Current Issues in Molecular Biology doi: 10.3390/cimb48070749

Authors: Mei Li Piaoran Fan Yirui Zhang Ranran Li Lemin Chen Donghui Zhang Lei Zhong

To meet the demand for controlled production of low-molecular-weight (Mw < 10 kDa) dextran with potential pharmaceutical applications, this study developed an efficient enzymatic preparation process using PC-Edex, a dextranase derived from Penicillium cyclopium CICC-4022. The effects of enzyme concentration, substrate concentration, temperature, and pH on the degradation of high-molecular-weight dextran were systematically investigated, and the optimal process conditions were established. A staged empirical control strategy based on the Malhotra model was developed to investigate and predict the behavior of dextran molecular weight changes during enzymatic hydrolysis. Under the optimized conditions, dextran with an Mw below 10 kDa was produced within 60 min, with the mass fraction of fragments smaller than 10 kDa reaching 94.56 ± 0.32% and the degradation rate exceeding 98.96 ± 0.15%. The resulting product exhibited a narrow molecular weight distribution (Mw/Mn = 1.528 ± 0.03) and adopted a compact random-coil conformation in aqueous solution. Multi-scale characterization results indicated that enzymatic degradation altered only the molecular weight of dextran, while the backbone structure, amorphous nature, and thermal stability were preserved. These findings present a robust and reproducible laboratory-scale process, which provides a reference for the industrial production of low-molecular-weight dextran for pharmaceutical purposes.

Current Issues in Molecular Biology doi: 10.3390/cimb48070748

Authors: Henna Ali Tyler Gengnagel Salem Birkholz Gowri Vadmal Elijah Torbenson Beau Hsia Abubakar Tauseef Peter T. Silberstein

Background/Aim: Fibroblast growth factor receptor 4 (FGFR4) is a tyrosine kinase involved in cell growth, proliferation, and angiogenesis. While FGFR1–3 are well studied in cancer, FGFR4 remains relatively understudied, and the distribution of its mutations across cancers and patient populations is not well defined. Materials and Methods: A retrospective pan-cancer analysis was performed using the AACR Project GENIE v12 database via cBioPortal. Tumors with somatic FGFR4 mutations were included, excluding copy number alterations and structural variants. Mutations were grouped by hotspot (amino acid 401) and major protein domains. Comparative analyses assessed cancer type distribution, demographics, mutation burden, and co-occurring genomic alterations using chi-square testing with multiple comparison correction. Results: A total of 4565 tumor samples (4283 patients) were analyzed. FGFR4 alterations were observed across diverse malignancies, most commonly non-small cell lung cancer, colorectal cancer, and melanoma. Mutations clustered primarily in the tyrosine kinase and immunoglobulin I-set domains, with no significant variation in distribution across cancer types. Sex was not associated with the mutation group, while race and ethnicity showed significant differences. The FGFR4 hotspot 401 group demonstrated a higher mutation burden, driven by a subset of hypermutated tumors, and showed enrichment for co-occurring alterations in chromatin remodeling, DNA repair, tumor suppressor, and receptor tyrosine kinase genes; however, sensitivity analyses indicated this association was largely attributable to mutation burden rather than a mutation-specific effect. Domain-based mutation groups had lower mutation burdens and fewer co-alterations. Conclusions: FGFR4 alterations occur across a broad range of cancers with consistent domain-level patterns. The hotspot 401 mutation shows a higher mutation burden and co-alteration frequency driven largely by a subset of hypermutated tumors, rather than acting as an isolated driver.

Current Issues in Molecular Biology doi: 10.3390/cimb48070747

Authors: Rogelio Frank Jiménez-Ortega Aldo Hugo de la Cruz-Montoya Nelly Patiño Rafael Velázquez-Cruz Alberto Hidalgo-Bravo

Osteoporosis (OP) and osteosarcoma (OS) are biologically distinct skeletal disorders that share dysregulated bone remodeling, inflammatory signaling, and microenvironmental interactions. This study performed an integrative transcriptomic analysis to identify shared osteoimmune regulatory programs in circulating monocytes from postmenopausal women with OP and OS tumors from Central Mexican cohorts. Two independent RNA-seq cohorts were analyzed separately and then integrated: circulating monocytes from postmenopausal women with OP and controls (7 OP and 7 controls), and donor-matched OS tissues (7 tumors and 7 healthy bone samples). Differential expression, module-based filtering, pathway enrichment, cross-cohort functional integration, directional concordance, and targeted interaction network analyses were performed. The OP cohort showed 169 differentially expressed genes, whereas the OS cohort showed 2135 genes. Module-based filtering retained 82 genes in OP and 278 in OS, with only six genes directly shared. However, pathway-level integration identified convergent signals involving PI3K-Akt, HIF-1 signaling, lipid and atherosclerosis, phagosome, endoplasmic reticulum protein processing, focal adhesion, proteoglycans in cancer, and cancer-related pathways. Directional analysis of 27 shared pathway-associated genes revealed predominantly discordant regulation, with CTNNB1 emerging as a central network node. These findings suggest that OP and OS converge through specific osteoimmune pathways rather than through a uniform shared gene program.

Current Issues in Molecular Biology doi: 10.3390/cimb48070746

Authors: Fan Huang Wenzheng Zhao Shiqing Wang Jun Li Ling Zeng Jingfang Zhu

This study investigated the content differences of major active ingredients in red, yellow, and white flowers of Carthamus tinctorius L. from Yumin County, Xinjiang, and evaluated their α-glucosidase inhibitory effects using high-performance liquid chromatography (HPLC), network pharmacology, molecular docking, and in vitro enzyme assays. HPLC analysis revealed that red safflower had the highest content of Hydroxysafflor yellow A (HSYA), while white safflower exhibited the highest levels of kaempferol and kaempferol-3-O-rutinoside. Network pharmacology identified 21 core targets and multiple pathways related to type 2 diabetes and insulin resistance. Molecular docking indicated that kaempferol-3-O-rutinoside had the lowest binding energy (−9.3 kcal/mol). However, in vitro assays showed that kaempferol exhibited the strongest α-glucosidase inhibition (half maximal inhibitory concentration (IC50) = 0.1768 mg/mL), followed by HSYA and kaempferol-3-O-rutinoside. Among extracts, white safflower demonstrated the highest inhibitory activity (IC50 = 0.5485 mg/mL). In conclusion, flower color significantly influences the chemical composition and hypoglycemic potential of safflower. White safflower extract exhibited the strongest α-glucosidase inhibitory activity among the three flower color extracts. This study identifies kaempferol as the key active ingredient for safflower’s α-glucosidase inhibitory activity, providing a scientific basis for developing Yumin safflower as hypoglycemic functional food.

Current Issues in Molecular Biology doi: 10.3390/cimb48070745

Authors: Zhaoxi Wang Shijie Chen Zhaoheng Wang Yong Sun Kun Wang Xuewen Kang

Intervertebral disc degeneration is a major pathological contributor to low back pain and functional impairment, yet its complex molecular mechanisms have not been fully elucidated. Heat shock proteins, as important molecular chaperones and core regulators of cellular stress responses, exhibit dual protective and pathogenic roles in the process of intervertebral disc degeneration. This review summarizes the expression changes and related regulatory networks of heat shock protein family members such as HSP70, HSP90, HSP27 and GRP78 in nucleus pulposus and annulus fibrosus cells, comprehensively discussing their involvement in the molecular mechanisms of intervertebral disc degeneration by influencing key processes such as cellular homeostasis, inflammatory responses, apoptosis, autophagy, and the synthesis and degradation of the extracellular matrix. Furthermore, this review highlights HSP-centered proteostasis regulation as an emerging therapeutic framework for IVDD and discusses how HSP modulation may be integrated with biomaterials, physical stimulation, and regenerative strategies. However, direct IVDD-specific evidence for certain HSP members remains limited, and this review also highlights current knowledge gaps and future research directions for HSP-centered proteostasis regulation.

Current Issues in Molecular Biology doi: 10.3390/cimb48070744

Authors: Sana Slimene Michelle David Daniel Dauzonne Renato Bensasson Marie-Joelle Virolle Fathi Moussa

Streptomyces coelicolor M145 (SC) is a model strain characterised by its highly active oxidative metabolism. This results in high levels of oxidative stress and the production of actinorhodin (ACT), a blue polyketide pigment with antioxidant properties. To determine whether oxidative stress triggers ACT biosynthesis in SC, we assessed the effect of adding five nitro-flavone (NF) derivatives to the growth medium. As NF molecules are well-known antioxidants, we hypothesized that their presence in the medium would moderate ACT production. The antioxidant efficiency of NF derivatives depends on their ability to donate electrons. This ability is linked to the nature of their 4’-substituent. The Quantitative Structure–Activity Relationship (QSAR) approach and Hammett constants predict the following order: OH > OCH3 > F > H > NO2. The results obtained demonstrate a clear dose–response relationship between the reduction in ACT production by SC and the antioxidant efficiency of these molecules. Therefore, these results strongly suggest that oxidative stress plays a pivotal role in initiating ACT biosynthesis. Furthermore, these results suggest that the direct spectrophotometric assay of ACT production by SC is a rapid, reliable, and inexpensive method of evaluating the in vivo antioxidant activity of any molecule, as predicted by the QSAR approach.

Current Issues in Molecular Biology doi: 10.3390/cimb48070742

Authors: Ana Cristina Moura Gualberto Brunna Letícia de Oliveira Santana Mariana Braccialli de Loyola Yasmim Sampaio da Costa Fábio Pittella-Silva

SETD5 (SET domain-containing 5) is a chromatin-associated regulator increasingly recognized as dysregulated in human malignancies; however, its contribution to tumor biology and tumor–immune interactions remain undefined. We performed an integrative pan-cancer multi-omics analysis to define the landscape of SETD5 dysregulation across cancer types. Transcriptomic, genomic, and epigenetic datasets were integrated to evaluate SETD5 alterations and molecular associations. Protein interaction and pathway enrichment analyses were conducted using STRING, GO, and KEGG, and immunogenomic profiling was used to interrogate associations between SETD5 expression, immune infiltration, and checkpoint programs. SETD5 was overexpressed across multiple malignancies, with low mutation frequency but recurrent copy-number gains. Promoter hypomethylation was detected in a subset of tumors with increased SETD5 expression, suggesting a possible association with epigenetic regulation. Pathway analyses linked SETD5 to macromolecule methylation and transcriptional regulation. SETD5 expression correlated positively with infiltration of macrophages, neutrophils, and dendritic cells, whereas associations with CD8+ and CD4+ T cells varied by tumor type. Tumor type-specific correlations were observed between SETD5 and immune checkpoints genes, including PD-L1 and TIM-3, suggesting an association with immunoregulatory tumor states. These findings identify SETD5 as a recurrently deregulated epigenetic regulator and highlight its potential role in transcriptional control and tumor immune modulation.

Current Issues in Molecular Biology doi: 10.3390/cimb48070743

Authors: Qingping Shi Shuang Gao Beiyan Chen Mingli Shen Jieru Han

The progression and immune escape of HCC are closely regulated by endoplasmic reticulum stress (ERS). However, the associated ceRNA regulatory networks and their prognostic value remain to be systematically elucidated. Here, we sought to establish a prognostic signature derived from an ERS-associated ceRNA network and to investigate its relationship with the tumor immune microenvironment. We integrated TCGA-LIHC transcriptomic data with the MSigDB ERS gene set to identify ERS-associated differentially expressed genes and construct a ceRNA regulatory network. Using a forward search strategy with 10-fold cross-validation, we screened candidate genes to select the optimal prognostic combination and constructed a multigene Cox regression signature. External validation was performed in the independent microarray cohort GSE14520. By integrating single-cell transcriptomics, CIBERSORT, ESTIMATE, TIDE, and drug sensitivity analyses, we revealed immune microenvironment characteristics associated with this signature. Based on the ceRNA network’s eight core ERS mRNAs, an optimal three-gene signature comprising STC2, CKS1B, and PSAT1 was selected via forward search. The signature demonstrated robust prognostic discrimination in the TCGA training cohort (C-index = 0.653) and was independently corroborated in the external GSE14520 dataset (C-index = 0.584, log-rank p = 0.008). The signature was confirmed as an independent prognostic indicator by multivariable Cox regression. Functional enrichment analysis demonstrated a marked accumulation of cell-cycle-related pathways in the high-risk group, notably DNA replication and the spindle assembly checkpoint. Single-cell transcriptomic profiling showed that STC2 and CKS1B were predominantly expressed by tumor epithelial cells, whereas CCL2 and ATF3 were mainly detected in macrophages and fibroblasts. Drug sensitivity analysis indicated that the high-risk group was more sensitive to drugs such as docetaxel and AZD5582, consistent with the upregulation of proliferation pathways in this group; in the low-risk group, VE-822 exhibited selective sensitivity. This study established a three-gene prognostic signature based on the ERS-associated ceRNA network. The signature demonstrated robust prognostic stratification capabilities in cross-platform validation and revealed molecular characteristics centered on uncontrolled cell-cycle progression, as well as an immunosuppressive microenvironment, in the high-risk group, providing an exploratory tool for prognostic assessment and treatment strategy selection in hepatocellular carcinoma (HCC).

