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Biomolecules
Volume 15
Issue 6
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Biomolecules, Volume 15, Issue 6 (June 2025) – 157 articles

Cover Story (view full-size image): Post-translational modifications (PTMs) critically regulate protein function, stability, and interactions in cellular signaling and disease. Although computational methods enable large-scale PTM site prediction, most models use linear sequence fragments alone, ignoring protein structural context and requiring separate models according to the PTM type. Here, we present MTPrompt-PTM, a multi-task framework combining prompt tuning with a structure-aware protein language model (S-PLM). It simultaneously predicts 13 PTM types via shared representations and task-specific prompts. This unified approach improves accuracy, efficiency, and generalizability across PTMs. Integrated knowledge distillation further enhances performance. MTPrompt-PTM pioneers multi-task protein property prediction by merging structural insights with adaptive prompt learning. View this paper
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13 pages, 1444 KiB  
Article
Cycloadditions as a Sweet Route to ‘Double C-Glycosylation’
by Kevin P. P. Mahoney, Rosemary Lynch, Rhea T. Bown, Sunil V. Sharma, Piyasiri Chueakwon, G. Richard Stephenson, David B. Cordes, Alexandra M. Z. Slawin and Rebecca J. M. Goss
Biomolecules 2025, 15(6), 905; https://doi.org/10.3390/biom15060905 - 19 Jun 2025
Viewed by 329
Abstract
Pharmaceuticals, such as the antibiotic erythromycin, and sodium-dependent glucose transporter (SGLT1 & SGTL2) inhibitors such as Bexagliflozin (diabetes) and Sotagliflozin (heart disease), are often sugar-decorated (glycosylated). Glycosylation is a key component of the binding motif in SGLT inhibitors and, in natural products, glycosylation [...] Read more.
Pharmaceuticals, such as the antibiotic erythromycin, and sodium-dependent glucose transporter (SGLT1 & SGTL2) inhibitors such as Bexagliflozin (diabetes) and Sotagliflozin (heart disease), are often sugar-decorated (glycosylated). Glycosylation is a key component of the binding motif in SGLT inhibitors and, in natural products, glycosylation often confers improved bioactivity and bioavailability. Whilst a single C-glycoside link between a sugar moiety and its aglycone core is a common feature in natural products isolated to date, only a small number, including the antibiotics granaticin and sarubicin, are covalently bonded twice to a single sugar moiety. The way in which this “double C-glycosylation” is naturally mediated is not yet known, yet has been speculated on. Here, we report the exploration and development of a potentially biomimetic procedure that utilises intermolecular cycloaddition chemistry to access new “double C-glycosylated” products and enables the creation of bridged polycyclic ethers from a common maltol-derived oxidopyrylium salt precursor. Full article
(This article belongs to the Section Chemical Biology)
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15 pages, 1207 KiB  
Review
Gene Fusions as Potential Therapeutic Targets in Soft Tissue Sarcomas
by Qiongdan Zheng, Tong Wang, Zijian Zou, Wenjie Ma, Zirui Dong, Jingqin Zhong, Wanlin Liu, Yu Xu, Tu Hu, Wei Sun and Yong Chen
Biomolecules 2025, 15(6), 904; https://doi.org/10.3390/biom15060904 - 19 Jun 2025
Viewed by 358
Abstract
Though having been discovered in one third of sarcomas, gene fusions are less studied in their roles as potential therapeutic targets, making conventional modalities the mainstream treatment options for sarcoma patients. Recent decades have witnessed encouraging progress in basic research delving into mechanisms [...] Read more.
Though having been discovered in one third of sarcomas, gene fusions are less studied in their roles as potential therapeutic targets, making conventional modalities the mainstream treatment options for sarcoma patients. Recent decades have witnessed encouraging progress in basic research delving into mechanisms underlying how gene fusions drive sarcomas; nevertheless, further translation to clinical application fails to keep abreast with the advances achieved in basic science. In this review, we will focus on key chromosomal translocation-driven sarcomas defined by characteristic hallmark fusion oncoproteins, including Ewing sarcoma with EWSR1–FLI1/ERG fusion, epithelioid hemangioendothelioma with WWTR1–CAMTA1/YAP1–TFE1 fusion, and others, to discuss the potential of directly targeting these fusion proteins as therapeutic targets in preclinical and clinical contexts. Full article
(This article belongs to the Special Issue Molecular Mechanisms and Genetics of Human Disease)
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21 pages, 842 KiB  
Review
Advances in Local Drug Delivery for Periodontal Treatment: Present Strategies and Future Directions
by Mayuka Nakajima, Mayuko Yanagawa, Honoka Takikawa, Truong Tran Thien, Lorena Zegarra-Caceres, Chunyang Yan and Koichi Tabeta
Biomolecules 2025, 15(6), 903; https://doi.org/10.3390/biom15060903 - 19 Jun 2025
Viewed by 482
Abstract
Periodontitis is a highly prevalent, irreversible inflammatory disease characterized by the destruction of tooth-supporting tissues, eventually leading to tooth loss. Conventional treatment involves the mechanical removal of the subgingival biofilm, which is a major cause of gingival inflammation. However, the inaccessibility of deep-seated [...] Read more.
Periodontitis is a highly prevalent, irreversible inflammatory disease characterized by the destruction of tooth-supporting tissues, eventually leading to tooth loss. Conventional treatment involves the mechanical removal of the subgingival biofilm, which is a major cause of gingival inflammation. However, the inaccessibility of deep-seated polymicrobial biofilms limits its effectiveness. Despite the adjunct use of systemic antimicrobials, their low site-specific bioavailability and systemic side effects remain concerns. Local drug administration offers a targeted alternative. However, the dynamic oral environment, which is characterized by continuous salivary and gingival crevicular fluid flow, poses challenges in maintaining therapeutic drug levels. Drug delivery systems (DDSs) provide technical solutions to overcome these limitations. With advancements in materials science and nanotechnology, diverse local DDS (LDDS) formulations tailored for periodontal applications have been developed. While traditionally focused on infection control, the application of LDDSs has expanded beyond antimicrobial therapy. Increasing attention has been paid to LDDS-based regenerative strategies, which aim to overcome the limitations of conventional regenerative therapies. This review aims to provide a comprehensive overview of the current and emerging DDS strategies in periodontal therapy, focusing on their applications in infection management and bone regeneration and discussing their limitations and prospects for clinical translation. Full article
(This article belongs to the Special Issue Biologics and Biomaterials for Periodontal Treatment: Current and Future Challenges)
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29 pages, 10703 KiB  
Article
Enhanced Therapeutic Efficacy of Omeprazole Nanosuspension in Ethanol-Induced Gastric Ulcer: A Focus on Oxidative Stress and Inflammatory Pathways
by Mody Albalawi and Sahar Khateeb
Biomolecules 2025, 15(6), 902; https://doi.org/10.3390/biom15060902 - 19 Jun 2025
Viewed by 414
Abstract
Gastric ulcer is a concerning condition that affects numerous individuals globally. Omeprazole (OMP), a well-known drug for treating stomach ulcers, has been associated with several adverse effects and limited solubility. The study aimed to create an omeprazole nanosuspension (OMP-NS) with improved solubility and [...] Read more.
