International Journal of Molecular Sciences

15 pages, 3291 KiB

Open AccessArticle

Structural Implications of H233L and H398P Mutations in Phospholipase Cζ: A Full-Atom Molecular Dynamics Study on Infertility-Associated Dysfunctions

by Fernando Hinostroza, Sofía Albornoz-Muñoz, Sebastián Vergara, Gabriela Urra, Ingrid Araya-Durán, Rafael A. Fissore, Fernando Danilo González-Nilo, Daniel Bustos and Ingrid Carvacho

Int. J. Mol. Sci. 2025, 26(10), 4706; https://doi.org/10.3390/ijms26104706 (registering DOI) - 14 May 2025

Abstract

Phospholipase Cζ (PLCζ), a sperm-specific enzyme, plays a critical role in mammalian fertilization. Mutations in PLCζ have been linked to male infertility, as they impair its ability to trigger calcium (Ca²⁺) oscillations necessary for egg activation and embryo development. During fertilization, [...] Read more.

Phospholipase Cζ (PLCζ), a sperm-specific enzyme, plays a critical role in mammalian fertilization. Mutations in PLCζ have been linked to male infertility, as they impair its ability to trigger calcium (Ca²⁺) oscillations necessary for egg activation and embryo development. During fertilization, PLCζ is introduced into the egg, where it hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol 1,4,5-trisphosphate and diacylglycerol, leading to Ca²⁺ release from the endoplasmic reticulum. Human infertility-associated mutations include H233L, H398P, and R553P, which disrupt PLCζ function. To elucidate the molecular consequences of the mutations, we employed full-atom molecular dynamics simulations to analyze structural perturbations and their impact on PIP₂ and Ca²⁺ binding. Our results reveal that H233L and H398P mutations significantly reduce interactions with PIP₂, disrupting hydrogen bonding and salt bridge formation, leading to misalignment of the substrate. Additionally, these mutations destabilize Ca²⁺ binding by altering its positioning within the active site. In contrast, the R553P mutation primarily affects intramolecular stability and enzyme dynamics without impairing substrate or ion binding. Free energy calculations indicate an increased affinity for PIP₂ in H233L and H398P mutants, leading to an aberrant substrate positioning and compromised hydrolysis. These structural insights help explain the egg activation failure and infertility of patients carrying these mutations. Full article

(This article belongs to the Special Issue Structural Dynamics of Macromolecules)

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23 pages, 1037 KiB

Open AccessReview

Transgene Mapping in Animals: What to Choose?

by Alexander Smirnov, Maksim Makarenko and Anastasia Yunusova

Int. J. Mol. Sci. 2025, 26(10), 4705; https://doi.org/10.3390/ijms26104705 (registering DOI) - 14 May 2025

Abstract

The phenomenal progress in biotechnology and genomics is both inspiring and overwhelming—a classic curse of choice, particularly when it comes to selecting methods for mapping transgene DNA integration sites. Transgene localization remains a crucial task for the validation of transgenic mouse or other [...] Read more.

The phenomenal progress in biotechnology and genomics is both inspiring and overwhelming—a classic curse of choice, particularly when it comes to selecting methods for mapping transgene DNA integration sites. Transgene localization remains a crucial task for the validation of transgenic mouse or other animal models generated by pronuclear microinjection. Due to the inherently random nature of DNA integration, reliable characterization of the insertion site is essential. Over the years, a vast number of mapping methods have been developed, and new approaches continue to emerge, making the choice of the most suitable technique increasingly complex. Factors such as cost, required reagents, and the nature of the generated data require careful consideration. In this review, we provide a structured overview of current transgene mapping techniques, which we have broadly classified into three categories: classic PCR-based methods (such as inverse PCR and TAIL-PCR), next-generation sequencing with target enrichment, and long-read sequencing platforms (PacBio and Oxford Nanopore). To aid in decision-making, we include a comparative table summarizing approximate costs for the methods. While each approach has its own advantages and limitations, we highlight our top four recommended methods, which we believe offer the best balance of cost-effectiveness, reliability, and simplicity for identifying transgene integration sites. Full article

(This article belongs to the Section Molecular Biology)

21 pages, 616 KiB

Open AccessReview

Biomarkers of Progression Independent of Relapse Activity—Can We Actually Measure It Yet?

by Gabriel Bsteh, Assunta Dal-Bianco, Nik Krajnc and Thomas Berger

Int. J. Mol. Sci. 2025, 26(10), 4704; https://doi.org/10.3390/ijms26104704 (registering DOI) - 14 May 2025

Abstract

Progression independent of relapse activity (PIRA) is increasingly recognized as a key driver of disability in multiple sclerosis (MS). However, the concept of PIRA remains elusive, with uncertainty surrounding its definition, underlying mechanisms, and methods of quantification. This review examines the current landscape [...] Read more.