Current Issues in Molecular Biology doi: 10.3390/cimb48070741

Authors: Xiangrong Meng Wentao Yang Yuan Liu Yongqi Zhang Die Luo Siyu Wei Yifan Guo Cen Qian

As the second largest order of Insecta, Lepidoptera is an important component of the ecosystems. However, there are few data on the mitochondrial genomes of Saturniidae. Here, the complete mitochondrial genome of Cricula andrei was sequenced and characterized. It was 15,324 bp in length, containing 13 protein-coding genes (PCGs), 22 tRNAs, 2 rRNAs and a control region (AT-rich region). The control region had an AT content of 90.40%, and the conserved ATAGA sequence guided a 19 bp poly-T. Amino acid composition analysis showed that Ile, Leu, Phe and Asn were the most frequent amino acids, and codon usage analysis revealed a preference for A/U-ending codons. Phylogenetic trees constructed by Bayesian inference and Maximum likelihood methods indicated that C. andrei is grouped with C. trifenestrata and supported the current taxonomic placement of Cricula within the family. The enrichment of the mitochondrial genome database of Lepidoptera will help to better understand the genetic and evolutionary relationships of lepidopteran populations, as well as related taxonomic issues.

Current Issues in Molecular Biology doi: 10.3390/cimb48070740

Authors: Federica Fogacci Giulia Fiorini Cristina Scollo Claudio Borghi Dino Vaira Arrigo Francesco Giuseppe Cicero

Helicobacter pylori (H. pylori) infection has been investigated as a potential contributor to extra-gastric vascular injury, although its cardiovascular relevance remains uncertain and context-dependent. This state-of-the-art narrative review synthesizes clinical, translational, and experimental evidence linking H. pylori infection to endothelial dysfunction and arterial stiffness, two complementary phenotypes of early vascular aging. Evidence is strongest for endothelial dysfunction, particularly in the presence of active or cytotoxin-associated gene A (CagA)-positive infection, extracellular-vesicle-mediated signaling, oxidative stress, impaired endothelial repair, selective attenuation of endothelium-dependent vasodilation, and short-term improvement after eradication. Associations with arterial stiffness are less consistent and appear more evident in selected settings characterized by younger age, inflammatory or metabolic vulnerability, and severe gastric injury. Conversely, serology-based studies and studies using late structural vascular endpoints frequently report null or discordant findings. Overall, H. pylori should not be considered a universal cardiovascular risk factor, but it may amplify early vascular injury in susceptible subgroups. Prospective studies using active-infection testing, virulence profiling, gastric histology, and prespecified vascular endpoints are needed to determine whether eradication produces sustained vascular benefit.

Current Issues in Molecular Biology doi: 10.3390/cimb48070739

Authors: Anna Vass József Király Erzsébet Szabó Gábor Kónya Ali Shammas Krisztián Szegedi Balázs Dezső Éva Juhász Gábor Halmos Zsuzsanna Szabó

Renal cell carcinoma (RCC) is frequently resistant to tyrosine kinase inhibitors (TKIs) such as sunitinib, limiting therapeutic efficacy. ATP-binding cassette (ABC) transporters, including ABCB1, ABCB5, and ABCG2, are important transporters implicated in multidrug resistance, influencing drug efflux and tumor progression. We aimed to evaluate the expression of ABCB1, ABCB5, and ABCG2 in human RCC tissues and human renal cancer cell lines CAKI-2 and A-498, and to investigate their potential association with sunitinib resistance and associated signaling pathways. Twenty paired tumorous and adjacent non-tumorous human kidney tissue samples were analyzed for ABC transporter gene expression using qRT-PCR. RCC cell lines CAKI-2 and A-498, including sunitinib-resistant derivatives, were treated with 40 µM sunitinib. The levels of transporters and key signaling proteins were assessed by Western blot. ABCG2 was consistently higher in tumorous tissues, and ABCB1 and ABCB5 showed grade-dependent increases in tumors. Resistant cells exhibited elevated ABCB1 and dynamic ABCB5 and ABCG2 expression patterns compared to sensitive cells, indicating an association between altered transporter expression and the resistant phenotype. Sunitinib treatment modulated signaling pathways, with differential activation of PI3K/Akt, NF-κB, and MAPK/ERK observed in sensitive versus resistant cells. Our findings suggest that altered ABCB1 and ABCG2 expression may be associated with the development of sunitinib resistance in RCC and may be linked to changes in key survival signaling pathways. These findings provide a basis for future functional studies investigating the role of ABC transporters in sunitinib resistance and may contribute to the development of personalized therapeutic strategies in RCC.

Current Issues in Molecular Biology doi: 10.3390/cimb48070738

Authors: Fuan Niu Yuting Dai Can Cheng Anpeng Zhang Huangwei Chu Jihua Zhou Bin Sun Xiao Gu Hua Wang Kaizhen Xie Fengzhen Shi Xueqing Zhang Bilian Hu Yue Qiu Xinyue Zhao Wei Tian Liming Cao

Japonica soft rice varieties possess excellent eating quality, and their cultivation area has been steadily expanding in recent years. This study aimed to analyze japonica soft rice varieties cultivated in the Yangtze River Delta region of China at the genome level and to provide a theoretical basis for optimizing disease resistance and other important traits. Genotypic characterization and evaluation of ten major japonica soft rice varieties from the Yangtze River Delta region were conducted using a genome-wide single nucleotide polymorphism (SNP) chip. The experimental results indicated that the soft rice varieties in the Yangtze River Delta region had a relatively high japonica component and were all classified as typical japonica rice varieties. Specifically, the highest (95.6%) and lowest (91.5%) proportions of japonica genomic segments were detected in Tai’an 1 and Zhehexiang 2, respectively. Japonica soft rice varieties from Shanghai exhibited a closer genetic distance to those from Jiangsu Province than to those from Zhejiang Province. Genomic identity was highest between Tai’an 1 and Nanjing 46 (87.9%) and lowest between Tai’an 1 and Jia 67 (74.4%). Based on the results of the chip assay, a total of twenty-six functional genes controlling key traits, such as yield, quality, and resistance to biotic and abiotic stresses, were identified in the ten analyzed varieties. Among them, Zhehexiang 2 carried the broad-spectrum blast resistance genes Pi2 and Pita, which is useful for improving the blast resistance of japonica soft rice varieties. The findings of this study provide genetic resources and carrier materials for the efficient molecular improvement of japonica soft rice varieties.

Current Issues in Molecular Biology doi: 10.3390/cimb48070737

Authors: Jinyue Duan Yi Wang Qisen Li Yujue Wang Jinrui Liu Yi Qi Yichi Zhang Changhao Fu Zhongyi Cong Can Wang Manman Su

Prostate stem cell antigen (PSCA), a pivotal member of the lymphocyte antigen-6 (Ly6) protein family, has been implicated in the tumorigenesis and neoplastic progression of diverse cancer types. In this study, we conducted a thorough investigation into the role of PSCA in the development of colorectal cancer (CRC). Survival analysis based on The Cancer Genome Atlas (TCGA) dataset demonstrated that elevated expression of PSCA was tightly correlated with unfavorable overall survival, inferior relapse-free survival, and worse post-progression survival among CRC patients. Additionally, PSCA exhibited significantly higher expression levels in colorectal cancer stem cell (CRC-SCs) relative to CRC cell lines. Loss-of-function assays using small interfering RNA (siRNA)-mediated silencing were performed to evaluate the effects of PSCA downregulation on the stemness properties of CRC-SCs, including proliferative capacity, invasive potential, and apoptotic rate, which were assessed by MTS assay, transwell invasion assay, and flow cytometry analysis, respectively. The results showed that silencing PSCA markedly suppressed the proliferation and invasion of CRC-SCs, while significantly promoting cellular apoptosis. RNA sequencing was performed to identify differentially expressed genes (DEGs) in the PSCA knockdown group compared to the negative control group. Follow-up analyses using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated that these DEGs were significantly enriched in the cell-substrate adherens junction term and the mitogen-activated protein kinase 9MAPK signaling pathway. Moreover, PSCA silencing substantially reduced the phosphorylation levels of the core MAPK signaling constituents, pBRAF and pERK1/2; conversely, PSCA overexpression prominently upregulated the expression of pBRAF and pERK1/2. In nude mice with CRC-SCs cancer xenograft tumors, treatment with PSCA siRNA significantly decreased tumor volume and weight, while also notably extending the survival time of the tumor-bearing mice compared to the control group. Collectively, these findings confirm that PSCA plays a critical oncogenic role in CRC cancer growth and malignant progression, suggesting its potential as a novel and promising therapeutic target for CRC.

Current Issues in Molecular Biology doi: 10.3390/cimb48070736

Authors: Chia-Yuan Lin Lok-I Chan Yu-Hsuan Chang Meng-Chun Lu Chia-Wen Tsai

The disruption of mitochondrial homeostasis is a trigger for insulin resistance. The loss of N-acetyltransferase 1 (Nat1) function, an insulin-sensitivity gene, contributes to mitochondrial dysfunction and insulin resistance. Carnosic acid (CA), a diterpene derived from rosemary, has demonstrated an anti-insulin-resistance effect. This study hypothesized that CA protects against TNF-α-induced insulin resistance in 3T3-L1 adipocytes by regulating mitochondrial dynamics, biogenesis, and function via Nat1. 3T3-L1 adipocytes were pretreated with CA for 12 h, followed by co-treatment with TNF-α for an additional indicated duration. Results showed that treatment of 3T3-L1 adipocytes with TNF-α decreases mitochondrial membrane potential (MMP) and PGC-1α protein levels and alters mitochondrial fission/fusion dynamics. Pretreatment with CA improved these effects. In parallel, CA prevented the TNF-α-induced reduction in Nat1 protein and improved insulin signaling by suppressing the phosphorylation of insulin receptor substrate-1 (IRS-1) at serine307, while restoring the phosphorylation of IRS-1 at tyrosine628 and Akt. Moreover, transfection with Nat1 siRNA inhibited the protective effect of CA against TNF-α-induced reductions in MMP, PGC-1α, and insulin signaling. In conclusion, CA ameliorated TNF-α-induced insulin resistance by reducing mitochondrial dysregulation by Nat1.

Current Issues in Molecular Biology doi: 10.3390/cimb48070735

Authors: Jieyao Ma Huiling Liu Yalan Wu Tingsheng Ma Huaming Xiao Wei Cai

Background/Objectives: Polygonatum kingianum Collett & Hemsl. (PK) is an edible medicinal herb with tonic effects. Its natural antioxidant peptides are valuable for functional food development, while their characteristics and mechanisms are unclear. This study aimed to identify PK antioxidant peptides and explore their antioxidant molecular mechanisms to support the utilization of PK active ingredients. Methods: Three peptide fractions (P1, P2, P3) were prepared from PK via defatting, alkali–acid precipitation and enzymatic hydrolysis. Nano-liquid chromatography coupled with Q Exactive mass spectrometry was used for peptide identification. Bioinformatic tools predicted peptide antioxidant activity, and molecular docking targeting the Kelch-like ECH-associated protein 1-nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) pathway verified peptide–target-binding affinity. Results: A total of 747, 1850 and 2537 peptides were identified from P1, P2 and P3, respectively, among which 119 peptides were screened out as potential antioxidant candidates. Docking analysis revealed 10 peptides with strong binding affinity to Keap1. These active peptides were short sequences of 3–5 residues enriched in hydrophobic and aromatic amino acids, which stably bound key residues within the Keap1 binding pocket. Conclusions: This study fills the research gap in PK peptidome profiling, clarifies structural signatures of candidate antioxidant peptides, and identifies high-affinity short peptides targeting the Keap1-Nrf2 antioxidant pathway. The established screening pipeline provides technical support for bioactive peptide mining and deep processing of PK resources.

Current Issues in Molecular Biology doi: 10.3390/cimb48070734

Authors: Ícaro S. Lopes Eduardo R. Fukutani Tiago F. Mota Bruno B. Andrade Mariana Araújo-Pereira Artur T. L. Queiroz

The omics sciences represent a revolution for clinical studies, offering integrative approaches to analyzing biological data with unprecedented depth. From the discovery of the double-helix structure of DNA to the CRISPR-Cas9 gene editing tool, passing through the evolution of sequencing platforms and the exponential advance of computing power and in silico tools, omics has progressed in its role of leading innovative solutions for old challenges in health sciences. In this review, we describe different omics, the history of their techniques and technologies, data analysis and up-to-date visualization tools used for clinical data in research and health systems. We also discuss how omics are currently being applied in diagnosis, precision and personalized medicine. For the future, omics vow to underpin the majority of decisions made by health professionals, allowing individualized treatments based on Big Data and personal biological information.

Current Issues in Molecular Biology doi: 10.3390/cimb48070733

Authors: Seher Saruhan Deniz Özdemir Can Ali Ağca

Myeloid cell leukemia-1 (MCL-1) is a cellular survival protein belonging to the Bcl-2 protein family and is overexpressed in human colorectal cancer (CRC). Background: This study aimed to reduce cancer progression by suppressing the MCL-1 protein using PROTAC MCL-1 Degrader-1 and Trametinib, with the aim of overcoming the apoptosis resistance caused by high MCL-1 expression levels in colorectal cancer cells. Methods: Therefore, we tested the cell viability, proliferation, mitochondrial membrane potential, cell cycle progression, and cell death potential of MCL1-specific PROTAC Mcl-1 Degrader-1 and the combination of PROTAC Mcl-1 Degrader-1 and Trametinib in colorectal cancer cell lines using different methods such as WST-8 assays, real-time cell analysis, MMP/JC-1 staining assays, flow cytometry, and Western blot analysis. Results: The results suggest that PROTAC Mcl-1 Degrader-1 is associated with dose- and time-dependent reductions in cell proliferation in colorectal cancer cells. Under the experimental conditions used in this study, PROTAC MCL-1 Degrader-1 showed limited effects on the migratory potential of colorectal cancer cells but was associated with changes in cell cycle distribution, including an increased proportion of HT-29 cells in the G2/M phase. In addition, combination treatment with PROTAC MCL-1 Degrader-1 and trametinib was associated with greater reductions in cell viability and proliferation than either monotherapy. The combination treatment was also accompanied by changes in mitochondrial membrane potential and increased apoptotic cell populations, with a higher proportion of early apoptotic cells observed in HT-29 and COLO-205 colorectal cancer cell lines. Conclusion: Our findings suggest that the reduction in MCL-1 protein levels observed following PROTAC Mcl-1 Degrader-1 treatment may contribute to its potential therapeutic relevance in colorectal cancer.

