (PTS), limiting the uptake of extracellular polyamines that may compensate for reduced biosynthesis. Recent studies have reshaped the concept of the mammalian PTS. Rather than a single plasma-membrane transporter, it is now viewed as a multicomponent and compartmentalized network involving cell-surface enrichment by heparan sulfate proteoglycans, caveolin-associated endocytosis in selected cellular contexts, endosomal or lysosomal escape mediated by P5B-type ATPases such as ATP13A3 and ATP13A2, vesicular storage mediated by SLC18B1, and acetylation-coupled export mediated by spermidine/spermine N1-acetyltransferase 1 (SAT1) and the SLC3A2-associated diamine exporter pathway. These findings provide a broader framework for understanding how tumor cells maintain high polyamine availability even when synthesis is pharmacologically inhibited. However, several fundamental questions remain unresolved. The direct PTS targets recognized by OAZ1 have not been identified, the structural basis by which polyamine-induced OAZ1 dimerization contributes to transport inhibition is still unclear, and the relationship between dysregulation of the OAZ1-PTS axis and therapeutic responses varies among tumor types. In this review, we summarize the molecular mechanisms of OAZ1 polyamine sensing, programmed frameshift translation, ODC inhibition and degradation, and feedback control of polyamine uptake. We also integrate recent advances on ATP13A2, ATP13A3, ATP13A4, SLC3A2, SLC18B1, SAT1, and related transport or export modules, and compare their alterations in neuroblastoma, lung cancer, prostate cancer, colorectal cancer, hepatocellular carcinoma, breast cancer, pancreatic cancer, leukemia, glioma, and oral squamous cell carcinoma. Particular attention is given to tumor-type specificity. MYCN-driven neuroblastoma appears to depend on both enhanced biosynthesis and compensatory uptake, lung and prostate cancer models provide functional evidence for OAZ1-dependent feedback repression of uptake, hepatocellular carcinoma highlights the immunological role of acetylated-polyamine efflux, and breast and pancreatic cancer studies suggest nonredundant contributions of ATP13 family members. Finally, we discuss translational strategies that aim to mimic or restore the dual negative-feedback function of OAZ1, including difluoromethylornithine (DFMO), AMXT1501-based polyamine blockade therapy, ATP13A3-directed intervention, targeting of AZIN1-OAZ1 antagonism, and modulation of SAT1-SLC3A2-associated acetylated-polyamine export. Future work should combine transport assays, interaction mapping, spatial omics, metabolomics, and immune profiling to define actionable biomarkers, such as OAZ1, AZIN1, ODC, ATP13A3, SLC3A2, SAT1, and tumor polyamine signatures. Such biomarkers will be important for selecting patients, monitoring target engagement, and designing combinations with chemotherapy, targeted therapy, or immune checkpoint blockade. A better understanding of the OAZ1-PTS axis may support biomarker-guided patient stratification and rational combination therapies targeting polyamine dependence and the immunometabolic tumor microenvironment.

