Due to its high sensitivity and non-destructive nature, Raman spectroscopy has become an essential analytical tool in biopharmaceutical analysis and drug development. Despite of the computational demands, data requirements, or ethical considerations, artificial intelligence (AI) and particularly deep learning algorithms has further advanced Raman spectroscopy by enhancing data processing, feature extraction, and model optimization, which not only improves the accuracy and efficiency of Raman spectroscopy detection, but also greatly expands its range of application. AI-guided Raman spectroscopy has numerous applications in biomedicine, including characterizing drug structures, analyzing drug forms, controlling drug quality, identifying components, and studying drug-biomolecule interactions. AI-guided Raman spectroscopy has also revolutionized biomedical research and clinical diagnostics, particularly in disease early diagnosis and treatment optimization. Therefore, AI methods are crucial to advancing Raman spectroscopy in biopharmaceutical research and clinical diagnostics, offering new perspectives and tools for disease treatment and pharmaceutical process control. In summary, integrating AI and Raman spectroscopy in biomedicine has significantly improved analytical capabilities, offering innovative approaches for research and clinical applications.

Mitochondria are fundamental organelles that play a crucial role in cellular energy metabolism, substance metabolism, and various essential cellular signaling pathways. The dysfunction of mitochondria is significantly implicated in the onset and progression of aging, neurodegenerative diseases, metabolic disorders, and tumors, thereby rendering mitochondria-targeted regulation, a vital strategy for disease prevention and treatment. The recently developed mitochondrial membrane chromatography (MMC) technique, which immobilizes mitochondrial proteins as a chromatographic separation medium, has shown great potential for efficiently screening mitochondria-targeted modulators from complex compound library. In contrast to traditional screening methods, MMC has no need to purify mitochondrial proteins and can preserve its in situ and physiological conformation. Consequently, it presents broader application prospects for screening mitochondrial modulators as well as investigating receptor-ligand interactions involving any target protein associated with mitochondria. This review aims to elucidate the critical role of mitochondria in the development and progression of major chronic diseases, discuss recent advancements and applications of MMC, and propose future directions for MMC in the identification of novel mitochondrial modulators.

Age-related macular degeneration (AMD) represents a predominant cause of blindness among older adults, with limited therapeutic options currently available. Oxidative stress, inflammation, and retinal pigment epithelium injury are recognized as key contributors to the pathogenesis of AMD. Regulated cell death plays a pivotal role in mediating cellular responses to stress, maintaining tissue homeostasis, and contributing to disease progression. Recent research has elucidated several regulated cell death pathways—such as apoptosis, ferroptosis, pyroptosis, necroptosis, and autophagy—that may contribute to the progression of AMD owing to cell death in the retinal pigment epithelium. These discoveries open new avenues for therapeutic interventions in patients with AMD. In this review, we provide a comprehensive summary and analysis of the latest advancements regarding the relationship between regulated cell death and AMD. Moreover, we examined the therapeutic potential of targeting regulated cell death pathways for the treatment and prevention of AMD, highlighting their roles as promising targets for future therapeutic strategies.

Drug-drug interactions (DDI) are a critical concern in drug development and clinical practice. A new molecular entity often requires numerous clinical DDI studies to assess potential risks in humans, which involves significant time, cost, and risk to healthy study participants. Consequently, there is growing interest in innovative techniques to improve the prediction of transporter-mediated DDI. Researchers in this field have focused on identifying endogenous molecules as biomarkers of transporter function. The development of biomarkers is notably more complex than that of exogenous drugs. Owing to their inherent selectivity, sensitivity, and ability to provide absolute quantification, liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are increasingly being employed for the quantitative investigation of new biomarkers. This review article presents recently developed bioanalytical approaches using LC-MS/MS for putative transporter biomarkers identified to date. Additionally, we summarize the published baseline endogenous levels of these potential biomarkers in a biological matrix to suggest a set of reference values for future research, thereby minimizing errors in biomarker-related data analyses or calculations.

