Wastewater-based epidemiology has emerged as a robust and effective tool for monitoring and assessing drug abuse trends. However, the analysis of drugs in wastewater remains challenging due to their ultra-trace concentrations and the inherent complexity of the wastewater matrix‌. In this study, a ‌magnetic graphene-imine-linked covalent organic polymer composite (MG-COP)‌ was ‌first prepared via a simple, mild, and scalable synthetic route and applied as a magnetic solid-phase extraction (MSPE)‌ adsorbent to simultaneously separate and enrich 14 target drugs of abuse and their metabolites in wastewater. The synthesized MG-COP demonstrated a high specific surface area, high thermal stability, and good magnetic separation properties. Subsequently, an efficient and rapid analytical method‌ was established by coupling MSPE with ultra-performance liquid chromatography-tandem mass spectrometry for the determination of 14 target analytes in wastewater. Under the optimized conditions, the method exhibited excellent sensitivity (limits of detection ranging from 1.0 to 10 ng L-1), high reproducibility (relative standard deviations within the range of 0.8-11.3%), and robust linearity with coefficient of determination (R2) ≥ 0.9932. Notably, the MSPE process required only 1 min for extraction‌ and a small sample volume of 10 mL. Furthermore, the established method was successfully applied to real wastewater samples, revealing methamphetamine, morphine, and codeine as the dominant drugs of abuse in the analyzed wastewater. The MG-COP-based MSPE method, characterized by simplicity, rapidity, and efficiency, provides an effective sample pretreatment approach for determining trace drugs of abuse in complex wastewater matrices.

Acute kidney injury (AKI) is a common and severe clinical complication with limited treatment options. TRIM21, an E3 ubiquitin ligase, was previously shown to be upregulated in ischemia/reperfusion (I/R)-induced AKI. This study further revealed its role in DNA damage repair. Here, we demonstrated that TRIM21 expression is elevated in human kidney biopsies, murine AKI models, and injured renal tubular epithelial cells (TECs), in parallel with increased DNA double-strand breaks (DSBs). TRIM21 knockout attenuated renal injury, improved renal function, and decreased TECs apoptosis. Mechanistically, TRIM21 was found to bind to INTS3 and facilitate its proteasomal degradation, suppressing DNA repair and promoting apoptosis. INTS3 overexpression alleviated cisplatin-induced DNA damage and apoptosis in TECs. Through virtual screening and activity evaluation, SB33-0223 was identified as a small-molecule inhibitor targeting the C-terminal PRYSPRY domain of the TRIM21 protein. By blocking the recruitment of antibody-bound substrates, SB33-0223 effectively prevents TRIM21-mediated ubiquitination and degradation of INTS3, thereby enhancing INTS3 stability and reducing DNA damage in vitro and in vivo. Our study discovered SB33-0223 as an inhibitor of TRIM21 for the first time, highlighting the TRIM21/INTS3 axis as a critical regulator of DNA repair in AKI, supporting SB33-0223 as a promising lead for targeted therapeutic intervention.

Drug abuse poses a severe threat to public health and societal security, necessitating the development of simple, fast, portable, and highly sensitive detection methods. Lateral flow assays (LFAs) have drawn considerable attention due to their ease of use, cost-effectiveness, and rapid results, making them suitable for point-of-care testing. This review summarizes recent advances in LFAs for detecting drugs of abuse in different matrices since 2017, covering (1) the evolution, configuration, and principles of LFA technology and sample preparation for LFAs; (2) recognition elements (e.g., antibodies and aptamers); (3) diverse nanomaterial labels, including colorimetric (e.g., gold nanoparticles and carbon nanotubes), luminescent (e.g., quantum dots, up-conversion nanoparticles, time-resolved fluorescence microspheres, and afterglow luminescent materials), magnetic, surface-enhanced Raman scattering, electrochemical, and multi-modal detection materials; and (4) intelligent analysis based on smartphone technology and artificial intelligence, along with an outlook on future development directions. This review is expected to provide useful insights for the future development and application of on-site LFAs in detecting illicit drugs and related small molecules.