Current Issues in Molecular Biology doi: 10.3390/cimb48070732

Authors: José Manuel Pérez de la Lastra Celia María Curieses Andrés Elena Bustamante Munguira Celia Andrés Juan Eduardo Pérez Lebeña

Polyphenols often oscillate between antioxidant and pro-oxidant behaviours depending on structural motifs and context, yet the field still relies heavily on test-tube antioxidant assays that poorly predict cellular and clinical outcomes. This review (2015-2025) integrates chemistry, enzymology, and human data to frame polyphenols as modulators of redox signalling rather than mere radical scavengers. We first formalize the catechol/o-quinone-hydroquinone/p-quinone cycle and the role of NQO1 and Keap1/NRF2 thresholds. We then examine bioavailability, conjugation, and microbiota-derived metabolites (metabotypes), highlighting when the “active” species is a conjugate or a microbial derivative. We discuss safety through quinone speciation and adduct chemistry, and connect food processing (e.g., PPO-driven browning) with shifts in quinone pools. Contextual “levers” (pH, O2, Fe/Cu, oxidases) can flip antioxidant to pro-oxidant outputs, sometimes beneficial via hormesis and redox preconditioning. In humans, randomised trials and prospective cohorts point in a broadly consistent direction, although primary composite endpoints have often proved null, and observational associations should not be read as equivalent to trial evidence. Heterogeneous results across studies are largely explained by dose, adherence, metabotypes, matrix, and endpoint selection. We propose a practical dose-response framework and a reporting checklist to improve interpretation and translation. Recasting polyphenols as tuneable redox-signalling agents clarifies apparent contradictions across models and suggests precision-nutrition strategies (metabotype-aware) and food design approaches (PPO and quinone speciation) with potential in healthy ageing, muscle, and brain.

Current Issues in Molecular Biology doi: 10.3390/cimb48070731

Authors: Asma Saeed Jamila Hijazi Zainab Bashir Pierre Khoueiry Assaad A. Eid Nadine Darwiche Maria Teresa Bengoechea-Alonso Johan Ericsson Borbala I. Mifsud Georges Nemer

Colorectal cancer (CRC) remains a leading cause of cancer morbidity worldwide, highlighting the need for improved therapies. Metformin, a widely used antihyperglycemic agent, has gained attention for its potential antitumor properties. In this study, we evaluated the effects of the tumor suppressor genes TP53 and CDKN1A on metformin responsiveness in a KRAS-mutant CRC in vitro model using HCT116 cells harboring a G13D mutation in KRAS. Using parental (p53+/+, p21+/+) and isogenic knockout cell lines, we assessed cell-cycle distribution and transcriptomic responses following metformin treatment. In parental wild-type cells, metformin exposure was associated with a dose- and time-dependent reduction in cell viability and an increased proportion of cells in the G0/G1 phase, with significance levels across treatment conditions ranging from p = 0.03 to p < 0.0001. Loss of p53 or p21 was associated with attenuated cellular responses to metformin, with p21-deficient cells responding primarily at higher doses and prolonged exposure (72 h). Transcriptomic profiling revealed extensive differential gene expression in parental cells (1399 DEGs), compared with more limited responses in p53−/− (270 DEGs) and p21−/− cells (32 DEGs). Differentially expressed genes associated with MAPK signaling (DUSP5) and inflammatory regulation (TNFAIP3) were observed across genotypes, whereas pathway enrichment of DNA replication and chromatin organization was specific to p53-deficient cells. These findings provide a transcriptomic and phenotypic characterization of genotype-dependent cellular responses to metformin and establish a basis for future mechanistic and functional validation studies.

Current Issues in Molecular Biology doi: 10.3390/cimb48070730

Authors: Tsong-Min Chang Ting-Ya Yang Huey-Chun Huang

Plerixafor is a clinically approved CXCR4 antagonist that mobilizes hematopoietic stem cells by disrupting CXCL12/CXCR4 retention signaling. However, its biochemical effects on melanocytes and pigmentation remain unexplored. We investigated how Plerixafor modulates CXCR4 signaling in melanocytes and evaluated its potential as a pro-melanogenic agent using in vitro and in vivo approaches. Human PIG1 melanocytes were treated with 10 nM Plerixafor with or without hydroquinone (HQ), followed by qPCR for MITF and tyrosinase expression, flow cytometry for CXCR4/CXCR7 and integrin profiling, transwell migration assays, β-arrestin siRNA knockdown, Western blotting, subcellular fractionation, and ChIP-qPCR for β-catenin binding to MITF regulatory regions. A murine HQ-induced depigmentation model was used to test topical Plerixafor on pigmentation, hair follicles, melanogenic gene expression, and systemic safety markers. Plerixafor significantly increased MITF and tyrosinase mRNA and enhanced melanocyte migration while counteracting HQ-induced suppression of melanogenic genes. In addition, it reduced cell-surface CXCR4 (consistent with β-arrestin-mediated receptor internalization) without altering CXCR7, c-KIT, or N-cadherin. β-Arrestin knockdown abolished Plerixafor-induced ERK phosphorylation and melanogenic responses, confirming β-arrestin dependence. Plerixafor promoted β-catenin nuclear translocation and direct β-catenin occupancy at MITF promoter/enhancer TCF/LEF motifs. In vivo, topical Plerixafor restored HQ-induced depigmentation, increased hair follicle number and melanin content, and upregulated cutaneous MITF and tyrosinase without hepatic, renal, or inflammatory toxicity. Plerixafor functions as a biased CXCR4 ligand in melanocytes, influencing the β-arrestin–β-catenin–MITF signaling axis to drive melanogenesis and re-pigmentation. These findings identify β-arrestin-dependent CXCR4 signaling as a tractable pharmacologic mechanism for therapeutic re-pigmentation in pigmentary disorders.

Current Issues in Molecular Biology doi: 10.3390/cimb48070729

Authors: Hawraa Zbeeb Chourouk Joumaa Giulia De Negri Atanasio Alberto Diaspro Laura Vergani

Insulin resistance (IR) is the underlying pathogenic mechanism for Type 2 Diabetes Mellitus, which is interconnected with Fatty Liver Disease. To investigate the molecular mechanisms by which different metabolic triggers contribute to hepatic IR onset, we exposed human HepG2 hepatocytes to varying glucose concentrations (25–50 mM), and/or insulin (1 nM), and a free fatty acid mixture (0.3 mM), mimicking moderate or severe hyperglycemia, hyperinsulinemia, and steatosis. Glucose consumption, glycogen and lipid droplet (LD) accumulation, gene expression, oxidative stress, and insulin signaling were assessed. Under severe hyperglycemia, both insulin and fatty acids decreased glucose consumption, whereas under moderate hyperglycemia, only insulin had this effect. Glycogen accumulation was increased across all treated conditions. Both insulin and fatty acids triggered steatosis and downregulated PPARγ and SIRT1 mRNA, with insulin increasing LD number, while FFAs increased both LD number and size. All conditions enhanced ROS production, resulting in oxidative stress, but with differences in antioxidant enzyme response. Insulin receptor expression was reduced by insulin and FFAs under moderate hyperglycemia but increased by these stimuli under severe hyperglycemia. Both stimuli enhanced AKT phosphorylation under both hyperglycemic conditions. We conclude that different triggers cooperate in promoting hepatic IR through distinct molecular and signaling mechanisms, suggesting that effective treatments may need to target multiple pathways simultaneously.

Current Issues in Molecular Biology doi: 10.3390/cimb48070728

Authors: Elena I. Dyachenko Lyudmila V. Bel’skaya

Saliva is a promising diagnostic fluid for studying diseases, including cancer. Saliva composition can reflect both local processes occurring in the oral cavity and systemic changes associated with distant tumors. This review examines changes in salivary electrolyte, amino acid, lipid, and cytokine profiles, tumor markers, and the oral microbiome in cancer. Collectively, these aspects reflect metabolic, inflammatory, immune, secretory, and tumor-associated processes. Metabolites can enter saliva via the salivary glands, systemic circulation, gingival fluid, extracellular vesicles, and oral cells, as well as directly from the site of disease during localized pathological processes. In tumors not localized in the oral cavity, changes in saliva composition are more often associated with systemic inflammation, altered oral microbiome, metabolic reprogramming, oxidative stress, and tumor-associated exosomes. Individual metabolites have limited specificity and cannot be used as independent diagnostic indicators. A comprehensive multimarker analysis of saliva is of greatest value. This approach can facilitate early diagnosis, identify the risk of disease development and progression, monitor therapy, and understand the biological changes associated with the pathological process, including tumors.

Current Issues in Molecular Biology doi: 10.3390/cimb48070727

Authors: Joselia Carlos Filip Konecny Maryia Ryskina John J. McGuire

Proteinase-activated receptor-2 (PAR2) regulates cardiac pressure and vascular function through context-dependent mechanisms that integrate G protein-coupled receptor signalling. We investigated the baseline cardiovascular phenotype of PAR2-deficient (PAR2−/−) mice and the acute in vivo effects of the PAR2 agonist trans-cinnamoyl-Leu-Ile-Gly-Arg-Leu-Orn-amide (tcLIGRLO). Left ventricular pressure–volume (PV) analysis revealed that PAR2−/− mice had elevated systolic and diastolic pressures with reduced end-diastolic volumes and increased ejection fraction while maintaining stroke volume and cardiac output. Thus, PAR2 deficiency produces a pressure-dominant cardiovascular state with increased mechanical work and preserved global function. In WT mice, tcLIGRLO dose-dependently reduced cardiac pressure generation, contractility, relaxation kinetics, and stroke work, whereas these effects were absent in PAR2−/− mice, indicating PAR2-dependent cardiac regulation. Despite these changes, global cardiac output remained largely preserved despite modest reductions in heart rate. In contrast, tcLIGRLO elicited strain-dependent vascular responses, including altered effective aortic elastance and reduced carotid blood flow in PAR2−/− mice. Ex vivo experiments showed that tcLIGRLO-induced contraction in PAR2−/− arteries required Gq-dependent signalling in this experimental context. These findings identify PAR2 as a context-dependent regulator of cardiac pressure and vascular tone, in which PAR2-dependent cardiac effects and PAR2-independent, Gq-mediated vascular responses reflect distinct but interacting signalling mechanisms.

Current Issues in Molecular Biology doi: 10.3390/cimb48070726

Authors: Xiaohang Gao Minghui Yang Jing Yang Yunpeng Zhong Chao Ma Guoliang Xu Lin Zhang Rong Zhang

Esophageal squamous-cell carcinoma (ESCC) remains a highly lethal malignancy, with long-term survival still unsatisfactory and an urgent need for clinically relevant molecular targets. Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is an RNA-binding protein with oncogenic activity in several tumor types, but the signaling events associated with its role in ESCC have not been fully defined. IGF2BP2 expression was examined in paired ESCC and adjacent non-tumor tissues by immunohistochemistry (10 paired samples) and qRT-PCR (16 paired samples). Associations with clinicopathological variables were evaluated in 108 TCGA ESCC cases. Stable lentiviral shRNA-mediated knockdown was established in KYSE30 and ECA109 cells. CCK-8, colony formation, Transwell, EdU, and flow cytometry assays were used to assess proliferation, clonogenic growth, migration, invasion, apoptosis, and cell-cycle distribution. EMT-associated markers were measured by Western blotting and qRT-PCR. RNA sequencing, KEGG enrichment analysis, and rescue experiments using the AKT activator SC79 were performed to explore downstream signaling. IGF2BP2 was upregulated in ESCC tissues at both the transcript and protein levels, and higher expression was associated with unfavorable clinicopathological characteristics. Silencing IGF2BP2 reduced proliferation, colony formation, migration, and invasion, promoted G0/G1 cell-cycle arrest, and increased apoptosis. EMT-associated marker analysis showed decreased Snail and increased Slug expression, indicating that these changes require cautious interpretation. RNA sequencing and KEGG analysis suggested enrichment of PI3K/AKT-related signaling after IGF2BP2 knockdown. IGF2BP2 silencing decreased PI3K and p-PI3K levels, whereas SC79 partly restored migratory and invasive capacities. IGF2BP2 contributes to malignant phenotypes in ESCC and may be associated with PI3K/AKT-related signaling. These findings indicate that IGF2BP2 is a candidate biomarker and potential therapeutic target, although further mechanistic and pharmacologic validation is required.