Natural products (NPs) are an important source of new drugs for the treatment of stroke. Identifying cellular targets for bioactive molecules is a major challenge and critical issue in the development of new drugs for stroke. Small-molecule probes play a unique role in target discovery. However, drawbacks to these probes include non-specificity, unstable activity, and difficulty in synthesis. Small-molecule probes based on NPs at least partially compensate for these shortcomings. NPs feature rich chemical and structural diversity, biocompatibility, and unique biological activities. These features could be exploited to provide new ideas and tools for target discovery. Small-molecule probes based on NPs provide a precise and direct search for interacting protein targets of NPs-active small molecules. This review explores the properties of small-molecule probes based on NPs and their applications in mechanistic studies of stroke and other diseases. We hope that this review will bring new perspectives to the mechanistic study of NPs-active small molecules and accelerate the translation of these ingredients into drug candidates for the treatment of stroke.

The critical role of protein disequilibrium in driving carcinogenesis has long been recognized. Though several inhibitors of heat shock protein (HSP) family members have entered clinical trials, none of them have been approved for clinical use as a result of inevitable toxicity, leading to the identification of safer therapeutic approaches sharing a similar efficacy relevant and urgent. Through delineating the role of HSP90 inhibitors in arresting cancer hallmarks, this paper identified HSP90 inhibition as an effective therapeutic strategy capable of concomitantly targeting multiple key transformed properties of cancers via modulating cellular proteostasis. Through interrogating intrinsic connections between proteostasis and redox homeostasis, this paper proposed cold atmospheric plasma (CAP) as a possible alternative of HSP90 inhibitors with little adverse effects. This paper extended the therapeutic spectrum of HSP90 inhibitors and CAP to inflammation-driven pathologies including autoimmune diseases, as inflammation is a manifestation of failed proteostasis. These insights may conceptually advance our understandings on the driving force of cancers that can be easily extended to other disorders originated from imbalanced proteostasis and abnormal inflammation. Tools proposed here for inhibiting HSP90 including CAP and its possible synergy with HSP90 inhibitors may shift the current treatment paradigm to a new avenue in oncology and other relevant fields.

β-elemene, a bioactive compound derived from traditional Chinese medicine (TCM), has been clinically used in cancer therapy. However, its molecular physicochemical properties require further optimization, and its precise anticancer mechanisms remain unknown. In modern drug development, structure-based drug design (SBDD) has significantly conserved resources, with computer-aided techniques such as molecular docking and molecule generation playing essential roles. A comprehensive review of existing molecular biology studies and virtual docking experiments led to the hypothesis that methyltransferase-like 3 (METTL3) may serve as a potential target of β-elemene. This discovery establishes a scientific foundation for integrating advanced, rational drug design strategies with β-elemene to enhance the therapeutic efficacy of TCM. Moreover, current artificial intelligence (AI)-based molecular generation models were examined, focusing on de novo molecular generation and lead optimization models. Their applications in the rational drug design of β-elemene were preliminarily explored to identify potential strategies for developing more potent anticancer derivatives by analyzing ligand-receptor interactions.

Mesoporous carbon nanoparticles (MCNs) have received considerable attention for biomedical applications due to their unique structural features, including high specific surface area, adjustable pore size, and remarkable biocompatibility. These properties have addressed key challenges such as inefficiencies in drug loading and release, minimizing the side effects associated with conventional treatments. In this review, the classification and the research progress of MCNs are summarized firstly, the preparation and modification techniques to enhance their functionality and properties are further reviewed, the main physicochemical properties are introduced as well, highlighting their contributions to MCNs in applications. In addition, the biomedical applications of MCNs are emphasized, including tumor therapy, tumor theranostics, antibacterial, delivery of active molecules and biological detection. Finally, the prospects and challenges of clinical application based on MCNs are analyzed to provide an effective reference and lay the foundation for further research.

To address the pressing issue of bacterial resistance, antibiotics with new mechanisms were urgently needed; yet, the majority of efforts centered on discovering novel structural compounds, often plagued by lengthy research timelines and unpredictability. In this study, we introduce an alternative strategy that rejuvenates outdated antibiotics through a unique delivery system. Specifically, we leveraged polymyxin B (PMB) and created a liposomal carrier encapsulating PMB and Fe²⁺, designated P/Fe@L-P. When administered to PMB-resistant Acinetobacter baumannii, P/Fe@L-P triggered a downregulation of Nrf2 and GPX4 proteins, accompanied by a significant surge in reactive oxygen species and malondialdehyde levels, signifying the induction of ferroptosis. This mechanism imparted potent antibacterial activity, with P/Fe@L-P achieving minimal inhibitory and bactericidal concentrations of 54 and 192 μM, respectively, outperforming free PMB (72 and 768 μM). In vivo evaluations in mice models further validated the superior efficacy of P/Fe@L-P over PMB in treating PMB-resistant Acinetobacter baumannii pneumonia. This work establishes a highly effective and practical “old drug, new trick” paradigm, potentially expediting the fight against the escalating threat of bacterial resistance.

