International Journal of Molecular Sciences

13 pages, 750 KB

Open AccessArticle

Sputum Biomarkers of Inflammation to Track Acute Respiratory Events in School-Age Children with Cystic Fibrosis

by Elad Ben-Meir, Lucy Perrem, Gyde Nissen, Michelle Shaw, Felix Ratjen and Hartmut Grasemann

Int. J. Mol. Sci. 2026, 27(3), 1616; https://doi.org/10.3390/ijms27031616 - 6 Feb 2026

Cystic fibrosis (CF) is characterized by neutrophil-driven airway inflammation and acute respiratory events (AREs) that contribute to progressive lung damage. AREs are clinically heterogeneous and often occur without measurable changes in lung function. This study aimed to evaluate the utility of molecular airway [...] Read more.

Cystic fibrosis (CF) is characterized by neutrophil-driven airway inflammation and acute respiratory events (AREs) that contribute to progressive lung damage. AREs are clinically heterogeneous and often occur without measurable changes in lung function. This study aimed to evaluate the utility of molecular airway inflammatory markers for detecting AREs in school-age children with CF. We performed a secondary analysis of a prospective observational study of children with CF (ages 6.7–16.8 years) followed for two years. Sputum samples were collected from 50 participants during stable visits and AREs. Concentrations of 14 inflammatory cytokines were measured using ELISA and multiplex assays. Associations with lung function (ppFEV₁ and lung clearance index [LCI]) and time to next ARE were assessed. A total of 179 sputum samples were analyzed, including 64 collected during AREs. Calprotectin, interleukin-8 (IL-8), and IL-1β were increased during AREs compared with stable visits, although concentrations frequently remained within ranges observed at stable visits. Other cytokines, including GM-CSF, IL-17A, IL-1α, TNF-α, and SPLUNC-1, were predictive of shorter time to subsequent AREs. No biomarker correlated with lung function measures. These findings indicate that airway inflammatory cytokine changes are associated with clinically diagnosed AREs but not with pulmonary function, supporting their potential role as complementary biomarkers in CF care. Full article

(This article belongs to the Special Issue New Research Insights in Cystic Fibrosis and CFTR-Related Diseases)

30 pages, 19932 KB

Open AccessArticle

Unraveling the Cross-Tissue Neuroimmune–Vascular Genetic Architecture of Migraine Using Integrated Multi-Omics, Single-Cell, and Spatial Transcriptomics: Prioritizing T-Cell Regulatory Networks and Peripheral Targets

by Chung-Chih Liao, Ke-Ru Liao and Jung-Miao Li

Int. J. Mol. Sci. 2026, 27(3), 1615; https://doi.org/10.3390/ijms27031615 - 6 Feb 2026

Abstract

Migraine is a complex neurovascular disorder in which immune signaling intersects with vascular and neural circuits, yet the tissue and cell-type context of common genetic risk remains incompletely defined. We integrated large-scale migraine genome-wide association study (GWAS) summary statistics with Genotype-Tissue Expression (GTEx) [...] Read more.

Migraine is a complex neurovascular disorder in which immune signaling intersects with vascular and neural circuits, yet the tissue and cell-type context of common genetic risk remains incompletely defined. We integrated large-scale migraine genome-wide association study (GWAS) summary statistics with Genotype-Tissue Expression (GTEx) v8 expression and splicing quantitative trait loci (eQTLs and sQTLs), Bayesian co-localization, single-cell RNA sequencing of peripheral blood mononuclear cells (PBMCs) from migraine cases and controls, a healthy single-cell multi-omics atlas (assay for transposase-accessible chromatin (ATAC) plus RNA), high-dimensional weighted gene co-expression network analysis (hdWGCNA), and embryo-level spatial transcriptomics. Genetic signals were enriched in peripheral arteries, heart, and blood, and gene-level enrichment highlighted mucosal–smooth muscle organs including the bladder and the cervix endocervix. Cell-type prioritization consistently implicated endothelial and vascular smooth muscle lineages, with additional support for inhibitory interneurons and bladder epithelium. In PBMC T cells, co-expression modules capturing cytotoxic/activation and T-cell receptor signaling programs contained migraine-prioritized genes, including PTK2B, nominating immune activation circuitry as a component of genetic susceptibility. Spatial projection further localized risk concordance to craniofacial/meningeal interfaces and visceral smooth muscle–mucosal structures. Together, these analyses delineate a systemic neuroimmune–vascular architecture for migraine and provide genetically anchored candidate pathways and targets for mechanistic and therapeutic follow-up. Full article

(This article belongs to the Special Issue Molecular Diagnosis and Treatment of Migraine)

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15 pages, 7126 KB

Open AccessArticle

Predicting Pathogenicity of TSHR Missense Variants of Uncertain Significance: An Integrative Computational Study

by Tassneem Awad Hajali, Islamia Ibrahim Ahmed Omer, Mohamad Y. Rezk and Hamdan Z. Hamdan

Int. J. Mol. Sci. 2026, 27(3), 1614; https://doi.org/10.3390/ijms27031614 - 6 Feb 2026

Abstract

Pathogenic variants in the thyroid-stimulating hormone receptor gene (TSHR) contribute to a wide spectrum of thyroid dysfunctions, ranging from congenital hypothyroidism to thyrotropin resistance. With the advancement of bioinformatics algorithms for variant effect prediction, assessing the pathogenic potential of variants has [...] Read more.