Current Issues in Molecular Biology doi: 10.3390/cimb48070725

Authors: Cristina Stefania Dumitru Alina Cristina Barb Antonia Armega Anghelescu Dorin Novacescu Marina Rakitovan Cristian Silviu Suciu Flavia Zara

Conventional TNM staging in head and neck squamous cell carcinoma (HNSCC) does not fully capture the molecular heterogeneity driving tumor aggressiveness. Identifying accessible biomarkers that complement staging is therefore a clinical priority. This retrospective study evaluated immunohistochemical VEGF expression in 50 histopathologically confirmed HNSCC cases and examined its association with TNM staging and clinicopathological parameters. VEGF expression was assessed using a semi-quantitative immunoreactivity score (IRS; range 0–12; median 5; IQR 3–8). High VEGF expression (IRS ≥ 4) was identified in 64% of cases and was significantly associated with advanced tumor stage (III–IV; p = 0.032), higher T stage (T3–T4; p = 0.041), and regional lymph node metastasis (p = 0.018). Multivariate logistic regression confirmed nodal status as an independent predictor of high VEGF expression (OR = 2.85; 95% CI 1.12–7.24; p = 0.028). These findings indicate that VEGF expression is associated with adverse pathological features and markers of tumor progression in HNSCC. In the absence of survival data, prognostic conclusions cannot be drawn; prospective studies with survival endpoints are required to establish the clinical utility of VEGF as a prognostic biomarker.

Current Issues in Molecular Biology doi: 10.3390/cimb48070724

Authors: Sara Senlle Cécile Nicole Patrícia M. A. Silva Odília Queirós Andrea Cunha

Oral squamous cell carcinoma (OSCC) represents a significant global health challenge characterized by high morbidity and mortality, frequently driven by therapeutic resistance and tumor aggressiveness. Metabolic reprogramming has emerged as a hallmark of OSCC, enabling tumor cells to sustain proliferation, survive under adverse microenvironmental conditions, and evade therapeutic stress. Recent advances in cancer metabolism have identified metabolic plasticity as a central determinant of OSCC progression and treatment failure, highlighting the need to integrate evidence on metabolic vulnerabilities and therapeutic opportunities. This narrative review aims to provide an updated overview of metabolic reprogramming in OSCC, with particular emphasis on the interplay between glycolysis, mitochondrial metabolism, glutamine metabolism, and fatty acid oxidation, and to discuss how these interconnected pathways may be therapeutically exploited. Although OSCC cells exhibit enhanced aerobic glycolysis, mitochondria remain functionally active and play critical roles in energy production, redox homeostasis, and metabolic adaptation. The therapeutic potential of targeting tumor metabolism is discussed, highlighting dichloroacetate (DCA) as a promising metabolic modulator capable of inhibiting pyruvate dehydrogenase kinase (PDK), restoring mitochondrial glucose oxidation, and partially reversing the glycolytic phenotype. The review also examines the current translational limitations of DCA, including toxicity, pharmacokinetic constraints, and compensatory metabolic adaptations that restrict its efficacy as a standalone therapy. Furthermore, potential synergistic strategies are explored, particularly the combination of DCA with paclitaxel, which enhances therapeutic efficacy through concurrent disruption of cytoskeletal integrity and metabolic homeostasis, thereby increasing cellular susceptibility to apoptosis and overcoming chemoresistance.

Current Issues in Molecular Biology doi: 10.3390/cimb48070723

Authors: Bhoomendra A. Bhongade Areeg Anwer Ali Mohamed El-Tanani Shakta Mani Satyam Sirajunisa Talath Adil Farooq Wali Syed Arman Rabbani Walaa Ibraheem Karolina Hoffmann Ashot Avagimyan Ioannis Ilias Sorina Ispas Viviana Maggio Anna Paczkowska Manfredi Rizzo

Metabolic reprogramming is a unifying characteristic of cancer and involves orchestrated changes in glucose, amino acid, lipid, and mitochondrial metabolism that go beyond the well-known Warburg effect. Evidence is accumulating that these metabolic states are actively remodeled by oncogene signaling and tumor suppressor loss, allowing cancer cells to sustain anabolic growth, redox homeostasis, and therapeutic stress. This review provides an overview of new findings on tumor metabolism, mechanisms, and the molecular networks governing this reprogramming. We discuss how the major oncogenic pathways, such as MYC, mTOR, HIF, and AMPK, reprogram metabolism using transcriptional, epigenetic, and post-translational control of metabolic flux. A focus is placed on mitochondrial bioenergetics, dynamics, and metabolite signaling such as cancer cell fitness and stress tolerance-defining factors. We also discussed metabolic crosstalk in the tumor ecosystem, including nutrient competition, metabolite coupling, and immunometabolic reprogramming to coordinate metabolism-mediated effects on tumorigenesis and therapeutic response. The review further considers the mechanistic basis for metabolism-targeted therapies, including pathway dependencies, adaptive responses, and micro-environmental context that constrain clinical benefit. Recent innovations such as spatial metabolomics, single-cell metabolic profiling, and systems-level models have unveiled significant intratumoral heterogeneity of metabolism, and they have provided important information about diverse vulnerabilities to therapeutic intervention. Accordingly, understanding the complex crosstalk between these metabolic networks is crucial to rationally designing combination strategies that selectively leverage cancer-specific metabolic liabilities with minimal toxicities against normal tissues.

Current Issues in Molecular Biology doi: 10.3390/cimb48070722

Authors: Hariharan Balasubramaniam Susiharan Govindasamy Srinivasan A. Santhana Krishna Kumar

N6-methyladenosine (m6A) is the most prevalent internal modification of eukaryotic mRNA and a central regulator of plant development and stress adaptation. The plant m6A writer complex requires two MT-A70 family proteins, the catalytic subunit MTA70 and its non-catalytic partner MTB, yet the evolutionary basis and structural logic underlying this functional division remain unresolved across land plant lineages. Here, we present an integrative computational analysis of MTA70 and MTB across 15 phylogenetically representative species spanning bryophytes, lycophytes, charophyte algae, monocots, and dicots. Phylogenomic reconstruction resolved three strongly supported clades, namely MTA70, MTB, and an intermediate MTA70-like group, demonstrating that catalytic-to-regulatory divergence predates the separation of major land plant lineages. MTA70 proteins exhibited strict conservation of gene structure, catalytic motifs, and domain architecture, reflecting selective constraint at functionally critical residues, whereas MTB showed extensive divergence in exon–intron organization and surface-exposed residues, consistent with relaxed structural constraints. AlphaFold2-based structural modeling and data-driven protein–protein docking predicted a stable MTA70–MTB heterodimer with a buried surface area of 1435 Å2 and a binding free energy of −8.1 kcal/mol, with Lys746 and Lys637 of MTB identified as primary interface hotspots by computational alanine scanning. Expression profiling across six species revealed preferential MTB accumulation in reproductive tissues, while promoter analysis identified statistically significant enrichment of jasmonate-responsive elements (TGACG-motif) in MTB promoters (Mann–Whitney U, p = 0.025) and a 3.4-fold higher abundance of ABA-responsive elements (ABRE) in MTB relative to MTA70, suggesting potential responsiveness to multiple phytohormone signals. Together, these findings establish an evolutionary and regulatory framework for MTB as a conserved scaffold coupling m6A deposition to developmental and environmental signaling in land plants.

Current Issues in Molecular Biology doi: 10.3390/cimb48070721

Authors: Raffaella Pagliaro Fabio Perrotta Stefano Sanduzzi Zamparelli Valerio Maria Carrozzo Alfredo Cipriano Michele Mondoni Giulia Maria Stella Andrea Bianco Filippo Scialò

Pulmonary fibrosis (PF), particularly idiopathic pulmonary fibrosis (IPF), is a progressive and often fatal interstitial lung disease characterised by complex genetic and molecular heterogeneity. Traditional diagnostic approaches, which rely on clinical, radiological and histopathological assessment, are frequently insufficient to capture the underlying biological diversity of the disease. The advent of next-generation sequencing (NGS) has substantially advanced the understanding of PF by enabling comprehensive genomic and transcriptomic profiling. NGS technologies, including whole-exome sequencing (WES), whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and targeted gene panels, have uncovered key genetic determinants. These include mutations in telomere-related genes (TERT, TERC, RTEL1) and surfactant-related genes (SFTPC, SFTPA2), as well as common variants like the MUC5B promoter polymorphism. These discoveries have clarified disease pathogenesis, revealed polygenic risk models, and may improve diagnostic accuracy, particularly in distinguishing overlapping interstitial lung disease (ILD) phenotypes. Beyond genetics, transcriptomic analyses have identified dysregulated pathways, including TGF-β, Wnt/β-catenin, and PI3K/Akt signalling, and have enabled the discovery of novel biomarkers for prognosis and therapeutic response. In selected clinical settings, NGS is beginning to support patient stratification and inform management decisions. Emerging applications, including liquid biopsy and integration with artificial intelligence, further expand the potential clinical utility of NGS. Despite challenges related to cost, data interpretation and standardisation, NGS represents a powerful research tool in PF.

Current Issues in Molecular Biology doi: 10.3390/cimb48070720

Authors: Dyah Iswantini Min Rahminiwati Trivadila Trivadila Novriyandi Hanif Siti Sadiah Rut Novalia Rahmawati Sianipar Riska Amelia Candra Rani Melati Sukma Susi Indariani Raisa Zahra Muthia Khansa

Chronic Obstructive Pulmonary Disease (COPD) is a chronic respiratory condition characterized by progressive deterioration in lung function and persistent symptoms, particularly dyspnea, which can lead to death. Inflammation is important in COPD pathogenesis because it causes persistent inflammatory responses in the respiratory tract. Therefore, this study aimed to investigate H2O extracts derived from Wedelia (Sphagneticola trilobata) and Sembung Rambat (Mikania micrantha), respectively, as anti-inflammatory agents and to analyze the metabolite profiles contained in the extracts. The results showed that Sembung Rambat H2O extract demonstrated significant inhibition of IL-2 (100 ± 0.00%), while Wedelia extract inhibited 74.68 ± 7.13% release of pro-inflammatory cytokine IL-6. Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS) analysis mainly identified the presence of amino acids, phenolic acids, organic acids, and terpenoids in both extracts. These results suggest the promising potential of Wedelia and Sembung Rambat extracts as herbal therapy for COPD.

Current Issues in Molecular Biology doi: 10.3390/cimb48070719

Authors: Nurtai Gubaidullin Gulnazym Ospankulova Aisarat Gajimuradova Alfiya Syzdykova Aibek Zhumalin Kalamkas Dairova Damilya Konysbayeva Viktoriya Gorbulya Kadyrzhan Makangali

Salinity stress affects not only the survival and productivity of halophytic plants, but also the composition, structure and processability of their biomass. In Salicornia spp., salt-induced regulation of ion transport, osmotic adjustment, reactive oxygen species signaling, antioxidant defense, and cell wall remodeling can directly influence residual salinity, water retention, texture, extractability, drying behavior, and oxidative stability of plant biomass. However, most existing transcriptomic studies of Salicornia and related halophytes have focused mainly on salt tolerance mechanisms, whereas the connection between stress-regulated molecular networks and processing-related biomass traits remains insufficiently systematized. This review addresses this gap by proposing a mechanistic framework that links salinity perception, ROS-mediated signaling, ABA and MAPK pathways, antioxidant gene families, transcription factor networks and processing-oriented quality traits. Special attention is given to enzymatic antioxidant systems, including SOD, CAT, APX, POD and components of the ascorbate-glutathione cycle, as well as to non-enzymatic defense mechanisms involving ascorbate, glutathione, phenolic compounds, carotenoids, proline and glycine betaine. The review also discusses the regulatory roles of WRKY, DREB/CBF, NAC, bZIP and MYB transcription factor families as molecular control points connecting salinity stress responses with downstream metabolic and structural traits. Network-based approaches, including WGCNA, pathway signatures and transcript panels, are considered more informative than single-gene markers for predicting complex quality traits in Salicornia biomass. In addition, recent genomic and computational strategies, including CRISPR/Cas-mediated functional validation, GWAS, genomic selection, multi-omics integration and AI-assisted modeling, are discussed as emerging tools for candidate-gene prioritization and predictive assessment of stress-dependent biomass quality. Overall, this review shifts the interpretation of Salicornia transcriptomics from a descriptive salt-tolerance model toward a mechanistic and application-oriented framework for improving halophytic raw materials for food, feed and bioprocessing applications.

Current Issues in Molecular Biology doi: 10.3390/cimb48070718

Authors: Muhammad Matloob Javed Abdullah A. Al-Doss Muhammad Altaf Khan Salem S. Alghamdi Basharat A. Dar Abdelhalim I. Ghazy

Salinity stress has a debilitating effect on crop productivity and triggers complex molecular changes in plants, and understanding these responses is important for improving stress tolerance. This study investigated transcriptomic changes in the salt-tolerant barley line CC89/Giza123 by analyzing gene expression in roots and leaves following exposure to 200 mM NaCl for 12 and 24 h. The number of differentially expressed genes (DEGs) showed a much stronger response in roots than in leaves. At 12 h, roots showed 1836 DEGs, and this number increased to 2696 at 24 h, whereas leaves showed 256 DEGS at 12 h, and 787 at 24 h. The presence of strong and early activation in roots appears to indicate that roots play a key role in the adaptive response to salinity stress initiation. GO and KEGG analyses of differentially expressed genes revealed tissue- and time-specific responses. Roots showed rapid activation of ribosome and secondary-metabolite pathways at 12 h, followed by shifts toward carbon fixation and energy-related pathways at 24 h. Leaves responded early by adjusting photosynthesis-antenna proteins and later expanded their response to defense-related and amino-acid biosynthesis pathways. Important salt-responsive genes were identified in both tissues, including protein kinases, protein phosphatases 2C (PP2Cs), phospholipases, aquaporins, detoxification enzymes, molecular chaperones, and Late Embryogenesis Abundant (LEA) proteins. These results highlight clear tissue-specific and time-dependent differences in how plants respond to salt stress, providing insights into the metabolic and regulatory mechanisms involved in salt tolerance. Overall, these results provide evidence that the response of barley to salinity is achieved by using coordinated and dynamic molecular changes in different tissues. The transcriptomic dataset is a useful source of candidate genes for further functional studies and is a significant resource for breeding and biotechnological approaches to the production of salt-tolerant cereal crops.