Infectious diseases severely threaten global public health security, necessitating rapid and highly sensitive diagnosis. This study presents a novel multiplex diagnostic platform combining transcription-mediated amplification (TMA) with the CRISPR-Cas12a2 system for rapid and highly sensitive detection of respiratory viruses. The assay uses an integrated microfluidic chip, which can simultaneously identify influenza A/B and respiratory syncytial viruses (RSV-A/B) with optimized CRISPR RNAs and isothermal amplification, achieving detection limits as low as 102 copies/μL within 60 min. The detection system showed excellent specificity; nonspecific reactions were not observed in the presence of nucleic acids from other respiratory pathogens. Clinical validation using nasopharyngeal swabs demonstrated high concordance with real-time quantitative reverse transcription polymerase chain reaction, with most positive samples detected within 40 min. The system eliminates DNA amplification steps, reduces contamination risk, and simplifies the workflow. Using two-step reactions on a centrifugal microfluidic chip, the TMA-CRISPR-Cas12a2 platform offers a promising integrated platform for multiplex respiratory pathogen screening, thereby supporting timely diagnosis and outbreak management.

Ochratoxin A (OTA) poses significant health risks and is prevalent in various plant-based foods, necessitating rapid and reliable on-site detection methods. In this study, a lateral flow aptasensor integrating magnetic solid-phase extraction (MSPE) with enzyme-catalyzed colorimetric signal transduction was developed for the rapid determination of OTA. To facilitate field deployment, a portable integrated MSPE device featuring adjustable rotation speed and duration, as well as automated magnetic separation, was designed to enable efficient sample pretreatment under on-site conditions. Polydopamine-coated magnetic beads were employed for MSPE due to their suitability for batch sample processing. Moreover, enzyme-catalyzed colorimetry was introduced as an alternative signal transduction strategy in lateral flow assays. The proposed aptasensor exhibited good specificity and satisfactory sensitivity, with a limit of detection of 0.30 ng/mL. Acceptable accuracy and precision were achieved, with recoveries of 91.1–97.2

Image-processing frameworks are critical for the absolute quantification of nucleic acids and proteins, in which accurate analysis of microreactors generated by digital PCR or digital ELISA is required. However, existing methods often suffer from reduced accuracy at high microreactor densities and limited generalizability. Here, we propose Microreactor-Vision, a deep learning framework that integrates an enhanced U-Net-based segmentation model with a lightweight convolutional neural network classifier. The framework achieves >99% quantitative accuracy and has been validated across multiple fluorescence channels and diverse microreactor architectures. Microreactor-Vision further demonstrates an ultra-low limit of detection and a broad dynamic range (10(-1)-10(4)spacecopies/mu L), with strong linearity between predicted and actual concentrations (coefficient of determination [R-2] > 0.99). To our knowledge, it achieves the highest detection throughput and accuracy reported to date while maintaining efficient inference speed. Microreactor-Vision offers a rapid, cost-effective, and robust solution for AI-driven absolute quantification, supporting translational and clinical biosensing applications.

Per- and poly-fluoroalkyl substances (PFAS) make up a large group (or class) consisting of thousands of synthetic chemicals.Chronic human exposure to trace amounts of some PFAS has been linked to adverse health effects (e.g., a higher incidence of breast cancer, renal disease, and thyroid disease). The current monitoring program in Canada targets only the most researched PFAS and the number of PFAS characterized in exposure assessments is still relatively low compared to the total number registered for commercial use, in addition to their transformation products in the environment. “Non-targeted analysis” (NTA) has emerged as a tool for identification and prioritization of chemical substances assessed for human exposure. Unlike “targeted analysis”, there are no clearly established processes for NTA method development and validation, and despite efforts having recently been made to harmonize NTA workflows, there are still inconsistencies that remain. While the sample preparation steps determine the types of chemicals that get extracted (e.g., via choice of elution solvent), standardized data acquisition and data analysis steps are required for reliable chemical identification and quantification without the use of reference standards. The goal of this study was the development of a general NTA protocol including appropriate quality assurance/quality control (QA/QC) elements to provide reproducible NTA results for the identification of PFAS in source and drinking water. Existing software tools (FluoroMatch, TraceFinder, and Compound Discoverer) are employed along with a developed retention time prediction model to improve confidence in chemical substance identification.