Myocardial infarction (MI) is the leading cause of cardiovascular disease-related death worldwide. Nonetheless, existing therapeutic approaches for MI are hampered by issues such as reliance on pharmacological agents and suboptimal patient adherence. Caffeic acid (CA) is a bioactive polyphenolic compound with important anti-inflammatory, anti-bacterial and anti-oxidant functions. Still, its specific role and mechanism in treating cardiovascular disease remain to be further studied. In recent years, a large number of studies have shown that the kelch-like ECH-associated protein 1/nuclear factor erythroid 2 related factor 2 (Keap1/Nrf2) pathway is a key factor in the occurrence and development of cardiovascular diseases. In this study, H₂O₂-induced oxidative stress model of H9c2 cells and left anterior descending branch (LAD) conjunctival induced acute myocardial infarction reperfusion (AMI/R) model were used to evaluate the protective effect of CA on the heart. The interaction between CA and Keap1 was analyzed by CA-labeled fluorescence probe, target fishing, isothermal titration calorimetry (ITC), protein crystallography and surface plasmon resonance (SPR). Our results suggested that CA binds Keap1 and degrades Keap1 in a p62-dependent manner, further promoting nuclear transcription of Nrf2 and thus effectively reducing oxidative stress. In addition, based on the three-dimensional eutectic structure, it was confirmed that CA directly targets Keap1 protein by interacting with residues M550 and N532, inducing conformation changes in Keap1 protein. We also found that the CA analog chlorogenic acid (GCA) can bind Keap1. In conclusion, this study elucidates a novel molecular mechanism and structural basis for the protective effects of CA against oxidative damage via the Keap1-Nrf2 pathway.

Sulfonylation is extensively used to label DNA and RNA, assess their interactions, and quantify components including nucleobases and nucleosides/nucleotides although the sulfonylation sites remain controversial. Here, we systematically investigated the sulfonylation of adenine (ade) and its nucleosides/nucleotides with 5-(dimethylamino)-naphthalene-1-sulfonyl chloride (DNS-Cl), 5-(diethylamino)-naphthalene-1-sulfonyl chloride (DEANS-Cl), and 5-((N,N-diethylleucyl)amino)-naphthalene-1-sulfonyl chloride (DELANS-Cl). Detailed spectral analysis with nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HRMS) showed similar sulfonylation behaviors among the reagents. For ade, its secondary amine in the imidazole ring (N⁹H) sulfonylated more readily than the exocyclic amino group (N⁶H₂). For adenosine and its nucleotides, the 2′-OH group in the ribosyl moiety was preferably sulfonylated, whereas the 3′-OH was the preferred site for 2′-deoxyadenosine and its nucleotides. Alkylation and amidation of the aromatic amino group in these 5-amino-naphthalene-1-sulfonyl chlorides did not influence the sulfonylation preferences. This offered a reliable approach and comprehensive details of such sites for ade and its nucleosides/nucleotides.

Colorectal cancer (CRC) is a prevalent gastrointestinal malignancy. However, the lack of diagnostic accuracy of traditional clinical serum biomarkers carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA19-9) results in patients being diagnosed at an advanced stage. Herein, we developed a novel method of ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) for relative quantification based on the non-specific enzyme pronase E and an isotope mass spectrometry (MS) probe 3-benzoyl/(benzoyl-2,3,4,5,6-d5)-2-oxothiazolidine-4-carboxylic acid (d0/d5-BOTC) to screen novel glycan biomarkers. We applied the method in a cohort of 102 serum samples (including 51 healthy volunteers (HV), 26 stage II CRC, and 25 stage III CRC) and 90 tissue samples (including 45 paracancerous tissue and 45 cancerous tissue). Results revealed that the serum levels of H5N4F, H5N4F3SA, H4N5F1SA, and H5N4SA2 in CRC patients were significantly different from those in HV (P < 0.01). The area under the curve values of H5N4F, H5N4F3SA, and H4N5F1SA in serum samples were 0.77, 0.71, and 0.91, respectively. The clinical diagnostic accuracies of these glycans ranged from 65% to 91%, which were significantly higher than those of CEA. Additionally, differential glycan profiles in tissues were further examined using the same method and compared with serum levels. H5N4F was found to be significantly down-regulated in all CRC groups (P < 0.0001), indicating strong specificity for CRC diagnosis. The glycans identified in this study are expected to serve as potential biomarkers for the early diagnosis of CRC, offering valuable reference points for clinical diagnosis and treatment.