Pathogenic variants in the thyroid-stimulating hormone receptor gene (TSHR) contribute to a wide spectrum of thyroid dysfunctions, ranging from congenital hypothyroidism to thyrotropin resistance. With the advancement of bioinformatics algorithms for variant effect prediction, assessing the pathogenic potential of variants has become increasingly important. This study aimed to investigate the pathogenic effects of TSHR variants classified as variants of uncertain significance (VUSs) in the gnomAD v4.1.0 database. TSHR variants listed in gnomAD v4.1.0 were retrieved and filtered to select missense VUSs based on ClinVar classifications. Multiple bioinformatics tools were used to assess the secondary and three-dimensional structures of the TSHR, as well as protein stability, evolutionary conservation, and molecular dynamics simulations. A total of 2760 TSHR variants were found in gnomAD v4.1.0, including 75 frameshifts, 80 splice-sites, 265 in the 3′ and 5′ untranslated regions, 422 synonymous, 892 others, and 1026 missense variants. Among these, 68 missense VUSs were identified and selected for bioinformatics analysis. Three variants (p.Cys29Trp, p.Leu57Pro, and p.Phe97Ser) were consistently predicted to be pathogenic by all the bioinformatics tools used. All three variants were located within the leucin-rich repeat domain extracellular region of the TSHR and within a highly conserved region across species. Molecular dynamics simulations for mutant proteins (p.Cys29Trp, p.Leu57Pro, and p.Phe97Ser) reveal structural instability in comparison to the wild protein. Comprehensive bioinformatics analysis revealed that three TSHR missense VUSs exhibited pathogenic potential. These variants may contribute to thyroid dysfunction by affecting the receptor’s structural and signalling integrity. Full article

(This article belongs to the Special Issue Genetic Testing and Tailored Treatment for Thyroid Disorders: Molecular Basis and New Trends)

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15 pages, 590 KB

Open AccessReview

Molecular Mechanisms of Accelerated Ageing in Geriatric Depression: Interplay of Telomere Attrition, Mitochondrial Dysfunction and Cellular Senescence

by Pratibha Revi Shanker and Rajkumar Dorajoo

Int. J. Mol. Sci. 2026, 27(3), 1613; https://doi.org/10.3390/ijms27031613 - 6 Feb 2026

Abstract

Late-life depression is a prevalent and debilitating disorder. It differs significantly from depression in younger adults and often co-occurs with cognitive decline and increased physical frailty. This narrative review explores the role of accelerated biological ageing in late-life depression. We examine evidence linking [...] Read more.

Late-life depression is a prevalent and debilitating disorder. It differs significantly from depression in younger adults and often co-occurs with cognitive decline and increased physical frailty. This narrative review explores the role of accelerated biological ageing in late-life depression. We examine evidence linking three interconnected processes, namely telomere attrition, mitochondrial dysfunction and cellular senescence, to the pathophysiology of late-life depression. Excessive attrition of telomeres may serve as a biomarker of accumulated stress and cellular ageing. Mitochondrial dysfunction not only reduces energy production but also promotes oxidative stress and inflammation that increase neuroinflammatory pathways and synaptic loss. Increased cellular senescence further induces senescence-associated secretory phenotype factors that drive chronic inflammation and neuronal loss. Together, these processes create a cycle of cellular stress, persistent inflammation and damage to brain circuits involved in late-life depression. We additionally highlight potential limitations in current findings and propose a roadmap for future research to better elucidate the mechanistic dysfunction of late-life depression. These include the need for evaluation in long-term prospective cohort studies, improved tools to better correlate blood-based markers with changes in disease-relevant brain tissues and regions, and trials that test treatment and lifestyle modifications that are targeted at ageing biomarkers. Full article

(This article belongs to the Special Issue Telomeres in Development, Senescence and Genome Instability: 2nd Edition)

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26 pages, 3427 KB

Open AccessArticle

The Dynamics of Blood-Count-Derived Inflammatory Indices in the Course of Systemic Treatment for Psoriasis: A Single Center Study

by Agnieszka Hołdrowicz, Daria Gierach-Michalska, Aleksandra Kośny, Radosław Zajdel and Agnieszka Żebrowska

Int. J. Mol. Sci. 2026, 27(3), 1612; https://doi.org/10.3390/ijms27031612 - 6 Feb 2026

Abstract

Psoriasis is a chronic inflammatory disease that affects up to 3% of the global population. In recent years, monoclonal antibodies targeting key cytokines underlying skin lesions and joint involvement in the course of psoriasis, i.e., TNF-α, IL-17, and IL-23, have been increasingly used [...] Read more.

Psoriasis is a chronic inflammatory disease that affects up to 3% of the global population. In recent years, monoclonal antibodies targeting key cytokines underlying skin lesions and joint involvement in the course of psoriasis, i.e., TNF-α, IL-17, and IL-23, have been increasingly used due to their high effectiveness and favorable safety profile. Numerous studies have been conducted analyzing the influence of cytokine inhibitors on non-specific inflammatory markers. However, only a limited number of studies on the effect of methotrexate (MTX) therapy on blood-count-derived inflammatory indices in patients with plaque psoriasis have been published so far. The study aims to analyze and compare the impact of methotrexate and biological drugs on the dynamics of selected blood-count-derived inflammatory indices in psoriatic patients. The analysis involved 182 patients receiving biological therapy, which resulted in a total of 219 treatment cycles (TCs) and 48 patients treated with therapeutic doses of MTX (48 TCs). In the biological subgroup, there were six TCs with an inhibitor of IL-12/23, 58 TCs with IL-17A inhibitors, 22 TCs with an inhibitor of IL-17AF, 113 TCs with IL-23 inhibitors, and 20 TCs with TNF-alfa inhibitors. A comparison between patients receiving biological treatment regardless of the drug and patients receiving MTX was conducted. Themajor factors determining the duration of MTX therapy were older age at the time of therapy initiation, a later onset of psoriasis, and a higher burden of comorbidities. Furthermore, the strongest impact on the average inflammatory state over time in patients treated with methotrexate was associated with comorbidities, male gender, and older age. Contrary to MTX therapy, patients receiving biological drugs were characterized by lower values of most assessed blood-count-derived inflammatory biomarkers at week 40 compared to baseline. It was confirmed that biologics and MTX treatment modify the dynamics of blood-count-derived inflammatory biomarkers in a different manner. Full article