Progression independent of relapse activity (PIRA) is increasingly recognized as a key driver of disability in multiple sclerosis (MS). However, the concept of PIRA remains elusive, with uncertainty surrounding its definition, underlying mechanisms, and methods of quantification. This review examines the current landscape of biomarkers used to predict and measure PIRA, focusing on clinical, imaging, and body fluid biomarkers. Clinical disability scores such as the Expanded Disability Status Scale (EDSS) are widely used, but may lack sensitivity in capturing subtle relapse-independent progression. Imaging biomarkers, including MRI-derived metrics (brain and spinal cord volume loss, chronic active lesions) and optical coherence tomography (OCT) parameters (retinal nerve fiber layer and ganglion cell-inner plexiform layer thinning), offer valuable insights, but often reflect both inflammatory and neurodegenerative processes. Body fluid biomarkers, such as neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP), are promising indicators of axonal damage and glial activation, but their specificity for PIRA remains limited. This review emphasizes the distinction between predicting PIRA—identifying individuals at risk of future progression—and measuring ongoing PIRA-related disability in real time. We highlight the limitations of current biomarkers in differentiating PIRA from relapse-associated activity and call for a clearer conceptual framework to guide future research. Advancing the precision and utility of PIRA biomarkers will require multimodal approaches, longitudinal studies, and standardized protocols to enable their clinical integration and to improve personalized MS management. Full article

(This article belongs to the Special Issue Multiple Sclerosis: From Molecular Pathology to Novel Therapeutic Approaches)

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16 pages, 2410 KiB

Open AccessReview

Audiovestibular Dysfunction in Patients with Hashimoto’s Disease: A Systematic Review

by Jiann-Jy Chen, Chih-Wei Hsu, Tien-Yu Chen, Chih-Sung Liang, Yen-Wen Chen, Bing-Yan Zeng and Ping-Tao Tseng

Int. J. Mol. Sci. 2025, 26(10), 4703; https://doi.org/10.3390/ijms26104703 (registering DOI) - 14 May 2025

Abstract

Although the inner ear is considered an immune-privileged organ because of the blood–labyrinth barrier, accumulating evidence has revealed an unexpected relation between Hashimoto’s disease and inner ear damage manifesting as audiovestibular dysfunction. Hashimoto’s disease can simultaneously affect both the auditory and vestibular systems, [...] Read more.

Although the inner ear is considered an immune-privileged organ because of the blood–labyrinth barrier, accumulating evidence has revealed an unexpected relation between Hashimoto’s disease and inner ear damage manifesting as audiovestibular dysfunction. Hashimoto’s disease can simultaneously affect both the auditory and vestibular systems, either through direct autoantibody attacks or through metabolic dysfunction associated with hypothyroidism. Currently, there is no consensus regarding tests or treatments for audiovestibular dysfunction related to Hashimoto’s disease. In this review, we summarize the currently available evidence regarding the characteristics, pathophysiology, diagnostic approaches, and treatment of audiovestibular dysfunction in patients with Hashimoto’s disease. Furthermore, we propose a specific steroid-plus-thyroxine treatment protocol to manage audiovestibular dysfunction associated with Hashimoto’s disease. This condition may respond to adequate treatment, potentially allowing reversibility if it is recognized and managed in a timely manner. Conversely, delayed diagnosis or failure to recognize the subtle presentation of audiovestibular dysfunction in patients with Hashimoto’s disease may lead to progressive hearing loss, immobility, and reduced quality of life. Based on the updated evidence in our review and our modified treatment protocol, we aim to provide new insights and therapeutic directions for clinicians managing audiovestibular dysfunction in patients with Hashimoto’s disease. Trial registration: PROSPERO CRD420250652982. Full article

(This article belongs to the Special Issue Hearing Loss: Molecular Biological Insights)

22 pages, 6185 KiB

Open AccessArticle

An Investigation of the Secretome Composition of Coriolopsis trogii Mafic-2001 and the Optimization of the Mafic-2001 Enzyme Cocktail to Enhance the Saccharification Efficacy of Chinese Distillers’ Grains

by Chengling Bao, Zhiyun Liu, Xiaoxia Zhong, Xiaofeng Guan, Yunhe Cao and Jinxiu Huang

Int. J. Mol. Sci. 2025, 26(10), 4702; https://doi.org/10.3390/ijms26104702 (registering DOI) - 14 May 2025

Abstract

The efficient degradation of lignocellulose is essential for valorizing agricultural waste and reducing environmental pollution. An efficient degradation process requires an enzyme cocktail capable of comprehensively deconstructing lignocellulosic components. In this study, the secretome of Coriolopsis trogii Mafic-2001 induced by rice straw was [...] Read more.

The efficient degradation of lignocellulose is essential for valorizing agricultural waste and reducing environmental pollution. An efficient degradation process requires an enzyme cocktail capable of comprehensively deconstructing lignocellulosic components. In this study, the secretome of Coriolopsis trogii Mafic-2001 induced by rice straw was examined, and the enzymatic composition and reaction conditions of Coriolopsis trogii were optimized. Mafic-2001 secreted an enzyme cocktail that included ligninolytic enzymes, cellulases, and hemicellulases. However, the relative abundances of endoglucanase (EG) and β-glucosidase (βG) were only 64.37% and 10.69%, respectively, compared with the relative abundance of cellobiohydrolase, which indicated a critical bottleneck in degradation efficiency. To overcome this limitation, the recombinant enzymes rEG1 and rβG1 were expressed in Pichia pastoris X-33. A functionally enhanced enzyme cocktail (rEG1–rβG1–Mafic-2001 = 0.05:0.09:0.86) was developed via a mixture design to achieve a reducing sugar yield of 2.77 mg/mL from Chinese distillers’ grains (CDGs). Structural analyses revealed that the optimized enzyme cocktail disrupted the reticulated fiber architecture of CDGs and attenuated the characteristic Fourier-transform infrared spectroscopy peaks of lignin, cellulose, and hemicellulose. This study elucidates the synergistic lignocellulose deconstruction mechanism of Mafic-2001 and establishes a precision enzyme-supplementation strategy for efficient CDG bioconversion, providing a scalable platform for the valorization of lignocellulosic biomass. Full article

