Biomolecules

22 pages, 1018 KB

Open AccessReview

Molecular Pathogenesis of Arrhythmogenic Cardiomyopathy: Mechanisms and Therapeutic Perspectives

by Eliza Popa and Sorin Hostiuc

Biomolecules 2025, 15(11), 1512; https://doi.org/10.3390/biom15111512 (registering DOI) - 25 Oct 2025

Arrhythmogenic cardiomyopathy (ACM) is a genetic cardiac disease characterized by a progressive loss of cardiomyocytes associated with fibrofatty myocardial replacement, resulting in a heightened risk of ventricular arrhythmias and sudden cardiac death. ACM is a common cause of sudden death in young individuals, [...] Read more.

Arrhythmogenic cardiomyopathy (ACM) is a genetic cardiac disease characterized by a progressive loss of cardiomyocytes associated with fibrofatty myocardial replacement, resulting in a heightened risk of ventricular arrhythmias and sudden cardiac death. ACM is a common cause of sudden death in young individuals, and exercise has been proven to be a factor in disease progression. Current therapeutic strategies, including lifestyle modification, antiarrhythmic pharmacological therapy, catheter ablation, and the placement of implantable cardioverter-defibrillators, remain primarily palliative options rather than addressing the underlying molecular substrate. The pathogenesis of ACM includes complex molecular and cellular mechanisms, linking genetic mutations to structural and electrical anomalies of the ventricle. The lack of targeted therapies contributes to a challenging approach to the disease. It highlights the need for a better understanding of the mechanisms that lead to myocardial remodeling and arrhythmic predisposition. With the help of animal models (especially murine) and induced pluripotent stem cells, there have been advances in understanding the molecular pathogenesis of ACM. In this review, we summarized some of the pathogenic molecular pathways involved in the development of ACM and emerging therapies targeted towards disease modification, not just prevention. Full article

(This article belongs to the Special Issue Cellular and Molecular Mechanisms in Cardiopathy and Therapeutic Strategies)

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18 pages, 4018 KB

Open AccessArticle

A Rapid, High-Throughput Method for the Construction of Mutagenesis Libraries

by Yuxin Lu, Shuting Meng, Xinyi Guan, Pengying He and Dongxin Zhao

Biomolecules 2025, 15(11), 1511; https://doi.org/10.3390/biom15111511 (registering DOI) - 25 Oct 2025

Abstract

As synthetic biology advances toward precise design, the construction of high-quality mutant libraries has become essential for large-scale functional screening. Traditional approaches, such as random and saturation mutagenesis, often suffer from low accuracy, high bias, and limited coverage. An ideal method should offer [...] Read more.

As synthetic biology advances toward precise design, the construction of high-quality mutant libraries has become essential for large-scale functional screening. Traditional approaches, such as random and saturation mutagenesis, often suffer from low accuracy, high bias, and limited coverage. An ideal method should offer controlled mutagenesis, comprehensive coverage, high throughput, operational simplicity, and controllable outcomes, enabling effective large-scale screening. Here, we developed a high-throughput, precisely controlled method for constructing a mutagenesis library based on chip-based oligonucleotide synthesis. Using PSMD10 as a model, we constructed a full-length amber codon scanning mutagenesis library with 93.75% mutation coverage. Among the five polymerases evaluated, KAPA HiFi HotStart, Platinum SuperFi II and Hot-Start Pfu DNA Polymerase demonstrated higher amplification efficiency and lower chimera formation rates, making them preferred enzymes for optimized library construction. Analysis of unmapped reads highlighted key technical factors, such as oligonucleotide synthesis errors and chimeric sequence formation caused by incomplete extension of DNA polymerase or synthesis across discontinuous templates during PCR. To improve efficiency and fidelity, we recommend refining PCR conditions and strengthening oligo synthesis quality control. We establish an efficient, scalable, precisely controlled mutagenesis library construction strategy tailored for high-throughput functional research and recommend using a high-fidelity, low-bias polymerase to ensure quality. Full article

(This article belongs to the Section Molecular Biology)

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14 pages, 503 KB

Open AccessArticle

Recreational Exercise and Inflammatory Patterns in Hashimoto’s Thyroiditis: Observations from a Cross-Sectional Study

by Marko Vuletić, Vanna Žnidar, Ana Barić Žižić, Sanda Sladić, Dean Kaličanin, Vesela Torlak Lovrić, Maja Cvek, Ante Punda and Vesna Boraska Perica

Biomolecules 2025, 15(11), 1510; https://doi.org/10.3390/biom15111510 (registering DOI) - 25 Oct 2025

Abstract

In this cross-sectional observational study, we investigated whether recreational exercise (RE) influences systemic inflammation in Hashimoto’s thyroiditis (HT) across different disease severity groups. We analyzed 403 participants from the Croatian Biobank of Patients with HT (CRO-HT), including 173 controls and 230 HT patients [...] Read more.

In this cross-sectional observational study, we investigated whether recreational exercise (RE) influences systemic inflammation in Hashimoto’s thyroiditis (HT) across different disease severity groups. We analyzed 403 participants from the Croatian Biobank of Patients with HT (CRO-HT), including 173 controls and 230 HT patients (euthyroid, levothyroxine [LT4]-treated, and hypothyroid). Serum levels of 92 inflammatory proteins were measured using the Olink^® Target 96 Inflammation panel, and exercise status was assessed via structured questionnaires. Linear regression revealed distinct protein associations depending on thyroid status. In controls, RE was associated with reduced MMP-10 and FGF-5, reflecting cardiovascular and muscle benefits. In euthyroid patients, RE was associated with decreased CXCL9 and TRAIL, implicating reduced type 1 inflammation and vascular risk. LT4-treated patients showed increases in IL-15RA and IL-24 with RE, suggesting improved muscle metabolism and anti-inflammatory effects. In hypothyroid patients, RE was associated with reduced CCL20 and increased HGF, while changes in TRANCE and TWEAK indicated mixed effects on bone and immune regulation. Notably, RE was associated with reduced CXCL9 and CCL20, two proteins previously linked to HT risk. Overall, RE is associated with distinct changes in inflammatory profiles across HT disease severity groups, with the most favourable responses observed in LT4-treated patients, suggesting synergy with hormone therapy. Full article

(This article belongs to the Special Issue Biomolecular Insights into Exercise-Induced Modulation of Immunometabolism)

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14 pages, 1659 KB

Open AccessArticle

Regulation of Klotho Production by Mineralocorticoid Receptor Signaling in Renal Cell Lines

by Elena Kohm, Martina Feger and Michael Föller

Biomolecules 2025, 15(11), 1509; https://doi.org/10.3390/biom15111509 (registering DOI) - 25 Oct 2025

Abstract

Through the mineralocorticoid receptor, aldosterone controls extracellular volume and arterial blood pressure by stimulating Na⁺ absorption and K⁺ secretion in epithelial cells of the kidney, colon, and several glands. Hyperaldosteronism promotes fibrosis and inflammation in epithelial and non-epithelial tissues, thereby favoring [...] Read more.