(This article belongs to the Special Issue Molecular Mechanisms and Therapeutic Targets in Skin Diseases)

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26 pages, 1826 KB

Open AccessReview

A New Complexity Layer: DNA Methylation and the Predictive Impact of Epigenetic Tests

by Giorgio Ladisa, Francesca Montenegro, Angela Picerno, Alessio Nigro, Antonella Cicirelli, Alessandra Stasi, Marco Fiorentino, Paola Pontrelli, Loreto Gesualdo and Fabio Sallustio

Int. J. Mol. Sci. 2026, 27(3), 1611; https://doi.org/10.3390/ijms27031611 - 6 Feb 2026

Abstract

The increasing complexity of disease mechanisms challenges accurate diagnosis, prevention, and early risk stratification. Beyond genetic predisposition, epigenetic regulation—particularly DNA methylation—represents a dynamic molecular interface linking environmental exposures, metabolic imbalance, inflammation, and disease development. DNA methylation is the most extensively studied epigenetic mechanism [...] Read more.

The increasing complexity of disease mechanisms challenges accurate diagnosis, prevention, and early risk stratification. Beyond genetic predisposition, epigenetic regulation—particularly DNA methylation—represents a dynamic molecular interface linking environmental exposures, metabolic imbalance, inflammation, and disease development. DNA methylation is the most extensively studied epigenetic mechanism and plays a central role in controlling gene expression across physiological and pathological conditions. In this review, we provide an integrated overview of DNA methylation biology and its involvement in inflammatory, metabolic, and oncological diseases, with a specific focus on pathways related to chronic inflammation and oxidative stress. We summarize evidence demonstrating how aberrant methylation patterns contribute to disease initiation and progression, highlighting recurrent epigenetic signatures affecting key regulatory genes. In parallel, we discuss current and emerging technologies for DNA methylation analysis, ranging from targeted methylation-specific assays to next-generation sequencing-based approaches, including nanopore adaptive sampling. Finally, we explore the translational potential of DNA methylation-based tests as predictive and preventive tools, emphasizing their ability to identify disease-associated molecular alterations before clinical onset. Overall, this evidence supports the integration of epigenetic profiling into future precision medicine strategies aimed at early risk assessment, prognosis refinement, and personalized prevention. Full article

(This article belongs to the Collection 30th Anniversary of IJMS: Updates and Advances in Biochemistry)

28 pages, 2858 KB

Open AccessReview

Enteric Infections, Dysbiosis, and Metabolic Dysfunction: The Role of Diarrheagenic Pathogens in Insulin Resistance

by Martin Zermeño-Ruiz, Filiberto Gutierrez-Gutierrez, Elsa Janneth Anaya-Ambriz, Emiliano Peña-Durán, Jesús Jonathan García-Galindo, Alfredo Huerta-Huerta, Araceli Lizbeth Quiñonez-Gallardo and Daniel Osmar Suárez-Rico

Int. J. Mol. Sci. 2026, 27(3), 1610; https://doi.org/10.3390/ijms27031610 - 6 Feb 2026

Abstract

Type 2 diabetes and insulin resistance are increasingly recognized as conditions influenced not only by genetic and lifestyle factors but also by infectious and microbial exposures. Diarrheagenic pathogens, including enterotoxigenic, enteroaggregative, and enterohemorrhagic Escherichia coli, as well as other enteric microorganisms, disrupt [...] Read more.

Type 2 diabetes and insulin resistance are increasingly recognized as conditions influenced not only by genetic and lifestyle factors but also by infectious and microbial exposures. Diarrheagenic pathogens, including enterotoxigenic, enteroaggregative, and enterohemorrhagic Escherichia coli, as well as other enteric microorganisms, disrupt the gut microbiota and compromise intestinal barrier integrity. These alterations promote dysbiosis, increased intestinal permeability, and systemic exposure to lipopolysaccharides and other microbial products, leading to metabolic endotoxemia and chronic low-grade inflammation. In parallel, pathogen-induced modulation of host immune responses contributes to adipose tissue inflammation, mitochondrial dysfunction, and impaired insulin signaling. This review summarizes current evidence linking diarrheagenic pathogens to insulin resistance, with emphasis on the microbiota–immune–metabolism axis. Understanding these interactions highlights novel perspectives on the pathogenesis of insulin resistance and suggests that targeted modulation of the gut microbiota or reduction in pathogen-driven inflammation may represent therapeutic opportunities to improve metabolic outcomes. Full article

(This article belongs to the Special Issue Microbiome-Immunity Crosstalk and Its Role in Health and Disease)

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22 pages, 958 KB

Open AccessArticle

Potential Neuroprotective Role of GLP-2 in Alzheimer’s Disease: Clinical Observations, Mechanistic Insights, and Comparison with GLP-1

by Maciej Czarnecki, Agnieszka Baranowska-Bik, Anna Litwiniuk, Małgorzata Kalisz, Anita Domańska, Anna Kurdyła and Wojciech Bik

Int. J. Mol. Sci. 2026, 27(3), 1609; https://doi.org/10.3390/ijms27031609 - 6 Feb 2026

Abstract

Alzheimer’s disease (AD) is the most common cause of dementia and is characterized by progressive cognitive decline, β-amyloid accumulation, tau pathology, oxidative stress, and neuroinflammation. Increasing evidence suggests that metabolic dysregulation may contribute to AD pathogenesis. Glucagon-like peptide-2 (GLP-2), an intestinal peptide hormone, [...] Read more.