Gastric ulcer is a concerning condition that affects numerous individuals globally. Omeprazole (OMP), a well-known drug for treating stomach ulcers, has been associated with several adverse effects and limited solubility. The study aimed to create an omeprazole nanosuspension (OMP-NS) with improved solubility and bioavailability. Additionally, the study investigated the potential therapeutic effects of OMP-NS on ethanol-induced gastric injury in rats, comparing it to traditional OMP therapy to identify novel therapeutic alternatives. The characterization of the OMP-NS was assessed using DLS, TEM, XRD, FTIR, UV spectrophotometric analysis, in vitro release studies, and entrapment efficiency (EE) assays. A total of 24 male Wistar albino rats (weighing 150–200 g, aged 8–10 weeks) were randomly divided into four groups (six rats/group). Gastric injury was induced using absolute ethanol (5 mL/kg), followed by oral administration of either OMP or OMP-NS (20 mg/kg) for 7 days. Data were analyzed using one-way ANOVA accompanied by the Bonferroni post hoc test or the Kruskal–Wallis test, based on data distribution, with significance set at p < 0.05. The OMP-NS demonstrated a Z-average diameter of 216.1 nm, a polydispersity index of 0.2, and a zeta potential of −19.6 mV. The particles were predominantly spherical with an average size of 67.28 nm. In vitro release studies showed 97.78% release at 8 h, with an EE% of 96.97%. Ethanol-induced gastric ulcers were associated with oxidative stress, characterized by elevated levels of NADPH, ROS, MDA, and NO, while the level of SOD was reduced. It was accompanied by increased inflammatory markers HMGB1, which subsequently increased TLR-2, MyD88, NF-κBp56, NLRP3, TNF-α, IL-1β, and IL-6 levels; conversely, a significant decrease in Nrf2/PPAR-γ/SIRT1 levels was observed. In contrast, OMP-NS administration significantly reduced oxidative stress and inflammatory markers, restored SOD activity, and upregulated protective pathways Nrf2/PPAR-γ/SIRT1 more effectively than conventional OMP therapy. In conclusion, OMP-NS represents a promising therapeutic strategy with notable anti-inflammatory and anti-ulcerogenic effects in ethanol-induced gastric ulcers. Full article
(This article belongs to the Section Cellular Biochemistry)
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17 pages, 1642 KiB  
Review
Ankyrin-G and Its Binding Partners in Neurons: Orchestrating the Molecular Structure of the Axon Initial Segment
by Xiaowei Zhu, Yanyan Yu, Zhuqian Jiang, Yoshinori Otani and Masashi Fujitani
Biomolecules 2025, 15(6), 901; https://doi.org/10.3390/biom15060901 - 19 Jun 2025
Viewed by 464
Abstract
The axon initial segment (AIS) is a specialized subcellular domain that plays an essential role in action potential initiation and the diffusion barrier. A key organizer of the AIS is Ankyrin-G, a scaffolding protein responsible for clustering voltage-gated ion channels, cell adhesion molecules [...] Read more.
The axon initial segment (AIS) is a specialized subcellular domain that plays an essential role in action potential initiation and the diffusion barrier. A key organizer of the AIS is Ankyrin-G, a scaffolding protein responsible for clustering voltage-gated ion channels, cell adhesion molecules (CAMs), and cytoskeletal components at this critical neuronal domain. Recent proteomic analyses have revealed a complex network of proteins in the AIS, emphasizing Ankyrin-G’s central role in its molecular architecture. This review discusses new findings in the study of AIS-associated proteins. It explains how Ankyrin-G and its binding partners (such as ion channels, CAMs, spectrins, actin, and microtubule-associated proteins including end-binding protein 3, tripartite motif-containing protein 46, and calmodulin-regulated spectrin-associated protein 2) organize their structure. Understanding the dynamic regulation and molecular interactions within the AIS offers insights into neuronal excitability and reveals potential therapeutic targets for axonal dysfunction–related diseases. Through these dynamic interactions, Ankyrin-G ensures the proper alignment and dense clustering of key channel complexes, thereby maintaining the AIS’s distinctive molecular and functional identity. By further unraveling the complexity of Ankyrin-G’s interactome, our understanding of AIS formation, maintenance, and plasticity will be considerably enhanced, contributing to the elucidation of the pathogenesis of neurological and neuropsychiatric disorders. Full article
(This article belongs to the Special Issue Cellular and Molecular Biology of Neurodevelopment)
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20 pages, 3130 KiB  
Article
Adaptation to Arginine Deprivation Leads to a More Aggressive, Therapy-Resistant Phenotype in HNSCC Cells
by Oleg Chen, Olena Vovk, Nikita Polishchuk, Oksana Mayevska, Galyna Shuvayeva, Melike Demir, Vasyl Lukiyanchuk, Leoni A. Kunz-Schughart, Anna Dubrovska and Oleh Stasyk
Biomolecules 2025, 15(6), 900; https://doi.org/10.3390/biom15060900 - 19 Jun 2025
Viewed by 377
Abstract
Purpose: The development of acquired resistance to arginine deprivation therapy (ADT) is a major barrier to its efficacy. This study aimed to elucidate the possible mechanisms underlying the resistance to ADT. Methods: We applied repeated ADT and established a subline SAS-R9 of the [...] Read more.
Purpose: The development of acquired resistance to arginine deprivation therapy (ADT) is a major barrier to its efficacy. This study aimed to elucidate the possible mechanisms underlying the resistance to ADT. Methods: We applied repeated ADT and established a subline SAS-R9 of the human head and neck squamous cell carcinoma (HNSCC) cells semi-resistant to arginine (Arg) deprivation in vitro. This subline was compared to the parental SAS cell lines for its relative clonogenic proliferation, aggregation, adhesion, and migration capacities. The transcriptomic changes were assessed by RNA sequencing. Signaling pathway alterations were confirmed by RT-PCR and Western blotting. Relative cell radioresistance was analyzed by radiobiological clonogenic survival assay. DNA double-strand break (DSB) repair was assessed by γH2A.X foci analysis. Results: SAS-R9 cells showed higher survival in response to ADT and radiotherapy, elevated clonogenic proliferation rate, cell–cell aggregation, and cell–matrix adhesion, along with increased epithelial–mesenchymal transition (EMT) markers and enhanced DNA DSB repair, potentially related to a more aggressive and therapy-resistant phenotype. Conclusions: While acute ADT has radiosensitizing potential, this new study suggests that long-term, repeated ADT is associated with cell selection and reprogramming, resulting in resistance to radiotherapy-induced DNA damage and higher tumor cell aggressiveness. Full article
(This article belongs to the Special Issue DNA Damage, Mutagenesis, and Repair Mechanisms)
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20 pages, 2917 KiB  
Review
HIF-1α: A Key Factor Mediating Tumor Cells from Digestive System to Evade NK Cell Killing via Activating Metalloproteinases to Hydrolyze MICA/B
by Quan Zhu, Shuyi Tang, Ting Huang, Chunjing Chen, Biyuan Liu, Chuyu Xiao, Liugu Chen, Wang Wang and Fangguo Lu
Biomolecules 2025, 15(6), 899; https://doi.org/10.3390/biom15060899 - 19 Jun 2025
Viewed by 414
Abstract
Malignant tumors of the digestive system are widespread and pose a serious threat to humans. Immune escape is an important factor promoting the deterioration of malignant tumors in the digestive system. Natural killer cells (NK cells) are key members of the anti-tumor and [...] Read more.
Malignant tumors of the digestive system are widespread and pose a serious threat to humans. Immune escape is an important factor promoting the deterioration of malignant tumors in the digestive system. Natural killer cells (NK cells) are key members of the anti-tumor and immune surveillance system, mainly exerting cytotoxic effects by binding to the activating receptor natural killer cell group 2D (NKG2D) on their cell surface with the corresponding ligands (major histocompatibility complex class I chain-related protein A/B, MICA/B) on the surface of tumor cells. Malignant tumors of epithelial origin usually highly express NKG2D ligands such as MICA, which can attract NK cells to kill tumor cells and also serve as an important basis for NK cell-based immunotherapy. Tumor cells highly express hypoxia-inducible factor-1α (HIF-1α), which promotes the expression of matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinases (ADAMs). These metalloproteinases hydrolyze MICA and other ligands on the surface of tumor cells to generate soluble molecules. These soluble ligands, when binding to NKG2D, cannot activate NK cells and also block the binding of NKG2D to MICA on the surface of tumor cells, enabling tumor cells to evade the killing effect of NK cells. Almost all organs in the digestive system originate from epithelial tissue, so the soluble ligands generated by the HIF-1α/MMPs or HIF-1α/ADAMs signaling pathways play a crucial role in evading NK cell killing. A comprehensive understanding of this immune escape process is helpful for a deeper understanding of the molecular mechanism of NK cell anti-tumor activity. This article reviews the molecular mechanisms of common digestive system malignancies evading NK cell killing, providing new insights into the mechanism of tumor immune escape. Full article
(This article belongs to the Section Molecular Medicine)
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33 pages, 10547 KiB  
Review
Prospects and Trends in Biomedical Microelectromechanical Systems (MEMS) Devices: A Review
by Lowell Welburn, Amir Milad Moshref Javadi, Luong Nguyen and Salil Desai
Biomolecules 2025, 15(6), 898; https://doi.org/10.3390/biom15060898 - 18 Jun 2025
Viewed by 477
Abstract
Designing and manufacturing devices at the micro- and nanoscales offers significant advantages, including high precision, quick response times, high energy density ratios, and low production costs. These benefits have driven extensive research in micro-electromechanical systems (MEMS) and nano-electromechanical systems (NEMS), resulting in various [...] Read more.