Abstract:Both type 2 diabetes mellitus (T2DM) and metabolic associated fatty liver disease (MAFLD) fall within the spectrum of metabolic diseases, and they exhibit a bi-directional causal relationship and robust reciprocal association. Their shared pathological cornerstone is insulin resistance (IR), which involves the interplay of mitochondrial dysfunction and chronic inflammation, forming a cascading pathological process of “IR—mitochondrial dysfunction—inflammation”. This largely explains the notable upward trend in T2DM-MAFLD co-occurrence observed over recent years. Exercise intervention, as a safe and effective non-pharmacological approach, can improve the pathological progression of these patients at multiple levels. Following the logical framework of “pathogenesis—efficacy comparison—molecular mechanisms—clinical translation”, this article systematically compares the efficacy and molecular mechanisms of moderate-intensity continuous training (MICT), resistance exercise, high-intensity interval training (HIIT), and combined training. MICT reduces intrahepatic triglycerides by promoting lipolysis and improving cardiorespiratory fitness; resistance exercise increases muscle mass and basal metabolic rate, offering unique advantages in preserving muscle while reducing fat and improving insulin sensitivity; HIIT is a time-efficient exercise modality that enhances patients’ cardiorespiratory fitness and insulin sensitivity by alternating brief periods of vigorous exertion with recovery periods, with a prominent short-term triglyceride-lowering effect; combined training produces synergistic effects, comprehensively improving glucolipid metabolism and showing the best long-term adherence. Mechanistically, exercise exerts its beneficial effects through three common pathways: (1) AMPK-mediated lipid oxidation and mitochondrial biogenesis; (2) IRS/PI3K/Akt-mediated insulin signaling sensitization; and (3) Nrf2/ARE anti-oxidation and TGF-β/Smads anti-fibrosis regulation. Different exercise modalities activate these pathways with distinct emphases: MICT most directly and persistently activates the AMPK pathway; resistance exercise uniquely improves IRS/PI3K/Akt signaling through muscle mass gain; HIIT induces the highest AMPK activation intensity and triggers unique lactate-mediated signaling regulation; combined training integrates the above multiple mechanistic advantages. For clinical translation, multidisciplinary team collaboration is essential to ensure safety and adherence; individualized prescriptions should be formulated according to the FITT-VP principle and patient phenotypes—frequency of 3-5 sessions/week of aerobic exercise combined with 2-3 sessions/week of resistance exercise; intensity of moderate-intensity (40%–<60% heart rate reserve (HRR))aerobic exercise and 60%-80% of one-repetition maximum (1-RM) for resistance exercise; time of at least 150 min/week of moderate-intensity aerobic exercise, 30-60 min per session; type of combined training as the preferred modality; total volume of ≥500-1 000 MET-min/week; and progression adjusted every 4-6 weeks—with real-time adjustments supported by wearable devices, ultimately forming a closed-loop management system from initial assessment to long-term follow-up. Notably, current studies have limitations such as small sample sizes and short intervention periods. Future research should focus on long-term follow-up, multi-omics biomarkers, and combined exercise-drug strategies. In conclusion, the systematic integration of structured, individualized, and sustainable exercise interventions into the multidisciplinary management pathway for patients with T2DM complicated by MAFLD is an urgent need in current clinical practice.

Abstract:Objective Oxygen partial pressure (pO?) is a critical indicator of the tumor microenvironment and plays an essential role in regulating tumor progression and therapeutic response, particularly for oxygen-dependent treatments such as photodynamic therapy (PDT). Although photoacoustic imaging (PAI) has shown great potential for functional and molecular imaging, conventional oxygenation imaging approaches based on hemoglobin absorption mainly provide relative oxygen saturation information and cannot directly quantify tissue oxygen partial pressure. Therefore, this study aimed to develop a fast laser sequence-based pump-probe photoacoustic tomography (PP-PAT) system for quantitative, non-invasive, and dynamic monitoring of oxygen partial pressure in tumor tissues.Methods The proposed PP-PAT system was developed based on the oxygen-sensitive triplet-state lifetime characteristics of methylene blue (MB). Upon optical excitation, MB molecules undergo transitions from the ground state to an excited state and subsequently form metastable triplet states through intersystem crossing. The lifetime of the triplet state is strongly affected by surrounding oxygen concentration due to dynamic oxygen quenching, providing a direct correlation between triplet-state kinetics and local oxygen partial pressure. A dual-wavelength pump-probe excitation scheme was implemented to selectively interrogate MB triplet-state dynamics. The pump pulse was used to initiate the triplet-state population, while the delayed probe pulse