Current Issues in Molecular Biology doi: 10.3390/cimb48070717

Authors: Ahmed Boalot Amira Younes Callie-Jo Woodward Afnan Alelaimi Ferhat Arabaci Donghyun Lee Claire H. Ozber Michal Heger Yazan S. Khaled

Pancreatic ductal adenocarcinoma (PDAC) is an invasive cancer with poor survival outcomes and limited treatment options. This systematic review aimed to identify and evaluate tissue-based biomarkers with potential applications in fluorescence-guided surgery (FGS), an approach that enables real-time intraoperative tumour visualisation to improve surgical precision. A systematic review was conducted in accordance with PRISMA guidelines and registered on PROSPERO (CRD420251056295). A comprehensive literature search was conducted across MEDLINE, Embase, and Cochrane Library databases from 1980 to January 2026 and was updated in March 2026. The EU Clinical Trials Registers were also searched for biomarker-targeted clinical trials. The TASC-T (Theranostic Target Selection Criteria) scoring system was used to assess biomarker suitability. EGFR, MSLN, CEA, and integrin αvβ6 scored highest, reflecting their strong potential as theranostic targets. MUC1 and uPAR also demonstrated high positivity and accessibility for imaging and therapy. While CA19-9 is a widely used serum biomarker, its role as a tissue target appears limited because of its high expression in benign conditions. EGFR and αvβ6 are both undergoing evaluation in phase II clinical trials and have emerged as promising targets for FGS in PDAC. This review highlights the need for standardised protocols and collaborative research to expand the clinical utility of these biomarkers. The methods and scoring criteria used in this review provide a transferable framework for identifying tissue biomarkers in other cancers and disease states.

Current Issues in Molecular Biology doi: 10.3390/cimb48070716

Authors: Weiyi Gao Meichun Qin Yi Zhu Fangming Song Xin Yang Wenchu Wang

With changes in lifestyle and dietary patterns, the incidence of prostate cancer in China has been increasing steadily, and it has become one of the most common malignancies in men. Chemotherapy remains a primary treatment for prostate cancer, but the subsequent development of drug resistance by tumor cells markedly compromises its therapeutic efficacy. Growth differentiation factor 15 (GDF15) has been shown to be closely associated with tumor cell proliferation and metastasis; however, its contribution to the drug resistance of prostate cancer cells within the tumor microenvironment has not been systematically investigated. In this study, we treated drug-resistant prostate cancer cells (PC3-TXR and DU145-TXR) with conditioned medium (CM) and examined GDF15 expression by Western blotting, real-time PCR, and ELISA. We then exposed drug-resistant cells to various concentrations of recombinant GDF15 (rGDF15) and CM, and assessed invasive and metastatic abilities as well as sensitivity to paclitaxel using Transwell and CCK-8 assays. We generated GDF15-knockout osteocytes using CRISPR-Cas9 technology and detected the expression of resistance-related signaling pathway components and epithelial–mesenchymal transition (EMT) markers. These findings were further validated through subcutaneous tumor formation assays in mice combined with immunohistochemistry to explore the mechanism by which GDF15 regulates drug resistance and metastasis in the crosstalk between drug-resistant prostate cancer cells and bone cells. Our results revealed that GDF15 expression was significantly upregulated in both drug-resistant prostate cancer cells and their surrounding microenvironment. rGDF15 enhanced the invasion and drug resistance of resistant cells, whereas knockout of GDF15 effectively inhibited these effects. Furthermore, we demonstrated that GDF15 regulates the biological behavior of drug-resistant cells by targeting and modulating the AKT signaling pathway and by promoting EMT initiation and progression. These findings clarify the molecular pathway through which GDF15 governs drug resistance in prostate cancer cells, offering a new direction for the treatment of tumor metastasis and biologically targeted therapy.

Current Issues in Molecular Biology doi: 10.3390/cimb48070715

Authors: Eugenia Yiannakopoulou

With a lifetime risk estimated to be 1 in 8 in industrialized countries, breast cancer is the most frequent type of cancer among women worldwide and the second leading cause of cancer deaths in women. More importantly, current evidence suggests that in women aged <45 years, breast cancer is unquestionably the leading cause of cancer-related deaths. Hormonal therapy has an established role in the treatment of breast cancer. Hormonal therapy aims at preventing the stimulation of mitogenic estrogen-dependent pathways. Hormonal therapy can be performed through blocking the production of estrogens or through blocking the action of estrogens upon tumor cells. The action of estrogens upon tumor cells can be blocked through selective estrogen receptor modulators (SERMs) or through selective estrogen receptor downregulators (SERDs). Estrogen receptor mutation (ESR1 mutation) is one of the common mechanisms by which breast cancer becomes resistant to additional therapies from SERMs or aromatase inhibitors. Fulvestrant, an injectable anti-estrogen, is the SERD commonly used. Fulvestrant has no agonistic activity and causes degradation of the estrogen receptor. This agent is more active in postmenopause than premenopause and is indicated in the treatment of advanced breast cancer in case of disease progression during or after tamoxifen. Oral SERDs are being rapidly developed to replace fulvestrant with the potential of higher efficacy and lower toxicities. Novel agents such as complete estrogen receptor antagonists (CERANs), proteolysis targeting chimeras (PROTACs), and selective estrogen receptor covalent antagonists (SERCAs) are also promising therapies. This manuscript focuses on recent advances in the development of drugs targeting the estrogen receptor.

Current Issues in Molecular Biology doi: 10.3390/cimb48070714

Authors: Linah H. Ali Hanaa A. Khalaf Sameera S. Hamed Ahmad M. Helaly Aya E. Maghrabia Doaa Ghorab Amal A. El Bakary

Paracetamol is a well-known analgesic drug. Studies linked gestational paracetamol use to disorders like autism and attention-deficit/hyperactivity disorder. This study aimed to evaluate the role of prenatal paracetamol exposure in 1-month-old offspring rats. Sixteen pregnant albino rats were divided into four groups: group I received saline; group II received sodium valproate 600 mg/kg i.p. on embryonic day 13; group III received paracetamol 100 mg/kg i.p. daily from embryonic day 13 to 21; group IV received paracetamol 300 mg/kg i.p. on embryonic day 13. Offspring were assessed for behavioral test parameters. Animal brains underwent histopathological and immunohistochemical analysis for brain-derived neurotrophic factor (BDNF). Offspring in groups II (valproic acid) and IV (single paracetamol dose) showed differences in neurobehavioral test parameters, supported by cerebellar and hippocampal pathology. BDNF-stained cerebellum and hippocampus sections from the valproic group and the single paracetamol group revealed focal staining in granular cerebellar cells and pyramidal hippocampal cells. These findings indicate that prenatal exposure to high doses of paracetamol induces neurodevelopmental pathology. The safety of paracetamol use during pregnancy requires further evaluation.

Current Issues in Molecular Biology doi: 10.3390/cimb48070713

Authors: Mohammad Amjad Hussain Mithila Kulkarni Reginald Samson Valder Suparna Laha

Leukemia is a hematopoietic defect, involving complex molecular and cellular alterations, in which dysregulated signaling through hematopoietic receptors, including c-MPL (c-Myeloproliferative Leukemia), has been implicated. The function of c-MPL is mostly regulated by the crosstalk and stoichiometry of its different isoforms. Although expression of c-MPL in hematological disorders has been studied, the regulation of its isoforms and their balance, functional roles, and mechanisms of action in conditions such as acute and chronic leukemia and myeloproliferative neoplasms remain poorly understood. The association of c-MPL isoforms with leukemia cell proliferation and aggressiveness was examined by immunophenotyping, immunofluorescence, RT-PCR, Western blotting and clonogenic assay. This study demonstrates that an increase in the ratio of c-MPL-Full Length (FL)/c-MPL-Truncated (TR), the conserved isoforms of c-MPL, influences tumorigenic markers such as Ki67, Caspase-3, and BCL-2, thereby promoting aggressiveness in leukemic cell lines. Furthermore, we have observed that with an increase in the c- MPL-FL/c-MPL-TR ratio, STAT5 activation increases, promoting the proliferative state of leukemic cells, thereby revealing c-MPL isoforms as a therapeutic target for leukemia. In this work, we observed an increased c-MPL expression in leukemic cell lines, but cell proliferation is independent of total c-MPL expression. Our study demonstrates the regulatory role of c-MPL isoforms, particularly c-MPL-FL, in increasing cell proliferation in leukemia cell lines. This finding is a step towards developing c-MPL isoform as a therapeutic target for leukemic conditions such as acute and chronic leukemias and myeloproliferative neoplasms, but it needs further investigation for complete validation.

Current Issues in Molecular Biology doi: 10.3390/cimb48070712

Authors: Sonia H. Navia Oscar Illescas Miguel Atl Silva-Magaña Imelda Juárez-Avelar Mariana Terrazas-Rodriguez Felipe Vaca-Paniagua Clara Estela Díaz Velásquez Libia Vega Luis I. Terrazas Miriam Rodríguez-Sosa

Intestinal dysbiosis is a hallmark of both inflammatory bowel conditions and colorectal cancer, yet the mechanisms by which inflammatory mediators alter microbial communities and may contribute to tumor development remain poorly understood. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine involved in innate immunity and the progression of inflammatory and neoplastic disorders. In this study, sequencing of the microbial 16S rRNA gene was performed to characterize the fecal microbiota profiles of wild-type (WT) and MIF-knockout (MIF-KO) BALB/c mice subjected to AOM/DSS-induced colitis-associated colorectal cancer (CAC). CAC induction resulted in marked microbial shifts, including increases in Muribaculaceae and Bacteroidota, in both WT and MIF-KO mice. Notably, MIF-KO CAC mice developed more severe disease compared with WT CAC mice. Furthermore, FMT experiments revealed that the fecal microbiota from MIF-KO donors was associated with increased tumor burden in WT recipients under CAC-inducing conditions compared with that in recipients colonized with WT-derived microbiota. Together, these findings suggest that MIF deficiency is associated with gut microbiota remodeling during CAC and support a potential relationship between the MIF-dependent host context, microbial composition and colorectal cancer severity.

Current Issues in Molecular Biology doi: 10.3390/cimb48070711

Authors: Di Tan Mengyi Qiao Qingqing Zhang Chuan Wang Xue Bai Fang Peng Dingyu Wu

Hepatic fibrosis is characterized by excessive extracellular matrix deposition and progressive impairment of liver structure and function. Ellagic acid (EA) has been reported to have antioxidant and anti-inflammatory properties, but its role in hepatic fibrosis remains incompletely understood. A CCl4-induced mouse model of hepatic fibrosis was established. EA was administered by oral gavage at 30, 60, or 120 mg/kg; colchicine was used as a positive control. Histopathological changes and collagen deposition were assessed using hematoxylin and eosin and Sirius Red staining, respectively. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations, as well as hepatic malondialdehyde (MDA) and hydroxyproline (Hyp) contents and total superoxide dismutase (T-SOD) activity, were determined. Immunohistochemistry and Western blotting were used to assess fibrosis-associated, EMT-associated, and PI3K/AKT-related proteins. Network pharmacology analysis was performed to identify candidate targets and pathways. EA treatment ameliorated histopathological injury and collagen deposition in CCl4-treated mice. EA, particularly at the high dose, reduced serum ALT and AST activities and hepatic MDA and Hyp contents while increasing hepatic T-SOD activity. EA treatment was also associated with lower serum IL-6 and higher IL-10 concentrations. In the EA-H group, α-SMA and collagen I (COL1A1) immunoreactivity were reduced; the abundance of N-cadherin, PI3K, and AKT was lower; and E-cadherin expression was higher. Network pharmacology identified PI3K/AKT-related signaling as a candidate pathway associated with the anti-fibrotic effects of EA. EA attenuated CCl4-induced hepatic fibrosis and was associated with lower oxidative stress, inflammation, and HSC activation. Direct PI3K/AKT inhibition was not established.

Current Issues in Molecular Biology doi: 10.3390/cimb48070710

Authors: Luisa Affatigato Sara Anselmo Anna Fricano Giuseppe Sancataldo Mariano Licciardi Alessio Incocciati Alessandra Bonamore Alberto Macone Alberto Boffi Valeria Militello

Ferritin, a physiological iron-storage protein, has emerged as a highly attractive platform for drug delivery owing to its biocompatibility, structural robustness, and intrinsic ability to encapsulate and protect therapeutic cargo within its hollow nanocage. Building upon previous studies that established the baseline characteristics of engineered ferritin mutants in comparison to the wild-type protein, this work specifically investigates and directly compares the thermal stability profiles of two distinct mutated variants. These variants of human H-chain ferritin, obtained through targeted site-directed mutagenesis, feature either four or six tryptophan residues per subunit, strategically positioned toward the inner cavity of the protein shell. These modifications were intended to enhance hydrophobic interactions with guest molecules while preserving the native quaternary architecture. Temperature-dependent changes in surface hydrophobicity and solvent accessibility were probed using the environment-sensitive fluorescent dye ANS, enabling a comparative assessment of the conformational behavior of the two mutants. Overall, this study highlights how targeted modulation of the internal cavity composition of ferritin can tune both its physicochemical properties and stability, providing insights relevant for the rational design of ferritin-based nanoplatforms for biomedical applications.