(This article belongs to the Section Biochemistry)

40 pages, 1702 KiB

Open AccessReview

P-Glycoprotein as a Therapeutic Target in Hematological Malignancies: A Challenge to Overcome

by Pablo Álvarez-Carrasco, Fernanda Morales-Villamil and Carmen Maldonado-Bernal

Int. J. Mol. Sci. 2025, 26(10), 4701; https://doi.org/10.3390/ijms26104701 (registering DOI) - 14 May 2025

Abstract

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by the ABCB1/MDR1 gene, is a major contributor to multidrug resistance in hematological malignancies. These malignancies, arising from hematopoietic precursors at various differentiation stages, can manifest in the bone marrow, circulate in the bloodstream, or infiltrate [...] Read more.

P-glycoprotein (P-gp), a transmembrane efflux pump encoded by the ABCB1/MDR1 gene, is a major contributor to multidrug resistance in hematological malignancies. These malignancies, arising from hematopoietic precursors at various differentiation stages, can manifest in the bone marrow, circulate in the bloodstream, or infiltrate tissues. P-gp overexpression in malignant cells reduces the efficacy of chemotherapeutic agents by actively expelling them, decreasing intracellular drug concentrations, and promoting multidrug resistance, a significant obstacle to successful treatment. This review examines recent advances in combating P-gp-mediated resistance, including the development of novel P-gp inhibitors, innovative drug delivery systems (e.g., nanoparticle-based delivery), and strategies to modulate P-gp expression or activity. These modulation strategies encompass targeting relevant signaling pathways (e.g., NF-κB, PI3K/Akt) and exploring drug repurposing. While progress has been made, overcoming P-gp-mediated resistance remains crucial for improving patient outcomes. Future research directions should prioritize the development of potent, selective, and safe P-gp inhibitors with minimal off-target effects, alongside exploring synergistic combination therapies with existing chemotherapeutics or novel agents to effectively circumvent multidrug resistance in hematological malignancies. Full article

(This article belongs to the Special Issue Advances in Cellular Immunotherapy for Hematological Malignancies)

28 pages, 764 KiB

Open AccessReview

A Brief Overview of the Epigenetic Regulatory Mechanisms in Plants

by Theodoros Tresas, Ioannis Isaioglou, Andreas Roussis and Kosmas Haralampidis

Int. J. Mol. Sci. 2025, 26(10), 4700; https://doi.org/10.3390/ijms26104700 (registering DOI) - 14 May 2025

Abstract

Plants continuously adapt to their environments by responding to various intrinsic and extrinsic signals. They face numerous biotic and abiotic stresses such as extreme temperatures, drought, or pathogens, requiring complex regulatory mechanisms to control gene activity and adapt their proteome for survival. Epigenetic [...] Read more.

Plants continuously adapt to their environments by responding to various intrinsic and extrinsic signals. They face numerous biotic and abiotic stresses such as extreme temperatures, drought, or pathogens, requiring complex regulatory mechanisms to control gene activity and adapt their proteome for survival. Epigenetic regulation plays a crucial role in these adaptations, potentially leading to both heritable and non-heritable changes across generations. This process enables plants to adjust their gene expression profiles and acclimate effectively. It is also vital for plant development and productivity, affecting growth, yield, and seed quality, and enabling plants to “remember” environmental stimuli and adapt accordingly. Key epigenetic mechanisms that play significant roles include DNA methylation, histone modification, and ubiquitin ligase complex activity. These processes, which have been extensively studied in the last two decades, have led to a better understanding of the underlying mechanisms and expanded the potential for improving agriculturally and economically important plant traits. DNA methylation is a fundamental process that regulates gene expression by altering chromatin structure. The addition of methyl groups to cytosines by DNA methylases leads to gene suppression, whereas DNA demethylases reverse this effect. Histone modifications, on the other hand, collectively referred to as the “histone code”, influence chromatin structure and gene activity by promoting either gene transcription or gene silencing. These modifications are either recognized, added, or removed by a variety of enzymes that act practically as an environmental memory, having a significant impact on plant development and the responses of plants to environmental stimuli. Finally, ubiquitin ligase complexes, which tag specific histones or regulatory proteins with ubiquitin, are also crucial in plant epigenetic regulation. These complexes are involved in protein degradation and play important roles in regulating various cellular activities. The intricate interplay between DNA methylation, histone modifications, and ubiquitin ligases adds complexity to our understanding of epigenetic regulation. These mechanisms collectively control gene expression, generating a complex and branching network of interdependent regulatory pathways. A deeper understanding of this complex network that helps plants adapt to environmental changes and stressful conditions will provide valuable insights into the regulatory mechanisms involved. This knowledge could pave the way for new biotechnological approaches and plant breeding strategies aimed at enhancing crop resilience, productivity, and sustainable agriculture. Full article