was applied to detect the transient triplet-state response. By optimizing the temporal interval between the pump and probe pulses, transient triplet-state differential (TTD) signals were extracted to enhance the specificity of oxygen-dependent molecular information and suppress background interference from conventional photoacoustic signals. The performance of the PP-PAT system was systematically evaluated through phantom and in vivo experiments. First, a single-tube phantom containing MB solution was used to investigate signal stability, reproducibility, and the influence of signal averaging on imaging quality. Subsequently, a dual-tube phantom model was established to simulate oxygen consumption during PDT, where one MB-containing tube was exposed to laser irradiation and the other served as a control. Finally, in vivo experiments were performed using a subcutaneous tumor model in nude mice. MB was locally injected into the tumor region before imaging to evaluate the feasibility of tumor oxygen partial pressure mapping using the proposed PP-PAT system.Results Phantom experiments demonstrated that the extracted TTD signals exhibited high stability and effectively reflected MB triplet-state dynamics while reducing non-specific background contributions. Signal averaging significantly improved the signal-to-noise ratio, and 50 repeated acquisitions provided an optimal compromise between image quality and acquisition efficiency. In the dual-tube phantom experiments, continuous PDT irradiation induced progressive oxygen depletion in the treated sample. The corresponding TTD decay rate gradually decreased, accompanied by an increase in the calculated triplet-state lifetime, indicating reduced oxygen quenching and decreased oxygen partial pressure. In contrast, the control sample showed relatively stable TTD kinetics and oxygenation levels during the same period. These results demonstrated that PP-PAT could quantitatively characterize oxygen consumption dynamics associated with photochemical reactions. In vivo experiments further demonstrated the capability of PP-PAT for spatially resolved oxygen partial pressure imaging in MB-injected tumor tissues. The reconstructed pO? maps revealed heterogeneous oxygen distributions within tumors, showing relatively hypoxic regions in the tumor core and higher oxygenation levels near the tumor boundary. This spatial oxygen distribution pattern was consistent with the typical physiological characteristics of solid tumors, where abnormal vascular structures and limited oxygen diffusion result in intratumoral oxygen gradients.Conclusion This study presents a fast laser sequence-based PP-PAT system capable of quantitative and non-invasive imaging of tissue oxygen partial pressure. By utilizing the oxygen-dependent triplet-state lifetime of MB and extracting TTD signals, the proposed method provides molecularly specific information related to oxygen dynamics beyond conventional photoacoustic oxygenation imaging. The system enables dynamic monitoring of oxygen consumption during PDT and reveals spatial oxygen heterogeneity within tumors. This technique provides a promising imaging approach for tumor hypoxia characterization, evaluation of oxygen-dependent therapeutic responses, and precision-guided cancer treatment.

Abstract:Alzheimer's disease (AD) is pathologically characterized by cerebral amyloid β-protein (Aβ) aggregation, neurofibrillary tangles and progressive cognitive deterioration. There is an urgent clinical demand for targeted therapeutic agents against AD, whereas the blood-brain barrier (BBB) acts as a critical physical barrier that blocks over 98% small-molecule drugs and nearly all biomacromolecules from entering brain parenchyma. Nanomedicine-based drug delivery systems (NDDSs) with tunable physicochemical properties can cross the BBB via multiple transcytosis pathways including adsorptive-mediated, receptor-mediated and transporter-mediated routes, opening a promising avenue for targeted AD treatment. A core academic viewpoint proposed herein is that robust in vitro endothelial penetration of nanocarriers cannot guarantee effective accumulation in brain target cells. Comprehensive evaluation of BBB-crossing delivery efficiency should not merely rely on in vitro permeability tests, but cover the full multi-step transcytosis cascade, cellular tropism in brain tissues and in vivo therapeutic outcomes. This review systematically sorts out diverse nanoplatforms applicable to BBB penetration for AD intervention. Inorganic nanomaterials such as gold and ceria nanoparticles possess large specific surface areas and intrinsic antioxidant capacity, which eliminate reactive oxygen species and hinder Aβ fibrillization. Liposomal formulations and solid lipid nanoparticles exhibit superior biocompatibility with biomimetic phospholipid bilayer architectures, capable of co-loading hydrophilic nucleic acids for Tau regulation and lipophilic Aβ inhibitors; relying on receptor-mediated transcytosis, they achieve sustained drug retention in the brain. Polymeric nanocarriers including nanogels and polyamidoamine dendrimers enable multi-target combinatorial therapy, and can be engineered to release cargo in response to inflammatory microenvironments, thereby