Alzheimer’s disease (AD) is the most common cause of dementia and is characterized by progressive cognitive decline, β-amyloid accumulation, tau pathology, oxidative stress, and neuroinflammation. Increasing evidence suggests that metabolic dysregulation may contribute to AD pathogenesis. Glucagon-like peptide-2 (GLP-2), an intestinal peptide hormone, has demonstrated neuroprotective effects in preclinical models, potentially through anti-inflammatory and anti-apoptotic mechanisms. However, its role in human neurodegenerative disorders remains insufficiently understood. This study aimed to compare plasma GLP-2 concentrations between individuals with AD and cognitively healthy controls and to examine associations between GLP-2 levels, cognitive impairment severity, and metabolic parameters. Sixty-one patients with clinically diagnosed AD and twenty-three cognitively unimpaired controls were recruited. Plasma total GLP-2 concentrations were assessed at baseline in all participants and additionally at 6 and 12 months in a subgroup of 34 AD patients. Cognitive function was evaluated using the Mini-Mental State Examination (MMSE) and the Clinical Dementia Rating (CDR) scale. Group comparisons, subgroup analyses based on AD severity, repeated-measures analyses, Spearman correlations, and multivariable linear regression models (including age and clinical group) were performed. Plasma GLP-2 concentrations were significantly higher in AD patients than in controls, with a moderate effect size (Cohen’s d ≈ 0.60). In severity-based subgroup analyses, both the mild and moderate-to-severe AD groups showed significantly higher GLP-2 levels than controls. Longitudinal analyses in AD patients (n = 34) showed no significant changes in GLP-2 concentrations over 12 months. Cognitive performance declined over time, with a significant reduction in MMSE from baseline to 6 months, whereas GLP-2 levels were not correlated with MMSE or CDR at any time point. GLP-2 levels correlated positively with body mass index (BMI), body weight, insulin, and HOMA-IR. In multivariable regression analysis, neither age nor clinical group independently predicted GLP-2 concentrations (both p > 0.05). Plasma GLP-2 concentrations were higher in patients with AD than in cognitively healthy controls; however, GLP-2 levels were not associated with cognitive performance or its progression over 12 months. GLP-2 was positively related to markers of adiposity and insulin resistance, suggesting stronger links to metabolic status than to cognitive severity. Further studies are needed to clarify whether GLP-2 alterations in AD reflect compensatory mechanisms, metabolic factors, or disease-related pathophysiology. Full article

(This article belongs to the Special Issue Advances in Neurodegenerative Diseases and Inflammation: Pathogenesis, Biomarkers and Treatments)

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27 pages, 1130 KB

Open AccessReview

Host Glycan–Lectin Interplay in SARS-CoV-2 Infection

by Hyeseong Oh, Vu Thi Thuy Tien, Showkot Ahmed, Jisoo Choi, Ki-Jun Ryu and Jinsung Yang

Int. J. Mol. Sci. 2026, 27(3), 1608; https://doi.org/10.3390/ijms27031608 - 6 Feb 2026

Abstract

Glycan-mediated processes can be critical determinants of viral attachment and entry, yet for enveloped RNA viruses, including SARS-CoV-2, their mechanistic roles remain incompletely defined. This review synthesizes current structural and functional evidence for glycan engagement during SARS-CoV-2 attachment and entry. We describe the [...] Read more.

Glycan-mediated processes can be critical determinants of viral attachment and entry, yet for enveloped RNA viruses, including SARS-CoV-2, their mechanistic roles remain incompletely defined. This review synthesizes current structural and functional evidence for glycan engagement during SARS-CoV-2 attachment and entry. We describe the general viral entry pathways and their reliance on glycan recognition, followed by the interactions of the SARS-CoV-2 spike glycoprotein with host glycans, including ABO(H) blood group antigens, sialylated glycans, and endogenous lectins. Based on structural biology, glycobiology, and virology, we focus on how the spike protein exploits both glycan motifs and lectin receptors to enhance attachment, promote cellular uptake, or modulate host tropism. We contextualize these mechanisms by comparing glycan dependencies across other human viruses, including the influenza virus, HIV, and norovirus. Finally, we provide a comparative virological perspective to derive broad evolutionary insights into how enveloped viruses exploit the host glycans. Full article

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24 pages, 1749 KB

Open AccessArticle

Artificial Intelligence-Enabled Integration Suggests TP53 Pathway Alterations as Prognostic Biomarkers in Populations with Disproportionate Health Burdens

by Fernando C. Diaz, Brigette Waldrup, Francisco G. Carranza, Sophia Manjarrez and Enrique Velazquez-Villarreal

Int. J. Mol. Sci. 2026, 27(3), 1607; https://doi.org/10.3390/ijms27031607 - 6 Feb 2026

Abstract

The incidence of early-onset colorectal cancer (EOCRC; <50 years) continues to increase, with the most rapid rises occurring among Hispanic/Latino (H/L) populations who remain underrepresented in molecular research. Because the TP53 signaling pathway is a key driver of colorectal tumorigenesis, this study aimed [...] Read more.

