International Journal of Molecular Sciences

46 pages, 3829 KB

Open AccessReview

Glutathione Biology in Neurodegenerative and Metabolic Diseases: Molecular Mechanisms, Pathophysiological Roles, and Therapeutic Perspectives

by Grażyna Gromadzka, Magdalena Kąkol, Magdalena Klimkiewicz and Maria Bendykowska

Int. J. Mol. Sci. 2026, 27(16), 7507; https://doi.org/10.3390/ijms27167507 - 21 Aug 2026

Abstract

Glutathione is an abundant intracellular low-molecular-weight thiol that contributes importantly to cellular redox homeostasis. Besides its well-established role in the antioxidant defense of the cell, glutathione regulates mitochondrial function, metabolism of toxicants, protein thiol oxidation/reduction, redox signaling, and immunity. Disturbances in glutathione metabolism [...] Read more.

Glutathione is an abundant intracellular low-molecular-weight thiol that contributes importantly to cellular redox homeostasis. Besides its well-established role in the antioxidant defense of the cell, glutathione regulates mitochondrial function, metabolism of toxicants, protein thiol oxidation/reduction, redox signaling, and immunity. Disturbances in glutathione metabolism have been shown to play a role in various diseases; however, it has become clear that changes in glutathione metabolism are a part of a complex, multifactorial process. In this review, we summarize current knowledge of the molecular mechanisms governing glutathione synthesis, recycling, compartmentalization, and biological functions, with particular emphasis on redox signaling, the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1) pathway, and reversible protein S-glutathionylation. We further examine how disturbances in glutathione homeostasis interact with mitochondrial dysfunction, chronic inflammation, metabolic stress, and impaired cellular signaling in Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, multiple sclerosis, Wilson’s disease, type 2 diabetes, and nonalcoholic fatty liver disease. We also evaluate current translational interventions targeting restoration of glutathione balance through glutathione supplementation, precursor supplementation, pharmacological modulation of endogenous antioxidant mechanisms, dietary interventions, and changes in lifestyle. Despite the fact that many interventions have been promising at the mechanistic and experimental level, there are still insufficient clinical data because of the problems associated with glutathione availability, tissue specificity, disease variability, and a lack of sufficiently powered clinical trials. The conclusion of this review is that glutathione should not be viewed as a universal therapeutic target; instead, glutathione should be perceived as an important factor contributing to cellular resilience and able to help other disease-specific interventions. Future progress in glutathione-based interventions will likely depend on integrating redox biomarkers, patient stratification, and precision medicine strategies to identify individuals most likely to benefit from targeted modulation of glutathione homeostasis. Full article

(This article belongs to the Collection New Advances in Molecular Toxicology)

19 pages, 2628 KB

Open AccessArticle

Association of ABHD12 Variants with the Spectrum of PHARC Syndrome Phenotypes

by Sara Romero-Vázquez, Cécile Méjécase, Ana Catalina Rodriguez-Martinez, Nicola Cronbach and Mariya Moosajee

Int. J. Mol. Sci. 2026, 27(16), 7506; https://doi.org/10.3390/ijms27167506 - 21 Aug 2026

Abstract

PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataracts) syndrome is a rare neurodegenerative inherited disorder characterized by a spectrum of these clinical phenotypes. Due to the considerable heterogeneity in the age of onset of the different clinical features and the similarities with [...] Read more.

PHARC (polyneuropathy, hearing loss, ataxia, retinitis pigmentosa and cataracts) syndrome is a rare neurodegenerative inherited disorder characterized by a spectrum of these clinical phenotypes. Due to the considerable heterogeneity in the age of onset of the different clinical features and the similarities with other conditions, it is often misdiagnosed. Variants in the ABHD12 gene have been identified as a genetic cause of PHARC syndrome and are predicted to result in loss-of-function or severely reduced protein synthesis. ABHD12 (α/β-hydrolase domain-containing protein 12) is responsible for the breakdown of very-long-chain lysophosphatidylserines (VLC-lyso-PS). When ABHD12 is dysfunctional, VLC-lyso-PS accumulates in the brain, producing inflammation. In this study, the clinical phenotypes of five patients with molecularly confirmed ABHD12 pathogenic variants from Moorfields Eye Hospital together with those of 64 patients reported in the literature were analyzed to investigate genotype–phenotype correlations. We report a novel variant c.985del p.(His329Thrfs*57) associated with PHARC syndrome. Moreover, patients with two null variants in ABHD12 manifest more of the clinical features of PHARC syndrome than those with at least one missense variant. Our study shows the importance of a complete clinical and genetic diagnosis to enable accurate diagnosis and genetic counselling for affected individuals. Full article

(This article belongs to the Special Issue Advances in Molecular Therapeutics for Retinal Disease)

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34 pages, 3460 KB

Open AccessArticle

Single-Exposure Prophylactic Transcranial Nano-Pulsed Laser Therapy Promotes Functional Resilience Following Mild Blast-Induced Neurotrauma

by Nikita Gupta, Katherine N. Sheffield, Mohammadhossein Khanmirzaei, Auston C. Grant, Jutatip Guptarak, Ian J. Bolding, Kathia M. Johnson, Rinat O. Esenaliev, Donald S. Prough and Maria-Adelaide Micci

Int. J. Mol. Sci. 2026, 27(16), 7505; https://doi.org/10.3390/ijms27167505 - 21 Aug 2026

Abstract

Blast-induced traumatic brain injury is a prevalent and underreported condition, particularly among military service members, for whom effective prophylactic interventions are lacking. Nano-pulsed laser therapy (NPLT) is a non-invasive neuromodulatory approach that delivers short pulses of near-infrared light to generate optoacoustic effects within [...] Read more.