Current Issues in Molecular Biology doi: 10.3390/cimb48070709

Authors: Jeongmin Lee Su Young Jung Hye Ok Kim Jae Min Lee Manish Kumar Singh Sung Soo Kim Tong In Oh Dong Choon Park Seung Geun Yeo

Chronic rhinosinusitis (CRS) is an inflammatory disease of the sinonasal mucosa whose pathogenesis is characterized by complex interactions of immunological and environmental factors. The maintenance of normal sinonasal function requires a balance of sinus ostial patency, mucociliary clearance, and mucus secretion, and disruption of this balance can lead to CRS. Although many studies have examined the pathophysiology of CRS, the role of reactive oxygen species (ROS) remains incompletely understood. In this review, we analyzed 22 studies of CRS that examined the effects of ROS on epithelial barrier function, local immune responses, and tissue remodeling. The results from in vitro studies, animal models, and human tissue analyses suggest that ROS are not merely by-products of inflammation, but appear to function as key mediators in the pathophysiology of CRS, particularly in the formation and persistence of the CRS phenotype with nasal polyps (CRSwNP). In particular, CRSwNP is characterized by increased activity of enzymes in the dual oxidase (DUOX) and NADPH oxidase (NOX) families, mitochondrial dysfunction, and decreased activity of superoxide dismutase (SOD) and peroxiredoxin 2 (PRDX2). At the molecular level, these alterations increase the generation of ROS and impair antioxidant defense. At the cellular level, these alterations disrupt the epithelial barrier, activate inflammasomes, increase pyroptosis, and induce the formation of neutrophilic and eosinophilic extracellular traps. These changes culminate in the epithelial–mesenchymal transition (EMT), with the formation of nasal polyps and tissue remodeling. Increased oxidative stress can also occur in CRS without nasal polyps (CRSsNP), but this phenotype appears to have relatively preserved antioxidant defense systems, which may partly explain the more limited structural remodeling. External stimuli, such as fungal proteases, bacterial toxins, and certain antibiotics, can also increase the production of ROS and may contribute to disease chronicity. Taken together, the level and pathophysiological roles of ROS differ in the two primary phenotypes of CRS. Further mechanistic studies are needed to clarify the specific alterations of redox pathways in these two phenotypes and to develop novel therapeutic strategies that target ROS.

Current Issues in Molecular Biology doi: 10.3390/cimb48070708

Authors: Yu Sun Po-Chih Hsu Chung-Che Tsai Tsui-Chin Peng Chan-Yen Kuo

The potential impact of propofol on head and neck cancer (HNC) progression and clinical outcomes, in relation to both direct effects on tumor biology and indirect effects mediated by perioperative immune and inflammatory responses, remains controversial. Most mechanistic evidence currently available for HNC originates from studies of oral squamous cell carcinoma. Accordingly, mechanistic findings are interpreted primarily as OSCC-derived evidence and should not be generalized to all HNC subsites without further validation. To address this, we critically examined propofol’s potential role as a perioperative anesthetic modulator of tumor biology and host responses. Preclinical studies have demonstrated that propofol suppresses proliferation, induces apoptosis, inhibits angiogenesis, and enhances 5-fluorouracil chemosensitivity. Conversely, other studies report enhanced epithelial–mesenchymal transition, migration, and invasion through SNAI1 upregulation, highlighting the context-dependent nature of propofol-mediated effects. The generalizability of these molecular observations to other HNC subsites, including laryngeal, hypopharyngeal, and oropharyngeal cancers, remains unclear. Clinically, propofol-based total intravenous anesthesia has been associated with reduced postoperative pulmonary complications and improved perioperative recovery. However, currently available HNC-specific studies have not demonstrated a consistent improvement in overall or recurrence-free survival. These discrepancies likely reflect tumor heterogeneity, variations in experimental design, perioperative confounding factors, and differences in host immune and inflammatory responses. This narrative review was based on literature identified through PubMed, Scopus, and Google Scholar, and integrates current mechanistic, immunological, and clinical evidence regarding propofol exposure during cancer surgery. Current evidence supports the perioperative benefits of propofol-based anesthesia; however, its long-term oncologic significance in HNC remains inconclusive and warrants further prospective, mechanism-driven, and biomarker-guided investigations.

Current Issues in Molecular Biology doi: 10.3390/cimb48070707

Authors: Dawid Jańczak Aleksandra Kornelia Maj Jakub Kędziorek Mateusz Antecki

Feline feces represent a difficult matrix for molecular diagnostics because stool-derived inhibitors and variable sample composition may compromise DNA recovery and downstream PCR amplification. This paired methodological study evaluated whether a pre-analytical chloroform:methanol cleanup step improves DNA extract quality, post-extraction amplifiability, and molecular detection of selected intestinal protozoa in cats. A total of 105 feline fecal samples were divided into paired aliquots and subjected either to direct automated DNA extraction or to extraction preceded by chloroform:methanol cleanup. DNA concentration and purity were assessed spectrophotometrically using DNA yield, A260/280, and A260/230 ratios. Extracts from both workflows were analyzed by PCR for Tritrichomonas foetus, Cryptosporidium spp., Pentatrichomonas hominis, and Giardia duodenalis. In addition, a post-extraction spike-recovery experiment was performed on selected paired extracts to directly evaluate PCR inhibition. Cleanup significantly increased DNA concentration and improved A260/280 and A260/230 ratios. It was also associated with additional Cryptosporidium and Giardia detections and significantly lower Cryptosporidium Ct values among paired positives. Spike-recovery testing showed that direct extracts markedly delayed amplification of an exogenous Cryptosporidium meleagridis 18S spike, whereas cleanup-based extracts showed significantly lower Ct delay and higher spike recovery. All Cryptosporidium-positive sequences were identified as Cryptosporidium felis. Sequencing of the six Giardia beta-giardin amplicons revealed four sequences assigned to assemblage F, one sequence assigned to assemblage B, and one sequence assigned to assemblage D. These findings support chloroform:methanol cleanup as a useful pre-analytical strategy for improving feline fecal DNA extract quality and reducing post-extraction PCR inhibition, although broader validation across additional extraction platforms and larger positive sample sets is required.

Current Issues in Molecular Biology doi: 10.3390/cimb48070706

Authors: Jiri Ruzicka Jean-Marie Ravel Jérôme Audoux Alexandre Boulat Julien Thévenon Kévin Yauy Marine Dancer Laure Raymond Yannis Lombardi Nicolas Philippe Michael GB Blum Nicolas Duforet-Frebourg Laurent Mesnard

Genome and exome sequencing have become central to diagnosing rare hereditary diseases, but each test returns thousands of variants that a clinical scientist must review by hand to find the one responsible for the patient’s condition. This manual interpretation is the main bottleneck in clinical genomics. To reduce it, we developed DiagAI, a machine-learning system that ranks the variants found in a patient and returns a short list of the most likely causal candidates. DiagAI combines three sources of evidence: a pathogenicity score from the Universal Pathogenicity Predictor (UP2), a model we trained to estimate how damaging a variant is on the five-tier scale of the American College of Medical Genetics and Genomics (ACMG); a phenotype-matching score from PhenoGenius, which weighs how well a gene’s known clinical features match the patient’s symptoms (encoded as Human Phenotype Ontology, or HPO, terms); and expert rules covering inheritance pattern and sequencing quality. We evaluated DiagAI on 966 exomes from adults investigated for kidney disease of unknown cause, of which 196 had a confirmed genetic diagnosis. We first tested UP2 on its own by ranking 62 confirmed disease-causing missense variants that were absent from its training data: UP2 placed the causal variant within the top 100 candidates in 87% of cases, compared with 61% for the widely used tool REVEL. Across the 196 diagnosed exomes, the full DiagAI shortlist contained the causal variant in 94.9% of cases when the patient’s symptoms were provided and in 90.8% when they were not, with a typical shortlist of about 10 variants. When symptoms were provided, the single top-ranked variant was the correct diagnosis in 74% of cases, versus 42% without symptoms, exceeding the performance of the established tools Exomiser and AI-MARRVEL on the same cohort. DiagAI produces compact, accurate shortlists that can reduce the manual interpretation workload as diagnostic sequencing volumes continue to grow.

Current Issues in Molecular Biology doi: 10.3390/cimb48070705

Authors: Ju-Seop Kang So-Jung Lim Ryun Kang So-Hyun Jeon Chang-Taek Oh Si-Young Jung Sang-Hoon Lee

Background: Alcoholic liver disease (ALD) represents a major global health burden, encompassing a spectrum of hepatic abnormalities ranging from steatosis to cirrhosis and hepatocellular carcinoma. Laennec, a human placenta-derived hydrolysate, was evaluated for its therapeutic effects on alcohol-induced fatty liver in an experimental animal model. Methods: Animals were pretreated with alcohol for 2 weeks, followed by the co-administration of alcohol and Laennec for 4 weeks. The study included four groups: normal control, alcohol-only, and low- and high-dose Laennec-treated groups. Results: Alcohol administration significantly elevated the serum ALT levels (3.17-fold vs. control), indicating hepatocellular injury, whereas Laennec treatment reduced the ALT levels in a dose-dependent manner. The AST levels increased in the alcohol-only group, although the change was not statistically significant; however, the AST levels decreased in the low-dose Laennec-treated groups. The AST/ALT ratio showed significantly dose-dependent recovery in the high-dose group. Laennec treatment attenuated hepatic lipid accumulation and inflammation, with the most pronounced effects observed in the high-dose group. Regarding alcohol-metabolizing enzymes, Laennec exhibited an inhibitory effect on alcohol-induced ADH activity, with no significant effect on ALDH. CYP2E1 activity was suppressed in a dose-dependent manner following Laennec administration. No significant changes were observed in the GPx activity. Additionally, high-dose Laennec significantly restored the catalase and STAT3 (Ser727) phosphorylation levels. Histopathological analysis (H&E staining) demonstrated marked reductions in lipid droplet accumulation and inflammatory cell infiltration in Laennec-treated groups compared to the alcohol-only group. Conclusion: These findings suggest that Laennec exerts hepatoprotective effects against alcohol-induced liver injury and steatosis, as evidenced by improvements in biochemical markers, enzyme activity, and histological features.

Current Issues in Molecular Biology doi: 10.3390/cimb48070704

Authors: Prakash Gangadaran Ramya Lakshmi Rajendran Muthu Subash Kavitha Byeong-Cheol Ahn

Exosomes are 30–150 nm extracellular vesicles that convey molecular information reflecting the physiological and pathological states of their source cells. In precision oncology, they function as a non-invasive “liquid biopsy,” enabling real-time monitoring of tumor dynamics and metastasis. However, extreme biofluid heterogeneity poses significant challenges for their isolation and analysis using conventional statistical approaches. This review aims to examine how artificial intelligence (AI), specifically machine learning and deep learning, transforms complex exosomal “noise” into actionable clinical insights. AI enhances exosome isolation, enables disease-specific biomarker identification, and predicts therapeutic responses with high precision. Integrating multi-omics data and single-exosome analysis enables AI-driven models to facilitate early cancer detection and therapeutic resistance monitoring. Despite challenges related to standardization and data privacy, the convergence of AI and exosome biology is poised to transform reactive cancer treatments into a proactive, personalized medical ecosystem. This approach also provides a framework for managing other complex systemic diseases.

Current Issues in Molecular Biology doi: 10.3390/cimb48070703

Authors: Zhangziyan Zhao Shanxue Jiang Haishu Sun

Existing anti-aging drugs are often limited by toxicity and resistance. In contrast, natural substances derived from food resources, edible plants, and agricultural by-products offer advantages such as low toxicity and suitability for dietary intake. Utilizing these resources aligns with sustainable development goals by promoting the valorization of food waste and functional food development; however, their complex composition makes traditional discovery inefficient and resource-intensive. Machine learning (ML) provides a powerful, sustainable in silico solution. By analyzing vast datasets, computational models can rapidly screen thousands of candidates, significantly reducing the chemical waste and time associated with traditional wet-lab screening. This review focuses on the current status of food-derived anti-aging bioactives and the emerging ML-based perspectives in this field. Key natural compounds and plant extracts are discussed, highlighting their dietary origins and mechanisms. Furthermore, we explore how advanced algorithms accelerate the identification of novel bioactives. Importantly, we address current translational gaps, including the need for explainable AI, ADME (Absorption, Distribution, Metabolism, and Excretion) prediction, and the standardization of complex mixtures. Overcoming these bottlenecks is essential for the sustainable development of effective, food-based anti-aging ingredients.

Current Issues in Molecular Biology doi: 10.3390/cimb48070702

Authors: Jie Pu Wang Guo Hongxin Li Zhihao Wang Tangda Mu Fuli Xie Jie Chen Jiawei Cao Chengfei Zhong Hongyu Li Qiang Tang

Stroke remains the second leading cause of death and the third leading cause of death and disability. With the growing incidence of strokes, the incidence of post-stroke cognitive impairment (PSCI) among patients is on a steady rise. These patients commonly present with impairments in memory, attention, executive function, comprehension, and judgment, substantially affecting their everyday lives. Magnetic resonance image (MRI) and cognitive assessment scales are widely employed as diagnostic tools for PSCI. They also have notable limitations. Furthermore, cerebrospinal fluid collection is relatively complex. Growing evidence suggests that blood-based biomarkers are closely involved in the onset and progression of PSCI, as bioassay techniques and equipment have become increasingly popular and optimized. Therefore, blood-based biomarkers play an essential role in the pathogenesis and progression of PSCI. This review concentrates on the research progress of blood-based biomarkers in PSCI to explore their significance for disease identification, with the aim of offering novel insights into the clinical diagnosis and therapy of PSCI.