(This article belongs to the Section Molecular Plant Sciences)

16 pages, 1107 KiB

Open AccessReview

Sweet Aging: Glycocalyx and Galectins in CNS Aging and Neurodegenerative Disorders

by Mohd Yaqub Mir and Adam Legradi

Int. J. Mol. Sci. 2025, 26(10), 4699; https://doi.org/10.3390/ijms26104699 (registering DOI) - 14 May 2025

Abstract

Aging and aging-related neurodegenerative disorders, such as Alzheimer’s disease, are characterized by chronic inflammation that progressively damages nervous tissue within the central nervous system (CNS). In addition to cytokines, lectin-like carbohydrate recognition molecules play a critical role in modifying cellular communication during inflammation. [...] Read more.

Aging and aging-related neurodegenerative disorders, such as Alzheimer’s disease, are characterized by chronic inflammation that progressively damages nervous tissue within the central nervous system (CNS). In addition to cytokines, lectin-like carbohydrate recognition molecules play a critical role in modifying cellular communication during inflammation. Among these, galectins—particularly anti-inflammatory galectin-1 and pro-inflammatory galectin-3—stand out due to their immunological functions and specificity for N-acetyllactosamine structures. Almost every cell type within the CNS can express and recognize galectins, influencing various essential cellular functions. N-acetyllactosamines, the ligand structures recognized by galectins, are found beneath sialylated termini in protein-linked oligosaccharides. During aging, protein-linked oligosaccharide structures become shorter, exposing N-acetyllactosamines on protein surfaces, which enhances their availability as binding sites for galectins. Genomic studies indicate that the number of galectin-1- and galectin-3-expressing microglial cells increases with age- or age-related disease (Alzheimer’s disease), reflecting an aging-associated rise in galectin concentrations within the CNS. This increase parallels a rise in free N-acetyllactosamine-like ligands, suggesting that galectin-N-acetyllactosamine interactions gain prominence and play a more significant role in aging-related CNS disorders. Understanding these interactions and their molecular implications offers potential avenues for targeted therapeutic strategies in combating aging-related CNS inflammation and neurodegeneration. Full article

(This article belongs to the Special Issue Neurodegeneration and Neuroinflammatory Processes in Age-Related Diseases)

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22 pages, 21306 KiB

Open AccessArticle

Fingolimod Prevents Neuroinflammation but Has a Limited Effect on the Development of Ataxia in a Mouse Model for SCA1

by Chen Yang, Nienke Gravendeel, Amy Chin On, Laura Post, Ryan van Bergen, Catarina Osorio and Martijn Schonewille

Int. J. Mol. Sci. 2025, 26(10), 4698; https://doi.org/10.3390/ijms26104698 (registering DOI) - 14 May 2025

Abstract

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder that predominantly affects the Purkinje cells (PCs) of the cerebellum, leading to cerebellar degeneration, motor dysfunction, and cognitive impairment. Sphingosine-1-phosphate (S1P) signaling, known to modulate neuroinflammation, has been identified as a potential therapeutic target [...] Read more.

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disorder that predominantly affects the Purkinje cells (PCs) of the cerebellum, leading to cerebellar degeneration, motor dysfunction, and cognitive impairment. Sphingosine-1-phosphate (S1P) signaling, known to modulate neuroinflammation, has been identified as a potential therapeutic target in SCA1. To investigate the therapeutic efficacy of the S1P modulator fingolimod, we treated a mouse model for SCA1, ATXN1[82Q]/+ mice during three different periods with fingolimod and assessed the effects. Potential therapeutic effects were monitored by tracking locomotion during the treatment period and examining PC morphology, connectivity, and markers for neuroinflammation post-mortem. Fingolimod treatment reduced astrocyte and microglial activation during all three treatment periods. We found no effect on calbindin levels or the thickness of the molecular layer, but fingolimod did improve the extent of the synaptic input of climbing fibers to PCs. While fingolimod improved important aspects of cellular pathology, we could only detect signs of improvement in the locomotion phenotype when treatment started at a later stage of the disease. In conclusion, fingolimod is able to mitigate neuroinflammation, preserve aspects of PC function in SCA1, and remediate part of the ataxia phenotype when treatment is appropriately timed. Although behavioral benefits were limited, targeting S1P pathways represents a potential therapeutic strategy for SCA1. Further studies are needed to optimize treatment regimens and assess long-term outcomes. Full article

(This article belongs to the Special Issue New Mechanisms and Therapeutics in Neurological Diseases—4th Edition)

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25 pages, 3487 KiB

Open AccessArticle

AI-Driven Drug Target Screening Platform Identified Oncogene CACNA2D1 Activated by Enhancer Infestation in Epstein-Barr Virus-Associated Nasopharyngeal Carcinoma

by Dittman Lai-Shun Chung, Geoffrey Ho Duen Leung, Songran Liu, Sarah Wing Yan Lok, Ying Xin, Yunfei Xia, Alex Zhavoronkov, Frank W. Pun, Wai-Tong Ng and Wei Dai

Int. J. Mol. Sci. 2025, 26(10), 4697; https://doi.org/10.3390/ijms26104697 (registering DOI) - 14 May 2025

Abstract

The management of nasopharyngeal cancer (NPC) is rapidly evolving, with immune checkpoint inhibitors emerging as a prominent treatment approach. However, drug development targeting specific molecular and cellular abnormalities in NPC has slowed. Recent advancements in artificial intelligence (AI) and bioinformatics, particularly those integrating [...] Read more.