Through the mineralocorticoid receptor, aldosterone controls extracellular volume and arterial blood pressure by stimulating Na⁺ absorption and K⁺ secretion in epithelial cells of the kidney, colon, and several glands. Hyperaldosteronism promotes fibrosis and inflammation in epithelial and non-epithelial tissues, thereby favoring loss of kidney and heart function. Mineralocorticoid receptor blockade therefore gains relevance especially in renal and cardiac disease. Kidney-derived Klotho is a powerful anti-aging protein with anti-fibrosis and anti-inflammatory effects providing cardio- and nephroprotection. We wondered whether Klotho expression and production is influenced by mineralocorticoid receptor agonists and antagonists. Using four renal cell lines, Madin-Darby canine kidney (MDCK), normal rat kidney, subtype 52E (NRK-52E), human kidney 2 (HK2) cells, and primary renal proximal tubule epithelial cells (RPTECs), and the four most frequently prescribed mineralocorticoid receptor blockers, spironolactone, eplerenone, finerenone, and esaxerenone, we assessed Klotho gene expression by qRT-PCR and Klotho protein by Western blotting. Aldosterone and eplerenone did not significantly affect Klotho expression in either cell line. Spironolactone enhanced Klotho expression in MDCK and NRK-52E cells and downregulated Klotho in HK2 cells and RPTECs. Novel non-steroidal mineralocorticoid receptor antagonist finerenone downregulated Klotho expression in MDCK, NRK-52E, and low-dose finerenone in HK2 cells. To conclude, common mineralocorticoid receptor antagonists are characterized by highly diverse effects on Klotho in four renal cell lines. Further studies are needed to define the role of mineralocorticoid receptor blockade for Klotho production. Full article

(This article belongs to the Special Issue New Insights into Autacoids in Disease)

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15 pages, 756 KB

Open AccessReview

Fab N-Glycosylation in IgG: Implications in Physiological and Pathological Immune Regulation

by Shuqi Chen, Feiyuan Yu, Binliang Huang, Ganbo Liang, Jieyi Xu, Yuning Lin and Qian Xu

Biomolecules 2025, 15(11), 1508; https://doi.org/10.3390/biom15111508 (registering DOI) - 25 Oct 2025

Abstract

Compared with classical Fc N-glycosylation, Fab N-glycosylation displays site heterogeneity and structural diversity. It contributes to immune regulation by modulating antibody stability, half-life, and antigen-binding activity, as well as by mediating blocking antibody effects. This review highlights the expression patterns and potential mechanisms [...] Read more.

Compared with classical Fc N-glycosylation, Fab N-glycosylation displays site heterogeneity and structural diversity. It contributes to immune regulation by modulating antibody stability, half-life, and antigen-binding activity, as well as by mediating blocking antibody effects. This review highlights the expression patterns and potential mechanisms of Fab N-glycosylated IgG in autoimmune diseases, pregnancy-induced immune tolerance, and tumor immune evasion, and discusses its structural and functional similarities to IgG4. Although Fab N-glycosylation plays an important role in both physiological and pathological conditions, the complexity of its glycan structures and variability in glycosylation sites hinder a precise understanding of its functional impacts. Clarifying these aspects is expected to emerge as a major focus in glycomics and antibody engineering research. Full article

(This article belongs to the Special Issue Advances in Cancer and Glycosylation)

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17 pages, 3259 KB

Open AccessArticle

Engineering a Thermostable Reverse Transcriptase for RT-PCR Through Rational Design of Pyrococcus furiosus DNA Polymerase

by Aleksandra A. Kuznetsova, Irina A. Grishina, Elena S. Mikushina and Nikita A. Kuznetsov

Biomolecules 2025, 15(11), 1507; https://doi.org/10.3390/biom15111507 (registering DOI) - 24 Oct 2025

Abstract

Engineering of a bifunctional enzyme that combines DNA-dependent DNA polymerase and reverse transcriptase (RT) activities is a highly promising biotechnological goal, as it would enable one-enzyme RT-PCR. For this purpose, we selected the high-fidelity Pyrococcus furiosus (Pfu) DNA polymerase as engineering scaffold. The [...] Read more.

Engineering of a bifunctional enzyme that combines DNA-dependent DNA polymerase and reverse transcriptase (RT) activities is a highly promising biotechnological goal, as it would enable one-enzyme RT-PCR. For this purpose, we selected the high-fidelity Pyrococcus furiosus (Pfu) DNA polymerase as engineering scaffold. The selection of amino acid residues for replacement was carried out based on a multi-sequence alignment of diverse DNA polymerases and literature data, which allowed us to target amino acids, which presumably are triggers of the RT activity appearance. Six mutant variants of the Pfu enzyme were created and their activity was analyzed. Through enzymatic screening, we identified the Pfu-M6 variant, which exhibits dual DNA-dependent and RNA-dependent DNA polymerase activity. This thermostable enzyme retains its inherent DNA polymerase function and has acquired the ability to catalyze reverse transcription under standard PCR conditions, which allows the created mutant form to be used for efficient amplification of DNA starting from an RNA template. Full article

(This article belongs to the Special Issue Exploring Thermophilic Enzymes: From Structural Foundations to Functional Enhancement)

28 pages, 3218 KB

Open AccessReview

The Metabolic Regulation of the NKG2D-Positive NK and T Cells and Their Role in Disease Progression

by Jiayi Tang, Yaqi Lu, Min Chen, Qifan Wu, Yifei Li, Yingqiao Qin, Shaomei Liang, Sulan Luo and Kunpeng Liu

Biomolecules 2025, 15(11), 1506; https://doi.org/10.3390/biom15111506 (registering DOI) - 24 Oct 2025

Abstract

Natural killer (NK) cells are the main cytotoxic lymphocytes of the natural immune system, which play an important role in tumor immune surveillance and anti-viral response. The surface receptor NKG2D can recognize NKG2D ligands on the surface of tumor or metabolism-stressed cells, thereby [...] Read more.

Natural killer (NK) cells are the main cytotoxic lymphocytes of the natural immune system, which play an important role in tumor immune surveillance and anti-viral response. The surface receptor NKG2D can recognize NKG2D ligands on the surface of tumor or metabolism-stressed cells, thereby activating immune responses and mediating cytotoxicity and anti-tumor activity of NK cells. However, NKG2D-positive NK cells are regulated by metabolites, and play a negative role in metabolic diseases. Various metabolites, including lipids, reactive oxygen species (ROS), glucose and amino acids, regulate NKG2D expression and NK cell activity and decide the immune microenvironment of pathological tissue. Thus, targeted therapies based on NKG2D-positive NK cell have entirely different strategies in the treatment of tumor or metabolic diseases. This article focuses on the metabolic regulation of NKG2D-positive NK cells and their opposite roles in disease progression, including of cancer and metabolic disease. In the future, in-depth studies of the regulatory mechanisms of the NKG2D signaling pathway by metabolites and the optimization of the safety and efficacy of targeted therapeutic strategies will lead to new breakthroughs in the treatment of tumors and metabolic diseases, providing patients with more effective treatment options. Full article

(This article belongs to the Section Biological Factors)

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24 pages, 1012 KB

Open AccessReview

The Role of Imaging in Monitoring Large Vessel Vasculitis: A Comprehensive Review

by Inês Sopa, Roberto Pereira da Costa, Joana Martins Martinho and Cristina Ponte

Biomolecules 2025, 15(11), 1505; https://doi.org/10.3390/biom15111505 (registering DOI) - 24 Oct 2025

Abstract

Giant cell arteritis (GCA) and Takayasu arteritis (TAK) are forms of primary large vessel vasculitis (LVV) affecting the aorta and its major branches. Timely diagnosis and accurate monitoring are essential to prevent irreversible damage. Current assessment strategies rely heavily on symptoms, physical examination, [...] Read more.