Blast-induced traumatic brain injury is a prevalent and underreported condition, particularly among military service members, for whom effective prophylactic interventions are lacking. Nano-pulsed laser therapy (NPLT) is a non-invasive neuromodulatory approach that delivers short pulses of near-infrared light to generate optoacoustic effects within cerebral tissue and has previously demonstrated therapeutic benefit following TBI. In this study, we evaluated whether a single pre-exposure application of NPLT could confer protection against neurological, cognitive, and cellular sequelae of mild blast injury. Adult male Sprague-Dawley rats were randomized to receive NPLT or Sham treatment 24 h prior to either Sham or mild blast exposure using the Advanced Blast Simulator. Neurological reflexes and vestibulomotor function were assessed on post-injury days (PIDs) 1–5, while cognitive performance was evaluated using the Morris Water Maze on PIDs 13–17. Histological analyses of microglia, astrocytes, and myelination were performed on PID 17. A single mild blast did not significantly alter gross neurological function but was associated with deficits in fine motor coordination and cognitive performance. Pre-exposure NPLT modestly attenuated blast-associated fine motor dysfunction, with a significant improvement compared with TBI on PID 4. In the Morris Water Maze, TBI animals exhibited significantly increased latency compared with Sham on PIDs 13 and 17, whereas NPLT + TBI animals did not significantly differ from Sham across the testing period, consistent with preservation of cognitive performance. Histological responses were regionally heterogeneous: NPLT alone produced distinct glial alterations, while NPLT + TBI animals exhibited a mixture of treatment- and injury-associated responses rather than uniform normalization to uninjured controls. NPLT did not prevent localized blast-associated reductions in corpus callosum myelin staining. In naive animals, NPLT significantly increased hippocampal brain-derived neurotrophic factor (BDNF) mRNA expression 24 h after treatment. A single pre-injury application of NPLT was associated with functional resilience following mild blast exposure despite persistent and regionally heterogeneous histopathological alterations. Increased hippocampal BDNF 24 h after NPLT, together with region-specific glial changes following NPLT in the absence of injury, demonstrates that a single treatment produces sustained molecular and cellular effects before blast exposure. These findings are consistent with the hypothesis that prophylactic NPLT establishes an altered pre-injury biological state that may modify the subsequent response to blast and support further investigation of NPLT as a prophylactic strategy and of the mechanisms underlying NPLT-associated preconditioning. Full article

(This article belongs to the Special Issue Progress in Photobiomodulation Therapy)

25 pages, 2475 KB

Open AccessArticle

PET Micro(nano)plastics Modulate Metformin–Albumin Binding and Species-Specific Bacterial Responses

by Hasan Saygin, Elif Aydin and Asli Baysal

Int. J. Mol. Sci. 2026, 27(16), 7504; https://doi.org/10.3390/ijms27167504 - 21 Aug 2026

Abstract

Metformin is a widely used antidiabetic drug that may enter biological and environmental systems together with micro/nanoplastics; however, their combined effects on protein interactions and microbial responses remain insufficiently understood. This study investigated how polyethylene terephthalate micro/nanoplastics (PET MNPs) influence metformin interactions with [...] Read more.

Metformin is a widely used antidiabetic drug that may enter biological and environmental systems together with micro/nanoplastics; however, their combined effects on protein interactions and microbial responses remain insufficiently understood. This study investigated how polyethylene terephthalate micro/nanoplastics (PET MNPs) influence metformin interactions with bovine serum albumin (BSA) and the subsequent responses of Escherichia coli and Staphylococcus aureus. BSA–metformin systems were conditioned with three PET MNP loads and increasing metformin concentrations. The resulting particle-depleted filtrates were evaluated using fluorescence spectroscopy, ultraviolet–visible spectroscopy, the Bradford assay, Rayleigh light scattering, turbidity, dithiothreitol-based oxidative potential, and reactive oxygen species (ROS) measurements. Bacterial growth, superoxide dismutase activity, glutathione-related thiol antioxidant response, lipid peroxidation, ROS generation, and biofilm formation were also assessed. PET MNP conditioning altered the fluorescence responses of tryptophan and tyrosine, modified BSA-associated absorbance, and produced non-linear changes in protein accessibility, aggregation-related scattering, turbidity, and oxidative indicators. The bacterial responses were species-specific. Escherichia coli showed increased bacterial growth under several exposure conditions, whereas Staphylococcus aureus exhibited reduced growth following metformin addition, particularly at the highest PET MNP load. Staphylococcus aureus also showed consistently elevated biofilm formation and a pronounced transient ROS increase under the high-PET, low-metformin condition. These findings indicate that upstream PET MNP conditioning can modify the physicochemical and biological properties of the filter-passing BSA–metformin phase, leading to concentration-dependent and species-specific bacterial responses. Full article

(This article belongs to the Special Issue Molecular Toxicology of Nano- and Microplastics: Mechanisms of Cellular Disruption and Biochemical Pathways)

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18 pages, 1976 KB

Open AccessArticle

Serum Periostin as a Complementary Biomarker of Frailty in Type 2 Diabetes: A Pilot Study Using the FRAIL Scale

by Sheila González-Salvatierra, Beatriz García-Fontana, Cristina García-Fontana, Luis Martínez-Heredia, José Francisco Rojas-Pérez, María Carmen Andreo-López, Antonia García-Martín and Manuel Muñoz-Torres

Int. J. Mol. Sci. 2026, 27(16), 7503; https://doi.org/10.3390/ijms27167503 - 21 Aug 2026

Abstract

Frailty is a multidimensional syndrome of reduced physiological reserve that is particularly prevalent in individuals with type 2 diabetes. Identifying objective biochemical markers to complement clinical screening tools, such as the FRAIL scale, is an increasingly important unmet need. Periostin, a matricellular protein [...] Read more.