Current Issues in Molecular Biology doi: 10.3390/cimb48070701

Authors: Fatouma Mohamed Abdoul-Latif Lamiae El Bouamri Badr Sellami Amal Bouribab Fatimazahra Guerguer Houda Mohamed Abdirahman Elmi Yahya Ali Ismae Ricardo Gil-Ortiz Samir Chtita

Objectives: Malaria remains a major global health burden, particularly in endemic regions such as Djibouti, where Plasmodium falciparum and Plasmodium vivax co-circulate, complicating disease control strategies. Increasing resistance to current antimalarial drugs reduces treatment effectiveness and highlights the urgent need for new, safe, and affordable therapeutic agents. This study aimed to identify potential inhibitors from Djiboutian medicinal plants using an integrated in silico approach targeting key proteins from both parasite species. Methods: A library of 222 phytoconstituents was screened against Plasmodium vivax FK506-binding protein 35 (PDB ID: 3IHZ) and Plasmodium vivax dihydrofolate reductase–thymidylate synthase (PDB ID: 1J3K) using molecular docking. Top-ranked compounds were further analyzed for binding interactions and evaluated for drug-likeness and pharmacokinetic properties using QikProp in Maestro v11.5. Selected protein–ligand complexes were subjected to 100 ns molecular dynamics simulations, and their stability was assessed using multiple descriptors, including structural deviation, flexibility, compactness, solvent exposure, and hydrogen bond persistence. Results: Several phytoconstituents exhibited strong binding affinities, with docking scores ranging from −6.09 to −7.54 kcal/mol, outperforming the reference drug artemisinin. Interaction analysis revealed key hydrogen bonds and hydrophobic contacts with essential active-site residues. ADMET predictions indicated favorable pharmacokinetic profiles, including high oral absorption, good membrane permeability, and low predicted toxicity. Molecular dynamics simulations demonstrated stable behavior for most complexes, with compound 121 showing enhanced stability in the 1J3K system and compound 123 exhibiting consistent dynamic stability in the 3IHZ system. In contrast, compound 82 displayed greater structural fluctuations despite maintaining stable hydrogen bond interactions. Conclusions: The integration of molecular docking, ADMET prediction, and molecular dynamics simulations identified compounds 121 and 123 as the most promising antimalarial candidates, exhibiting an optimal balance of binding affinity, favorable pharmacokinetic properties, and dynamic stability. These findings highlight the potential of Djiboutian medicinal plants as a valuable source of novel antimalarial agents and provide a strong computational foundation for future experimental validation.

Current Issues in Molecular Biology doi: 10.3390/cimb48070700

Authors: Tomasz Kowalczyk Anna Merecz-Sadowska Belma Konuklugil İbrahim Seyda Uras Radosław Zajdel Patricia Rijo Przemysław Sitarek

The development of multi-targeted therapeutic agents is increasingly recognized as essential for treating multifactorial diseases. Butyrolactone I and butyrolactone III, γ-butyrolactone derivatives isolated from the marine fungus Aspergillus terreus, represent structurally related natural products with largely unexplored polypharmacological potential. This study employed a comprehensive in silico approach combining ADMET profiling, quantum chemical calculations, molecular docking, and molecular dynamics simulations to evaluate their therapeutic potential across multiple pharmacological targets. Physicochemical analysis revealed favorable drug-like properties for both compounds, with complete compliance with Lipinski’s Rule of Five, high predicted gastrointestinal absorption (>80%), and acceptable toxicity profiles (toxicity class 4, LD50 = 2000 mg/kg). Neither compound showed hepatotoxic, neurotoxic, cardiotoxic, carcinogenic, or mutagenic liabilities. Frontier molecular orbital analysis (DFT/B3LYP/6-31G(d,p)) revealed comparable HOMO energies (−6.054 and −6.059 eV), with butyrolactone III exhibiting enhanced kinetic stability based on a larger HOMO–LUMO gap (4.662 eV vs. 4.443 eV) and higher chemical hardness (η = 2.331 eV vs. 2.222 eV). Molecular docking against four therapeutic targets revealed target-selective binding profiles: butyrolactone III demonstrated binding affinity toward acetylcholinesterase exceeding donepezil (−9.0 vs. −8.3 kcal/mol), while butyrolactone I exhibited MDM2 binding affinity slightly exceeding nutlin-3a (−7.8 kcal/mol). Both compounds showed moderate interactions with COX-2 and topoisomerase IV. Molecular dynamics simulations validated the stability of AChE complexes (RMSD < 2.0 Å) and the MDM2–butyrolactone I complex (RMSD: 0.69 ± 0.09 Å), while the MDM2–butyrolactone III complex exhibited significant instability (RMSD up to 3.55 Å), highlighting the critical role of the prenyl group in MDM2 recognition. These findings, consistent with, though not a direct experimental validation of, previously published in vitro data, support the evaluation of butyrolactone III as a scaffold for neuroprotective agents and butyrolactone I as a p53 pathway modulator for cancer therapy, illustrating the potential value of fungal metabolites in multi-target drug discovery and the role of integrated computational approaches in prioritizing candidates for subsequent experimental testing.

Current Issues in Molecular Biology doi: 10.3390/cimb48070699

Authors: Nazenin Karakoç Sema Agüloğlu Fincan Barış Enez Veysi Ortakaya

Microbial α-amylases are widely used in industrial biotechnology due to their catalytic efficiency, stability, and cost-effective production. Identifying sources of newly isolated bacterial strains with desirable biochemical properties remains important for improving industrial enzyme applications. In this study, a newly isolated strain, Bacillus simplex BCHCNZ282B, isolated from soil samples collected in Ergani (Diyarbakır, Turkey), was investigated for its extracellular α-amylase production. The enzyme production conditions were optimized, followed by purification involving ammonium sulfate precipitation, dialysis, and final purification by starch-affinity chromatography. The purified enzyme was biochemically characterized in terms of optimal activity conditions; stability; kinetic parameters; and the effects of metal ions, inhibitors, and detergents. Optimal α-amylase production was obtained at 35 °C and pH 7.0 after 36 h of incubation. The optimum activity of the purified enzyme was attained at 50 °C and pH 7.0. Sequential purification resulted in a six-fold increase in specific activity and a final recovery yield of 19%. The enzyme’s molecular mass was 70 kDa. Increased enzyme activity was obtained in the presence of Mg2+ and Mn2+, while EDTA was inhibitory to enzyme activity. The purified enzyme maintained high relative activity from pHs 7.0 to 9.0 and was active at all temperatures studied. In addition, enzyme activity was observed even in the presence of some of the tested detergents. The α-amylase produced by B. simplex BCHCNZ282B possessed many biochemical properties, such as stability in a wide range of pH values, high-temperature activity, and resistance to several detergents, which indicate many potential biotechnological applications of the enzyme. Nevertheless, further research on genomic characterization and structural analysis should be conducted to establish the industrial application of the enzyme.

Current Issues in Molecular Biology doi: 10.3390/cimb48070698

Authors: Rebecca Schwarz Britt Hofmann Ulrich Gergs Uwe Kirchhefer Joachim Neumann

Cantharidin (CANT) and sodium fluoride inhibit serine/threonine phosphatase (PP) 1 and PP2A in the heart. We hypothesized that Cant and sodium fluoride could weaken the reduction in force by carbachol in the presence of serotonin in human right atrial preparations (HAP). We measured contractility in HAP, and for comparison, in left atria from wild type mice (CD1, WT), mice with human 5-HT4-serotonin receptor cardiac overexpression (5-HT4-TG), or in mice with human H2-histamine receptor cardiac overexpression (H2-TG). Isoprenaline (1 µM), histamine (1 µM), and serotonin (1 µM) raised contraction in HAP. Likewise, isoprenaline (1 µM), histamine (1 µM), and serotonin (1 µM) raised contractility in the left atria of WT, H2-TG, and 5-HT4-TG. Carbachol (1 µM), a muscarinic cholinoceptor agonist, diminished the force of contraction in a time-dependent manner after prior stimulation of force by serotonin, isoprenaline, or histamine in HAP but also in left atria from 5-HT4-TG, WT, and H2-TG. These anti-β-adrenergic, anti-serotoninergic and anti-histaminergic effects of carbachol on force of contraction were attenuated by 100 µM or 30 µM cantharidin or 3 mM sodium fluoride in HAP and in left atrial preparations from WT, H2-TG, and 5-HT4-TG, respectively. We conclude that muscarinic cholinoceptor activation may exert a negative inotropic effect in the HAP by stimulating PP1 and/or PP2A, which had previously been inhibited by isoprenaline or histamine or serotonin.

Current Issues in Molecular Biology doi: 10.3390/cimb48070697

Authors: Jing-Quan Zheng Chin-Hua You Tung-Yu Tiong Bo-Jung Chen Jou-Chun Chou

Current research primarily focuses on post-treatment resistance, leaving the immediate impact of cisplatin on the tumor–immune interaction poorly understood. Tumor cells were co-cultured with immune cells to assess immune cell activation. The correlation with RAB27A/B and CD274 expression levels in non-small cell lung cancer (NSCLC) were analyzed using TCGA. The relationship between RAB27A/B and the tumor microenvironment was assessed by TIMER2.0. Cisplatin significantly increased PD-L1 mRNA levels and surface protein expression, detected by qPCR, flow cytometry and confocal microscopy. In the co-culture assay, cisplatin-induced cell surface PD-L1 protein levels inhibited the activation of Jukat T-cells. Cisplatin also upregulated genes associated with exosome secretion and increased total exosome particle counts and secretion per cell. Pretreatment with GW4869, an exosome release inhibitor, significantly enhanced cisplatin sensitivity in HCC827 cells. While GW4869 reduced intracellular RAB27A/B and EV PD-L1 levels, it did not alter cellular PD-L1 expression. Bioinformatic analysis further revealed that high RAB27A/B expression correlates with poor 5-year survival in NSCLC and is negatively associated with CD8+ T-cell activation, while positively correlating with cancer-associated fibroblasts. Both RAB27A and RAB27B gene expression levels positively correlated with CD274. Conclusions: RAB27A and RAB27B may be crucial proteins in modulating cisplatin-induced exosomal PD-L1 levels and the tumor microenvironment.

Current Issues in Molecular Biology doi: 10.3390/cimb48070696

Authors: Hakki Coskun Zuhal Tuncbilek Husnu Cagri Genc Gulcihan Cinar Kaya Ayca Tas

Gastric cancer remains a major cause of cancer-related mortality worldwide, highlighting the need for an improved understanding of its molecular mechanisms. Purinergic and glutamatergic signaling pathways, immune checkpoint molecules, and oxidative stress are thought to contribute to tumor biology; however, their combined evaluation in gastric cancer is limited. Methods: Gene expression levels of glutamate receptor ionotropic N-Methyl-D-aspartate 2A (GRIN2A), purinergic receptor P2X1 (P2RX1), and programmed death-ligand 1 (PD-L1/CD274) were analyzed using real-time PCR, while serum protein levels were determined by ELISA. Total antioxidant status (TAS) and total oxidant status (TOS) were also measured to assess systemic oxidative balance. Results: Gene expression analysis revealed an increased expression trend for P2RX1 in the gastric cancer group compared with healthy controls, whereas GRIN2A and CD274 (PD-L1) expression levels showed numerical decreases. Serum concentrations of GRIN2A and PD-L1, as well as TAS and TOS levels, were numerically higher in patients with gastric cancer; however, none of these differences reached statistical significance (p > 0.05). Likewise, serum P2RX1 levels did not differ significantly between groups. Receiver operating characteristic (ROC) analysis demonstrated area under the curve (AUC) values close to 0.5 for all evaluated parameters, indicating limited discriminatory and diagnostic performance. Conclusions: These findings suggest that these molecular markers may reflect disease-related biological alterations rather than serve as independent circulating diagnostic biomarkers in gastric cancer.

Current Issues in Molecular Biology doi: 10.3390/cimb48070695

Authors: Roberta Chow Patricia Armati

This review aims to provide a translational link between clinical evidence in the application of photobiomodulation for treatment of painful conditions in medicine and dentistry and neurophysiological effects of photobiomodulation therapy (PBMt). PBMt is gaining increasing acceptance as a therapeutic modality for pain management, particularly within dental practice. However, a clearer understanding of its mechanisms of action remains essential for broader clinical adoption and integration into mainstream healthcare. Central to the therapeutic effects of PBMt for pain is its interaction with neurons, which possess unique structural and functional characteristics. Advances in our understanding of the cellular architecture of dorsal root and trigeminal ganglion neurons highlight the importance of the stem axon and axon initial segment (AIS), a specialized region adjacent to the axon hillock that is now recognized as the principal site of action potential initiation. These developments have important implications for understanding the biological effects of PBMt and its clinical application. This review synthesizes evidence demonstrating that PBM influences cytoskeletal organization, mitochondrial structure and function, and intracellular signaling pathways, with downstream effects on neuronal excitability and nerve conduction. By integrating findings from cellular, neurophysiological, and clinical studies, the review examines how these mechanistic effects may contribute to pain modulation and analgesia.