The incidence of early-onset colorectal cancer (EOCRC; <50 years) continues to increase, with the most rapid rises occurring among Hispanic/Latino (H/L) populations who remain underrepresented in molecular research. Because the TP53 signaling pathway is a key driver of colorectal tumorigenesis, this study aimed to clarify its prognostic significance in FOLFOX-treated EOCRC across ancestry groups. We analyzed 2515 colorectal cancer (CRC) cases (266 H/L, 2249 non-Hispanic White [NHW]) stratified by ancestry, age at onset, and FOLFOX exposure. Fisher’s exact, chi-square, and Kaplan–Meier’s analyses were applied, and multi-dimensional data integration was performed using AI-HOPE and AI-HOPE-TP53, conversational artificial intelligence platforms enabling natural language-driven exploration of clinical, genomic, and therapeutic features. TP53 pathway alterations were common in both H/L (85%) and NHW (83%) FOLFOX-treated patients. Among late-onset NHW cases, FOLFOX treatment was associated with higher TP53 mutation frequencies and lower ATM and CDKN2A mutation rates compared with untreated counterparts, while CHEK2 alterations were significantly less frequent in late-onset H/L patients. Missense mutations were the predominant alteration type across groups. These findings suggest that TP53 pathway alterations may be associated with ancestry- and treatment-specific clinical patterns in EOCRC and illustrate how AI-enabled integrative analytic frameworks can facilitate hypothesis generation and prioritize candidate biomarkers for future validation in precision oncology. Full article

(This article belongs to the Special Issue Oncogenes and Tumor Suppressors: From Basic Science to Therapeutic Applications)

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15 pages, 1020 KB

Open AccessArticle

New Considerations Around the Singular Water Temperature Explaining the Anomalous Behavior of Liquid Phase

by Domenico Mallamace, Giovanni Romanelli, Roberto Senesi and Francesco Mallamace

Int. J. Mol. Sci. 2026, 27(3), 1606; https://doi.org/10.3390/ijms27031606 - 6 Feb 2026

Abstract

The water thermodynamics is characterized by polydispersity, which determines its structural and dynamic properties. This is due to the specifics of its characteristic bond: the hydrogen bond (HB). The isobars of the two fundamental thermodynamic functions, the isothermal compressibility ( [...] Read more.

The water thermodynamics is characterized by polydispersity, which determines its structural and dynamic properties. This is due to the specifics of its characteristic bond: the hydrogen bond (HB). The isobars of the two fundamental thermodynamic functions, the isothermal compressibility (

K_{T} (P . T)

) and the isobaric expansivity (

α_{P} (P, T)

), show the presence of a temperature

T^{*} ≃ 315 \pm 5

K where both have a singular behavior. In this work, by carefully considering the thermal properties of the isobars of density

ρ

, specific heat

C_{P}

and the self-diffusion

D_{S}

, we suggest the universality characteristics of this temperature. In addition, by analyzing the average intermolecular distance

d_{O O}

, in the same area of the P-T phase diagram, we demonstrate that such realities are due in the supercooled liquid state to the ratio between its two characteristic phases: the low-density liquid (LDL due to HB) and the HDL (which entirely characterizes the remaining parts of the phase diagram). Full article

(This article belongs to the Section Physical Chemistry and Chemical Physics)

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15 pages, 5420 KB

Open AccessArticle

Probing the Feasibility of Single-Cell Fixed RNA Sequencing from FFPE Tissue

by Xiaochen Liu, Katherine Naughton, Samuel D. Karsen, Patricia Bentley, Lori Duggan, Neha Chaudhary, Kathleen M. Smith, Lucy Phillips, Dan Chang and Naim A. Mahi

Int. J. Mol. Sci. 2026, 27(3), 1605; https://doi.org/10.3390/ijms27031605 - 6 Feb 2026

Abstract

Single-cell RNA sequencing (scRNA-seq) provides a comprehensive understanding of cellular complexity; however, its requirement for fresh or frozen samples limits its flexibility. To overcome this limitation to effectively leverage clinical samples, Chromium Fixed RNA Profiling on formalin-fixed paraffin-embedded (FFPE) tissue blocks (scFFPE-seq) was [...] Read more.

Single-cell RNA sequencing (scRNA-seq) provides a comprehensive understanding of cellular complexity; however, its requirement for fresh or frozen samples limits its flexibility. To overcome this limitation to effectively leverage clinical samples, Chromium Fixed RNA Profiling on formalin-fixed paraffin-embedded (FFPE) tissue blocks (scFFPE-seq) was developed to perform single-nucleus RNA sequencing from nuclei isolated from FFPE. In this study, we utilized fresh tissue samples from colon, ileum, and skin to assess the viability of scFFPE-seq compared to these fresh samples. We were able to recover unique cell types from challenging FFPE tissues and validated scFFPE-seq findings through Hematoxylin and Eosin (H&E) images. The results demonstrated that scFFPE-seq effectively captured the single-cell transcriptome in FFPE tissues, obtaining comparable cell abundance, cell type annotation, and pathway characterization to those in fresh tissues. Overall, the study presents strong evidence of the potential of scFFPE-seq to enhance scientific knowledge by enabling the generation of high-quality, sensitive single-nucleus RNA-seq data from preserved tissue samples. This technique unlocks the vast archives of FFPE samples for extensive retrospective genomic studies. Full article

(This article belongs to the Special Issue New Insights in Translational Bioinformatics: Second Edition)

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9 pages, 1765 KB

Open AccessCommunication

Quercetin Dilates Retinal Arterioles via Nitric Oxide-Dependent Mechanisms in Rats

by Asami Mori, Akihiro Sakurai, Sarina Takimoto, Kenji Sakamoto and Tsutomu Nakahara

Int. J. Mol. Sci. 2026, 27(3), 1604; https://doi.org/10.3390/ijms27031604 - 6 Feb 2026

Abstract

The purpose of this study was to investigate whether quercetin, a flavonoid abundantly found in onion leaves and other plant foods, induces the dilation of retinal blood vessels in rats. The time-course changes in retinal arteriolar diameter were measured using a retinal circulation [...] Read more.