Frailty is a multidimensional syndrome of reduced physiological reserve that is particularly prevalent in individuals with type 2 diabetes. Identifying objective biochemical markers to complement clinical screening tools, such as the FRAIL scale, is an increasingly important unmet need. Periostin, a matricellular protein involved in tissue remodeling, bone metabolism, and chronic complications of diabetes, has emerged as a potential candidate. In this cross-sectional study of 137 adults with type 2 diabetes (65 ± 8 years), participants were classified as robust, pre-frail, or frail according to FRAIL scores. Frailty correlated positively with age (p < 0.001), BMI (p = 0.006), waist circumference (p = 0.01), and diabetes duration (p = 0.040), and negatively with TBS (p = 0.001), HDL-c (p = 0.040), and eGFR (p = 0.009). A stronger positive correlation was observed with serum periostin (p < 0.001). Frail individuals showed higher periostin levels than pre-frail (p = 0.006) and robust participants (p = 0.008), independent of age. Periostin demonstrated moderate discriminatory capacity for frailty status (AUC = 0.710; p < 0.001), while adding periostin to clinical variables resulted in a numerical increase in overall model discrimination (AUC = 0.900 vs. 0.858). A threshold of >1307 pmol/L yielded 77.8% sensitivity and 66.7% specificity. These preliminary findings indicate that periostin is significantly associated with frailty in type 2 diabetes and may represent a complementary biochemical biomarker of frailty status, warranting further validation in longitudinal studies. Full article

(This article belongs to the Special Issue Molecular Studies in Aging, 2nd Edition)

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34 pages, 7503 KB

Open AccessArticle

Molecularly Imprinted Polymers Based on Cyclodextrin Derivatives and Chitosan for Selective Extraction of Drugs and Dyes

by Linara Kopnova, Alexander Kopnov, Igor Zlotnikov and Elena Kudryashova

Int. J. Mol. Sci. 2026, 27(16), 7502; https://doi.org/10.3390/ijms27167502 - 21 Aug 2026

Abstract

A series of molecularly imprinted polymers (MIPs) based on hydroxypropyl-β-cyclodextrin (HPCD) crosslinked with 1,6-hexamethylene diisocyanate (HMD) or toluene diisocyanate (TDI), as well as hybrid chitosan–HPCD polymers crosslinked with genipin, were synthesized using levofloxacin and fluorescein as template molecules. The structure and spatial organization [...] Read more.

A series of molecularly imprinted polymers (MIPs) based on hydroxypropyl-β-cyclodextrin (HPCD) crosslinked with 1,6-hexamethylene diisocyanate (HMD) or toluene diisocyanate (TDI), as well as hybrid chitosan–HPCD polymers crosslinked with genipin, were synthesized using levofloxacin and fluorescein as template molecules. The structure and spatial organization of the obtained materials were characterized by FTIR spectroscopy, FTIR microscopy mapping, and ζ-potential measurements. The influence of pH, crosslinker content, and template structure on sorption performance was investigated. All MIPs exhibited maximum sorption at pH 3.0. The highest sorption capacity toward levofloxacin was achieved for the LV–Chit–HPCD–Genipin_MIP (74.8 mg/g), whereas the fluorescein-imprinted FL–HPCD–TDI_MIP (1:1) demonstrated the highest sorption capacity (135.5 mg/g) and selectivity coefficient (84.1). Dynamic column experiments confirmed efficient analyte extraction, reducing the analyte concentration by more than 90% after ten loading cycles. All synthesized MIPs exhibited excellent regenerability, with less than 3% loss of sorption efficiency after ten consecutive sorption–desorption cycles. The applicability of the developed sorbents to real matrices was demonstrated using milk and blood plasma samples after minimal sample preparation. Fluorescein extraction efficiencies reached 97.6% and 93.6% for milk and plasma, respectively. The obtained results demonstrate that HPCD-based MIPs combine high sorption capacity, exceptional selectivity, operational stability, and applicability to complex biological matrices, making them promising materials for selective sample preparation, analyte preconcentration, and controlled drug delivery systems. Full article

(This article belongs to the Special Issue Cyclodextrins: Properties and Applications, 4th Edition)

18 pages, 2165 KB

Open AccessArticle

Decoding Tumor–Immune Interactions in Hepatocellular Carcinoma Through Network-Centered Identification of CXCR2

by Saleh A. Almatroodi, Tarique Sarwar and Arshad Husain Rahmani

Int. J. Mol. Sci. 2026, 27(16), 7501; https://doi.org/10.3390/ijms27167501 - 21 Aug 2026

Abstract

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide and exhibits considerable biological heterogeneity in both molecular and clinical characteristics. The diverse molecular alterations and clinical manifestations of HCC indicate substantial heterogeneity across patient subgroups. This study aimed to identify novel [...] Read more.

Hepatocellular carcinoma (HCC) is one of the most prevalent cancers worldwide and exhibits considerable biological heterogeneity in both molecular and clinical characteristics. The diverse molecular alterations and clinical manifestations of HCC indicate substantial heterogeneity across patient subgroups. This study aimed to identify novel therapeutic targets and predictive biomarkers associated with HCC using an integrative bioinformatics approach. High-throughput genomic datasets were obtained from the UCSC Xena browser to retrieve mRNA HTSeq-count data from the TCGA-HCC cohort. Gene co-expression network (GCN), protein–protein interaction network (PPIN), and enrichment analyses were performed to identify key dysregulated genes and their biological significance. Integrated network analyses identified three dysregulated hub genes, namely CXCR2, TLR2, and TLR4. Genomic alterations in these genes were further evaluated across tumor samples in the TCGA-HCC cohort. Kaplan–Meier (KM) survival analysis demonstrated that lower CXCR2 mRNA expression was significantly associated with poorer overall survival (OS) and recurrence-free survival (RFS). Furthermore, TIMER and UALCAN analyses revealed significant associations between CXCR2 expression and tumor purity, as well as immune cell infiltration levels, including T cells, macrophages, dendritic cells (DCs), and neutrophils. These findings suggest that CXCR2 is significantly associated with the immune microenvironment of HCC and represents a potential prognostic biomarker whose biological role warrants further mechanistic investigation. Full article