Natural products (NPs) make a major contribution to drug development, offering a huge molecule pool for drug leads. Nevertheless, the pharmaceutical industry and academy have declined their enthusiasm to NPs research since the great challenges in elucidating the complex component and intricate mechanism of NPs. Here, we introduce an efficient fragment-based target research (FBTR) approach for pharmacology study and optimization of NPs. Focusing on the core fragment within the molecules of NPs, we screen the outstanding activity that be triggered, and corresponding target. Finally, drug optimization was carried out around the molecules that obtaining the activity-related core fragment and verified both in vitro and in vivo. With this approach, we obtained an optimized NPs named Erigeron breviscapus polyphenols (EBP) with definite target. After optimization, EBP plus (EBPP) not only trigger immunogenic cell death (ICD) of glioblastoma (GBM) cells effectively by targeting to Cys105 amino acid site of Fas-associating protein with a novel death domain (FADD) protein, but also prolong the survival of GBM mice by an average of 17.6 days. Significantly, our investigation presents an approach for addressing challenges in NPs development and opening up new opportunities for drug discovery. Our findings demonstrate the utility of FBTR in exploring the function of NPs, revealing the target, and advancing drug optimization for stronger clinical translation.

Liraglutide (Lira), a glucagon-like peptide-1 (GLP-1) receptor agonist approved for diabetes and obesity, has shown significant potential in treating metabolic dysfunction-associated steatotic liver disease (MASLD). However, its systematic molecular regulation and mechanisms remain underexplored. In this study, a mouse model of MASLD was developed using a high-fat diet (HFD), followed by Lira administration. Proteomics and glycoproteomics were analyzed using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS), while potential molecular target analysis was conducted via quantitative real-time polymerase chain reaction (qPCR) and Western blotting. Our results revealed that Lira treatment significantly reduced liver weight and serum markers, including alanine aminotransferase (ALT) and others, with glycosylation changes playing a more significant role than overall protein expression. The glycoproteome identified 255 independent glycosylation sites, emphasizing the impact of Lira on amino acid, carbohydrate metabolism, and ferroptosis. Simultaneously, proteomic analysis highlighted its effects on lipid metabolism and fibrosis pathways. 21 signature molecules, including 7 proteins and 14 N-glycosylation sites (N-glycosites), were identified as potential targets. A Lira hydrogel formulation (Lira@fibrin (Fib) Gel) was developed to extend drug dosing intervals, offering enhanced therapeutic efficacy in managing chronic metabolic diseases. Our study demonstrated the importance of glycosylation regulation in the therapeutic effects of Lira on MASLD, identifying potential molecular targets and advancing its clinical application for MASLD treatment.

Allergic conjunctivitis is a common ocular surface condition. Although corticosteroids are potent anti-inflammatory agents for its management, their use is often restricted by potential side effects. Conventional eye drops face challenges such as short retention time and poor corneal permeability, resulting in low drug bioavailability. To overcome these limitations, we developed a preservative-free synthetic high-density lipoprotein (sHDL) nanodisc eye drop containing dexamethasone palmitate. This novel formulation enhances drug stability and extends retention time on the ocular surface. In a mouse model of ovalbumin (OVA)-induced allergic conjunctivitis, the nanodisc eye drop significantly alleviated symptoms while reducing corticosteroid concentration, demonstrating excellent safety and biocompatibility. This innovative approach shows great promise for the treatment of allergic conjunctivitis and may lay the groundwork for new therapeutic strategies in anterior ocular disease management.

The nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome is downregulated in hepatocellular carcinoma (HCC), and its stability is regulated by ubiquitination. However, the regulatory mechanisms underlying NLRP3 deubiquitination and its role in HCC metastasis remains unclear. We demonstrated that ubiquitin-specific protease 50 (USP50) directly interacts with NLRP3, exhibiting deubiquitinase (DUB) activity through specific cleavage of K48-linked polyubiquitination chains to stabilize NLRP3 by preventing proteasomal degradation. Clinically, we observed that low NLRP3 and high β-catenin levels were negatively correlated in HCC specimens. Subsequent mechanistic exploration confirmed that NLRP3 exerts negative regulation on β-catenin by binding with glycogen synthase kinase 3 beta (GSK3β), reversing the downstream epithelial-mesenchymal transition (EMT) process, and inhibiting HCC metastasis. Notably, USP50 was found to activate NLRP3 inflammasome by promoting nuclear factor-kappa B (NF-κB) signaling, consequently enhancing proinflammatory cytokines. Furthermore, USP50 overexpression negatively regulated β-catenin, reversed EMT process and inhibited HCC metastasis in vivo. In conclusion, USP50 has emerged as a key player in regulating the NLRP3 inflammasome and inhibiting HCC metastasis by reversing the EMT process. As a result, it presents itself as a promising therapeutic target for HCC in the clinical setting. The intricacies of this regulatory mechanism, as revealed by our study, provide valuable insights into the understanding and potential interventions for HCC.

Dry eye (DE), a multifactorial ocular surface disease, is predominantly characterized by inflammation as a central pathological factor. Ursolic acid (UA), a pentacyclic triterpenoid with well-documented anti-inflammatory properties, was evaluated in this study for its therapeutic effects on ocular surface dysfunction associated with DE and its underlying mechanisms. A hyperosmotic stress model (500 mOsM) using human corneal epithelial cells (HCEs) and an animal model of DE was established to assess UA's protective effects on both cellular and organismal levels. Comprehensive assessments, including phenol-red cotton tests and slit-lamp examinations, were performed to evaluate ocular surface damage in the DE mouse model. Potential UA-related targets and their relevance to DE pathology were identified through database mining. Protein-protein interaction (PPI) network construction and pathway enrichment analysis using the Metascape platform highlighted core targets and signaling pathways. Molecular docking simulations using AutoDock and PyMOL further elucidated the interaction modes between UA and its targets. To validate the molecular mechanisms underlying UA's therapeutic effects, integrative analyses were conducted using single-cell sequencing data from the Single Cell Portal and RNA sequencing of tissue samples. The results demonstrated that UA eye drops significantly preserved ocular surface functional units and alleviated DE symptoms, through modulation of the epidermal growth factor receptor (EGFR)/rat sarcoma (RAS)/rapidly accelerated fibrosarcoma (RAF)/mitogen-activated protein kinase (MAPK) kinase 1 (MAP2K1)/MAPK1 signaling pathway, as supported by network pharmacological analysis. Single-cell sequencing localized the distribution of key pathway proteins to the anterior ocular segment, particularly the cornea. In vivo experiments confirmed the therapeutic efficacy of UA eye drops via the EGFR/RAS/RAF/MAP2K1/MAPK1 pathway. Collectively, these findings underscore the potential of UA eye drops as a promising therapeutic approach for managing ocular surface disorders in DE.

Abnormal bile acid (BA) metabolism has been implicated in the pathogenesis of central nervous system (CNS) diseases, but its role in epilepsy remains unclear. In this study, we investigated the relationship between gut microbiota-driven dysregulation of BA metabolism and seizure-induced ferroptotic neuronal death in epilepsy. Our targeted metabolomic analysis revealed elevated levels of deoxycholic acid (DCA) in the serum and cerebrospinal fluid (CSF) of epileptic patients, which correlated with cognitive impairment. In a pentylenetetrazol (PTZ)-induced mouse model of epilepsy, 16S ribosomal RNA (16S rRNA) sequencing showed significant alterations in gut microbiota composition. Importantly, fecal microbiota transplantation (FMT) from healthy mice into epileptic mice significantly reduced seizure activity and improved cognitive function, primarily by normalizing serum and brain levels of secondary bile acids (SBAs), including DCA. Both in vitro and in vivo experiments demonstrated that DCA promotes ferroptosis in hippocampal neurons by activating the farnesoid X receptor (FXR). This activation triggered the nuclear factor erythroid 2-related factor 2 (Nrf2)-heme oxygenase-1 (HO-1) signaling pathway, known to be involved in oxidative stress and cell death regulation. Our findings suggest that the upregulation of DCA, through its effects on FXR and HO-1, plays a critical role in the progression of epilepsy by inducing ferroptosis in hippocampal neurons. Targeting the DCA-FXR-HO-1 axis may provide a novel therapeutic strategy for treating seizures and associated cognitive deficits in epilepsy.