The purpose of this study was to investigate whether quercetin, a flavonoid abundantly found in onion leaves and other plant foods, induces the dilation of retinal blood vessels in rats. The time-course changes in retinal arteriolar diameter were measured using a retinal circulation evaluation system based on a high-resolution digital fundus camera developed in our laboratory. The intravenous administration of quercetin (10–100 µg/kg/min) increased the retinal arteriolar diameter in a dose-dependent manner. This vasodilatory effect of quercetin was almost completely suppressed through an intravitreal pretreatment with N_ω-nitro-l-arginine methyl ester (l-NAME), a nitric oxide (NO) synthase inhibitor. In contrast, the systemic intravenous infusion of quercetin did not cause significant changes in the systemic blood pressure and heart rate. These results suggest that NO production plays an important role in the quercetin-induced dilation of retinal arterioles. Quercetin, which is abundantly present in several plant foods and possesses antioxidant properties, may be a useful agent for the prevention of various ocular diseases associated with visual impairment caused by reduced retinal blood flow. Full article

(This article belongs to the Special Issue Artificial Intelligence in Ophthalmology: A New Era for Retinal Disease Detection and Treatment)

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15 pages, 2576 KB

Open AccessArticle

Intestinal Protective Effects of a Pomegranate Peel Extract in In Vitro and Ex Vivo Studies

by Lucia Recinella, Alessandra Acquaviva, Annalisa Bruno, Davide Ciaramellano, Angelica Pia Centulio, Melania Dovizio, Cristina Milillo, Massimo Mozzon, Daniele Generali, Gianluca Genovesi, Giustino Orlando, Annalisa Chiavaroli, Claudio Ferrante, Patrizia Ballerini, Luigi Brunetti and Sheila Leone

Int. J. Mol. Sci. 2026, 27(3), 1603; https://doi.org/10.3390/ijms27031603 - 6 Feb 2026

Abstract

Recovery of nutritional and bioactive molecules by pomegranate peel (PP) has found wide applications in food and pharmaceutical industries. We investigated protective effects of a PP extract (PPE) from Mediterranean (Mazara del Vallo, Italy) on intestinal inflammation by using in vitro and ex [...] Read more.

Recovery of nutritional and bioactive molecules by pomegranate peel (PP) has found wide applications in food and pharmaceutical industries. We investigated protective effects of a PP extract (PPE) from Mediterranean (Mazara del Vallo, Italy) on intestinal inflammation by using in vitro and ex vivo models. Reactive oxygen species (ROS) and lactate dehydrogenase (LDH) levels, as well as tight junction protein-1 (ZO-1) expression, were determined in lipopolysaccharide (LPS)-injured Caco-2 cells treated with PPE. We evaluated anti-inflammatory and antioxidant effects of PPE in isolated colon specimens of adult male mouse (C57/BL6) stimulated by LPS. Cyclooxygenase-2 (COX-2), nuclear factor-kB (NF-kB), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), as well as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX), and inducible nitric oxide synthase (i-NOS) gene expression was determined. We also characterized phytochemical composition of the extract through chromatographic (HPLC-UV) and spectrophotometric techniques. PPE was rich in punicalagins A and B, along with other polyphenols such as hydroxytyrosol (HT), catechin, p-coumaric acid, and rosmarinic acid. In Caco-2 cells, PPE reduced ROS generation and LDH release, restoring intestinal barrier integrity by upregulating ZO-1 expression. In addition, PPE increased SOD, CAT, and GPX and suppressed COX-2, NF-kB, TNF-α, IL-1β and i-NOS LPS-induced gene expression in colon. PPE mitigates inflammation and oxidative stress, restoring intestinal barrier function. The beneficial effects induced by the extract could be related to the pattern of polyphenolic composition, with particular regard to HT, rosmarinic acid, p-coumaric acid, catechin, as well as punicalagins A and B. Full article

(This article belongs to the Special Issue Anti-Inflammatory and Anti-Oxidant Effects of Extracts from Plants)

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15 pages, 14226 KB

Open AccessBrief Report

High Glucose-Induced Alterations in Regucalcin Expression in Podocytes and Their Potential Consequences

by Olga Żołnierkiewicz and Dorota Rogacka

Int. J. Mol. Sci. 2026, 27(3), 1602; https://doi.org/10.3390/ijms27031602 - 6 Feb 2026

Abstract

Regucalcin (RGN) is a multifunctional regulator of intracellular calcium signaling, implicated in cellular homeostasis and stress responses. While aberrant RGN activity has been associated with diabetic kidney disease (DKD), existing studies have primarily focused on its role in proximal tubular cells. Whether RGN [...] Read more.