Giant cell arteritis (GCA) and Takayasu arteritis (TAK) are forms of primary large vessel vasculitis (LVV) affecting the aorta and its major branches. Timely diagnosis and accurate monitoring are essential to prevent irreversible damage. Current assessment strategies rely heavily on symptoms, physical examination, and inflammatory markers, which lack sensitivity and specificity, particularly in patients treated with IL-6 inhibitors. This narrative review provides a comprehensive overview of the role of imaging in monitoring LVV. Ultrasound, magnetic resonance imaging, and positron emission tomography better reflect disease activity and treatment response compared to conventional clinical and laboratory measures. Notably, emerging imaging-based tools such as the OMERACT GCA Ultrasound Score, the Takayasu Ultrasound Index, and the TAK Integrated Disease Activity Index (TAIDAI) are promising treat-to-target instruments. While computed tomography is primarily used to assess structural damage, conventional angiography now plays a more limited role, mainly reserved for procedural planning and haemodynamic evaluation. A key challenge remains: interpreting persistent vascular abnormalities, which may indicate active disease, vascular remodelling, or irreversible damage. Standardisation of imaging protocols and interpretation is needed, alongside further research on the prognostic value of imaging for relapse risk. This review supports a multimodal, patient-tailored approach in which imaging is central to the long-term management of LVV. Full article

(This article belongs to the Special Issue Biomarkers for Vascular Disease II)

21 pages, 10601 KB

Open AccessArticle

Hypoxia-Induced Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Regulate Macrophage Polarization and Enhance Angiogenesis to Promote Diabetic Wound Healing

by Yongfeng Su, Junda Lu, Feiyuan Liang and Jianwen Cheng

Biomolecules 2025, 15(11), 1504; https://doi.org/10.3390/biom15111504 (registering DOI) - 24 Oct 2025

Abstract

Background: Diabetic wound healing has always been a clinical challenge with minimal response or efficacy to standard treatment. This study aims to assess the therapeutic potential of hypoxia-induced extracellular vesicles (hy-EVs) produced by human umbilical cord mesenchymal stem cells (HUCMSCs) to treat [...] Read more.

Background: Diabetic wound healing has always been a clinical challenge with minimal response or efficacy to standard treatment. This study aims to assess the therapeutic potential of hypoxia-induced extracellular vesicles (hy-EVs) produced by human umbilical cord mesenchymal stem cells (HUCMSCs) to treat diabetic wounds. Methods: HUCMSCs were isolated from umbilical cord tissue, cultured under hypoxic conditions to induce the release of extracellular vesicles (EVs) and compared with normoxia-induced extracellular vesicles (n-EVs). We assessed the functions of hy-EVs on human skin fibroblasts (HSFs) and human umbilical vein endothelial cells (HUVECs) in vitro. Simultaneously, we analyzed the pro-angiogenic effects of hy-EVs, their effects on macrophage polarization, and their ability to scavenge endogenous reactive oxygen species (ROS). In addition, a diabetic wound model was established to assess the curative effect of hy-EVs in diabetic wound healing. Results: We found by in vitro study that hy-EVs markedly improved the functional activities of HSFs, thus significantly promoting wound repair. Remarkably, it was determined that hy-EVs greatly enhanced the proliferation and migration ability as well as the angiogenic ability of HUVECs, while promoting the expression of hypoxia-inducible factor-1α (HIF-1α), vascular endothelial-generation-associated factor A (VEGFA), and platelet endothelial adhesion molecule (CD31), which suggested that hy-EVs can effectively activate the HIF-1α pathway to promote angiogenesis. Above all, we found that hy-EVs promoted the expression of CD206 while decreasing the expression of CD86, suggesting that hy-EVs could induce macrophages to shift from M1-type (pro-inflammatory) to M2-type (anti-inflammatory), thereby modulating the inflammatory response. Additionally, hy-EVs inhibited ROS production in both HSFs and HUVECs to reduce oxidative stress. In vivo results showed that hy-EVs enhanced collagen deposition and angiogenesis, modulated macrophage polarization, and inhibited immune response at the wound spot, which significantly enhanced diabetic wound healing. Conclusions: Our study shows that hy-EVs significantly promote angiogenesis through activation of the HIF-1α pathway, modulate macrophage polarization and attenuate cellular oxidative stress, possibly through delivery of specific miRNAs and proteins. Our discoveries offer a key theoretical basis and potential application to develop novel therapeutic strategies against diabetes-related tissue injury. Full article

(This article belongs to the Section Molecular Medicine)

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18 pages, 2809 KB

Open AccessSystematic Review

The Prognostic Role of STAT5B Across Cancer Types and Comparative Analysis with STAT5A: A Systematic Review

by Christine Maninang, Jinghong Li and Willis X. Li

Biomolecules 2025, 15(11), 1503; https://doi.org/10.3390/biom15111503 (registering DOI) - 24 Oct 2025

Abstract

Background: The signal transducer and activator of transcription 5 (STAT5) proteins, STAT5A and STAT5B, are highly homologous transcription factors with distinct roles in cancer biology. While STAT5A has been characterized as a context-dependent modulator of tumor progression, the prognostic significance of STAT5B remains [...] Read more.

Background: The signal transducer and activator of transcription 5 (STAT5) proteins, STAT5A and STAT5B, are highly homologous transcription factors with distinct roles in cancer biology. While STAT5A has been characterized as a context-dependent modulator of tumor progression, the prognostic significance of STAT5B remains less clear. Here, we conducted a systematic meta-analysis of STAT5B to evaluate its association with overall survival across cancers and to compare its prognostic role with that of STAT5A, as reported previously. Methods: Microarray datasets from the Prognoscan database were analyzed for STAT5B expression and overall survival. Hazard ratios (HRs) were estimated using Cox proportional hazards models, and results from 42 datasets were synthesized by meta-analysis. Subgroup analyses were performed by cancer type, and heterogeneity was assessed using Cochran’s Q Test and I² statistics. Results: Pooled analysis showed that high STAT5B expression was significantly associated with favorable overall survival (lnHR = −0.4009; 95% CI: −0.6007 to −0.2011; p < 0.0001), albeit with notable heterogeneity (I² = 64%). Subgroup analyses indicated that STAT5B was particularly protective in lung cancers (lnHR = −0.5170; p = 0.0042) and hematologic malignancies (lnHR = −0.6988; p < 0.0001). In contrast, STAT5A demonstrated divergent effects, conferring favorable survival in breast cancer but poorer outcomes in hematologic cancers. Conclusions: Elevated STAT5B expression is associated with improved survival in multiple cancers, supporting a potential tumor-suppressive role distinct from STAT5A. These findings underscore the importance of isoform-specific STAT5 evaluation in cancer prognosis and suggest that STAT5B may serve as a potential biomarker and therapeutic target. Full article

(This article belongs to the Section Bioinformatics and Systems Biology)

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20 pages, 16153 KB

Open AccessArticle

STAMBP Accelerates Progression and Tamoxifen Resistance of Breast Cancer Through Deubiquitinating ERα

by Zhihuai Wang, Likai Gu, Mei Yang, Yi Zhou, Xihu Qin and Chen Xiong

Biomolecules 2025, 15(11), 1502; https://doi.org/10.3390/biom15111502 (registering DOI) - 24 Oct 2025

Abstract

Breast cancer (BRCA) remains a global health burden, with endocrine-resistant ER-positive BRCA posing therapeutic challenges. This study investigates STAMBP’s role in breast cancer progression and evaluates its potential as a therapeutic target. Through siRNA library screening in ER-positive cell lines, we identified STAMBP [...] Read more.