Abstract Immunotherapy has revolutionized cancer treatment, yet many patients show non-sensitivity. Here, we collected treatment-naïve samples from 190 esophageal squamous cell carcinoma (ESCC) patients undergoing anti-programmed death 1 (PD1) immunotherapy for proteome, phosphoproteome, and immunohistochemistry (IHC) analysis. Proteome-based stratification of ESCC identifies three proteomic subtypes (G-I-G-III) related to immunotherapy response and different molecular features, revealing that patients with high mitochondrial complex I protein expression show sensitivity to anti-PD1 immunotherapy. High mitochondrial complex I protein expression of ESCC cells or patient-derived organoids increases sensitivity to CD8 + T cell-mediated killing in the co-culture systems. Phosphoproteomic data analysis reveals YAP1 activation impairs immunotherapy efficacy. Inhibiting YAP1 or increasing mitochondrial complex I levels bolsters immunotherapy effectiveness in ESCC allograft tumors. Finally, we develop a highly accurate predictive model (AUC ≥ 0.90) by the signatures of mitochondrial complex I-mediated anti-tumor immune response and validate it in independent cohorts. This study provides a rich resource for investigating the mechanisms and indicators of immunotherapy in ESCC.

The pervasive threat of organophosphorus pesticides (OPs) contamination and associated poisoning incidents demands detection strategies that go beyond simple quantification, specifically targeting the identification of unknown analogs within matrices. Herein, we report a novel chemiluminescent (CL) sensing platform driven by rationally designed, enzyme-specific probes (ACh-CL and BCh-CL) that exploit distinct steric and electronic interactions with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Unlike conventional assays, we constructed the classification models by integrating distinct signal patterns across three concentration gradients with machine learning models, including XGBoost and Random Forest. Based on that, we successfully discriminated OPs from non-OPs and non-pesticide analogs (NPAs) in samples of unknown category and concentration. Achieving high sensitivity (low to 0.1 ng/mL) and rapid response (10 min), the method showed good anti-interference in vegetables, soil, water and serum, with recoveries of 74.21 %–113.8 %. Overall, this CL technique-driven, data-enhanced protocol enables high-throughput screening and risk assessment in food safety and environmental monitoring.

Hepatocellular carcinoma (HCC) is a common type of primary liver cancer and is considered the third leading cause of cancer-related deaths worldwide. The high aggressiveness and resistance to therapies exhibited by HCC present significant challenges to global public health. As the primary metabolic organ in the human body, the liver undergoes substantial metabolic reprogramming during carcinogenesis, affecting various metabolic pathways including those involved in carbohydrates, lipids, and amino acids. Notably, disruptions in amino acid metabolism play a critical role in the initiation and progression of HCC, helping to sustain its malignant characteristics. This review aims to provide an in-depth analysis of the alterations observed in aromatic amino acids metabolism, branched chain amino acids (BCAAs) metabolism, glutamine metabolism, and other amino acid metabolism processes, including serine, arginine, and methionine, along with the expression patterns of associated metabolic enzymes. Furthermore, it discusses potential therapeutic approaches and their clinical relevance, offering insights and strategies for improving HCC diagnosis and treatment in the future.

Five tetrahydroisoquinoline alkaloids (1-5) were separated from the 90% ethanol extract of Piper nigrum. Compounds 2 and 3 previously chemically synthesized were isolated from natural sources for the first time and their NMR data were reported comprehensively in the present paper. Compound 1 was firstly isolated from plants, while compounds 4 and 5 were isolated from Piperaceae family for the first time. Additionally, the chemotaxonomic significance of the obtained compounds was discussed.