(This article belongs to the Special Issue Advances in Molecular and Cellular Pathology of Cancer Research)

16 pages, 1542 KB

Open AccessArticle

Identification of Growth-Related Single-Nucleotide Polymorphisms in the cAMP-Dependent Protein Kinase Type I Regulatory Subunit-like Gene of the Oriental River Prawn, Macrobrachium nipponense

by Yuefan Zhang, Hao Dong, Xiaofan Fang, Hui Qiao, Wenyi Zhang, Yiwei Xiong, Sufei Jiang and Shubo Jin

Int. J. Mol. Sci. 2026, 27(16), 7500; https://doi.org/10.3390/ijms27167500 - 21 Aug 2026

Abstract

Macrobrachium nipponense is an economically important freshwater crustacean in China, and growth-related traits are major determinants of its commercial value. Identifying single nucleotide polymorphisms (SNPs) associated with growth-related traits may provide useful molecular markers for the genetic improvement of growth performance. Previous studies [...] Read more.

Macrobrachium nipponense is an economically important freshwater crustacean in China, and growth-related traits are major determinants of its commercial value. Identifying single nucleotide polymorphisms (SNPs) associated with growth-related traits may provide useful molecular markers for the genetic improvement of growth performance. Previous studies have suggested that the cAMP-dependent protein kinase type I regulatory subunit-like gene (Mn-PKA-R1) is involved in the regulation of growth and molting in M. nipponense. In the present study, a cultured full-sib family comprising 120 females and 120 males was used to screen SNPs within the coding region of Mn-PKA-R1, and evaluate their associations with body weight, body length, full length, and abdominal width. Seven SNPs were identified in exon 12, including five nonsynonymous missense variants and two synonymous variants, whereas no SNPs were detected in the other exons examined. The mean effective number of alleles (N_e), observed heterozygosity (H_o), expected heterozygosity (H_e), Nei’s gene diversity (Nei), and polymorphic information content (PIC) were 1.3561, 0.3048, 0.2350, 0.2340, and 0.1962, respectively, in females, compared with 1.3118, 0.2714, 0.1868, 0.1861, and 0.1512, respectively, in males. Four SNPs were significantly associated with growth-related traits in females: C+82748G and A+82750G were associated with body weight and abdominal width, T+82810G was associated with abdominal width, and A+83324G was associated with body weight, full length, and abdominal width (p < 0.05). No significant genotype–trait associations were detected among the SNP loci that could be statistically evaluated in males. Pairwise r² values ranged from 0.001 to 0.693 in females and from 0.000 to 0.351 in males, with generally higher values observed in females within the investigated full-sib family. Females also showed slightly higher mean genetic polymorphism parameters than males, while significant SNP–trait associations were detected only in females within this family. These findings provide candidate SNPs for further validation of their potential utility in marker-assisted selection in M. nipponense. Full article

(This article belongs to the Special Issue Molecular Genetics and Genomics of Aquatic Crustaceans)

26 pages, 94016 KB

Open AccessArticle

LMF-CP: An Interpretable Multimodal Late-Fusion Framework for Compound Carcinogenicity Prediction

by Yingjie Zhu, Liujie He and Xinjie Liang

Int. J. Mol. Sci. 2026, 27(16), 7499; https://doi.org/10.3390/ijms27167499 - 21 Aug 2026

Abstract

Accurately predicting the carcinogenicity of compounds is of great significance for drug discovery, clinical drug safety, and chemical risk assessment. Traditional methods for assessing carcinogenicity rely on animal testing, which suffers from limitations such as time-consuming processes, high costs, significant interspecies differences, and [...] Read more.

Accurately predicting the carcinogenicity of compounds is of great significance for drug discovery, clinical drug safety, and chemical risk assessment. Traditional methods for assessing carcinogenicity rely on animal testing, which suffers from limitations such as time-consuming processes, high costs, significant interspecies differences, and low predictive throughput. In recent years, computational modeling-based prediction methods (such as Quantitative Structure–Activity Relationships, QSAR) have made some progress, but they still face challenges such as insufficient molecular feature information and poor model interpretability. To overcome these barriers, the multimodal deep learning framework LMF-CP (Late Multimodal Fusion of Carcinogenicity Prediction) is proposed to enhance the performance and interpretability of compound carcinogenicity prediction. First, to comprehensively characterize the structural and physicochemical properties of compounds, a multimodal representation system based on four molecular modalities is constructed, namely SMILES sequences, molecular fingerprints, molecular images, and molecular graph structures. Specifically, Text Convolutional Neural Network (TextCNN), Multi-Layer Perceptron (MLP), Visual Geometry Group Network (VGGNet), as well as Molecular Graph Attention Network (MGAT) are employed to process this information, respectively. Second, to integrate information from different molecular representations, a late-stage fusion strategy based on Lasso stacking is employed. On the test set, LMF-CP achieves an area under curve (AUC) of 0.828, an accuracy (ACC) of 0.782, an F1 score of 0.786, a sensitivity (SEN) of 0.786, and a specificity (SPE) of 0.779. In addition, this paper combines Shapley Additive Explanations (SHAP) analysis with Bemis–Murcko scaffold analysis to interpret the model results from two perspectives. Finally, a visual online platform for predicting the carcinogenicity of compounds is designed, providing a convenient tool for the rapid assessment of compound carcinogenicity and structural interpretation. Full article