Breast cancer (BRCA) remains a global health burden, with endocrine-resistant ER-positive BRCA posing therapeutic challenges. This study investigates STAMBP’s role in breast cancer progression and evaluates its potential as a therapeutic target. Through siRNA library screening in ER-positive cell lines, we identified STAMBP as a key regulator of ERα signaling and observed its upregulation in BRCA samples. (fold changes > 2, sample sizes = 30, p < 0.001), particularly in ER-positive subtypes. Prognostic analysis demonstrated that STAMBP overexpression correlates with poor clinical outcomes in ER-positive BRCA patients (p < 0.05). In vitro functional assays showed STAMBP promoted proliferation, metastasis, and epithelial–mesenchymal transition of ER-positive cells by regulating the activity of ERα signaling. Mechanistically, the deubiquitinase STAMBP directly reduces the K48-linked polyubiquitination levels of ERα, enhancing its protein stability and activating downstream oncogenic signaling. STAMBP knockdown restored tamoxifen sensitivity in endocrine-resistant BRCA cells by reducing ERα stability. This study has certain limitations, including the absence of pharmacological validation and reliance on small, single-center clinical cohorts, which should be addressed in future research to further substantiate the clinical relevance of targeting STAMBP in BRCA. Collectively, our findings identified STAMBP as a prognostic marker and demonstrated its dual role in driving ER-positive BRCA malignancy and mediating endocrine resistance. Targeting STAMBP may represent an innovative approach to improve endocrine therapeutic efficacy in ER-positive BRCA. Full article

(This article belongs to the Special Issue Breast Cancer: Molecular Pathology, Drug Resistance and Novel Treatment Strategies)

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28 pages, 770 KB

Open AccessReview

Leveraging Artificial Intelligence and Modulation of Oxidative Stressors to Enhance Healthspan and Radical Longevity

by Donald D. Haines, Stephen Christopher Rose, Fred M. Cowan, Fadia F. Mahmoud, Albert A. Rizvanov and Arpad Tosaki

Biomolecules 2025, 15(11), 1501; https://doi.org/10.3390/biom15111501 (registering DOI) - 24 Oct 2025

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Abstract

This review explores the transformative potentials of artificial intelligence (AI) in promoting healthspan and longevity. Healthspan focuses on enhancing quality of life free from chronic conditions, while longevity defines current lifespan limits within a particular species and encompasses biological aging at multiple levels. [...] Read more.

This review explores the transformative potentials of artificial intelligence (AI) in promoting healthspan and longevity. Healthspan focuses on enhancing quality of life free from chronic conditions, while longevity defines current lifespan limits within a particular species and encompasses biological aging at multiple levels. AI methodologies—including machine learning, deep learning, natural language processing, robotics, and data analytics—offer unprecedented tools to analyze complex biological data, accelerate biomarker discovery, optimize therapeutic interventions, and personalize medicine. Notably, AI has facilitated breakthroughs in identifying accurate biomarkers of biological age, developing precision medicine approaches, accelerating drug discovery, and enhancing genomic editing technologies such as CRISPR. Further, AI-based analysis of endogenous cytoprotection, especially the activity of molecules such as heme oxygenase, with particular application to hemolytic diseases. AI-driven robotics and automated monitoring systems significantly improve elderly care, lifestyle interventions, and clinical trials, demonstrating considerable potential to extend both healthspan and lifespan. However, the integration of AI into longevity research poses ethical and societal challenges, including concerns over privacy, equitable access, and broader implications of extended human lifespans. Strategic interdisciplinary collaboration, transparent AI methodologies, standardized data frameworks, and equitable policy approaches are essential to responsibly harness AI’s full potential in transforming longevity science and improving human health. Full article

(This article belongs to the Special Issue New Insights into Mechanisms and Therapeutics for Cardiovascular Disorders: 2nd Edition)

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21 pages, 1196 KB

Open AccessReview

Understanding Atopic Dermatitis: Pathophysiology and Management Strategies

by Heng Chai, Wing Sum Siu, Hui Ma and Yuzhen Li

Biomolecules 2025, 15(11), 1500; https://doi.org/10.3390/biom15111500 - 24 Oct 2025

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Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin condition characterized by itching, redness, and dryness, significantly impacting the quality of life of affected individuals. With a rising prevalence across diverse demographics, understanding AD is crucial due to its systemic nature and association with [...] Read more.

Atopic dermatitis (AD) is a chronic inflammatory skin condition characterized by itching, redness, and dryness, significantly impacting the quality of life of affected individuals. With a rising prevalence across diverse demographics, understanding AD is crucial due to its systemic nature and association with comorbidities such as asthma and allergic rhinitis, as well as its psychosocial implications. The pathophysiology of AD involves a complex interplay of genetic predispositions and environmental triggers, leading to dysbiosis and increased susceptibility to superinfection. Clinically, AD manifests variably across age groups, with distinct presentations in pediatric and adult populations. Diagnosis is primarily based on clinical assessment criteria, supplemented by differential diagnoses and, when necessary, skin tests for allergies. Current management strategies encompass topical therapies, including moisturizers, corticosteroids, and calcineurin inhibitors, alongside systemic treatments such as antihistamines, immunosuppressants, and biologics. Lifestyle modifications, including trigger avoidance and effective skin care routines, are essential components of comprehensive care. Emerging novel therapies targeting specific biomarkers are currently under investigation in clinical trials, offering promising avenues for more effective management. However, challenges remain in optimizing treatment protocols and addressing the multifaceted nature of AD. In conclusion, this review highlights the need for continued research and awareness regarding atopic dermatitis. A multidisciplinary approach to management is essential to enhance patient outcomes and address the complexities of this prevalent and impactful condition. Full article

(This article belongs to the Special Issue Unraveling the Complexity of Autoimmune and Autoinflammatory Skin Diseases: From Molecular Pathways to Targeted Therapies)

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23 pages, 1718 KB

Open AccessArticle

miR-155-5p Silencing Does Not Alter BTLA Molecule Expression in CLL T Cells: Implications for Targeted Immunotherapy

by Agata Kosmaczewska, Lidia Ciszak, Anna Andrzejczak, Anna Tomkiewicz, Anna Partyka, Zofia Rojek-Gajda, Irena Frydecka, Dariusz Wołowiec, Tomasz Wróbel, Agnieszka Bojarska-Junak, Jacek Roliński and Lidia Karabon

Biomolecules 2025, 15(11), 1499; https://doi.org/10.3390/biom15111499 - 24 Oct 2025

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Abstract

Given that we have demonstrated that miR-155-5p is increased in CLL PBMCs and that its reduction with inhibitory siRNA partially restores the immune checkpoint BTLA protein level in CLL B cells, risk stratification for using anti-miR-155-based immunotherapy in CLL seems reasonable, particularly with [...] Read more.

Given that we have demonstrated that miR-155-5p is increased in CLL PBMCs and that its reduction with inhibitory siRNA partially restores the immune checkpoint BTLA protein level in CLL B cells, risk stratification for using anti-miR-155-based immunotherapy in CLL seems reasonable, particularly with its potential impact on T cells. Therefore, we aimed to assess the role of miR-155-5p in the epigenetic modification of BTLA levels in CLL T cells, especially since we observed that BTLA expression unfavorably promotes increased proliferative activity and IL-4 secretion in T cells, thus suggesting BTLA malfunction in the CLL T cell subset. Transfection of PBMCs with an inhibitor of miR-155-5p (INH) led to about a ten-fold down-regulation of miR-155-5p levels compared to control siRNA (NC) both in CLL patients and healthy individuals (HC), as assessed by RT-qPCR. Additionally, we did not find any significant differences in BTLA protein expression in T cells after silencing miR-155-5p in either examined group. We demonstrated for the first time that immunotherapy approaches based on systemic administration of anti-miR-155-5p therapeutics would be a favorable strategy in CLL, since they do not affect BTLA expression in T cell populations and could benefit CLL patients with impaired BTLA levels on CLL cells. Full article

(This article belongs to the Special Issue Epigenetic Drivers of Cancer Metastasis: From Methylation to Non-Coding RNAs)

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20 pages, 8232 KB

Open AccessArticle

Abietic Acid Induces DNA Damage and Cell Apoptosis in Lung Cancer Cells Through Targeting TOP2A

by Zhiyu Zhu, Jie Gu, Zehua Liao, Mengting Chen, Yun Wang, Jingyi Song, Jing Xia, Xinbing Sui, Shuang Lin and Xueni Sun

Biomolecules 2025, 15(11), 1498; https://doi.org/10.3390/biom15111498 - 24 Oct 2025

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Abstract

Background: This study investigated the therapeutic effects and underlying mechanisms of abietic acid, an abietane diterpene extracted from Pimenta racemosa var. grissea, against lung cancer. Methods: Initially, cell viability, colony formation, flow cytometry, and mitochondrial membrane potential detection were conducted [...] Read more.