Designing and manufacturing devices at the micro- and nanoscales offers significant advantages, including high precision, quick response times, high energy density ratios, and low production costs. These benefits have driven extensive research in micro-electromechanical systems (MEMS) and nano-electromechanical systems (NEMS), resulting in various classifications of materials and manufacturing techniques, which are ultimately used to produce different classifications of MEMS devices. The current work aims to systematically organize the literature on MEMS in biomedical devices, encompassing past achievements, present developments, and future prospects. This paper reviews the current research trends, highlighting significant material advancements and emerging technologies in biomedical MEMS in order to meet the current challenges facing the field, such as ensuring biocompatibility, achieving miniaturization, and maintaining precise control in biological environments. It also explores projected applications, including use in advanced diagnostic tools, targeted drug delivery systems, and innovative therapeutic devices. By mapping out these trends and prospects, this review will help identify current research gaps in the biomedical MEMS field. By pinpointing these gaps, researchers can focus on addressing unmet needs and advancing state-of-the-art biomedical MEMS technology. Ultimately, this can lead to the development of more effective and innovative biomedical devices, improving patient care and outcomes. Full article
(This article belongs to the Special Issue Novel Materials for Biomedical Applications: 2nd Edition)
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26 pages, 2952 KiB  
Review
Signal Peptides: From Molecular Mechanisms to Applications in Protein and Vaccine Engineering
by Shuai Zhang, Zhihui He, Hui Wang and Jingbo Zhai
Biomolecules 2025, 15(6), 897; https://doi.org/10.3390/biom15060897 - 18 Jun 2025
Viewed by 550
Abstract
Signal peptides (SPs) are short amino acid sequences located at the N-terminus of nascent proteins and are widely present across various life forms. They play crucial roles in protein synthesis, transmembrane transport, and intracellular signal transduction. With the rapid advancement of bioinformatics, studies [...] Read more.
Signal peptides (SPs) are short amino acid sequences located at the N-terminus of nascent proteins and are widely present across various life forms. They play crucial roles in protein synthesis, transmembrane transport, and intracellular signal transduction. With the rapid advancement of bioinformatics, studies have revealed that the functions of SPs are far more complex than previously understood. In recombinant protein expression systems, the rational design and optimization of SPs are essential for enhancing the expression efficiency and secretion level of exogenous proteins. Meanwhile, the application value of SPs in vaccine development has attracted increasing attention. This review summarizes the structural characteristics, functional mechanisms, and applications of SPs in recombinant protein production and SP-based vaccines. It also discusses their biological roles, the significance of engineering optimization strategies, and the current challenges, aiming to provide theoretical support and practical guidance for improving recombinant protein yield and advancing SP-based vaccine development. Full article
(This article belongs to the Section Molecular Biology)
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15 pages, 4868 KiB  
Article
Genome-Wide Identification of DNA Methyltransferases (Dnmts) in Fish and Its Potential Roles During Sex Change in Blackhead Seabream
by Sixin Guo, Binwei Duan, Jianchao Chen, Mingyang Cui, Canbei You, Hanyin Wei, Xiazi Huang, Li Deng and Kai Zhang
Biomolecules 2025, 15(6), 896; https://doi.org/10.3390/biom15060896 - 18 Jun 2025
Viewed by 221
Abstract
DNA methylation, also known as 5-methylcytosine, is an epigenetic modification that has crucial functions in multiple important biological processes in fish, such as gonadal development. The cellular DNA methylation level is tightly regulated by DNA methyltransferases (Dnmt). However, detailed investigations of this family [...] Read more.
DNA methylation, also known as 5-methylcytosine, is an epigenetic modification that has crucial functions in multiple important biological processes in fish, such as gonadal development. The cellular DNA methylation level is tightly regulated by DNA methyltransferases (Dnmt). However, detailed investigations of this family in fish are very scarce. In this study, our results confirmed that teleost genomes contain 4 to 16 Dnmt genes, with diversity likely resulting from a combination of whole-genome duplication (WGD), tandem duplication, and gene loss. Differences were observed in tissue distribution, transcription abundance, and protein structure of Dnmt duplicates, supporting their subfunctionalization or neofunctionalization after duplication. Interestingly, we found that fish Dnmt3b duplicates likely have acquired the functions of mammalian Dnmt3l, which may compensate for the absence of fish Dnmt3l. Furthermore, transcriptome analysis and qPCR results indicated that DNA methyltransferase genes (Dnmt1, Dnmt3aa, Dnmt3ab, Dnmt3ba, and Dnmt3bb.1) possibly play important roles in the natural sex change of protandrous hermaphrodite blackhead seabream (Acanthopagrus schlegelii) and inferred that global remodeling of gonadal DNA methylation, regulated by DNA methyltransferase genes, was closely associated with sex change in sequentially hermaphroditic fishes. Overall, our results may help provide a better understanding of the evolution and function of DNA methyltransferases in fish. Full article
(This article belongs to the Section Molecular Genetics)
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21 pages, 10870 KiB  
Article
A Novel Copper Ionophore Nanoshuttle (Winged Cu) for Inducing Cuproptosis in B16 Melanoma Cells
by Yuhuan Wu, Ziyao Chang, Wenhao Wang, Chuanbin Wu, Xin Pan and Zhengwei Huang
Biomolecules 2025, 15(6), 895; https://doi.org/10.3390/biom15060895 - 18 Jun 2025
Viewed by 336
Abstract
Cuproptosis, a newly discovered copper-dependent programmed cell death pathway, represents a promising approach for anticancer therapy. However, the efficacy of cuproptosis critically depends on intracellular copper accumulation. Traditional copper ionophores have limited therapeutic efficacy due to their reliance on serum copper levels. Therefore, [...] Read more.
Cuproptosis, a newly discovered copper-dependent programmed cell death pathway, represents a promising approach for anticancer therapy. However, the efficacy of cuproptosis critically depends on intracellular copper accumulation. Traditional copper ionophores have limited therapeutic efficacy due to their reliance on serum copper levels. Therefore, the development of novel copper ionophores to enhance intracellular copper levels is urgently needed. In this study, we targeted a melanoma model and pioneered the application of Bis(2-hydroxyethyl)dithiocarbamic acid copper(II) [Cu(HEDTC)2] as a highly efficient copper ionophore for inducing cuproptosis in B16 melanoma cells. Compared to conventional copper ionophores, Cu(HEDTC)2 exhibits superior intracellular copper delivery efficiency, thereby enhancing the induction of cuproptosis. We further constructed a Cu(HEDTC)2@Soluplus-nanomicelle (CS NM) system designed to disrupt copper ion homeostasis in tumor cells and amplify cuproptosis. In this system, Cu(HEDTC)2, as a novel copper ionophore, significantly enhanced the copper level in B16 melanoma cells. Upon cellular internalization, CS NM underwent degradation and released copper ions, which subsequently triggered cuproptosis by causing abnormal aggregation of mitochondrial lipoylated proteins. This study provides a new experimental foundation and potential therapeutic strategy for cuproptosis-based cancer treatment. Full article
(This article belongs to the Section Chemical Biology)
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18 pages, 4606 KiB  
Article
Dynamic 3D-Network Coating Composite Enables Global Isolation of Phosphopeptides, Stepwise Separation of Mono- and Multi-Phosphopeptides, and Phosphoproteomics of Human Lung Cells
by Linlin Liu, Zhenhua Chen, Danni Wang, Weida Liang, Binbin Wang, Chenglong Xia, Yinghua Yan, Chuanfan Ding, Xiaodan Meng and Hongze Liang
Biomolecules 2025, 15(6), 894; https://doi.org/10.3390/biom15060894 - 18 Jun 2025
Viewed by 292
Abstract
Protein phosphorylation is one of the most common and important post-translational modifications (PTMs) and is highly involved in various biological processes. Ideal adsorbents with high sensitivity and specificity toward phosphopeptides with large coverage are therefore essential for enrichment and mass spectroscopy-based phosphoproteomics analysis. [...] Read more.