Regucalcin (RGN) is a multifunctional regulator of intracellular calcium signaling, implicated in cellular homeostasis and stress responses. While aberrant RGN activity has been associated with diabetic kidney disease (DKD), existing studies have primarily focused on its role in proximal tubular cells. Whether RGN is expressed in podocytes and how its expression responds to diabetic-like stimuli remain largely unexplored. Podocyte injury under diabetic conditions is a critical event in DKD pathogenesis. Therefore, in this study, we aimed to investigate whether podocytes express RGN and how its expression is affected under high-glucose (HG) conditions. To address these questions, we employed quantitative real-time PCR, Western blotting, fluorescence-based protein staining, and immunohistochemical analysis of renal sections. Our results confirmed RGN expression in podocytes and revealed its dysregulation at both the mRNA and protein levels under HG conditions. Additionally, we identified the subcellular localization of RGN and a significant association with sarco/endoplasmic reticulum calcium ATPase (SERCA), a key enzyme regulating endoplasmic reticulum (ER) calcium storage and the ER stress response. Altered RGN expression in podocytes exposed to HG concentrations may contribute to the progression of DKD, possibly through the disruption of intracellular calcium homeostasis. Full article

(This article belongs to the Special Issue Chronic Kidney Disease: From Molecular Mechanisms to Therapeutic Approaches)

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16 pages, 4745 KB

Open AccessArticle

The I148M PNPLA3 Variant Forces Progressive Portal MASLD by Spatially Perturbing Metabolic Pathways Across Liver Zones

by Erika Paolini, Marica Meroni, Miriam Longo, Sara Badiali, Marco Maggioni, Anna Ludovica Fracanzani and Paola Dongiovanni

Int. J. Mol. Sci. 2026, 27(3), 1601; https://doi.org/10.3390/ijms27031601 - 6 Feb 2026

Abstract

Genetics strongly impacts the course of metabolic dysfunction-associated steatotic liver disease (MASLD), with the I148M Patatin like phospholipase domain containing 3 (PNPLA3) variant representing the main modifier. Fat accumulation in the hepatic lobule, strongly enhanced by this SNP, may be influenced [...] Read more.

Genetics strongly impacts the course of metabolic dysfunction-associated steatotic liver disease (MASLD), with the I148M Patatin like phospholipase domain containing 3 (PNPLA3) variant representing the main modifier. Fat accumulation in the hepatic lobule, strongly enhanced by this SNP, may be influenced by the liver’s zonation. Therefore, we applied spatial transcriptomics to investigate the metabolic processes across portal (PZ)-central (CZ) zones in I148M PNPLA3 carriers. Visium CytAssist technology was applied to liver biopsies from MASLD patients sharing similar disease severity, who were wild-type (WT) or homozygous for the I148M variant (Discovery cohort, n = 4). The distribution of steatosis, inflammation, and fibrosis was assessed in the liver biopsies of MASLD patients, stratified according to the I148M variant (validation cohort, n = 100). At the Visium-LOUPE browser, we spatially mapped PZ and CZ hepatocytes (HEPs), revealing higher lipid turnover, glucose signaling, and lower mitochondrial activity in I148M-PZ-HEPs compared to 148M-CZ-HEPs. Thus, the I148M variant could unbalance the physiological hepatic zonation boosting steatosis development in PZ, consequently inducing mitochondrial dysfunction. The unsupervised analysis confirmed the altered metabolic pattern among CZ and PZ in patients carrying the variant. Interestingly, PNPLA3 expression was higher in I148M-PZ, which also showed an enrichment of non-parenchymal cells, thus possibly explaining the more severe injury in this area. Finally, in the validation cohort, we observed a pronounced PZ distribution of steatosis, inflammation, and fibrosis in I148M PNPLA3 subjects compared to WT, confirming the spatial data. The I148M variant contributes to the metabolic switching across different hepatic zones and represents a new clinical perspective by defining a specific histological pattern of MASLD. Full article

(This article belongs to the Special Issue 25th Anniversary of IJMS: Updates and Advances in Molecular Pathology, Diagnostics, and Therapeutics)

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17 pages, 980 KB

Open AccessArticle

Peripheral Syndecan-3 and Neurofilament Light Chain as Complementary Blood Biomarkers for Alzheimer’s Disease

by Anett Hudák, Annamária Letoha and Tamás Letoha

Int. J. Mol. Sci. 2026, 27(3), 1600; https://doi.org/10.3390/ijms27031600 - 6 Feb 2026

Abstract

Reliable and disease-specific blood biomarkers are critically needed for Alzheimer’s disease (AD), particularly in early stages when interventions are most effective. Although phosphorylated tau and neurofilament light chain (NfL) are widely used, their diagnostic specificity has been reported to decrease in elderly populations [...] Read more.

Reliable and disease-specific blood biomarkers are critically needed for Alzheimer’s disease (AD), particularly in early stages when interventions are most effective. Although phosphorylated tau and neurofilament light chain (NfL) are widely used, their diagnostic specificity has been reported to decrease in elderly populations with multimorbidities. Syndecan-3 (SDC3), a heparan sulfate proteoglycan implicated in amyloid and tau aggregation, has recently emerged as a mechanistically relevant biomarker candidate. In this clinically realistic cohort study, we examined 46 participants, including 23 clinically diagnosed AD patients and 23 age-matched non-AD individuals with psychiatric and/or metabolic comorbidities. SDC3 expression was quantified in peripheral blood mononuclear cells (PBMCs), while soluble SDC3 and NfL were measured in plasma. Both PBMC-expressed and plasma SDC3 levels were elevated in AD compared with non-AD participants and showed a strong intercorrelation, whereas plasma NfL was likewise increased in AD. Individually, PBMC-SDC3, plasma SDC3, and NfL demonstrated moderate discriminatory performance. However, multivariable models integrating SDC3 (PBMC or plasma), NfL, and age achieved substantially improved discrimination (AUC > 0.8). SDC3 did not correlate with NfL, consistent with a biological signal distinct from neuroaxonal injury and reflective of peripheral immune–metabolic remodeling. Together, these findings identify SDC3 as a blood-based biomarker associated with systemic immune remodeling that complements established neuronal markers in a clinically realistic AD versus non-AD comparison. While exploratory, this study supports further investigation of SDC3 within integrated, multi-domain biomarker strategies in larger and independent cohorts. Full article