Background: This study investigated the therapeutic effects and underlying mechanisms of abietic acid, an abietane diterpene extracted from Pimenta racemosa var. grissea, against lung cancer. Methods: Initially, cell viability, colony formation, flow cytometry, and mitochondrial membrane potential detection were conducted to determine the impact of abietic acid on lung cancer cells. Subsequently, the antitumor mechanisms of abietic acid were predicted using network pharmacology and validated via immunofluorescence, reactive oxygen species (ROS) detection, molecular docking, gene knockdown techniques and Western blotting. Finally, an in vivo xenograft model assessed its tumor-suppressive potential, with Hematoxylin–Eosin (H&E) staining, Western blotting, and immunohistochemistry performed to examine pathological changes and protein expression alterations. Results: The proliferation of lung cancer cells was significantly inhibited by abietic acid. Additionally, abietic acid induced apoptosis and reduced mitochondrial membrane potential. Network pharmacology and Gene Ontology (GO) enrichment analysis revealed that the DNA damage response was a key biological process affected by abietic acid. Further results demonstrated that abietic acid induces DNA damage in lung cancer cells through targeting DNA topoisomerase II alpha (TOP2A). In vivo studies confirmed the antitumor efficacy of abietic acid and its low systemic toxicity. Conclusions: Abietic acid demonstrated significant antitumor effects in lung cancer cells by downregulating TOP2A, which induced DNA damage and apoptosis, revealing its clinical potential. Full article

(This article belongs to the Topic Natural Products and Drug Discovery—2nd Edition)

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17 pages, 3580 KB

Open AccessArticle

Novel Synthetic Strategies Towards Analogues of Cadaside and Malacidin Antibiotic Peptides

by Katharina Webhofer, Darsha Naidu, Milandip Karak, Stephen A. Cochrane, Christopher J. Morris and Rachael Dickman

Biomolecules 2025, 15(11), 1497; https://doi.org/10.3390/biom15111497 - 23 Oct 2025

Viewed by 266

Abstract

With antibiotic resistance becoming an increasingly pressing issue, the search for novel antimicrobial drugs is more important than ever before. The recently discovered calcium-dependent lipopeptides cadaside A/B and malacidin A/B have promising activity against resistant Gram-positive bacteria. With limited reports of synthetic routes [...] Read more.

With antibiotic resistance becoming an increasingly pressing issue, the search for novel antimicrobial drugs is more important than ever before. The recently discovered calcium-dependent lipopeptides cadaside A/B and malacidin A/B have promising activity against resistant Gram-positive bacteria. With limited reports of synthetic routes towards these peptides available in the literature, especially for cadasides, we herein report a novel on-resin synthesis strategy. We used this strategy to produce fifteen simplified malacidin and cadaside analogues. In addition, both minimum inhibitory concentration and thin layer chromatography assays were conducted to determine antimicrobial activity and advance our understanding of these peptides’ structure–activity relationships. Full article

(This article belongs to the Special Issue Antimicrobial Peptides in Nature: Inspiration for Rationally Designed Antimicrobials)

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15 pages, 1732 KB

Open AccessArticle

Divergent Conversion Efficiencies of Mycobacterium sp. 191574 for Various Phytosterols and Their Underlying Mechanisms

by Zifu Ni, Yingjing Bi, Zihao Wang, Yun Han, Yanlan Bi, Linshang Zhang and Shangde Sun

Biomolecules 2025, 15(11), 1496; https://doi.org/10.3390/biom15111496 - 23 Oct 2025

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Abstract

Steroid drugs have a broad range of applications in medicine. The microbial degradation of phytosterols for the synthesis of steroid drug intermediates holds significant potential for industrial applications. In this study, the transformation efficiency and underlying mechanisms of different phytosterols in Mycobacterium sp. [...] Read more.

Steroid drugs have a broad range of applications in medicine. The microbial degradation of phytosterols for the synthesis of steroid drug intermediates holds significant potential for industrial applications. In this study, the transformation efficiency and underlying mechanisms of different phytosterols in Mycobacterium sp. 191574 were investigated. Among the tested compounds, β-sitosterol exhibited the highest conversion efficiency, followed by mixed sterols, while stigmasterol showed the lowest efficiency. Proteomic analysis identified key enzymes involved in sterol metabolism. Further molecular docking experiments revealed that acyl-CoA synthetase (A0A0T1W1C0) and hydrolase (A0A0T1W815) may act as rate-limiting enzymes contributing to the low conversion rate of stigmasterol. Significant differences in hydrogen bonding patterns and three-dimensional spatial structures between these enzymes and sterol-derived ligands were observed, resulting in reduced binding affinity of stigmasterol. This study provides a theoretical basis for the optimization of sterol biotransformation processes and offers a foundation for improving the biological production efficiency of sterol-based pharmaceutical intermediates. Full article

(This article belongs to the Section Natural and Bio-derived Molecules)

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25 pages, 1760 KB

Open AccessReview

Cisplatin-Induced Skeletal Muscle Atrophy: Biomolecular Mechanisms and the Protective Role of Exercise-Induced Myokines

by Miaomiao Xu and Xiaoguang Liu

Biomolecules 2025, 15(11), 1495; https://doi.org/10.3390/biom15111495 - 23 Oct 2025

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Abstract

Cisplatin is a widely used chemotherapy drug for the treatment of various cancers; however, its clinical use is often accompanied by skeletal muscle atrophy, which not only impacts patients’ physical health but also significantly diminishes their quality of life. The mechanisms underlying cisplatin-induced [...] Read more.

Cisplatin is a widely used chemotherapy drug for the treatment of various cancers; however, its clinical use is often accompanied by skeletal muscle atrophy, which not only impacts patients’ physical health but also significantly diminishes their quality of life. The mechanisms underlying cisplatin-induced muscle atrophy are complex and involve a series of molecular biological processes, including oxidative stress, inflammation, protein degradation, and muscle cell apoptosis. Recent studies have suggested that exercise intervention can significantly alleviate cisplatin-induced muscle damage by modulating exercise-induced myokines. Myokines, such as muscle-derived cytokines (e.g., IL-6, irisin) and other related factors, can mitigate muscle atrophy through anti-inflammatory, antioxidative, and muscle-synthesis-promoting mechanisms. This review explores the molecular mechanisms of cisplatin-induced skeletal muscle atrophy, examines the potential protective effects of exercise intervention, and highlights the role of exercise-induced myokines in this process. The findings suggest that exercise not only alleviates chemotherapy-induced muscle atrophy by improving metabolic and immune status but also activates myokines to promote muscle regeneration and repair, offering a promising adjunctive therapy for cisplatin-treated patients. Full article

(This article belongs to the Section Molecular Biology)

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13 pages, 1636 KB

Open AccessArticle

The Identification of a Sub-Micromolar Peptide-Based Protein Arginine Methyltransferase 1 (PRMT1) Inhibitor from a Plate-Based Screening Assay

by Tina M. Sawatzky, Sarah A. Mann, Jordan Shauna Tucker, Aida A. Bibart, Corey P. Causey and Bryan Knuckley

Biomolecules 2025, 15(11), 1494; https://doi.org/10.3390/biom15111494 - 23 Oct 2025

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Abstract

Post-translational modifications (PTMs) expand the structural diversity of proteins beyond the standard amino acids, influencing protein-protein interactions. Protein methylation, a prevalent PTM, involves the transfer of methyl groups from S-adenosylmethionine (SAM) to lysine and arginine residues. Arginine methylation is catalyzed by the Protein [...] Read more.