Protein phosphorylation is one of the most common and important post-translational modifications (PTMs) and is highly involved in various biological processes. Ideal adsorbents with high sensitivity and specificity toward phosphopeptides with large coverage are therefore essential for enrichment and mass spectroscopy-based phosphoproteomics analysis. In this study, a newly designed IMAC adsorbent composite was constructed on the graphene matrix coated with mesoporous silica. The outer functional 3D-network layer was prepared by free radical polymerization of the phosphonate-functionalized vinyl imidazolium salt monomer and subsequent metal immobilization. Due to its unique structural feature and high content of Ti4+ ions, the resulting phosphonate-immobilized adsorbent composite G@mSiO2@PPFIL-Ti4+ exhibits excellent performance in phosphopeptide enrichment with a low detection limit (0.1 fmol, tryptic β-casein digest) and superior selectivity (molar ratio of 1:15,000, digest mixture of β-casein and bovine serum albumin). G@mSiO2@PPFIL-Ti4+ displays high tolerance to loading and elution conditions and thus can be reused without a marked decrease in enrichment efficacy. The captured phosphopeptides can be released globally, and mono-/multi-phosphopeptides can be isolated stepwise by gradient elution. When applying this material to enrich phosphopeptides from human lung cell lysates, a total of 3268 unique phosphopeptides were identified, corresponding to 1293 phosphoproteins. Furthermore, 2698 phosphorylated peptides were found to be differentially expressed (p < 0.05) between human lung adenocarcinoma cells (SPC-A1) and human normal epithelial cells (Beas-2B), of which 1592 were upregulated and 1106 were downregulated in the cancer group. These results demonstrate the material’s superior enrichment efficiency in complex biological samples. Full article
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15 pages, 748 KiB  
Review
Exercise-Mediated Skeletal Muscle-Derived IL-6 Regulates Bone Metabolism: A New Perspective on Muscle–Bone Crosstalk
by Chenyu Zhu, Xiaoqing Ding, Min Chen, Jie Feng, Jun Zou and Lingli Zhang
Biomolecules 2025, 15(6), 893; https://doi.org/10.3390/biom15060893 - 18 Jun 2025
Viewed by 398
Abstract
Skeletal muscles and bones maintain musculoskeletal system function through their collaborative interaction, whereby muscles regulate bone metabolism via mechanical coupling. An increasing number of studies have shown that various cytokines secreted by skeletal muscles during exercise closely regulate the balance of bone homeostasis. [...] Read more.
Skeletal muscles and bones maintain musculoskeletal system function through their collaborative interaction, whereby muscles regulate bone metabolism via mechanical coupling. An increasing number of studies have shown that various cytokines secreted by skeletal muscles during exercise closely regulate the balance of bone homeostasis. Interleukin-6 (IL-6), one of the first muscle-secreted factors to be discovered, not only plays an important role in regulating the function of the muscle itself but also regulates bone metabolic processes in a bidirectional manner through multiple complex signal transduction pathways, thereby affecting the balance between bone formation and bone resorption. The exact mechanism by which IL-6 regulates bone metabolism is not fully understood, and there are few summaries on how exercise affects bone metabolism through IL-6 from skeletal muscles. Accordingly, this study will take skeletal muscle-derived IL-6 as an entry point to explore how the cross-organ regulatory activities of the muscles targeting bones during exercise affect bone metabolic processes. This study also aims to improve the mechanism of muscle–bone crosstalk under the effect of exercise and provide a theoretical basis and clinical diagnosis and treatment ideas from multiple perspectives for exercise to improve bone health. Full article
(This article belongs to the Special Issue Feature Papers in Cellular Biochemistry)
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54 pages, 2627 KiB  
Review
Calcium Signaling Dynamics in Vascular Cells and Their Dysregulation in Vascular Disease
by Chang Dai and Raouf A. Khalil
Biomolecules 2025, 15(6), 892; https://doi.org/10.3390/biom15060892 - 18 Jun 2025
Viewed by 525
Abstract
Calcium (Ca2+) signaling is a fundamental regulatory mechanism controlling essential processes in the endothelium, vascular smooth muscle cells (VSMCs), and the extracellular matrix (ECM), including maintaining the endothelial barrier, modulation of vascular tone, and vascular remodeling. Cytosolic free Ca2+ concentration [...] Read more.
Calcium (Ca2+) signaling is a fundamental regulatory mechanism controlling essential processes in the endothelium, vascular smooth muscle cells (VSMCs), and the extracellular matrix (ECM), including maintaining the endothelial barrier, modulation of vascular tone, and vascular remodeling. Cytosolic free Ca2+ concentration is tightly regulated by a balance between Ca2+ mobilization mechanisms, including Ca2+ release from the intracellular stores in the sarcoplasmic/endoplasmic reticulum and Ca2+ entry via voltage-dependent, transient-receptor potential, and store-operated Ca2+ channels, and Ca2+ elimination pathways including Ca2+ extrusion by the plasma membrane Ca2+-ATPase and Na+/Ca2+ exchanger and Ca2+ re-uptake by the sarco(endo)plasmic reticulum Ca2+-ATPase and the mitochondria. Some cell membranes/organelles are multifunctional and have both Ca2+ mobilization and Ca2+ removal pathways. Also, the individual Ca2+ handling pathways could be integrated to function in a regenerative, capacitative, cooperative, bidirectional, or reciprocal feed-forward or feed-back manner. Disruption of these pathways causes dysregulation of the Ca2+ signaling dynamics and leads to pathological cardiovascular conditions such as hypertension, coronary artery disease, atherosclerosis, and vascular calcification. In the endothelium, dysregulated Ca2+ signaling impairs nitric oxide production, reduces vasodilatory capacity, and increases vascular permeability. In VSMCs, Ca2+-dependent phosphorylation of the myosin light chain and Ca2+ sensitization by protein kinase-C (PKC) and Rho-kinase (ROCK) increase vascular tone and could lead to increased blood pressure and hypertension. Ca2+ activation of matrix metalloproteinases causes collagen/elastin imbalance and promotes vascular remodeling. Ca2+-dependent immune cell activation, leukocyte infiltration, and cholesterol accumulation by macrophages promote foam cell formation and atherosclerotic plaque progression. Chronic increases in VSMCs Ca2+ promote phenotypic switching to mesenchymal cells and osteogenic transformation and thereby accelerate vascular calcification and plaque instability. Emerging therapeutic strategies targeting these Ca2+-dependent mechanisms, including Ca2+ channel blockers and PKC and ROCK inhibitors, hold promise for restoring Ca2+ homeostasis and mitigating vascular disease progression. Full article
(This article belongs to the Special Issue Calcium Signaling in Cell Function and Dysfunction)
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20 pages, 6538 KiB  
Article
Influence of the Maillard Reaction on the Properties of Gelatin/Zein Nanofibers Loaded with Dihydromyricetin Prepared by Electro-Blowing Spinning
by Hui Xiang, Runtian Wu, Man Xiao, Jianhui An, Longchen Shang, Yexing Tao and Lingli Deng
Biomolecules 2025, 15(6), 891; https://doi.org/10.3390/biom15060891 - 18 Jun 2025
Viewed by 295
Abstract
This study investigated gelatin/zein nanofibers loaded with dihydromyricetin (0–20%, relative to protein weight), before and after the Maillard reaction (60 °C with 50% relative humidity for 6 h). Scanning electron microscopy and diameter distribution analysis indicated that dihydromyricetin incorporation increased the fiber diameter [...] Read more.