Mitophagy, the selective autophagic elimination of mitochondria, is essential for maintaining mitochondrial quality and cell homeostasis. Impairment of mitophagy flux, a process involving multiple sequential intermediates, is implicated in the onset of numerous neurodegenerative diseases. Screening mitophagy inducers, particularly understanding their impact on mitophagic intermediates, is crucial for neurodegenerative disease treatment. However, existing techniques do not allow simultaneous visualization of distinct mitophagic intermediates in live cells. Here, we introduce an artificial intelligence-assisted fluorescence microscopic system (AI-FM) that enables the uninterrupted recognition and quantification of key mitophagic intermediates by extracting mitochondrial pH and morphological features. Using AI-FM, we identify a potential mitophagy modulator, Y040-7904, which enhances mitophagy by promoting mitochondria transport to autophagosomes and the fusion of autophagosomes with autolysosomes. Y040-7904 also reduces amyloid-β pathologies in both in vitro and in vivo models of Alzheimer's disease. This work offers an approach for visualizing the entire mitophagy flux, advancing the understanding of mitophagy-related mechanisms and enabling the discovery of mitophagy inducers for neurodegenerative diseases.

Quercetin, a ubiquitous dietary flavonoid, has garnered significant scientific interest for its potential as an ergogenic aid in endurance sports. This interest is predicated on robust preclinical evidence demonstrating its potent antioxidant, anti-inflammatory, and mitochondrial biogenesis-stimulating properties. However, a persistent disconnect remains between promising laboratory findings and the equivocal, inconsistent, and often modest results reported in human trials with athletes. This review critically and systematically evaluates the scientific literature concerning quercetin's purported antioxidant and fatigue-resisting properties in the context of endurance training. We dissect the primary molecular mechanisms through which quercetin is proposed to act, including the activation of the nuclear factor erythroid 2–related factor 2 (Nrf2) antioxidant response pathway, modulation of the peroxisome proliferator–activated receptor-gamma coactivator-1α (PGC-1α)/sirtuin-1 (SIRT1) axis for mitochondrial biogenesis, and inhibition of the nuclear factor kappa-light-chain-enhancer of activated B cells inflammatory signaling cascade. The core of this review is a critical analysis of the human performance and recovery data, juxtaposing studies that show benefit with those that report null effects. We synthesize the key controversies that dominate the field, focusing on the critical confounding roles of poor bioavailability, participant training status, supplementation dosage, and timing. The evidence suggests that quercetin's most reliable effects may lie in accelerating recovery from exercise-induced muscle damage (EIMD) and reducing soreness, rather than directly enhancing maximal endurance performance. Its primary value may be as a “training adaptogen” that modulates cellular stress responses, thereby improving fatigue resistance over time. We conclude that quercetin's poor oral bioavailability is the principal barrier that has likely confounded the majority of human research to date. Future research must prioritize the use of high-bioavailability formulations to definitively ascertain whether the impressive preclinical benefits of quercetin can be translated into meaningful, practical applications for endurance athletes.

Exosomes, emerging as ideal non-invasive biomarkers for disease diagnosis and monitoring, have seldom been explored based on metabolite levels. In this study, we designed and synthesized a pH-responsive phase-transition bifunctional affinity nanopolymer (pH-BiAN) that could efficiently and homogeneously separate exosomes from urine. Specifically, poly-4-vinylpyridine (P4VP) was chosen as the pH-responsive polymer and simultaneously modified with two exosome-affinity components CD63 aptamer and distearoyl phosphoethanolamine (DSPE) through a one-step amide reaction at room temperature. By utilizing two distinct but synergistic affinity mechanisms-the immune affinity between CD63 aptamer and exosomal CD63 proteins, and hydrophobic interactions between the DSPE and the exosomal lipids-pH-BiAN can enable efficient and specific exosome separation. Moreover, during the urine exosome capture procedure, the pH-BiAN outperforms conventional solid exosome separation materials by remaining soluble in the urine sample, significantly enhancing mass transfer and contact efficiency. After exosome capture, pH-BiAN can quickly aggregate and convert to solid upon pH adjustment, allowing for easy centrifugation separation. Afterwards, multiple machine learning models were established by combining liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) untargeted metabolomics for isolated exosomes, and the clinical accuracy of the training and test sets was more than 0.919, which could well distinguish early osteoarthritis patients from healthy people.