Post-translational modifications (PTMs) expand the structural diversity of proteins beyond the standard amino acids, influencing protein-protein interactions. Protein methylation, a prevalent PTM, involves the transfer of methyl groups from S-adenosylmethionine (SAM) to lysine and arginine residues. Arginine methylation is catalyzed by the Protein Arginine Methyltransferase (PRMT) family to yield mono- and dimethylarginine forms. PRMT1, the isozyme responsible for the majority of asymmetric dimethylation (ADMA) is implicated in various diseases, including cancer. Here, we report the synthesis and screening of a second-generation peptide library to identify novel PRMT1 substrates. The library, based on histone peptides, incorporated varying sequences of amino acids, facilitating substrate specificity studies. Screening identified 7 peptide sequences as exceptional PRMT1 substrates, which were confirmed by kinetic analysis. Consensus sequences revealed key recognition elements for PRMT1 catalysis, suggesting roles for small non-polar side chains and specific residues near the substrate arginine. Furthermore, we developed a peptide-based PRMT1 inhibitor by substituting the substrate arginine with a chloroacetamidine warhead. The inhibitor exhibited sub-micromolar inhibitory potency against PRMT1, surpassing previous peptide-based inhibitors. Our findings contribute to understanding PRMT1 substrate specificity and provide a scaffold for developing potent inhibitors targeting PRMT1 in diseases, including cancer. Full article

(This article belongs to the Special Issue Advances and Novel Strategies for Studying Underexplored Post-Translational Modifications (PTMs))

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16 pages, 4220 KB

Open AccessArticle

Long Non-Coding RNAs Contribute to Glucose Starvation-Induced Dedifferentiation in Lung Adenocarcinoma

by Aparamita Pandey, Pasquale Saggese, Adriana Soto, Estefany Gomez, Martín Alcaraz, Jr. and Claudio Scafoglio

Biomolecules 2025, 15(11), 1493; https://doi.org/10.3390/biom15111493 - 23 Oct 2025

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Abstract

Nutrient deprivation causes dedifferentiation in solid tumors, driving an aggressive phenotype. We previously showed that glucose starvation-induced dedifferentiation is driven by epigenetic changes induced by a deficit of alpha-ketoglutarate (α-KG). Deficient activity of α-KG-dependent histone demethylases leads to unbalanced hypermethylation of histone 3 [...] Read more.

Nutrient deprivation causes dedifferentiation in solid tumors, driving an aggressive phenotype. We previously showed that glucose starvation-induced dedifferentiation is driven by epigenetic changes induced by a deficit of alpha-ketoglutarate (α-KG). Deficient activity of α-KG-dependent histone demethylases leads to unbalanced hypermethylation of histone 3 on lysine 27 (H3K27) by methyltransferase EZH2. H3K27 hypermethylation is a key mechanism of starvation-induced dedifferentiation. Here, we investigate a new aspect of this mechanism and show that epitranscriptomic changes are also induced by glucose restriction. Specifically, hypermethylation of select long non-coding RNAs leads to their upregulation under glucose deprivation as a consequence of reduced activity of the RNA demethylase FTO. We identified LINC00662 as an lncRNA required for EZH2 recruitment to target gene promoters induced by low glucose. These findings characterize the epigenetic response to glucose restriction beyond histone methylation, revealing that RNA methylation of lncRNAs such as LINC00662 represents a parallel mechanism converging on EZH2. Full article

(This article belongs to the Section Molecular Biology)

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11 pages, 379 KB

Open AccessArticle

Exploring the Association of FTO rs9939609 with Type 2 Diabetes, Fasting Glucose and HbA1c in a Southeastern Mexican Region of Predominant Mayan Genetic Background

by Nicolas Fragoso-Bargas, Litzy Naomi Toloza-Couoh, Irma Quintal-Ortiz, Guillermo Valencia-Pacheco and Nina Valadez-Gonzalez

Biomolecules 2025, 15(11), 1492; https://doi.org/10.3390/biom15111492 - 23 Oct 2025

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Abstract

Type 2 diabetes (T2D) is a multifactorial disease characterized by chronic hyperglycemia. The FTO variant rs9939609 has been widely associated with obesity, but emerging evidence suggests a broader role for T2D risk across diverse populations. However, Mayan ancestry individuals remain underrepresented in genetic [...] Read more.

Type 2 diabetes (T2D) is a multifactorial disease characterized by chronic hyperglycemia. The FTO variant rs9939609 has been widely associated with obesity, but emerging evidence suggests a broader role for T2D risk across diverse populations. However, Mayan ancestry individuals remain underrepresented in genetic studies. Thus, we evaluated the association of rs9939609 with T2D, fasting glucose, and HbA1c in a southeastern Mexican region with prevalent Mayan ancestry. Birthplace was used as a proxy for ancestry, although no formal ancestry assessment was conducted. The A allele was associated with increased risk for T2D in both additive (OR = 1.88 [1.08–3.40], p = 0.031) and dominant (OR = 2.09 [1.08–4.15], p = 0.032) models, even after adjusting for age, sex, BMI, waist circumference, and waist/hip ratio. The A allele was also associated with increased fasting glucose and HbA1c levels in the dominant model. Mediation analysis suggested that T2D may mediate the effect of rs9939609-A on glucose traits. rs9939609-A may be a risk factor for T2D and elevated glucose levels in this population. However, the results should be interpreted with caution due to the limited sample size, which may result in under- or overestimation of effect sizes. Full article

(This article belongs to the Special Issue Molecular Mechanisms and Therapeutic Strategies for Metabolic Dysfunction: From Molecules to Population Health)

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11 pages, 394 KB

Open AccessArticle

Association Between Heavy Metals Exposure and Elevated High-Sensitivity C-Reactive Protein: Mediating Role of Body Mass Index

by Seong-Uk Baek and Jin-Ha Yoon

Biomolecules 2025, 15(11), 1491; https://doi.org/10.3390/biom15111491 - 23 Oct 2025

Viewed by 192

Abstract

Heavy metal exposure is linked to obesity and systemic inflammation. This study explored the mediating role of body mass index (BMI) in the association of heavy metal exposure with high-sensitivity C-reactive protein (hs-CRP). Blood levels of mercury (Hg), cadmium (Cd), and lead (Pb) [...] Read more.

Heavy metal exposure is linked to obesity and systemic inflammation. This study explored the mediating role of body mass index (BMI) in the association of heavy metal exposure with high-sensitivity C-reactive protein (hs-CRP). Blood levels of mercury (Hg), cadmium (Cd), and lead (Pb) were assessed in a nationwide sample of 4521 adults. Linear regressions were employed to examine associations among blood heavy metal levels, BMI, and hs-CRP levels. Mediation analyses were conducted to estimate the indirect effect of exposure to each heavy metal on the elevation of hs-CRP through an increase in BMI. The median (Q1; Q3) values for blood levels of heavy metals were 3.15 (2.10; 4.84) for Hg (μg/L), 0.95 (0.63; 1.38) for Cd (μg/L), and 1.67 (1.28; 2.21) for Pb (μg/dL). Blood Hg level was associated with both BMI (adjusted β: 0.73; 95% CI [confidence interval]: 0.51; 0.96) and a log-transformed hs-CRP level (adjusted β: 0.07; 95% CI: 0.02; 0.13). Blood Cd and Pb levels showed no clear associations with BMI and hs-CRP. The indirect effect of Hg exposure on hs-CRP via BMI was 0.069 (95% CI: 0.037; 0.102), and that of the direct effect was 0.003 (95% CI: −0.001; 0.007), with BMI accounting for 95.7% (95% CI: 88.6%, 102.0%) of the total association between Hg levels and hs-CRP levels. Hg exposure was linked to increased hs-CRP levels, with elevated BMI explaining most of this association. This research offers insights into the mechanisms through which Hg contributes to systemic inflammation, underscoring the potential role of BMI as a key mediator. Full article

(This article belongs to the Section Biological Factors)

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14 pages, 2090 KB

Open AccessReview

Cuproptosis as a Potential Therapeutic Target for Steatotic Liver Disease

by Yujie Pan, Cheng Luo, Qitao Guo, Qifei Duan, Ziyan Wu and Yan Li

Biomolecules 2025, 15(11), 1490; https://doi.org/10.3390/biom15111490 - 23 Oct 2025

Viewed by 116

Abstract

Steatotic liver disease (SLD) has become one of the most prevalent chronic liver diseases, representing a significant health burden worldwide. The complex pathogenesis of SLD results in a lack of specific therapeutic targets and effective drug treatment modalities. Copper (Cu) is a trace [...] Read more.