(This article belongs to the Special Issue Computational Strategies in Toxicology)

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15 pages, 3964 KB

Open AccessArticle

Engineering of a Carbonic Anhydrase from Hydrogenimonas thermophila Through Fusion Tags and Surface Mutagenesis Enhances Solubility While Revealing Stability–Function Relationships

by Colleen Varaidzo Manyumwa, Carsten Jers and Ivan Mijakovic

Int. J. Mol. Sci. 2026, 27(16), 7498; https://doi.org/10.3390/ijms27167498 - 21 Aug 2026

Abstract

Protein solubility can limit enzyme performance in industrial applications. This is the case for some carbonic anhydrases (CAs), key enzymes for CO₂ capture and utilization. In this study, we investigated an α-class CA from the thermophilic bacterium Hydrogenimonas thermophila (HtCA), which was [...] Read more.

Protein solubility can limit enzyme performance in industrial applications. This is the case for some carbonic anhydrases (CAs), key enzymes for CO₂ capture and utilization. In this study, we investigated an α-class CA from the thermophilic bacterium Hydrogenimonas thermophila (HtCA), which was predominantly expressed as an insoluble protein in Escherichia coli. Surface analysis using Molecular Operating Environment (MOE) revealed extensive hydrophobic regions, suggesting a basis for its poor solubility. To improve solubility, three C-terminal fusion tags were evaluated (Gb1, ng3-NEXT, and T7B9). All tagged variants showed markedly increased soluble expression as determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis. To reduce surface hydrophobicity, selected residues were substituted with charged amino acids. Most variants displayed improved solubility, and V136D showed enhanced thermostability, retaining 76% activity after exposure to 90 °C for an hour. However, the F177D variant completely lost all enzymatic activity, highlighting the importance of evaluating both solubility and catalytic function during protein engineering. Molecular dynamics simulations supported the experimental findings, revealing that thermostable variants exhibited reduced structural fluctuations and favorable free-energy landscapes, while the inactive F177D mutant sampled a broader conformational space and higher-energy conformations, consistent with decreased structural stability and loss of catalytic activity. Full article

(This article belongs to the Special Issue The Characterization and Application of Enzymes in Bioprocesses, 2nd Edition)

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25 pages, 2404 KB

Open AccessArticle

Beyond the Score: Fixed-Budget Benchmarking of Virtual Screening Integration Strategies for Decision-Centric Drug Discovery

by Elisabetta Grazia Tomarchio, Rocco Buccheri and Antonio Rescifina

Int. J. Mol. Sci. 2026, 27(16), 7497; https://doi.org/10.3390/ijms27167497 - 21 Aug 2026

Abstract

Virtual screening (VS) workflows often combine structure- and ligand-based methods; however, their value depends on the number of compounds that can be tested. We benchmarked 20 fixed-budget strategies derived from molecular docking (GNINA CNN score), maximum common substructure (MCS) similarity, and a calibrated [...] Read more.

Virtual screening (VS) workflows often combine structure- and ligand-based methods; however, their value depends on the number of compounds that can be tested. We benchmarked 20 fixed-budget strategies derived from molecular docking (GNINA CNN score), maximum common substructure (MCS) similarity, and a calibrated machine-learning (ML)-QSAR classifier across five pharmacologically diverse targets. Individual methods, best-rank and worst-rank fusion, mean-rank consensus, and sequential funnels were evaluated at 1%, 5%, and 10% library fractions, with every strategy selecting the same number of compounds. ML-QSAR was the strongest standalone method, recovering 47.6%, 81.6%, and 84.4% of actives at the three cutoffs. At the 1% budget, ML-QSAR achieved the highest mean hit recovery (47.6% recall; 99.2% precision). At 5% and 10%, best-rank fusion of QSAR and MCS produced the highest mean recall (83.2% and 86.4%). Among the sequential workflows, QSAR → MCS achieved the highest 1% hit recovery (45.2 ± 3.3% recall), whereas docking-first funnels consistently underperformed under the default, non-optimized conditions evaluated in this study. Target-level results showed substantial variability in MCS-containing workflows and limited benefits from adding docking without target-specific optimization. Under matched assay budgets, a validated ligand-based predictor or a simple two-method rank-fusion scheme provided the highest observed mean hit recovery without requiring elaborate integration. Full article

(This article belongs to the Special Issue Beyond Docking Scores: Decision-Centric Computer-Aided Drug Discovery)

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15 pages, 6404 KB

Open AccessArticle

Molecular Dissection of the SlBAG9 Promoter from Tomato and Its Thermo-Regulatory Activity

by Fan Fei, Fan Yang, Yucheng Peng, Menghan Zhu, Sihan Li, Hailong Jiang and Haidong Ding

Int. J. Mol. Sci. 2026, 27(16), 7496; https://doi.org/10.3390/ijms27167496 - 21 Aug 2026

Abstract

The Bcl-2-associated athanogene (BAG) gene family plays vital roles in plant growth, development, and biotic and abiotic stress responses. Previous work has demonstrated that tomato SlBAG9, a group II BAG member, negatively regulates plant thermotolerance. However, the regulatory mechanisms governing SlBAG9 expression [...] Read more.