suppressing excessive microglial activation and protecting neuronal mitochondria. Beyond conventional nanocarriers, this work elaborates two cutting-edge BBB-crossing delivery platforms: biomimetic nanosystems and metal-organic frameworks (MOFs). Biomimetic nanoparticles camouflaged with erythrocyte, platelet or macrophage membranes, as well as natural exosomes, evade immune clearance, prolong systemic circulation and inherently home to inflammatory lesions. Serving as “nano-decoys”, they neutralize Aβ neurotoxins and remodel cerebral inflammatory microenvironments simultaneously. MOFs feature high porosity and customizable pore channels for co-delivery of multiple therapeutics, and can be integrated with near-infrared photothermal and photooxidation modalities to facilitate focal brain lesion therapy. This review highlights a transformative paradigm shift in the field of BBB-targeted AD therapy: research focus has shifted from simply maximizing cerebral drug penetration toward active modulation and functional restoration of the BBB. Impaired BBB transporters intrinsically impede endogenous Aβ clearance. Accordingly, BBB-regulating nanocarriers are designed to remodel the low-density lipoprotein receptor-related protein 1 (LRP1) trafficking cascade, redirecting endocytic vesicles from lysosomal degradation to non-degradable transcytosis and restoring the intrinsic Aβ efflux capacity of the BBB. Distinct from conventional strategies that only exert local lesion inhibition, this systemic clearance strategy eliminates cerebral Aβ deposits by accelerating peripheral excretion.

Abstract:Glycosylation constitutes a critical, highly intricate, and diverse co- and post-translational modification characterized by the covalent attachment of sugar moieties to proteins, lipids, or small RNAs. Complex glycans are ubiquitously present across nearly all living organisms. Despite their prevalence, our comprehension of glycan diversity remains limited, likely due to the inherent challenges associated with elucidating their structural complexity. Among post-translational modifications, protein glycosylation is notably prevalent, involving the enzymatic transfer of oligosaccharides to specific amino acid residues by glycosyltransferases. This modification plays a pivotal role in modulating protein function, including participation in various biological and biochemical recognition processes. G protein-coupled receptors (GPCRs), encoded by approximately one thousand genes, share a conserved architecture comprising seven transmembrane helices interconnected by three intracellular and three extracellular loops. Representing the largest family of human membrane proteins, GPCRs regulate a vast array of physiological and pathological processes, thereby constituting the most extensive class of therapeutic targets. In mammalian systems, the majority of GPCRs undergo glycosylation predominantly at their extracellular N-terminus or extracellular loops, primarily through N-linked and O-linked glycosylation. These covalent carbohydrate modifications exert profound effects on multiple facets of GPCR biology, including endoplasmic reticulum folding and quality control, membrane trafficking and surface expression, receptor internalization and recycling or degradation, ligand binding and signal transduction, biased signaling, and receptor dimerization. Notably, glycosylation can exert both positive and negative regulatory influences on these processes. Dysregulation of GPCR glycosylation has been implicated in a range of pathological conditions, such as cancer, diabetes, and neurological disorders, underscoring its physiological and pathological significance. Aberrant glycosylation patterns may lead to dysfunctional receptor signaling, thereby contributing to disease progression. Consequently, elucidating the precise roles of glycosylation in GPCR function not only enhances fundamental understanding of receptor biology but also facilitates the development of novel therapeutic strategies targeting glycosylation pathways or specific GPCR glycoforms. Although the functional consequences of glycosylation have been investigated for several decades, delineating their exact structural underpinnings has remained challenging due to the intrinsic flexibility and heterogeneity of carbohydrate structures. Recent advances in structural biology, particularly single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography, have catalyzed a paradigm shift in our understanding of GPCR glycosylation. The integration of these sophisticated structural techniques with biochemical approaches has redefined glycosylation as a complex structural element integral to receptor function. These developments provide a molecular framework for comprehending how glycans influence receptor pharmacology and open new avenues for the rational design of glyco-engineered biologics and allosteric modulators targeting specific extracellular motifs. This mini-review concentrates on glycosylation within the GPCR superfamily, summarizing the impact of glycosylation modifications on receptor function and regulatory mechanisms, recent progress in the structural biology of GPCR glycosylation, and prospective directions for future research in this field.