This study investigated gelatin/zein nanofibers loaded with dihydromyricetin (0–20%, relative to protein weight), before and after the Maillard reaction (60 °C with 50% relative humidity for 6 h). Scanning electron microscopy and diameter distribution analysis indicated that dihydromyricetin incorporation increased the fiber diameter from 692 ± 133 to 922 ± 121 nm, while the nanofibers maintained a uniform morphology following the Maillard reaction. Fourier transform infrared spectroscopy revealed that dihydromyricetin formed hydrogen bonds with protein molecules. X-ray diffraction results indicate that dihydromyricetin was uniformly dispersed within the gelatin/zein nanofibers. The addition of dihydromyricetin improved the thermal stability of the nanofibers. Furthermore, after the Maillard reaction, the nanofibers with dihydromyricetin demonstrated enhanced water resistance. Mechanical testing revealed that nanofibers containing 20% dihydromyricetin after the Maillard reaction exhibited a considerably higher elastic modulus of approximately 90 MPa. In addition, nanofibers containing dihydromyricetin exhibited notable antioxidant activity and antibacterial properties against Escherichia coli and Staphylococcus aureus. In summary, gelatin/zein nanofibers containing high concentrations of dihydromyricetin exhibited favorable physical and functional properties, supporting their suitability as effective delivery systems for dihydromyricetin in active packaging applications. Full article
(This article belongs to the Section Natural and Bio-derived Molecules)
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24 pages, 464 KiB  
Review
Protein–Protein Interactions in Base Excision Repair
by Govardhan Rathnaiah and Joann B. Sweasy
Biomolecules 2025, 15(6), 890; https://doi.org/10.3390/biom15060890 - 18 Jun 2025
Viewed by 368
Abstract
The Base Excision Repair (BER) pathway involves a highly coordinated series of protein–protein interactions that facilitate the recognition, excision, and repair of damaged bases. Key enzymes such as DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), polynucleotide kinase-phosphatase (PNKP), DNA polymerase b (Pol β), ligase [...] Read more.
The Base Excision Repair (BER) pathway involves a highly coordinated series of protein–protein interactions that facilitate the recognition, excision, and repair of damaged bases. Key enzymes such as DNA glycosylases, apurinic/apyrimidinic endonuclease 1 (APE1), polynucleotide kinase-phosphatase (PNKP), DNA polymerase b (Pol β), ligase IIIα (LigIIIα), poly (ADP-ribose) polymerases PARP1 and PARP2, and X-ray repair cross-complementing protein 1 (XRCC1) catalyze BER in a tightly regulated molecular network. These interactions ensure the seamless handoff of DNA intermediates between the core enzymes of the BER pathway. Understanding the details of protein–protein interactions in BER provides valuable insights into the molecular underpinnings of DNA repair processes. In this review, we focus on protein–protein interactions between the components of the single-nucleotide BER (SN-BER) pathway and other proteins that interact with BER components and regulate the coordination of the pathway. We also briefly discuss the interactions of other proteins that interact with the components of SN-BER based on functional evidence. Full article
(This article belongs to the Special Issue Molecular Mechanisms in DNA and RNA Damage and Repair)
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14 pages, 2189 KiB  
Article
Application of Free and Encapsulated DNA Tracers in Surface Water Studies in Lithuanian Climatic Conditions
by Dominyka Švedaitė, Anastasija Kriučkova, Augustas Morkvėnas, Vitalijus Karabanovas, Gintautas Stankūnavičius, Vigilija Klima, Jaunius Urbonavičius and Rūta Ivanec-Goranina
Biomolecules 2025, 15(6), 889; https://doi.org/10.3390/biom15060889 - 18 Jun 2025
Viewed by 288
Abstract
The applicability of free and encapsulated DNA as tracers in surface water studies in Lithuanian climatic conditions was evaluated. Tracer DNA synthesis and analysis were performed using real-time polymerase chain reaction (RT-PCR). Alginate and chitosan were used to obtain the microcapsules with DNA, [...] Read more.
The applicability of free and encapsulated DNA as tracers in surface water studies in Lithuanian climatic conditions was evaluated. Tracer DNA synthesis and analysis were performed using real-time polymerase chain reaction (RT-PCR). Alginate and chitosan were used to obtain the microcapsules with DNA, and their sizes were determined using an atomic force microscopy. The Murlė stream in the city of Vilnius was chosen for field experiments using the prepared tracers. It was found that both types of tracers may be applied to surface water studies, but the relative concentration recovery of encapsulated DNA tracers is 3–6 times higher than that of free DNA tracers. It was concluded that the alginate/chitosan capsules protect DNA from the sandy layer in Murlė stream, direct UV exposure and other environmental factors that could degrade DNA. To our knowledge, this is the first report about free and encapsulated DNA tracer application in surface water studies in Lithuania. Full article
(This article belongs to the Section Biomacromolecules: Proteins, Nucleic Acids and Carbohydrates)
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11 pages, 1227 KiB  
Communication
Improving the Catalytic Activity and Thermostability of FAST-PETase with a Multifunctional Short Peptide
by Jun Yang, Binyang Deng, Pingan Liao, Siyu Lin, Liqi Zheng, Xing Yang, Fei Wang, Chao Zhai and Lixin Ma
Biomolecules 2025, 15(6), 888; https://doi.org/10.3390/biom15060888 - 18 Jun 2025
Viewed by 323
Abstract
Previous reports indicated that self-assembling amphipathic peptide S1v1 (AEAEAHAH)2 significantly enhances the soluble expression, thermostability, and activity of the target proteins when fused to them. In order to obtain high-efficiency enzymes for the large-scale degradation of polyethylene terephthalate (PET), this multifunctional peptide [...] Read more.
Previous reports indicated that self-assembling amphipathic peptide S1v1 (AEAEAHAH)2 significantly enhances the soluble expression, thermostability, and activity of the target proteins when fused to them. In order to obtain high-efficiency enzymes for the large-scale degradation of polyethylene terephthalate (PET), this multifunctional peptide was fused to the N- and C-terminus of FAST-PETase, a variant of Ideonella sakaiensis PETase (IsPETase), with a PT-linker (TTVTTPQTS) harbored between the target protein and the multifunctional peptide. Consistent with previous reports, S1v1 increased the solubility of FAST-PETase slightly. Moreover, it increased the activity of FAST-PETase dramatically. The amount of terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalic acid (MHET) released from PET substrate after 24 h of digestion at 50°C by fusion enzymes bearing N- and C-terminal S1v1 tag was approximately 2.9- and 4.6-fold that of FAST-PETase, respectively. Furthermore, the optimal temperature and thermostability of the fusion proteins increased in comparison with FAST-PETase. The present study provides a novel strategy to improve the depolymerization efficiency of FAST-PETase. Full article
(This article belongs to the Section Biomacromolecules: Proteins, Nucleic Acids and Carbohydrates)
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19 pages, 947 KiB  
Article
Early-Life Adversity and Epigenetic Aging: Findings from a 17-Year Longitudinal Study
by Emily Barr, Maude Comtois-Cabana, Andressa Coope, Sylvana M. Coté, Michael S. Kobor, Chaini Konwar, Sonia Lupien, Marie-Claude Geoffroy, Michel Boivin, Nadine Provençal, Nicole L. A. Catherine, Jessica K. Dennis and Isabelle Ouellet-Morin
Biomolecules 2025, 15(6), 887; https://doi.org/10.3390/biom15060887 - 18 Jun 2025
Viewed by 420
Abstract
Youth exposed to early-life adversity (ELA) are at greater risk for poorer physical and mental health outcomes in adolescence and adulthood. Although the biological mechanisms underlying these associations remain elusive, DNA methylation (DNAm) has emerged as a potential pathway. DNAm-based measures of epigenetic [...] Read more.