The Bcl-2-associated athanogene (BAG) gene family plays vital roles in plant growth, development, and biotic and abiotic stress responses. Previous work has demonstrated that tomato SlBAG9, a group II BAG member, negatively regulates plant thermotolerance. However, the regulatory mechanisms governing SlBAG9 expression remain poorly understood. In this study, we isolated and characterized the authentic 1486 bp full-length promoter (P1) of SlBAG9 from the tomato genome. Building upon our previous transcript-level observations, we provide here a detailed functional characterization of this promoter at the cellular and tissue level. In silico analysis identified several key cis-acting regulatory elements, including abscisic acid-responsive elements (ABRE), anaerobic response elements (ARE), and a heat shock element (HSE1). We used stable transgenic tomato plants carrying SlBAG9pro::GUS to verify that the full-length promoter was capable of driving the expression of β-glucuronidase reporter gene (GUS) in transgenic tomato plants, showing GUS staining was detectable in the roots, stems, leaves, flowers, fruits, and seeds, with the highest activity in red-ripe fruits. Notably, GUS activity was significantly upregulated by high temperature (HT) but not by PEG, NaCl, ABA, or cold treatments. To further dissect the HT-responsive regulatory module, we generated three 5′-terminal deletion fragments (−386 bp, P2; −239 bp, P3; and −113 bp, P4) and fused them to GUS. Under HT stress, the smallest deletion P4 showed negligible GUS activity, whereas P1, P2, and P3 retained significant activity. Furthermore, site-directed deletion of the HSE1 element in the full-length context (MU-P1) abolished HT inducibility, confirming that HSE1 serves as a critical positive HT-responsive element. Collectively, these findings confirm and extend our observations that SlBAG9 is a stress-responsive gene, and the characterized HSE1-dependent promoter module represents a promising candidate for genetic engineering aimed at enhancing thermotolerance in crops. Full article

(This article belongs to the Special Issue Advances in Plant Breeding and Biotechnology: From Lab to Field)

35 pages, 1512 KB

Open AccessReview

Lactate as a Master Regulator of Immune Suppression: From Metabolic Waste to Epigenetic Checkpoint in Colorectal Cancer

by Beiyan Chen, Shuang Gao, Xin Chen, Qingping Shi, Mingli Shen and Jieru Han

Int. J. Mol. Sci. 2026, 27(16), 7495; https://doi.org/10.3390/ijms27167495 - 21 Aug 2026

Abstract

Colorectal cancer, especially the microsatellite-stable subtype, which accounts for 85% to 95% of cases, resists immune checkpoint inhibitors largely due to metabolic reprogramming in the tumor microenvironment. Lactate has evolved from a waste product into a central immunosuppressive regulator. Oncogenic KRAS and BRAF [...] Read more.

Colorectal cancer, especially the microsatellite-stable subtype, which accounts for 85% to 95% of cases, resists immune checkpoint inhibitors largely due to metabolic reprogramming in the tumor microenvironment. Lactate has evolved from a waste product into a central immunosuppressive regulator. Oncogenic KRAS and BRAF mutations drive aerobic glycolysis, causing glucose deprivation and massive lactate accumulation in the tumor microenvironment. Lactate suppresses immunity through three parallel mechanisms. It signals via GPR81 to recruit polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and inhibit T-cell function. It contributes to histone H3K18 lactylation, which silences effector genes including IFN-γ and GZMB while upregulating PD-L1 expression. It also acidifies the microenvironment to pH 6.0–6.5, directly impairing NK and T-cell activity. Concurrent lipid abundance stabilizes the MCT4 lactate exporter, forming a bidirectional feed-forward loop that amplifies lactate effects. Spatial metabolic heterogeneity creates distinct immune battlefields, with a supportive ‘metabolic oasis’—a concept proposed in this review—at the invasive front and a deeply immunosuppressive core. Thus, lactate acts as an epigenetic and signaling hub that bridges oncogenic mutations, metabolic competition and immune evasion. Targeting lactate metabolism through LDHA or MCT4 inhibition, modulation of histone lactylation, or disruption of lactate-lipid crosstalk, when combined with classical immune checkpoint blockade and guided by spatial biomarkers, offers a promising strategy to overcome immunotherapy resistance in this challenging subtype. Full article

(This article belongs to the Section Molecular Immunology)

18 pages, 1700 KB

Open AccessReview

Rhizobacteria-Mediated Reprogramming of Phytohormone Landscapes for Mitigating Salinity Stress in Plants

by Arghyadeepa Moharana, Lochan Dhruw, Armita Chakraborty, Preeti Pashwan, Sanjida Sultana Keya, Md. Mezanur Rahman, Archita Singh, Mamta Bhardwaj, Lam-Son Phan Tran and Aarti Gupta

Int. J. Mol. Sci. 2026, 27(16), 7494; https://doi.org/10.3390/ijms27167494 - 21 Aug 2026

Abstract

Salinity stress is one of the major stressors that limits yield potential in field crops. Salinity-led imbalances in ionic and water potential, as well as oxidative damage, impair photosynthesis. Plant-growth-promoting rhizobacteria (PGPRs) have been demonstrated to mitigate salinity-stress-induced damage through various mechanisms such [...] Read more.

Salinity stress is one of the major stressors that limits yield potential in field crops. Salinity-led imbalances in ionic and water potential, as well as oxidative damage, impair photosynthesis. Plant-growth-promoting rhizobacteria (PGPRs) have been demonstrated to mitigate salinity-stress-induced damage through various mechanisms such as biofilm and exopolysaccharide production, modulation of plant root architecture or molecular signaling involving modulation of sodium/potassium efflux transporters. PGPRs are known to induce biosynthesis and signaling of various phytohormones in plants. PGPR-derived phytohormones can in turn regulate molecular signaling involved in maintaining ion fluxes, preventing salinity-induced senescence, and reinforcing plant root architecture, thereby maintaining plant growth and development under saline conditions. In this review, we provide comprehensive advances on how PGPRs modulate and integrate biosynthesis and/or signaling of various phytohormones, such as auxins, cytokinins, gibberellin, ethylene, abscisic acid, salicylic acid, jasmonates, brassinosteroids and strigolactones, to reshape plant architecture, physiological and biochemical responses in plants under salinity. We integrate molecular evidence with morpho-physiological studies and propose a phytohormone-centric framework to select strains that optimize growth, ion homeostasis and plant stress resilience under salinity. Full article