The management of nasopharyngeal cancer (NPC) is rapidly evolving, with immune checkpoint inhibitors emerging as a prominent treatment approach. However, drug development targeting specific molecular and cellular abnormalities in NPC has slowed. Recent advancements in artificial intelligence (AI) and bioinformatics, particularly those integrating multi-omics data, offer a more effective alternative to traditional in vitro screening methods for identifying clinically actionable targets in NPC. Through a combination of multi-omics analyses and AI-driven screening, we identified CACNA2D1 as a novel cancer-cell-specific therapeutic target in NPC. Our research indicates that exploiting Epstein–Barr virus (EBV) tethering increases H3K27 acetylation near the CACNA2D1 promoter. Analysis of clinical specimens revealed significant upregulation of CACNA2D1 at both the transcriptional and translational levels (p-value < 0.01). Functional studies demonstrated that the mouse tumour size shrank by one-third upon the depletion of CACNA2D1, and there was an 85% reduction in cancer cell growth through the blockage of enhancers, while the presence of CACNA2D1 conferred a survival advantage during NPC tumour development. These findings highlight the potential of CACNA2D1 as a promising target for therapeutic intervention in NPC. Full article

(This article belongs to the Special Issue Recent Advances in Molecular Targets for Treatment and Drug Delivery in Tumors)

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15 pages, 4471 KiB

Open AccessArticle

Biosynthesized Calcium Peroxide Nanoparticles as a Multifunctional Platform for Liver Cancer Therapy

by Sen Wu, Siqi Li, Xin Xia, Gen Zhang and Ting Wang

Int. J. Mol. Sci. 2025, 26(10), 4696; https://doi.org/10.3390/ijms26104696 (registering DOI) - 14 May 2025

Abstract

To overcome the limitations associated with chemically synthesized nanoparticles in cancer therapy, researchers have increasingly focused on developing nanoparticles with superior biocompatibility and prolonged tumor retention using biosynthetic methods. In this study, we first identified the presence of calcium peroxide nanoparticles (CaO₂ [...] Read more.

To overcome the limitations associated with chemically synthesized nanoparticles in cancer therapy, researchers have increasingly focused on developing nanoparticles with superior biocompatibility and prolonged tumor retention using biosynthetic methods. In this study, we first identified the presence of calcium peroxide nanoparticles (CaO₂ NPs) in the blood of individuals who had ingested calcium gluconate. Furthermore, the dropwise addition of calcium gluconate to human serum resulted in the spontaneous self-assembly of CaO₂ NPs. Next, following tail vein injection of fluorescently labeled CaO₂ NPs into subcutaneous tumor-bearing nude mice, we observed that the nanoparticles exhibited prolonged accumulation at the tumor sites compared to other organs through visible-light imaging. Immunofluorescence staining demonstrated that CaO₂ NPs co-localized with vesicular transport-associated proteins, such as PV-1 and Caveolin-1, as well as the albumin-binding-associated protein SPARC, suggesting that their transport from tumor blood vessels to the tumor site is mediated by Caveolin-1- and SPARC-dependent active transport pathways. Additionally, the analysis of various organs in normal mice injected with CaO₂ NPs at concentrations significantly higher than the experimental dose showed no apparent organ damage. Hemolysis assays indicated that hemolysis occurred only at calcium concentrations of 300 µg/mL, whereas the experimental concentration remained well below this threshold with no detectable hemolytic activity. In a subcutaneous tumor-bearing nude mouse model, treatment with docetaxel-loaded CaO₂ NPs showed a 68.5% reduction in tumor volume compared to free docetaxel (DTX) alone. These novel biosynthetic CaO₂ NPs demonstrated excellent biocompatibility, prolonged retention at the tumor site, safety, and drug-loading capability. Full article

(This article belongs to the Section Molecular Nanoscience)

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19 pages, 9476 KiB

Open AccessReview

Development of Liquid Chromatography on Monolithic Supports—From First Concepts to Real Analytical and Preparative Techniques

by Tomislav Friganović and Djuro Josić

Int. J. Mol. Sci. 2025, 26(10), 4695; https://doi.org/10.3390/ijms26104695 (registering DOI) - 14 May 2025

Abstract

In this review, we trace the evolution of liquid chromatography from the pioneering work of Tennikova and Svec to the current monolithic polymethacrylate supports for performing liquid chromatography with biological macromolecules and nanoparticles, which offer rapid, high-throughput separations. By using interconnected channels with [...] Read more.