Youth exposed to early-life adversity (ELA) are at greater risk for poorer physical and mental health outcomes in adolescence and adulthood. Although the biological mechanisms underlying these associations remain elusive, DNA methylation (DNAm) has emerged as a potential pathway. DNAm-based measures of epigenetic age have been associated with ELA, indicating accelerated aging. According to the stress sensitization hypothesis, prenatal adversity may further heighten sensitivity to subsequent stressors in childhood and adolescence. This study examined the associations between ELA and six epigenetic aging measures, considering both the timing of adversity and the participant’s sex. Data were drawn from the Quebec Longitudinal Study of Child Development, with two cumulative indices of ELA derived from prospectively collected data: the Perinatal Adversity and the Child and Adolescent Adversity indices. Higher Perinatal Adversity scores were associated with accelerated DunedinPACE scores. No significant associations were found between ELA and the other epigenetic clocks, nor did we find support for the stress sensitization hypothesis—though a sex-specific trend emerged among girls. The findings suggest that DunedinPACE may be more sensitive to variations in ELA than other clocks. Future research should systematically investigate sex-dimorphic associations between ELA and epigenetic aging, with particular attention to the impact of perinatal adversity. Full article
(This article belongs to the Special Issue Molecular Advances in Mechanism and Regulation of Lifespan and Aging)
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35 pages, 820 KiB  
Review
Dairy Propionibacteria: Probiotic Properties and Their Molecular Bases
by Franca Rossi, Serena Santonicola, Valerio Giaccone, Alessandro Truant and Giampaolo Colavita
Biomolecules 2025, 15(6), 886; https://doi.org/10.3390/biom15060886 - 17 Jun 2025
Viewed by 529
Abstract
This review summarizes the current knowledge on the probiotic characteristics of dairy propionibacteria, represented by Propionibacterium freudenreichii and some Acidipropionibacterium species commonly consumed through raw milk cheese. For example, in Swiss-type cheeses, P. freudenreichii is added as a starter culture. Some strains of [...] Read more.
This review summarizes the current knowledge on the probiotic characteristics of dairy propionibacteria, represented by Propionibacterium freudenreichii and some Acidipropionibacterium species commonly consumed through raw milk cheese. For example, in Swiss-type cheeses, P. freudenreichii is added as a starter culture. Some strains of P. freudenreichii have been included in mixed probiotic commercial preparations or used to produce tablets from fermented culture media containing bioactive substances such as short-chain fatty acids (SCFAs), bifidogenic molecules, and vitamins. Acidipropionibacterium acidipropionici and A. jensenii strains have mainly been evaluated as health and productivity promoters in farm animals. For P. freudenreichii, the molecular mechanisms behind its probiotic action have been well elucidated, and recently, novel potential applications have been demonstrated in animal models. P. freudenreichii strains have been shown to mitigate inflammatory bowel diseases (IBDs) and mucositis and prevent necrotizing enterocolitis (NEC) in newborns. Their immunomodulation capacity has alleviated symptoms of food allergies, obesity, diabetes, colorectal cancer (CRC), and infections. Moreover, P. freudenreichii inhibited osteoclastogenesis in a rheumatoid arthritis model. Most observed effects are mediated by proteins on the cell surface or contained in extracellular vesicles (EVs) such as the surface layer (S-layer) protein SlpB, DlaT, and GroEL. No safety issues have been reported for these bacteria. However, investigations into transferable antibiotic resistance traits are still needed, and clinical trials are required to evaluate their effectiveness as probiotics for humans. Full article
(This article belongs to the Special Issue Prebiotics and Probiotics in Health and Disease: Looking at the Future)
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43 pages, 3064 KiB  
Review
Cardiac Glycosides: From Natural Defense Molecules to Emerging Therapeutic Agents
by Arturo Ponce, Catalina Flores-Maldonado and Ruben G. Contreras
Biomolecules 2025, 15(6), 885; https://doi.org/10.3390/biom15060885 - 17 Jun 2025
Viewed by 533
Abstract
Cardiac glycosides (CGs), a class of plant- and animal-derived compounds historically used to treat heart failure, have garnered renewed interest for their diverse pharmacological properties beyond Na+/K+-ATPase (NKA) inhibition. Recent studies reveal that CGs modulate key signaling pathways—such as [...] Read more.
Cardiac glycosides (CGs), a class of plant- and animal-derived compounds historically used to treat heart failure, have garnered renewed interest for their diverse pharmacological properties beyond Na+/K+-ATPase (NKA) inhibition. Recent studies reveal that CGs modulate key signaling pathways—such as NF-κB, PI3K/Akt, JAK/STAT, and MAPK—affecting processes central to cancer, viral infections, immune regulation, and neurodegeneration. In cancer, CGs induce multiple forms of regulated cell death, including apoptosis, ferroptosis, pyroptosis, and immunogenic cell death, while also inhibiting angiogenesis, epithelial–mesenchymal transition, and cell cycle progression. They demonstrate broad-spectrum antiviral activity by disrupting viral entry, replication, and mRNA processing in viruses such as HSV, HIV, influenza, and SARS-CoV-2. Immunologically, CGs regulate Th17 differentiation via RORγ signaling, although both inhibitory and agonistic effects have been reported. In the nervous system, CGs modulate neuroinflammation, support synaptic plasticity, and improve cognitive function in models of Alzheimer’s disease, epilepsy, and multiple sclerosis. Despite their therapeutic potential, clinical translation is hindered by narrow therapeutic indices and systemic toxicity. Advances in drug design and nanocarrier-based delivery are critical to unlocking CGs’ full potential as multi-target agents for complex diseases. This review synthesizes the current knowledge on the emerging roles of CGs and highlights strategies for their safe and effective repurposing. Full article
(This article belongs to the Section Natural and Bio-derived Molecules)
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17 pages, 989 KiB  
Article
Age Trajectories of O2 Saturation and Levels of Serum Bicarbonate or End-Tidal CO2 Across the Life Course of Women and Men: Insights from EHR and PSG Data
by Leping Li, Min Shi, David M. Umbach and Zheng Fan
Biomolecules 2025, 15(6), 884; https://doi.org/10.3390/biom15060884 - 17 Jun 2025
Viewed by 200
Abstract
To elucidate the changes in gas exchange across the life course, we estimated the age trajectories of O2 saturation, CO2 (as either end-tidal or serum bicarbonate), resting heart rate, and resting respiratory rate from age 2 yr onward in female and [...] Read more.
To elucidate the changes in gas exchange across the life course, we estimated the age trajectories of O2 saturation, CO2 (as either end-tidal or serum bicarbonate), resting heart rate, and resting respiratory rate from age 2 yr onward in female and male patients separately. We utilized two sources’ data: electronic health records (EHR) representing ambulatory visits of approximately 53,000 individuals and sleep clinic polysomnogram (PSG) records representing an additional ~21,000. We used linear regression to estimate age-group-specific mean response levels for women and men. We compared estimated female–male differences between pre- and post-pubertal children and between pre- and post-menopausal periods among adults. Women between 15 and 45 years had higher O2 saturation and lower serum bicarbonate levels or end-tidal CO2 levels than men of similar ages. For O2 saturation and for both measures of CO2, the female–male difference was larger on average among adults at pre-menopausal ages than those at post-menopausal ages. Women had higher O2 saturation throughout their lives than men; however, the difference disappeared in the elderly. Women between menarche and menopause had significantly lower end-tidal CO2 and serum bicarbonate than men of similar ages. After menopause, however, women appeared to have higher mean levels of both end-tidal CO2 and serum bicarbonate than men. Full article
(This article belongs to the Section Biological Factors)
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12 pages, 925 KiB  
Article
Pre-Amplification of Cell-Free DNA: Balancing Amplification Errors with Enhanced Sensitivity
by Wei Yen Chan, Ashleigh Stewart, Russell J. Diefenbach, Elin S. Gray, Jenny H. Lee, Richard A. Scolyer, Georgina V. Long and Helen Rizos
Biomolecules 2025, 15(6), 883; https://doi.org/10.3390/biom15060883 - 17 Jun 2025
Viewed by 355
Abstract
Circulating tumour DNA (ctDNA) is a promising biomarker for personalised oncology. However, its clinical utility is limited by detection sensitivity, particularly in early-stage disease. T-Oligo Primed Polymerase Chain Reaction (TOP-PCR) is a commercial amplification approach utilising an efficient “half-adapter” ligation design and a [...] Read more.