(This article belongs to the Special Issue Plant Stress Biology)

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26 pages, 21363 KB

Open AccessArticle

Identification of Cellular Senescence-Related Hub Genes in Rheumatoid Arthritis from Bioinformatics Analysis Through Machine Learning up to Verifications in Mouse Macrophages and Tests in Patients

by Dandan Wang, Linkun Tian, Qingshan Ma, Zhengdong Zhang, Yi Wang, Junhao Fang, Hairong Xu, Qi Chen, Hongdian Chen, Fangyuan Wang, Qiaoyan Zhang, Quanlong Zhang and Luping Qin

Int. J. Mol. Sci. 2026, 27(16), 7493; https://doi.org/10.3390/ijms27167493 - 21 Aug 2026

Abstract

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and joint destruction. Cellular senescence contributes to chronic inflammation, yet key senescence-associated regulators in RA remain unclear. This study aimed to identify RA senescence-related signatures and their roles. Using GSE89408 as [...] Read more.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and joint destruction. Cellular senescence contributes to chronic inflammation, yet key senescence-associated regulators in RA remain unclear. This study aimed to identify RA senescence-related signatures and their roles. Using GSE89408 as the training cohort, we screened hub genes by intersecting differentially expressed and senescence-related genes via WGCNA and three machine learning algorithms, with GSE55457 for external validation. Immune infiltration, regulatory network, subtyping and drug prediction were analyzed. Clinical and in vitro assays validated RIPK2 expression and function in the macrophage senescence-like phenotype, with preliminary signaling exploration. Three senescence-related hub genes (TNFAIP6, SLC2A3, RIPK2) were identified. The derived nomogram showed robust diagnostic performance (AUC = 0.988). Hub genes correlated strongly with myeloid cells, especially macrophages. Two immunologically distinct RA subtypes were identified. RIPK2 was upregulated in clinical samples; its inhibition attenuated LPS-induced macrophage senescence-like changes and inflammation. Preliminary data suggested RIPK2 may act via the NF-κB pathway. This study identifies RA senescence-associated signatures, revealing RIPK2 linking innate immunity to macrophage senescence-like changes, offering novel insights into pathogenesis and supporting it as a candidate biomarker and therapeutic target. Full article

(This article belongs to the Section Molecular Informatics)

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21 pages, 3270 KB

Open AccessArticle

Amino Oxidase Hard Protein Corona with Metabolic-Triggered Intracellular Biocatalysis

by Federica Tonolo, Mary Bortoluzzi, Graziano Rilievo, Alessandro Cecconello, Aura Cencini, Lavinia Rutigliano, Maria Pia Rigobello, Maria Luisa Di Paolo, Alberto Macone, Pasquale Fino, Enzo Agostinelli, Massimiliano Magro and Fabio Vianello

Int. J. Mol. Sci. 2026, 27(16), 7492; https://doi.org/10.3390/ijms27167492 - 21 Aug 2026

Abstract

A hard protein corona was engineered onto tannic acid-modified magnetic nanoparticles (SAMN@TA), a magnetic and luminescent core–shell nano-carrier, using Bovine Serum Amine Oxidase (BSAO), an enzyme catalyzing the oxidation of polyamines and producing the corresponding aldehydes and hydrogen peroxide. The absorption and intracellular [...] Read more.

A hard protein corona was engineered onto tannic acid-modified magnetic nanoparticles (SAMN@TA), a magnetic and luminescent core–shell nano-carrier, using Bovine Serum Amine Oxidase (BSAO), an enzyme catalyzing the oxidation of polyamines and producing the corresponding aldehydes and hydrogen peroxide. The absorption and intracellular bioactivity of the self-assembled multimodal SAMN@TA@BSAO were investigated on an intestinal barrier model built with human colorectal adenocarcinoma (Caco-2) cells. The tailored BSAO corona possessed fouling resistance and, at the same time, was able to activate the clathrin-mediated endocytosis (CME) mechanism. Despite its size and intrinsic complexity, the nano-vehicle was effectively transported across the cell layer, safely transiting across the cell cytoplasm and reaching the lumen. As a function of intracellular polyamine concentration, the system’s biological activity induced intracellular oxidative stress, leading to the activation of the Keap1/Nrf2 oxidative protection pathway. The SAMN@TA@BSAO effect was well described by a dose–response curve with an EC50 of around 30 µg mL⁻¹ and a programmable killing efficiency (>50.0%), recalling the feasibility of a low molecular weight drug administration. The present study contributes to the nascent knowledge on engineering protein corona as a key to rationally design nanomaterials for biomedical applications. Full article

(This article belongs to the Collection Feature Papers in Molecular Biophysics)

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36 pages, 1469 KB

Open AccessReview

Interactions Between Antioxidants: How Useful Are In Vitro Studies for Understanding the Action of Antioxidants in Complex Systems?

by Izabela Sadowska-Bartosz and Grzegorz Bartosz

Int. J. Mol. Sci. 2026, 27(16), 7491; https://doi.org/10.3390/ijms27167491 - 21 Aug 2026

Abstract

Interactions between antioxidants are believed to be fundamental for proper nutrition and health benefits. This review discusses various types of antioxidant interactions in vitro, their mechanisms, and their dependence on assay conditions and evaluates the validity of extrapolations of results obtained from cell-free [...] Read more.