(This article belongs to the Special Issue Latest Research in Alzheimer’s Disease)

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21 pages, 5259 KB

Open AccessArticle

3,4-Dihydroxybenzaldehyde Exerts Anti-Alzheimer’s Effects by Inhibiting Aβ Protofibril Assembly and Activating Antioxidant Defense Mechanisms

by Zhourong Zhao, Lin Yang, Zhuo Zhang, Jia Song, Chao Zhang and Xiaohua Duan

Int. J. Mol. Sci. 2026, 27(3), 1599; https://doi.org/10.3390/ijms27031599 - 6 Feb 2026

Abstract

3,4-Dihydroxybenzaldehyde (DBD) is a polyphenolic active constituent derived from Gastrodia elata. Its characteristic phenolic structure is associated with diverse bioactivities, such as anti-inflammatory, antioxidant, and cardioprotective effects. However, its role and underlying mechanisms in combating Alzheimer’s disease (AD) remain inadequately elucidated. In [...] Read more.

3,4-Dihydroxybenzaldehyde (DBD) is a polyphenolic active constituent derived from Gastrodia elata. Its characteristic phenolic structure is associated with diverse bioactivities, such as anti-inflammatory, antioxidant, and cardioprotective effects. However, its role and underlying mechanisms in combating Alzheimer’s disease (AD) remain inadequately elucidated. In this study, we employed computational and experimental approaches to investigate the anti-AD effects of DBD. Molecular dynamics simulations revealed that DBD binds to Aβ fibrils via π–π stacking, hydrophobic interactions, and hydrogen bonds, suggesting its potential to disrupt Aβ fibril stability and thereby inhibit aggregation. In vivo experiments in an AD C. elegans model demonstrated that 2 mM DBD treatment significantly delayed paralysis and extended lifespan. It also improved locomotor activity and pharyngeal pumping rates, while reducing lipofuscin accumulation. These results collectively suggest that DBD promotes healthspan-associated phenotypes. Broad-targeted metabolomics analysis indicated that DBD significantly altered the metabolic profile of the worms. Further mechanistic investigations suggested that the protective effects of DBD are associated with the activation of the DAF-16/FOXO and SKN-1/Nrf2 signaling pathways, accompanied by enhanced resistance to oxidative and thermal stress in nematodes. These findings suggest that DBD exhibits anti-AD potential through multimodal mechanisms, which involve interference with Aβ toxicity and reinforcement of cellular defense. This study supports DBD as a candidate compound and provides a rationale for its further investigation. Full article

(This article belongs to the Section Molecular Neurobiology)

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24 pages, 2189 KB

Open AccessArticle

Integrating Metabolic and Gene Expression Profiling of Glucosinolate Biosynthesis Under Drought Stress in Brassica oleracea

by Hajer Ben Ammar, Souhir Kabtni, Donata Arena, Marwen Amari, Nicolas Al Achkar, Ferdinando Branca and Sonia Marghali

Int. J. Mol. Sci. 2026, 27(3), 1598; https://doi.org/10.3390/ijms27031598 - 6 Feb 2026

Abstract

Drought stress induces pronounced metabolic and transcriptional reprogramming of glucosinolate (GLS) biosynthesis in Brassica oleracea. An integrative approach combining HPLC-based quantification of individual GLSs, quantitative real-time PCR of core biosynthetic and regulatory genes, correlation-based network analysis, and in silico promoter characterization was [...] Read more.

Drought stress induces pronounced metabolic and transcriptional reprogramming of glucosinolate (GLS) biosynthesis in Brassica oleracea. An integrative approach combining HPLC-based quantification of individual GLSs, quantitative real-time PCR of core biosynthetic and regulatory genes, correlation-based network analysis, and in silico promoter characterization was applied to evaluate drought responses across genetically diverse accessions. Drought triggered strong, accession-specific shifts in GLS composition, with sinigrin content increasing from 35.9% to 55.1% in BR1 and glucoerucin reaching up to 80.2% in CCP1, while indolic GLSs such as glucobrassicin and neoglucobrassicin accounted for >75% of total GLSs in CV2 and CCP3. Hierarchical clustering separated accessions into four distinct drought response clusters independent of morphotype. Correlation analysis revealed drought-induced rewiring of GLS interdependencies, characterized by strengthened positive associations among aliphatic GLSs (r > 0.75). Gene expression profiling identified conserved MYB-centered regulatory modules (MYB28, MYB29, MYB34, MYB122) alongside strong accession-specific induction of CYP79F1 (up to 6.3-fold), FMOGS-OX5 (up to 4.8-fold), and ST5a (up to 5.1-fold). Promoter analysis revealed enrichment of ABA- and stress-responsive cis-regulatory elements. These findings delineate a genotype-dependent regulatory framework underlying GLS plasticity and identify quantitative metabolic and transcriptional markers relevant for breeding drought-resilient Brassica cultivars. Full article

(This article belongs to the Special Issue Molecular Research on Plant Breeding)

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