Circulating tumour DNA (ctDNA) is a promising biomarker for personalised oncology. However, its clinical utility is limited by detection sensitivity, particularly in early-stage disease. T-Oligo Primed Polymerase Chain Reaction (TOP-PCR) is a commercial amplification approach utilising an efficient “half-adapter” ligation design and a single-primer-based PCR strategy. This study evaluated the clinical value and application of cell-free DNA (cfDNA) pre-amplification. cfDNA amplification with TOP-PCR preserved DNA size profiles and resulted in a 22 bp size increase due to the half-adaptor ligation. Gene target amplification rates varied, showing lower efficiency for the GC-rich TERT promoter amplicon and higher efficiency for the BRAF and TP53 amplicons. Optimised pre-amplification (20 ng cfDNA input and 5–7 cycles of PCR) enhanced ctDNA detection sensitivity and expanded sample availability for the detection of multiple tumour-informed mutations. Importantly, PCR errors emerged in pre-amplified cfDNA samples, underscoring the necessity for negative controls and the establishment of stringent mutation positivity thresholds. Full article
(This article belongs to the Special Issue Tumor Genomics and Liquid Biopsy in Cancer Biology)
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21 pages, 13615 KiB  
Article
Real-Time SPR Biosensing to Detect and Characterize Fast Dissociation Rate Binding Interactions Missed by Endpoint Detection and Implications for Off-Target Toxicity Screening
by William Martelly, Rebecca L. Cook, Chidozie Victor Agu, Lydia R. Gushgari, Salvador Moreno, Sailaja Kesiraju, Mukilan Mohan and Bharath Takulapalli
Biomolecules 2025, 15(6), 882; https://doi.org/10.3390/biom15060882 - 17 Jun 2025
Viewed by 327
Abstract
Accurate detection of biomolecular interactions is essential in many areas, from the detection of the presence of biomarkers in the clinic to the development of therapeutic drugs and biologics in biopharma to the understanding of various biological processes in basic research. Traditional endpoint [...] Read more.
Accurate detection of biomolecular interactions is essential in many areas, from the detection of the presence of biomarkers in the clinic to the development of therapeutic drugs and biologics in biopharma to the understanding of various biological processes in basic research. Traditional endpoint approaches can suffer from false-negative results for biomolecular interactions with fast kinetics. By contrast, real-time detection techniques like surface plasmon resonance (SPR) monitor interactions as they form and disassemble, reducing the risk of false-negative results. By leveraging cell-free expressed proteins captured on either glass or SPR biosensors and using two different commercial antibodies with variable off-rates that both target HaloTag antigens as a model, we compare and contrast results from a fluorescence endpoint assay versus real-time sensor-integrated proteome on chip (SPOC®) SPR-based detection. In this study, we illustrate the limitations of the representative immunofluorescent endpoint assay when investigating transient interactions characterized by fast dissociation rates. We highlight the importance of choosing reagents well suited to the selected assay, as well as the importance of considering binding kinetics and protein ligand conformational states when interpreting results from binding assays, especially for applications as critical as the off-target screening of therapeutics. Full article
(This article belongs to the Section Molecular Biophysics: Structure, Dynamics, and Function)
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30 pages, 3197 KiB  
Review
LncRNA–Protein Interactions: A Key to Deciphering LncRNA Mechanisms
by Zuoneng Wang, Muhammad Aftab, Zigang Dong, Yanan Jiang and Kangdong Liu
Biomolecules 2025, 15(6), 881; https://doi.org/10.3390/biom15060881 - 17 Jun 2025
Viewed by 530
Abstract
Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators in a multitude of biological processes. However, their functional basis, particularly structure-based functional characteristics, remains elusive. lncRNAs exert their influence primarily through intricate interactions with various cellular components. Among these, interactions with proteins have [...] Read more.
Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators in a multitude of biological processes. However, their functional basis, particularly structure-based functional characteristics, remains elusive. lncRNAs exert their influence primarily through intricate interactions with various cellular components. Among these, interactions with proteins have garnered increasing attention. Recent research highlights the significance of the interactions with proteins as a plausible mechanism underlying lncRNA functions. Here, we delve into the interactions between lncRNAs and RNA-binding proteins (RBPs), explore their implications in cellular processes, and examine bioinformatic and experimental approaches for characterizing these interactions. We introduce an innovative ISD strategy to decipher the mysterious mechanism of lncRNAs. Through reviewing the recent advances in the study of proteins and their complexes, we incorporate the ISD strategy into our integrated structural analysis pipeline for comprehensively understanding the structure-function relationship of lncRNAs. Advances in the development of innovative therapeutic approaches based on lncRNA-protein interactions (LPIs) are reviewed accordingly. Full article
(This article belongs to the Section Molecular Biophysics: Structure, Dynamics, and Function)
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19 pages, 790 KiB  
Review
Not Just PA28γ: What We Know About the Role of PA28αβ in Carcinogenesis
by Paolo Cascio
Biomolecules 2025, 15(6), 880; https://doi.org/10.3390/biom15060880 - 16 Jun 2025
Viewed by 214
Abstract
The ubiquitin-proteasome pathway performs a strictly controlled degradation of specific protein substrates within the eukaryotic cell. This catabolic mechanism allows the rapid removal of proteins damaged in any way, and therefore potentially capable of compromising cellular homeostasis, as well as the constant turnover [...] Read more.
The ubiquitin-proteasome pathway performs a strictly controlled degradation of specific protein substrates within the eukaryotic cell. This catabolic mechanism allows the rapid removal of proteins damaged in any way, and therefore potentially capable of compromising cellular homeostasis, as well as the constant turnover of all cellular proteins, in order to balance their synthesis and thus maintain the correct levels of proteins required by the cell at any given time. Consequently, the ubiquitin-proteasome system plays a fundamental role in regulating essential cellular processes, such as the cell cycle, apoptosis, immune responses, and inflammation, whose dysregulation or malfunction can lead to neoplastic transformation. Not surprisingly, therefore, alterations in the activity and regulatory mechanisms of the proteasome are common not only in various types of tumors, but often represent a contributing cause of oncogenesis itself. Among proteasome modulators, PA28γ, due to its function in promoting cell growth and proliferation, while inhibiting apoptosis and cell-mediated immune responses, has received great attention in recent years for its well established pro-tumoral activity. Conversely, the role played in oncogenesis by the second paralogue of the PA28 family of proteasome activators, namely PA28αβ, is less clearly defined, which is also related to the lower level of general understanding of its cellular activities and biological functions. However, increasing experimental evidence has demonstrated that PA28αβ also plays a non-secondary role in the process of neoplastic transformation and tumor growth, both by virtue of its regulatory function on class I cell-mediated immune responses and through activity promoting cell duplication and growth. This review aims to summarize the current knowledge and evidence on the molecular mechanisms and cellular functions through which PA28αβ may support development and growth of cancer. Full article
(This article belongs to the Special Issue Mechanisms, Biology and Regulation of Proteasomes—in Memory of Professor Alfred L. Goldberg (1942–2023))
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37 pages, 4082 KiB  
Review
Probiotics and Cancer: Mechanistic Insights and Organ-Specific Impact
by Md Faruque Ahmad, Fakhruddin Ali Ahmad, Abdulrahman A. Alsayegh, Md. Zeyaullah, Ahmad O. Babalghith, Hani Faidah, Faiyaz Ahmed, Anjum Khanam, Boshra Mozaffar, Nahla Kambal and Farkad Bantun
Biomolecules 2025, 15(6), 879; https://doi.org/10.3390/biom15060879 - 16 Jun 2025
Viewed by 612
Abstract
Probiotics have been revealed in various studies to modulate the gut microbiome and have a substantial impact on cancers, comprising oesophageal, lung, liver, and colorectal cancer. These properties are endorsed by a diverse mechanism, including the modulation of the gut microbiome; preventing the [...] Read more.