Interactions between antioxidants are believed to be fundamental for proper nutrition and health benefits. This review discusses various types of antioxidant interactions in vitro, their mechanisms, and their dependence on assay conditions and evaluates the validity of extrapolations of results obtained from cell-free assays and assays of cellular antioxidant activity to in vivo and food systems. In vitro studies revealed that the type of interaction (additive, antagonistic, or synergistic) depends not only on the identity of compounds but also on their absolute concentrations, concentration ratio, type of assay, reaction medium, and the method of analysis of results. Moreover, reactions of antioxidants in simple model systems, employing synthetic indicators, do not fully reflect their reactivity in food systems and organisms. Although in vitro studies may be a useful and necessary step for explaining the mechanisms of interactions between antioxidants, their importance for explaining the phenomena observed in vivo seems limited, as the organismal effects of compounds referred to as antioxidants depend rather on their actions on specific signaling pathways than on their direct antioxidant action. Nevertheless, they may provide useful information for food preservation, allowing for the reduction of the amounts of antioxidant additives. Full article

(This article belongs to the Special Issue Advanced Research in Antioxidant Activity: Second Edition)

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30 pages, 8172 KB

Open AccessArticle

17β-Estradiol Modulates Cancer Cell–Fibroblast Communication via Autophagy-Mediated Extracellular Vesicle Secretion and Promotes Poor Prognosis in Non-Small Cell Lung Cancer

by Rosa Vona, Camilla Cittadini, Barbara Ascione, Lucrezia Gambardella, Katia Fecchi, Lucia Bertuccini, Annalisa Tocci, Lorenzo D’Ambrosio, Maria Cristina Gagliardi, Federica Felicetti, Elena Ortona, Paola Nisticò, Anna Maria Mileo and Paola Matarrese

Int. J. Mol. Sci. 2026, 27(16), 7490; https://doi.org/10.3390/ijms27167490 - 21 Aug 2026

Abstract

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Smoking is the primary etiological factor, but growing evidence suggests the involvement of estrogen in its development and progression, although its role remains unclear. This study explores: (i) the effects induced [...] Read more.

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related mortality. Smoking is the primary etiological factor, but growing evidence suggests the involvement of estrogen in its development and progression, although its role remains unclear. This study explores: (i) the effects induced by estrogen, namely, 17β-estradiol (E2), alone or in combination with a mixture of inflammatory cytokines (Mix), in two human NSCLC cell lines, A549 and Calu1, and (ii) whether and how tumor cells can modulate the activation of normal lung fibroblasts. We found that E2 significantly enhances migration, invasion, and epithelial–mesenchymal transition in NSCLC cells, as well as their resistance to cisplatin, particularly in combination with Mix. Pharmacological inhibition of ERβ reversed the E2-induced effects, implicating ERβ in E2-mediated signaling. Furthermore, E2 increased autophagic flux and induced a shift toward secretory autophagy and the release of extracellular vesicles, which activated normal lung fibroblasts, as demonstrated by the increased expression of α-SMA, FAP, PDGFR-β, and PDPN. The clinical relevance of these data was supported by computational analyses revealing an elevated expression of the Mix gene signature, including TGF-β, IL-6, IL-8, CCXL-16, and ERβ, which was associated with shorter overall survival in NSCLC patients. This molecular profile was linked to the elevated expression of secretory autophagy genes and cancer-associated fibroblast markers. Validation in three large clinical cohorts (TCGA-LUNG, OAK and POPLAR) strengthens the clinical relevance of this E2-related pro-tumor axis while suggesting a promising therapeutic avenue for NSCLC patients. Full article

(This article belongs to the Special Issue Sex and Gender Medicine: New Horizons in Human Health and Disease)

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27 pages, 5523 KB

Open AccessArticle

Structure-Guided Discovery Reveals Recurrent Bioactive Peptide Architectures Across Coleoptera

by Thaís Caroline Gonçalves, João Alfredo Teodoro and Danilo T. Amaral

Int. J. Mol. Sci. 2026, 27(16), 7489; https://doi.org/10.3390/ijms27167489 - 21 Aug 2026

Abstract

Bioactive peptides are an important source of therapeutic molecules and molecular scaffolds involved in defense, signaling, and immune regulation. Despite the extraordinary diversity of Coleoptera, the structural landscape of beetle-derived bioactive peptides remains largely unexplored, limiting our understanding of their evolutionary diversity and [...] Read more.

Bioactive peptides are an important source of therapeutic molecules and molecular scaffolds involved in defense, signaling, and immune regulation. Despite the extraordinary diversity of Coleoptera, the structural landscape of beetle-derived bioactive peptides remains largely unexplored, limiting our understanding of their evolutionary diversity and biotechnological potential. Here, we performed a large-scale structural survey of predicted toxin-like peptide scaffolds across publicly available Coleoptera transcriptomes by integrating transcriptome mining, peptide maturation prediction, physicochemical characterization, AlphaFold 3 structural modeling, structural similarity analyses, and interpretable machine learning. We identified 291 candidate peptides, of which 155 contained canonical signal peptides and 273 produced mature peptides within the expected size range of known bioactive peptides. Structural analyses revealed that, despite extensive sequence diversity, many candidates were organized into a comparatively restricted repertoire of compact cysteine-rich architectures, indicating that structural similarity is retained across peptides exhibiting substantial primary-sequence variation. Comparative structural analyses further identified recurrent protein architectures shared across multiple beetle lineages, while machine learning prioritization integrated structural and biochemical descriptors to identify high-confidence candidates for future functional characterization. These analyses establish the first structural atlas of predicted toxin-like peptides across Coleoptera and demonstrate that structure-guided transcriptome mining provides a powerful framework for uncovering recurrent bioactive peptide scaffolds that would remain largely undetected using sequence-based approaches alone. Beyond expanding our understanding of peptide evolution in beetles, this resource is a foundation for future structural, functional, and biotechnological exploration of bioactive peptides in underexplored animal groups. Full article

(This article belongs to the Section Biochemistry)

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