This research presents a digital microfluidic (DMF) array chip for real-time quantitative polymerase chain reaction (qPCR) assay. Combining cleanroom-based chip fabrication process with a facile, cleanroom-free rework method, the DMF chip exhibits high droplet-manipulating performance, flexible structural design, and low implementation cost. Meanwhile, consistent real-time PCR results can be acquired from repeatedly used DMF substrates, demonstrating the applicability of the rework strategy on PCR assay, and potential of widespread application of DMF technology on miniaturized and automated nucleic acid detection.

The quality assessment of therapeutic antibodies in harvested cell culture fluid (HCCF) is significantly hindered by matrix-related large-sized impurities such as vesicles and cellular debris. This study introduces a novel neonatal Fc receptor (FcRn)-immobilized magnetic macroporous material, magMZIF-8@PDA-FcRn, which was custom-designed as size-exclusion magnetic solid phase extraction (MSPE) adsorbent to address these challenges. The magMZIF-8@PDA-FcRn composites exhibited a macroporous structure, with pore sizes ranging from 120 to 240 nm, selectively permitted the entry of immunoglobulin G (IgG) molecules while effectively excluding large-sized impurities. Enhanced by an exterior polydopamine (PDA) layer, these composites demonstrate improved acidic stability and robust performance under processing conditions. The macropores are densely lined with FcRn ligands, which facilitated the selective capture of IgG. Compared to nonporous materials, the magMZIF-8@PDA-FcRn composites demonstrated superior size-exclusion capabilities, achieving a 36.6 % reduction in particle count and a significant decrease in particles larger than 300 nm in the eluate. This effectively mitigated the impact of large-sized impurities on subsequent chromatographic analysis, with significantly slower increase in column pressure. This innovative material was successfully employed as a size-exclusion MSPE adsorbent for the extraction and quality assessment of IgG with potential long circulation half-life in HCCF samples, demonstrated its applicability.

Cancer cells frequently undergo energy metabolic stress induced by the increased dynamics of nutrient supply. Hepatocyte nuclear factor 4A (HNF4A) is a master transcription factor (TF) in hepatocytes that regulates metabolism and differentiation. However, the mechanism underlying how HNF4A functions in cancer progression remains unclear due to conflicting results observed in numerous studies. To address the roles of HNF4A in hepatocellular carcinoma (HCC), we investigated the regulatory functions of HNF4A in HCC cells under different glucose supply conditions. We found that HNF4A exhibited tumor-suppressive effects on the proliferation and migration of HCC cells in glucose-sufficient conditions and tumor-promotive effects on HCC cells in glucose-insufficient conditions. Further investigation revealed that this diverse function of HNF4A was dependent upon the AMPK pathway activity. Similarly, the prognosis predicted by HNF4A was also correlated with whether the AMPKa expression levels were low or high in clinical HCC patients. Multiomics approaches consisting of proteomics and ChIP-seq revealed that key HNF4A target genes, including NEDD4 and RPS6KA2, are involved in the diverse function of HNF4A in HCC in response to the AMPK activity status. Specifically, HNF4A could bind to the promoter region of NEDD4 and RPS6KA2, and upregulating their expression. Our study has demonstrated the relationship between and synergism of AMPK and HNF4A in the progression of HCC under diverse nutrient conditions.

Gentamicin (GEN) residues in food products, arising from its extensive use in veterinary medicine, have prompted stringent regulations and a demand for simple, rapid detection methods. In this study, aptamer-modified magnetic covalent organic frameworks (COFs), denoted as TpDBD@Fe₃O₄@apt, were synthesized and employed as adsorbents in magnetic solid-phase extraction (MSPE) for the highly specific capture and enrichment of GEN residues in milk. Several factors influencing MSPE efficiency were systematically evaluated and optimized. Coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), the developed method exhibited good linearity within the linear range (R2 ≥ 0.999), low detection limits (0.48-0.54 ng/mL), satisfactory recoveries (92.7 %-108.8 %), and high precision (RSDs ≤10.8 %). The proposed method was successfully applied to real milk samples, providing an efficient solution for the detection of GEN residues.