In this review, we trace the evolution of liquid chromatography from the pioneering work of Tennikova and Svec to the current monolithic polymethacrylate supports for performing liquid chromatography with biological macromolecules and nanoparticles, which offer rapid, high-throughput separations. By using interconnected channels with a tailored channel diameter, monoliths minimize the diffusion limitations typical of particle-based systems. Radial flow designs and optimized channel architectures enable the direct loading of complex biological fluids, reducing the need for sample preparation and optimizing the purification of large biomolecules and nanoparticles such as proteins, nucleic acids, extracellular vesicles, and viruses. Recent work has integrated monoliths into immunoaffinity and enzyme reactor platforms, streamlining analytical workflows and preparative applications in vaccine production and gene therapy. The ongoing advances in monolithic materials, channel geometry, and continuous processing hold promise for even greater efficiency and scalability in future applications. Full article

(This article belongs to the Section Biochemistry)

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13 pages, 1623 KiB

Open AccessArticle

Long-Term Real-World Outcomes and Insights of Biologic Therapies in Chronic Rhinosinusitis with Nasal Polyps

by Reut Book, Anna Lazutkin and Ron Eliashar

Int. J. Mol. Sci. 2025, 26(10), 4694; https://doi.org/10.3390/ijms26104694 (registering DOI) - 14 May 2025

Abstract

Chronic rhinosinusitis with nasal polyps is a Type 2 inflammatory disease associated with a significant burden on quality of life. While biological therapies have shown efficacy in randomized controlled trials, data on long-term real-world outcomes remain limited. This retrospective cohort study evaluated the [...] Read more.

Chronic rhinosinusitis with nasal polyps is a Type 2 inflammatory disease associated with a significant burden on quality of life. While biological therapies have shown efficacy in randomized controlled trials, data on long-term real-world outcomes remain limited. This retrospective cohort study evaluated the clinical efficacy, safety, and treatment dynamics of biologics, particularly anti-IL-4 (dupilumab), over a five-year period at a tertiary medical center. Fifty-two patients with CRSwNP meeting the EPOS/EUFOREA eligibility criteria were included. Clinical parameters, including nasal polyp score, SNOT-22, and olfactory function, were assessed across follow-up intervals. Anti-IL-4 therapy demonstrated the most consistent and sustained improvements in all clinical parameters, with a significant proportion of patients maintaining response beyond 36 months. A subset of patients underwent interval extension of dupilumab injections without loss of efficacy. Subdomain analysis of the SNOT-22 questionnaire revealed improvements predominantly in nasal and emotional domains. Treatment response, assessed according to the EUFOREA criteria, favored anti-IL-4 over anti-IL-5 and anti-IgE. Side effects were infrequent and mostly mild. These findings support the durable effectiveness of biologics in real-world CRSwNP management and suggest that tapering down the injection intervals may be a feasible strategy for selected patients. Further studies are needed to refine treatment response definitions and optimize patient-specific therapeutic approaches. Full article

(This article belongs to the Special Issue Chronic Rhinosinusitis: Aetiology, Immunology and Treatment, 3rd Edition)

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23 pages, 6147 KiB

Open AccessArticle

Multivariate Spectroscopic Analysis of Protein Secondary Structures in Gingival Crevicular Fluid: Insights from FTIR Amide III Band Across Oral Disease Stages

by Pavel Seredin, Tatiana Litvinova, Yuri Ippolitov, Dmitry Goloshchapov, Yaroslav Peshkov, Boknam Chae, Raul O. Freitas and Francisco C. B. Maia

Int. J. Mol. Sci. 2025, 26(10), 4693; https://doi.org/10.3390/ijms26104693 (registering DOI) - 14 May 2025

Abstract

This study applies multivariate data analysis to deconvolute the spectral profiles of the Amide III region in the infrared spectra of gingival crevicular fluid (GCF). This reveals the impact of major oral diseases, such as dental caries and periodontal diseases, on the transformation [...] Read more.

This study applies multivariate data analysis to deconvolute the spectral profiles of the Amide III region in the infrared spectra of gingival crevicular fluid (GCF). This reveals the impact of major oral diseases, such as dental caries and periodontal diseases, on the transformation of the secondary structure of GCF proteins. A two-stage analytical approach was employed: first, principal component analysis (PCA) was performed to establish the main factors of variation in the data, followed by pairwise comparisons of the samples based on the results of the Amide III profile deconvolution. The analysis also accounted for comorbidities, such as oncological and gastrointestinal diseases. This approach allowed for the identification of subtle differences in the composition and conformation of the secondary structure of GCF proteins while accounting for the superposition of multiple influencing factors. This methodology was effective in identifying biomarkers of oral diseases in GCF. For the first time, it has been demonstrated that the relative content of the β-sheet-associated component in the spectral profile of the secondary structure element of the protein fraction of GCF serves as a statistically significant marker for dental caries, regardless of the presence or absence of other diseases. Additionally, a significant decrease in the relative content of α-helix structures was observed in GCF from patients with oncological diseases. The changes in the spectral profile of the Amide III band of GCF identified in this study have not been previously detected using molecular spectroscopy, correlated with the secondary structure of proteins, or analyzed using multivariate analysis methods. Full article

(This article belongs to the Section Biochemistry)

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24 pages, 8106 KiB

Open AccessArticle

Multi-Omics Analysis of the Epigenetic Effects of Inflammation in Murine Type II Pneumocytes

by Jenna A. Fernandez, Qiyuan Han, Andrew T. Rajczewski, Thomas Kono, Nicholas A. Weirath, Alexander S. Lee, Abdur Rahim and Natalia Y. Tretyakova

Int. J. Mol. Sci. 2025, 26(10), 4692; https://doi.org/10.3390/ijms26104692 (registering DOI) - 14 May 2025

Abstract

Chronic inflammation plays a central role in the pathogenesis of lung diseases including asthma, long COVID, chronic obstructive pulmonary disease (COPD), and lung cancer. Lipopolysaccharide (LPS) is a potent inflammatory agent produced by Gram-negative bacteria and also found in cigarette smoke. Our earlier [...] Read more.