Abstract:CRISPR-Cas-based biosensing systems have become important platforms for rapid nucleic acid detection because of their programmable sequence recognition and high specificity. In many diagnostic workflows, the CRISPR reaction is coupled with polymerase chain reaction or isothermal pre-amplification to enrich low-abundance targets before readout. This design improves sensitivity, but it also introduces additional primers and enzymes, requirements for temperature control and reaction compatibility, and a higher demand for contamination management. These factors make the whole assay less convenient for integrated and field-deployable point-of-care testing. Developing pre-amplification-free CRISPR assays is therefore not simply a pursuit of shorter protocols, but a way to simplify molecular diagnosis at the system level, especially in settings where rapid decisions, closed workflows, and minimal manual operation are required. Microfluidic devices provide an important engineering route for this purpose. Their small reaction volumes, precise fluid manipulation, high-throughput partitioning, and compatibility with portable readout make it possible to integrate target confinement, reaction control, signal acquisition, and quantitative analysis on a chip. Droplet microfluidics and microwell arrays can divide a sample into large numbers of independent microreactors, enabling digital counting of rare recognition events. Continuous-flow, centrifugal, and paper-based microfluidic formats further provide options for automated operation, low-cost fabrication, and on-site use. In this context, microfluidics is not only a signal-enhancement method, but also a platform that connects CRISPR molecular recognition with practical assay implementation. This review summarizes recent progress in microfluidic-based pre-amplification-free CRISPR-Cas biosensing for rapid detection. We focus on four related technical directions. crRNA engineering, including spacer-length tuning, multi-crRNA design, chemical modification, and allosteric regulation, can improve recognition kinetics, nuclease stability, and mismatch discrimination. Digital microfluidic detection based on droplets or microwell arrays converts single-molecule recognition events into countable positive partitions, thereby improving quantitative capability without target pre-amplification. Physical signal transduction interfaces couple CRISPR activity to electrochemical, electrochemiluminescent, fluorescent, or surface-enhanced Raman scattering readouts, making weak molecular signals easier to detect with portable instruments. On-chip cascade signal amplification strategies further enhance output intensity through enzymatic reactions, DNA circuits, nanomaterials, or cross-domain amplification modules while avoiding direct amplification of the target nucleic acid. Together, these strategies show that the performance of a pre-amplification-free assay depends not only on biochemical recognition, but also on how the reaction is confined, amplified, transduced, and operated. In addition to describing these mechanisms, this review compares different strategies in terms of sensitivity improvement, operational complexity, cost, and suitability for point-of-care testing. Representative applications in food authenticity identification and rapid pathogen screening are also discussed to illustrate how these platforms perform in practical scenarios. Finally, we analyze the remaining challenges, including the detection of low-abundance targets in complex matrices, on-chip sample preparation, device-to-device reproducibility, long-term reagent storage, and standardization. Future development will likely depend on tighter integration of sample processing, CRISPR reaction, signal readout, and data interpretation, ultimately moving pre-amplification-free CRISPR diagnostics toward automated and user-friendly sample-to-answer systems.