Steatotic liver disease (SLD) has become one of the most prevalent chronic liver diseases, representing a significant health burden worldwide. The complex pathogenesis of SLD results in a lack of specific therapeutic targets and effective drug treatment modalities. Copper (Cu) is a trace element that plays a critical role in various physiological processes, particularly hepatic metabolism. Meanwhile, Cu overload can induce cellular toxicity, which is generally explained by its capacity to induce oxidative damage. In 2022, a novel form of programmed cell death, designated as cuproptosis, was identified. In essence, excess Cu ions bind to the lipoylated components of the tricarboxylic acid cycle, resulting in proteotoxic stress and subsequent cell death. The role of cuproptosis in the pathologies of Cu overload-induced diseases has gained considerable attention. However, the association between SLD and Cu overload, particularly cuproptosis, remains to be elucidated. This review provides a concise overview of cuproptosis. The significance of Cu overload in SLD, as well as the potential association between cuproptosis and SLD, is explored. This review aims to offer insights into the potential of cuproptosis as a therapeutic target for SLD. Full article

(This article belongs to the Section Cellular Biochemistry)

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18 pages, 1079 KB

Open AccessReview

Structural Insights into the SARS-CoV-2 Spike Protein and Its Implications for Antibody Resistance

by Yuichiro Yamamoto and Kohji Noguchi

Biomolecules 2025, 15(11), 1489; https://doi.org/10.3390/biom15111489 - 22 Oct 2025

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Abstract

The COVID-19 pandemic, caused by SARS-CoV-2, has profoundly affected global health and the economy. The emergence of variants with spike mutations, particularly within the receptor-binding domain (RBD), has reduced the efficacy of many neutralizing antibodies (nAbs), and recent variants, including KP.3 and other [...] Read more.

The COVID-19 pandemic, caused by SARS-CoV-2, has profoundly affected global health and the economy. The emergence of variants with spike mutations, particularly within the receptor-binding domain (RBD), has reduced the efficacy of many neutralizing antibodies (nAbs), and recent variants, including KP.3 and other circulating strains, show partial escape from infection- or vaccine-induced immunity. To overcome this, developing broad-spectrum nAbs that target the conserved S2 subunit of the spike protein is crucial. Unlike the highly mutable RBD, the S2 region remains structurally conserved, providing a promising foundation for universal protection. Deeper insight into S2 structure and function, together with advances in bispecific antibody design, could facilitate the development of next-generation therapeutics resilient to viral evolution. This review examines the structural evolution of the SARS-CoV-2 spike, focusing on the therapeutic potential of S2-targeting antibodies and strategies to overcome antibody resistance. Full article

(This article belongs to the Special Issue Genetic and Structural Analyses of Coronaviruses: Insights into SARS-CoV-2 and Beyond)

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28 pages, 2591 KB

Open AccessReview

Standard Sample Preparation for Serial Femtosecond Crystallography

by Christina Schmidt, Kristina Lorenzen, Joachim Schulz and Huijong Han

Biomolecules 2025, 15(11), 1488; https://doi.org/10.3390/biom15111488 - 22 Oct 2025

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Abstract

The development of serial crystallography (SX), including serial synchrotron crystallography (SSX) at synchrotron sources and serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs), has facilitated the collection of high-resolution diffraction data from micron-sized crystals, providing unique insights into the structures and dynamics [...] Read more.

The development of serial crystallography (SX), including serial synchrotron crystallography (SSX) at synchrotron sources and serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs), has facilitated the collection of high-resolution diffraction data from micron-sized crystals, providing unique insights into the structures and dynamics of biomolecules at room temperature. Standard samples are essential for the commissioning of new XFEL instruments and the validation of experimental setups. In this review, we summarize currently used standard proteins and describe representative microcrystal preparation workflows for four widely adopted models, lysozyme, myoglobin, iq-mEmerald, and photoactive yellow protein (PYP), drawing on established methodologies and accumulated experience from their applications at the European XFEL. By consolidating existing knowledge and integrating protocols that have been systematically refined and optimized through our experimental efforts, this review aims to provide practical guidance for the serial crystallography community, thereby enhancing reproducibility and ensuring consistent experimental performance across facilities. Full article

(This article belongs to the Special Issue Innovative Biomolecular Structure Analysis Techniques)

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20 pages, 8173 KB

Open AccessArticle

Non-Vesicular Extracellular Particle (NVEP) Proteomes from Diverse Biological Sources Reveal Specific Marker Composition with Varying Enrichment Levels

by Wasifa Naushad, Bryson C. Okeoma, Carlos Gartner, Yulica Santos-Ortega, Calvin P. H. Vary, Lakmini S. Premadasa, Alessio Noghero, Jack T. Stapleton, Ionita C. Ghiran, Mahesh Mohan and Chioma M. Okeoma

Biomolecules 2025, 15(11), 1487; https://doi.org/10.3390/biom15111487 - 22 Oct 2025

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Abstract

Extracellular particles (EPs), an umbrella term encompassing membrane-enclosed extracellular vesicles (EVs) and non-vesicular extracellular particles ([NVEPs], previously described as extracellular condensates [ECs]) contain a complex cargo of biomolecules, including DNA, RNA, proteins, and lipids, reflecting the physiological state of their cell of origin. [...] Read more.

Extracellular particles (EPs), an umbrella term encompassing membrane-enclosed extracellular vesicles (EVs) and non-vesicular extracellular particles ([NVEPs], previously described as extracellular condensates [ECs]) contain a complex cargo of biomolecules, including DNA, RNA, proteins, and lipids, reflecting the physiological state of their cell of origin. Identifying proteins associated with EPs that regulate host responses to physiological and pathophysiological processes is of critical importance. Here, we report the findings of our study to gain insight into the proteins associated with NVEPs. We used samples from human semen, the rat brain, and the rhesus macaque (RM) brain and blood to assess the physical properties and proteome profiles of NVEPs from these specimens. The results show significant differences in the zeta potential, concentration, and size of NVEPs across different species. We identified 938, 51, and 509 total proteins from NVEPs isolated from rat brain tissues, RM blood, and human seminal plasma, respectively. The species-specific protein networks show distinct biological themes, while the species-conserved protein interactome was identified with six proteins (ALB, CST3, FIBA/FGA, GSTP1, PLMN/PLG, PPIA) associated with NVEPs in all samples. The six NVEP-associated proteins are prone to aggregation and formation of wide, insoluble, unbranched filaments with a cross-beta sheet quaternary structure, such as amyloid fibrils. Protein-to-function analysis indicates that the six identified proteins are linked to the release of dopamine, immune-mediated inflammatory disease, replication of RNA viruses, HIV-HCV co-infection, and inflammation. These interesting findings have created an opportunity to evaluate NVEPs for their potential use as biomarkers of health and disease. Additional in-depth studies are needed to clarify when and how these proteins sustain their physiological role or transition to pathogenic roles. Full article

(This article belongs to the Collection Feature Papers in 'Biomacromolecules: Proteins')

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21 pages, 2194 KB

Open AccessArticle

Multi-Omics Integration Reveals Electroacupuncture Ameliorates Cognitive Impairment in Alzheimer’s Disease via Gut–Brain Axis

by Shuai Zhang, Xinyuan Liu, Shuyu Xu, Weixian Li, Jie Song, Qing Tian and Yanjun Du

Biomolecules 2025, 15(11), 1486; https://doi.org/10.3390/biom15111486 - 22 Oct 2025

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Abstract

Background: Alzheimer’s disease (AD) lacks effective therapeutic strategies. Electroacupuncture (EA) offers promising neuroprotective effects, but its underlying mechanisms remain unclear. Objective: To explore the mechanisms of EA’s neuroprotective effects on AD via microbiome and metabolome integration. Methods: Utilizing a well-established model of AD, [...] Read more.