Probiotics have been revealed in various studies to modulate the gut microbiome and have a substantial impact on cancers, comprising oesophageal, lung, liver, and colorectal cancer. These properties are endorsed by a diverse mechanism, including the modulation of the gut microbiome; preventing the metabolism of carcinogenic substances; exertion of anti-inflammatory action, immunopotentiator properties, and antioxidant activities; prevention of tumour growth; and decreasing the adverse effects of chemotherapy. There are promising perspectives regarding the new and developing field of probiotic research in relation to cancer treatment. This review demonstrates the recent findings of probiotics efficacy in cancer prevention and treatment and organ-specific impact along with protection from chemotherapy-induced side effects. The present evidence specifies that strategic probiotics application may be an effective complementary approach for the management of numerous kinds of cancer; still, additional studies and clinical trials are required to comprehend the relationships between cancer and probiotics. Full article
(This article belongs to the Section Natural and Bio-derived Molecules)
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20 pages, 3969 KiB  
Article
Analysis of Novel DNA Adducts Derived from Acetaldehyde
by Yuuki Betsuyaku, Mina Motohashi, Akira Sassa, Takeji Takamura-Enya and Yukari Totsuka
Biomolecules 2025, 15(6), 878; https://doi.org/10.3390/biom15060878 - 16 Jun 2025
Viewed by 273
Abstract
Alcohol consumption is a known risk factor for esophageal and liver cancers. Recently, it was reported that mutation signatures characterized by T:A to C:G mutations (SBS16), which are suggested to be associated with alcohol intake, are frequently detected in esophageal, liver, and stomach [...] Read more.
Alcohol consumption is a known risk factor for esophageal and liver cancers. Recently, it was reported that mutation signatures characterized by T:A to C:G mutations (SBS16), which are suggested to be associated with alcohol intake, are frequently detected in esophageal, liver, and stomach cancers among the Japanese population. However, the scientific evidence linking alcohol consumption to SBS16 remains lacking. Acetaldehyde (AA), a carcinogenic metabolite of alcohol, is considered a key contributor to alcohol-related cancer development. Although the guanine adducts associated with alcohol exposure have been reported as part of its carcinogenic mechanism, an adenine adduct, N6-ethyl-deoxyadenosine (N6-ethyl-dA), a potential contributor to the SBS16 mutation pattern, was recently identified using a mass spectrometry-based DNA adductome approach. However, the mutagenicity assessment of N6-ethyl-dA using primer extension assays and the supF gene mutation test showed that this adenine adduct is not mutagenic. To identify another candidate as a driver adduct for SBS16, a DNA adductome approach was conducted, leading to the identification of a novel adenine adduct, 3-(2′-deoxyribos-1′-yl)-7,9-dimethyl-3,9-dihydro-7H-[1,3,5]oxadiazino[4,3-i]purine (N1-oxydiethylidene-dA), in which two AA molecules are bound to an adenine base. Moreover, N1-oxydiethylidene-dA was detected in mouse livers, and its levels increased following ethanol administration, suggesting that alcohol may contribute to SBS16 induction via the formation of N1-oxydiethylidene-dA. Full article
(This article belongs to the Special Issue Recent Advances in Adduct Science)
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14 pages, 1314 KiB  
Article
Analytical and Clinical Validation of a Plasma Fibroblast Growth Factor 21 ELISA Kit Using an Automated Platform in Steatotic Liver Disease
by Makito Tanaka, Shingo Tanaka, Ryo Kobayashi, Ryosei Murai and Satoshi Takahashi
Biomolecules 2025, 15(6), 877; https://doi.org/10.3390/biom15060877 - 16 Jun 2025
Viewed by 225
Abstract
Steatotic liver disease is a global health challenge that requires reliable and noninvasive diagnostic biomarkers. This research aimed to validate the analytical and clinical performance of a fibroblast growth factor 21 (FGF21) enzyme-linked immunosorbent assay (ELISA) kit using an automated immunoassay analyzer. Plasma [...] Read more.
Steatotic liver disease is a global health challenge that requires reliable and noninvasive diagnostic biomarkers. This research aimed to validate the analytical and clinical performance of a fibroblast growth factor 21 (FGF21) enzyme-linked immunosorbent assay (ELISA) kit using an automated immunoassay analyzer. Plasma FGF21 levels were measured using a commercial ELISA kit on an automated immunoassay analyzer. Validation included intra- and inter-assay precision, dilution linearity, spike recovery, lower limit of quantification (LLOQ), interference testing, and sample stability analysis. Clinical evaluation involved 97 patients who underwent abdominal ultrasound-based attenuation imaging for the diagnosis of hepatic steatosis. The assay demonstrated high analytical precision, with intra- and inter-assay coefficients of variation <15% and an LLOQ of 3.260 pg/mL. Dilution linearity, spike recovery, and interference tests confirmed the reliability of the assay, whereas stability tests highlighted the minimal effect of freeze-thaw cycles and storage conditions. Clinically, FGF21 levels correlated with attenuation coefficient (r = 0.44). Diagnostic performance indicated 84% sensitivity and 81% specificity at defined FGF21 thresholds for the diagnosis of hepatic steatosis. This research confirmed the reliable analytical and clinical performance of the FGF21 ELISA kit, reinforcing its potential as a diagnostic biomarker of hepatic steatosis. Full article
(This article belongs to the Section Molecular Biomarkers)
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25 pages, 1271 KiB  
Article
New Insights into the Sex Chromosome Evolution of the Common Barker Frog Species Complex (Anura, Leptodactylidae) Inferred from Its Satellite DNA Content
by Lucas H. B. Souza, Juan M. Ferro, Helena M. Milanez, Célio F. B. Haddad and Luciana B. Lourenço
Biomolecules 2025, 15(6), 876; https://doi.org/10.3390/biom15060876 - 16 Jun 2025
Viewed by 362
Abstract
Satellite DNAs (satDNAs) play a crucial role in understanding chromosomal evolution and the differentiation of sex chromosomes across diverse taxa, particularly when high karyotypic diversity occurs. The Physalaemus cuvieri–Physalaemus ephippifer species complex comprises at least seven divergent lineages, each exhibiting specific karyotypic signatures. [...] Read more.
Satellite DNAs (satDNAs) play a crucial role in understanding chromosomal evolution and the differentiation of sex chromosomes across diverse taxa, particularly when high karyotypic diversity occurs. The Physalaemus cuvieri–Physalaemus ephippifer species complex comprises at least seven divergent lineages, each exhibiting specific karyotypic signatures. The group composed of Ph. ephippifer, Lineage 1B of ‘Ph. cuvieri’ (L1B), and a lineage resulting from their secondary contact is especially intriguing due to varying degrees of sex chromosome heteromorphism. In this study, we characterized the satellitome of Ph. ephippifer in order to identify novel satDNAs that may provide insights into chromosomal evolution, particularly concerning sex chromosomes. We identified 62 satDNAs in Ph. ephippifer, collectively accounting for approximately 10% of the genome. Notably, nine satDNA families were shared with species from distantly related clades, raising questions about their potential roles in anurans genomes. Among the seven satDNAs mapped via fluorescent in situ hybridization, PepSat3 emerged as a strong candidate for the centromeric sequence in this group. Additionally, PepSat11 and PepSat24 provided evidence supporting a translocation involving both arms of the W chromosome in Ph. ephippifer. Furthermore, a syntenic block composed of PepSat3, PcP190, and PepSat11 suggested an inversion event during the divergence of Ph. ephippifer and L1B. The variation in signal patterns of satDNAs associated with nucleolar organizer regions (NORs) highlights the complexity of NOR evolution in this species complex, which exhibits substantial diversity in this genomic region. Additionally, our findings for PepSat30-350 emphasize the importance of validating the sex-biased abundance of satDNAs. Full article
(This article belongs to the Special Issue Molecular Insights into Sex and Evolution)
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