Chronic inflammation plays a central role in the pathogenesis of lung diseases including asthma, long COVID, chronic obstructive pulmonary disease (COPD), and lung cancer. Lipopolysaccharide (LPS) is a potent inflammatory agent produced by Gram-negative bacteria and also found in cigarette smoke. Our earlier study revealed that the intranasal exposure of A/J mice to LPS for 7 days altered gene expression levels in alveolar Type II epithelial cells (AECIIs), which serve as precursors to lung adenocarcinoma and are also preferentially targeted by SARS-CoV-2. In the present work, we employed a comprehensive multi-omics approach to characterize changes in DNA methylation/hydroxymethylation, gene expression, and global protein abundances in the AECIIs of A/J mice following the sub-chronic exposure to LPS and after a 4-week recovery period. Exposure to LPS led to hypermethylation at regulatory elements within the genome such as enhancer regions and expression changes in genes known to play a role in lung cancer tumorigenesis. Changes in protein abundance were consistent with an inflammatory phenotype and also included tumor suppressor proteins. Integration of the multi-omics data resulted in a model where LPS-driven inflammation in AECIIs triggers epigenetic changes that, along with genetic mutations, may contribute to lung cancer development. Full article

(This article belongs to the Special Issue Molecular Research of Multi-omics in Cancer)

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32 pages, 26719 KiB

Open AccessReview

Importance of Advanced Detection Methodologies from Plant Cells to Human Microsystems Targeting Anticancer Applications

by Mostafa M. Gouda, Eman R. Elsharkawy, Yong He and Xiaoli Li

Int. J. Mol. Sci. 2025, 26(10), 4691; https://doi.org/10.3390/ijms26104691 (registering DOI) - 14 May 2025

Abstract

The growing global demand for phytochemicals as bioactive sources is prompting scientists to develop methods that link their sensory properties to their mechanisms of action in cancer treatment. Recent techniques for tracking the actions of small plant metabolites (SPMs) from single-cell plant sources [...] Read more.

The growing global demand for phytochemicals as bioactive sources is prompting scientists to develop methods that link their sensory properties to their mechanisms of action in cancer treatment. Recent techniques for tracking the actions of small plant metabolites (SPMs) from single-cell plant sources to their molecular anticancer biomarkers could provide valuable insights in this field. Among the critical methods discussed in this review are the real-time tracking of cell components through stable isotope probing (Sis) and microspectroscopy, which has attracted the attention of biotechnologists. Additionally, the precise pathways required for studying new insights into functional materials are discussed, based on high-resolution and accurate technologies, which could aid their functional categorization. Notably, the molecules under study have recently garnered attention for their anticancer applications due to advancements in effective evaluation techniques that surpass traditional methods. In December 2020, the Food and Drug Administration (FDA) authorized 89 SPMs as safe anticancer natural molecules. In conclusion, by combining spatiotemporal techniques and SPMs’ mechanisms, they could facilitate the development of more exceptional, bio-efficient materials. Full article

(This article belongs to the Special Issue Cheminformatics in Drug Discovery and Green Synthesis)

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13 pages, 593 KiB

Open AccessReview

Current Methods in Synovial Fluid Microbiota Characterization: A Systematic Review

by Elena Bardi, Daniele D’Arrigo, Chiara Pozzi, Andrea Gatti, Luca Bertolino, Alberto Favaro, Maria Rescigno and Tommaso Bonanzinga

Int. J. Mol. Sci. 2025, 26(10), 4690; https://doi.org/10.3390/ijms26104690 (registering DOI) - 14 May 2025

Abstract

Evidence suggests that a cross-talk between the gut microbiota and joint health exists in a paradigm known as the gut–joint axis. Recent studies have also reported the presence of microorganisms potentially involved in the pathogenesis and progression of arthritis in synovial joints, previously [...] Read more.