Background: Alzheimer’s disease (AD) lacks effective therapeutic strategies. Electroacupuncture (EA) offers promising neuroprotective effects, but its underlying mechanisms remain unclear. Objective: To explore the mechanisms of EA’s neuroprotective effects on AD via microbiome and metabolome integration. Methods: Utilizing a well-established model of AD, Senescence-Accelerated Mouse Prone 8 (SAMP8), EA intervention was performed. 16S ribosomal RNA (rRNA) sequencing and serum metabolomics were conducted on SAMP8 mice, SAMP8 mice after EA intervention, and their normal control group Senescence-Accelerated Mouse Resistant 1 (SAMR1) mice. Results: SAMP8 mice were subjected to electroacupuncture (EA) treatment at the Baihui (GV20) and Shenshu (BL23) acupoints for 15 min daily over a period of four weeks. EA enhanced cognitive function and reduced neuronal damage in AD models. The treatment lowered pro-inflammatory cytokines (TNF-α, IL-1β) and AD-related pathologies (tau, Aβ_1-42). EA also rebalanced gut microbiota by increasing beneficial Gastranaerophilales while decreasing harmful Proteobacteria. Additionally, it restored purine and phenylpropanoid metabolism by regulating key metabolites. Importantly, EA reduced levels of specific metabolites linked to pro-inflammatory bacteria (Sphingomonas, Massilia, Escherichia-Shigella), simultaneously decreasing their abundance. These findings highlight EA’s multi-target effects on neuroinflammation, gut microbiota, and metabolic pathways in AD. Notably, the interactions between EA-regulated key metabolites and AD-related targets, predicted via PubChem and ChEMBL databases, remain computational and have not been validated by experimental studies. Conclusions: EA exerts neuroprotective effects in AD via modulation of gut microbiota and metabolic pathways, representing a novel non-pharmacological therapeutic strategy. Full article

(This article belongs to the Section Biological Factors)

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23 pages, 3304 KB

Open AccessArticle

Fingolimod Improves Anxiety-like Behavior and Modulates Sphingosine-1-Phosphate Receptors Gene Expression in a Diabetic Mouse Model

by Przemysław Leonard Wencel, Kamilla Blecharz-Klin, Agnieszka Piechal, Justyna Pyrzanowska, Dagmara Mirowska-Guzel and Robert Piotr Strosznajder

Biomolecules 2025, 15(11), 1485; https://doi.org/10.3390/biom15111485 - 22 Oct 2025

Viewed by 213

Abstract

Background: Type 2 diabetes mellitus (T2DM) is a rapidly expanding worldwide health issue associated with impairments in memory and executive functions. The bioactive sphingolipid sphingosine-1-phosphate (S1P) regulates cell death/survival and the inflammatory response by acting on S1P receptors (S1PRs). Unfortunately, the role of [...] Read more.

Background: Type 2 diabetes mellitus (T2DM) is a rapidly expanding worldwide health issue associated with impairments in memory and executive functions. The bioactive sphingolipid sphingosine-1-phosphate (S1P) regulates cell death/survival and the inflammatory response by acting on S1P receptors (S1PRs). Unfortunately, the role of S1PRs signaling in the T2DM brain remains elusive. Methods: The effect of fingolimod (FTY720, S1PRs modulator) on the behavior and expression profile of genes encoding S1PRs, sphingosine kinases (SPHK1 and 2), glucose transporters, proteins engaged in insulin signaling, sirtuin 1 (SIRT1), and proinflammatory cytokines in the brain cortex and hippocampus of diabetic mice was examined. Results: We observed a significant reduction in S1pr1, Sirt1, and insulin-like growth factor-1 (Igf1) gene expression that was accompanied by elevation of Sphk2, S1pr3, Il6, and Tnf in T2DM mice. Moreover, animals showed anxiety-like behavior and memory deficits. Fingolimod administration recovered downregulated S1pr1, Sirt1, and Igf1 expression and upregulated Slc2a4 (GLUT-4) and Ide (insulin-degrading enzyme). Furthermore, FTY720 reduced the elevated expression of Il6 and Tnf. Fingolimod also exerted an anxiolytic effect in T2DM. Conclusions: Results indicate an important role of S1PR modulation in T2DM. Moreover, fingolimod affected mRNA levels of proteins engaged in glucose metabolism/insulin signaling and improved the behavior of diabetic mice. Full article

(This article belongs to the Section Molecular Biology)

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20 pages, 2934 KB

Open AccessArticle

Protective Effects and Potential Mechanisms of D-Aspartate on Testicular Damage Induced by Polystyrene Microplastics

by Sara Falvo, Giulia Grillo, Imed Messaoudi, Nada Fradi, Gabriella Chieffi Baccari, Maria Maddalena Di Fiore, Alessandra Biasi, Maria Rosaria Ambruosi, Alessandra Santillo and Massimo Venditti

Biomolecules 2025, 15(11), 1484; https://doi.org/10.3390/biom15111484 - 22 Oct 2025

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Abstract

Polystyrene Microplastics (PS-MPs) affect testicular activity, as evidenced by increased oxidative stress, apoptosis, and autophagy activation, impairing steroidogenesis and spermatogenesis. The present study investigates, for the first time in vivo, the potential protective effect of D-aspartate (D-Asp) against PS-MPs-induced damage on the testicular [...] Read more.

Polystyrene Microplastics (PS-MPs) affect testicular activity, as evidenced by increased oxidative stress, apoptosis, and autophagy activation, impairing steroidogenesis and spermatogenesis. The present study investigates, for the first time in vivo, the potential protective effect of D-aspartate (D-Asp) against PS-MPs-induced damage on the testicular function of adult rats. D-Asp, well-known stimulator of testosterone biosynthesis and spermatogenesis progression, possesses pharmacological properties, including antioxidant and anti-apoptotic ones. The results showed that PS-MP’s adverse effects on testicular activity were reversed by D-Asp treatment. Mechanistically, D-Asp inhibited testicular oxidative stress by modulating the protein levels of CAT, SOD1, SOD2, and 4-HNE; affecting TBARS levels; and reducing apoptosis, as suggested by CYT C analysis and a TUNEL assay. Furthermore, D-Asp administration mitigated PS-MPs-induced autophagy activation by modulating the expression of LC3BI, LC3BII, and p62 proteins. Finally, the amino acid counteracts PS-MPs damage on steroidogenesis and spermatogenesis by restoring normal levels of steroidogenic (StAR, 3β-HSD, and 17β-HSD) and spermatogenic (PCNA and SYCP3) markers. This study encourages further research to understand the potential value of the amino acid in improving human testicular health and male fertility. Full article

(This article belongs to the Section Biological Factors)

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