Evidence suggests that a cross-talk between the gut microbiota and joint health exists in a paradigm known as the gut–joint axis. Recent studies have also reported the presence of microorganisms potentially involved in the pathogenesis and progression of arthritis in synovial joints, previously believed to be sterile. This systematic review describes in detail the methodologies employed to characterize the microbiota in human synovial fluid (SF). A literature search was conducted in PubMed, Embase, and Web of Science up to 5 February 2025. Nine studies aimed to characterize the SF microbiome using next-generation sequencing or polymerase chain reaction. Eight studies detected bacterial DNA in SF. However, significant heterogeneity and incomplete reporting in methodologies, including sample collection and preparation, contamination management, DNA extraction and amplification, sequencing technology, targeted 16S rRNA or ITS regions, and bioinformatics processing, limit the comparability and significance of findings. Given the potential implications for understanding arthritis mechanisms and developing targeted treatments, a standardized methodological and reporting approach in SF microbiota characterization is needed to enhance the reproducibility and the relevance of results. Full article

(This article belongs to the Special Issue Highlights in Pathophysiology of the Musculoskeletal System, 2nd Edition)

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21 pages, 4074 KiB

Open AccessArticle

A Structural In Silico Analysis of Novel Epitopes from Toxoplasma gondii Proteins for the Serodiagnosis of Toxoplasmosis

by Angelis del Valle Benitez Betancourt, Tamires Lopes Silva, Débora Karolla de Freitas Oliveira, Nilson Nicolau-Junior, João Luis Garcia, Ricardo Toshio Fujiwara, Tiago Wilson Patriarca Mineo and José Roberto Mineo

Int. J. Mol. Sci. 2025, 26(10), 4689; https://doi.org/10.3390/ijms26104689 (registering DOI) - 14 May 2025

Abstract

Toxoplasmosis is a widely spread zoonosis worldwide, considered one of the most important parasitic infections that affect global public health, and usually, it is not correctly diagnosed. Serological tests for the diagnosis of Toxoplasma gondii infection have limitations in differentiating acute from chronic [...] Read more.

Toxoplasmosis is a widely spread zoonosis worldwide, considered one of the most important parasitic infections that affect global public health, and usually, it is not correctly diagnosed. Serological tests for the diagnosis of Toxoplasma gondii infection have limitations in differentiating acute from chronic infection, which is important to determine the appropriate clinical management and treatment, mainly in pregnant women and immunocompromised individuals infected by this parasite. The present study aimed to characterize immunogenic epitopes from T. gondii immunodominant antigens, as SAG1(SRS29B), SAG2A (SRS34A), GRA1, GRA2, GRA3, GRA5, GRA6, GRA7, MAG1, BSR4, and CCp5A, by investigating if these parasite components might emerge as alternatives to improve the diagnosis of toxoplasmosis. A detailed comparative in silico analysis was used for this purpose. Once the protein sequences were retrieved from the ToxoDB database, different parameters were calculated, including physicochemical characteristics, accessibility values, and antigenicity. Multiple sequence alignment, 3D structures modeling, and the validation of 3D structures were also performed among all 11 peptides. Considering the results from the combination of all parameters analyzed, it can be hypothesized that the linear epitopes from SAG1, GRA3, and BSR4 proteins were found to be stable and hydrophilic, with a significant antigenicity score, and accessibility on the protein surface. Also, these three selected peptides were able to detect anti-T. gondii antibodies in serum samples from pigs infected by tachyzoites, when compared with control serum samples, obtained from the same naïve animals and tested by ELISA, demonstrating remarkable difference in terms of reactivity. Taken together, as our study addresses a critical challenge in the serodiagnosis of toxoplasmosis, particularly in gestational and congenital infections, where false-positive and false-negative results often arise from the use of native or recombinant antigens of T. gondii, our findings highlight the potential of synthetic peptides derived from novel epitopes of this parasite as alternative tools for the development of more accurate immunodiagnostic assays for toxoplasmosis. Full article

(This article belongs to the Special Issue Parasite Biology and Host-Parasite Interactions: 2nd Edition)

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17 pages, 8363 KiB

Open AccessArticle

A Biomimetic Adhesive/Kaolin Material with Strong Adhesion, Sealing, and Active Coagulation Function for Arterial Hemostasis

by Wanli Zhang, Junqin Mao, Aiping Yang, Tao Shen, Qiuyu Zeng, You Tang, Yan Du, Guoyu Lv, Heng Zheng and Hong Li

Int. J. Mol. Sci. 2025, 26(10), 4688; https://doi.org/10.3390/ijms26104688 (registering DOI) - 14 May 2025

Abstract

Effective prehospital hemostasis is pivotal for improving survival outcomes in arterial hemorrhage. Existing hemostatic materials have limitations in terms of both efficiency and portability, particularly under complex wound conditions. This study engineered a kaolin-reinforced biomimetic adhesive composite powder (DTG-K), which achieved dual hemostatic [...] Read more.

Effective prehospital hemostasis is pivotal for improving survival outcomes in arterial hemorrhage. Existing hemostatic materials have limitations in terms of both efficiency and portability, particularly under complex wound conditions. This study engineered a kaolin-reinforced biomimetic adhesive composite powder (DTG-K), which achieved dual hemostatic mechanisms: mechanical occlusion via strong interfacial adhesion (lap-shear strength of 58.3 kPa) and biochemical activation of the intrinsic coagulation pathway through factor XII. In vitro evaluations confirmed its exceptional hemocompatibility, with a 0.93% hemolysis rate. When applied to femoral artery hemorrhage models in Bama swine, DTG-K demonstrated complete hemostasis within 30 s through synergistic physical sealing and biochemical coagulation. These findings demonstrate the potential of DTG-K as an innovative strategy for managing life-threatening arterial hemorrhage in prehospital emergency scenarios. Full article

(This article belongs to the Section Materials Science)

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