Current Issues in Molecular Biology doi: 10.3390/cimb48070694

Authors: Tzong-Shi Wang I-Shiang Tzeng Yi-Chyan Chen Mao-Liang Chen

Neuroinflammation plays an essential role in the pathogenesis of several associated brain diseases, including neurodegenerative disorders (Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS)), and traumatic brain injury (TBI). In these diseases, persistent microglial and astrocyte aggregates, elevated proinflammatory cytokines, and oxidative stress drive neuronal injury and cognitive disability. Rho GTPases, in particular the Rho family members Ras homolog family member A (RhoA), Ras-related C3 botulinum toxin substrate 1 (Rac1), and cell division control protein 42 homolog (CDC42), regulate neuroinflammation, cytoskeletal dynamics, immune responses, and the maintenance of BBB integrity. These proteins are involved in many neuropathological diseases due to dysregulation, making them interesting therapeutic targets. Bioactives used in herbal care have attracted interest for their ability to influence neuroinflammation and even their anti-neurodegenerative activity. Studies show that flavonoids, alkaloids, polyphenols, and other botanical compounds alter Rho GTPase activity, which, in turn, leads to decreased inflammation. This review critically summarizes current evidence regarding phytochemical regulation of Rho GTPase signaling in neurodegenerative disorders such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), with particular emphasis on the underlying molecular mechanisms, context-dependent signaling responses, and current translational challenges. Furthermore, existing knowledge gaps and future research priorities are discussed to facilitate the development of mechanism-based therapeutic strategies targeting Rho GTPases.

Current Issues in Molecular Biology doi: 10.3390/cimb48070693

Authors: Yi Luo Ailing Hu Jingya Lu Wenshu Yuan Yu Tan Takuji Yamaguchi Zenji Kawakami Yasushi Ikalashi Yoshinao Harada Hiroyuki Kobayashi

Boiogito (BOT), a traditional Kampo herbal medicine, has been reported to exhibit anti-inflammatory and anti-obesity properties. However, its potential role in protecting adipocyte function under inflammatory conditions at different stages of adipocyte development remains unclear. This study investigated the effects of BOT on adipogenesis and tumor necrosis factor-α (TNF-α)-induced inflammatory responses in differentiating and mature 3T3-L1 adipocytes. In this study, 3T3-L1 preadipocytes were induced to differentiate and exposed to TNF-α in the presence or absence of BOT during differentiation or after full adipocyte maturation. Lipid accumulation was assessed by Oil Red O staining, while adipokine secretion and inflammatory cytokine production were evaluated by ELISA. The expression of adipogenic markers and inflammatory signaling molecules was analyzed using quantitative PCR and Western blotting. TNF-α significantly inhibited the expression of adipogenesis-related factors at the transcriptional level in adipocytes, reduced lipid accumulation and adiponectin expression, and enhanced inflammatory cytokine production. BOT treatment dose-dependently attenuated these effects, restoring adipogenic capacity and suppressing inflammatory responses in both differentiating and mature adipocytes. Mechanistically, BOT reduced TNF-α-induced activation of the NF-κB pathway, as evidenced by decreased phosphorylation of NF-κB p65 and IκB. These findings demonstrate that BOT preserves adipocyte function and mitigates inflammation-associated adipocyte dysfunction throughout adipocyte development. The protective effects of BOT may contribute to the regulation of obesity-associated metabolic inflammation, partly through modulation of NF-κB signaling.

Current Issues in Molecular Biology doi: 10.3390/cimb48070692

Authors: Negar Taghavi Pourianazar

Background: Head and neck squamous cell carcinoma (HNSCC) remains a highly aggressive malignancy with poor clinical outcomes. Although poly(ADP-ribose) polymerase (PARP) inhibitors have shown promising activity in tumors with homologous recombination deficiency, their efficacy in BRCA wild-type HNSCC remains limited. Reactive oxygen species (ROS)-induced DNA damage may increase cellular dependence on DNA repair pathways and thereby enhance sensitivity to PARP inhibition. This study investigated whether ROS-mediated DNA damage could sensitize BRCA wild-type HNSCC cells to the PARP inhibitor olaparib. Methods: BRCA wild-type HSC-3 and SCC-25 HNSCC cell lines were exposed to H2O2 to induce oxidative stress. Intracellular ROS levels were quantified using DCFDA assays, DNA double-strand breaks were evaluated by γ-H2AX ELISA, PARP activity was assessed by ELISA, and cell viability was determined using MTT assays. Expression levels of DNA repair genes (PARP1, PARP2, BRCA1, BRCA2, RAD51, and MLH1), checkpoint kinases (ATM, ATR, and CHK1), the homologous recombination regulator FANCD2, and redox defense genes (NQO1, GPX4, and SLC7A11) were analyzed by qRT-PCR. Therapeutic selectivity was assessed using HGF-1 normal human gingival fibroblasts as a normal cell control. Apoptosis was measured through caspase-3/7 activity assays, and drug interactions were evaluated using the Chou–Talalay method. Results: H2O2 treatment increased intracellular ROS levels in both cell lines, accompanied by significant induction of DNA damage as demonstrated by elevated γ-H2AX levels. ROS induction markedly enhanced olaparib sensitivity, significantly reducing IC50 values in both HSC-3 and SCC-25 cells. Combined H2O2 and olaparib treatment produced strong synergistic cytotoxicity, suppressed DNA repair, checkpoint kinase, and redox defense gene expression, and increased caspase-3/7 activity compared with control cells. Importantly, the combination demonstrated selective cytotoxicity toward cancer cells, with normal HGF-1 cells retaining significantly higher viability. Conclusions: ROS-induced DNA damage significantly enhances the anti-tumor activity of olaparib in BRCA wild-type HNSCC cells through a functional synthetic lethal-like interaction involving the simultaneous collapse of DNA repair capacity, checkpoint activation, and oxidative stress buffering, culminating in apoptosis induction. These findings support the rationale for combining ROS-generating therapies with PARP inhibitors in HNSCC treatment.

Current Issues in Molecular Biology doi: 10.3390/cimb48070691

Authors: Patricia Fernanda Schuck Gustavo da Costa Ferreira Hércules Rezende Freitas

The sodium-coupled citrate transporter NaCT (SLC13A5) imports extracellular citrate into cells. In the CNS, SLC13A5 is described to be expressed predominantly in neurons. Cytosolic citrate levels rely on citrate generated in mitochondria and imported from other CNS cells, regulating intermediary metabolism and supplying acetyl-CoA for lipid synthesis and histone acetylation. Despite evidence for NaCT’s role in neurometabolic homeostasis, its transcriptional behavior across Alzheimer’s disease (AD) progression and across astrocyte subtypes remains uncharacterized at single-cell resolution. We analyzed single-nucleus RNA sequencing data from 1,378,211 nuclei across 84 donors in the Seattle Alzheimer’s Disease Brain Cell Atlas (SEA-AD) Middle Temporal Gyrus dataset to profile SLC13A5 and seven citrate metabolism genes across a continuous AD pseudoprogression score. SLC13A5 expression was restricted to astrocytes (~20% prevalence) and concentrated in the Astro 2 supertype (24.0%), a homeostatic subtype characterized by low C3 (1.6%) and CD44 (5.5%), which expanded with pseudoprogression (Spearman rho = +0.345, FDR < 0.001). The A1-reactive Astro 3 supertype, where SLC13A5 prevalence was 0.87%, declined concordantly (rho = −0.393). Opposing compositional and transcriptional forces produced apparent stability in overall SLC13A5 prevalence. SLC13A3 and ACO1 showed progressive donor-level declines correlating with Braak stage and Thal phase (rho range: −0.307 to −0.349, FDR < 0.01). APOE4 carriers exhibited lower SLC13A5 prevalence specifically within Astro 2 nuclei (median 17.6% vs. 25.9%; Wilcoxon p = 0.025), though this association did not survive multivariate regression. No difference in Astro 2 SLC13A5 expression was detected between cognitively resilient and expected-AD donors with equivalent high Braak burden (p = 0.888). Contrary to the prevailing description of NaCT as a neuronal transporter, SLC13A5 transcript in the SEA-AD MTG dataset was detected almost exclusively in astrocyte nuclei, concentrated in the homeostatic Astro 2 subtype, and maintained as this subtype expanded with advancing AD pathology. Because these are nuclear transcript measurements, they delimit where SLC13A5 mRNA is detectable rather than establishing the cellular site of NaCT protein or activity, which requires in situ validation.

Current Issues in Molecular Biology doi: 10.3390/cimb48070690

Authors: Ömer Kokaçya Percin Pazarci Halil Mahir Kaplan

Background/Objectives: Malignant melanoma is characterized by constitutive PI3K/Akt/mTOR and MAPK activation, driving aggressive behavior and therapeutic resistance. Alpha-lipoic acid (αLA), a naturally occurring dithiol compound with an established clinical safety profile, has shown anticancer potential; however, its integrated molecular mechanisms in melanoma remain poorly defined. This study aimed to comprehensively evaluate the cytotoxic and mechanistic effects of αLA in B16F10 murine melanoma cells. Methods: Antiproliferative effects were assessed by MTT assay at four concentrations (250, 500, 750, 1000 µM) over 48 h. Protein levels of apoptotic markers (Bax, Bcl-2, Caspase-3, AIF), kinase signaling components (p-Akt, p-mTOR, p-ERK, p-JNK), ER stress markers (GRP78, GADD153/CHOP), and cell cycle regulator Wee1 were quantified by ELISA at a specifically selected sub-lethal concentration of 750 µM (inducing ~38% growth inhibition). Results: αLA dose-dependently inhibited B16F10 proliferation. At 750 µM, it triggered robust intrinsic apoptotic signaling, evidenced by a nearly 10-fold shift in the Bax/Bcl-2 ratio and greater than 9-fold Caspase-3 activation. Elevated AIF suggested profound mitochondrial stress and the potential priming of concurrent caspase-independent cell death mechanisms. αLA suppressed survival signaling by reducing p-Akt (44%), p-mTOR, p-ERK, and p-JNK. Treatment triggered lethal ER stress via GRP78 and GADD153/CHOP upregulation and upregulated Wee1, suggesting the induction of stress-responsive checkpoint signaling. The simultaneous CHOP upregulation and p-Akt suppression highlight a concurrent dysregulation of stress and survival pathways, suggesting a potential pro-apoptotic interplay. Conclusions: αLA exerts potent multi-target anticancer effects by inducing a broad spectrum of associated molecular changes, including the suppression of PI3K/Akt/mTOR and MAPK networks, induction of ER stress, engagement of cell cycle checkpoints, and activation of the mitochondrial Bax/Bcl-2/Caspase-3 axis. Importantly, these correlative findings do not establish proven pathway dependencies. Nevertheless, this concurrent dysregulation positions αLA as a potential disruptor of inter-pathway resilience underlying drug resistance.

Current Issues in Molecular Biology doi: 10.3390/cimb48070689

Authors: Julia Inglot Dorota Bartusik-Aebisher Joanna Katarzyna Strzelczyk Angelika Myśliwiec Klaudia Dynarowicz Dorota Hudy Oliwia Trzaskoś Jacek Tabarkiewicz Aleksandra Kawczyk-Krupka Magdalena Moś David Aebisher

Glioblastoma multiforme (GBM), isocitrate dehydrogenase (IDH)-wildtype, is the most aggressive primary malignant tumor of the central nervous system, characterized by poor prognosis and high recurrence rates despite standard multimodal treatment. This study investigates the molecular response of glioblastoma cells to 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT), focusing on gene expression changes associated with apoptosis, ferroptosis, and oxidative stress. Human glioblastoma T98G cells were treated with 5-ALA followed by light irradiation, and gene expression was analyzed using RT-qPCR. PDT induced moderate upregulation of pro-apoptotic genes (BAX, CASP3, FAS) alongside increased expression of the anti-apoptotic gene BCL2, indicating simultaneous activation of cell death and survival pathways. Ferroptosis-related genes showed mixed responses, with slight upregulation of ACSL4 and downregulation of GPX4, suggesting increased susceptibility to lipid peroxidation. The most significant change was observed in GCH1 expression, reflecting activation of oxidative stress response mechanisms. However, none of the observed changes reached statistical significance, likely due to the limited sample size. These findings demonstrate that PDT induces a complex and dual biological response in glioblastoma cells, involving both cytotoxic and adaptive mechanisms. This may limit therapeutic efficacy and contribute to treatment resistance. The results support the rationale for combining PDT with targeted molecular therapies aimed at inhibiting antioxidant defenses and anti-apoptotic pathways. Additionally, personalized therapeutic strategies based on tumor molecular profiles may enhance treatment outcomes. Further studies with larger sample sizes and functional validation are required to confirm these preliminary observations.

Current Issues in Molecular Biology doi: 10.3390/cimb48070688

Authors: He Wang Shiyi Lun Hu Ding Zhimeng Li Xian Wu Huiyang Yuan Bo Yang Guoxin Ji Huan Wang Shumin Wang

Epilepsy is a common chronic neurological disorder characterized by recurrent seizures. Gastrodia elata, the dried tuber of G. elata Bl. (Orchidaceae), is a valuable medicinal and edible botanical resource. This study optimized the preparation of Yellow Rice Wine-Processed G. elata (YPGE) and investigated its antiepileptic effects and underlying mechanisms in a pentylenetetrazol (PTZ)-kindled rat model. Processing parameters were optimized using single-factor experiments combined with an analytic hierarchy process (AHP)-entropy weight method (EWM) weighting strategy and Box–Behnken design–response surface methodology. The optimal parameters were determined as 18% alcohol by volume, 72 °C drying temperature, and 32 h drying time. Compared with unprocessed G. elata (GE), YPGE exhibited 0.54-, 0.13-, 1.87-, and 3.58-fold increases in the contents of gastrodin (GAS), G. elata polysaccharides (GEPs), p-hydroxybenzyl alcohol (p-HBA), and total parishins (TP), respectively, and demonstrated significantly enhanced in vitro antioxidant activity (IC50 values of 2.604, 2.719, and 4.046 mg/mL for DPPH, ABTS, and hydroxyl radicals). In vivo, both GE and YPGE significantly reduced seizure severity, decreased inflammatory cytokines (TNF-α, IL-1β), alleviated oxidative stress (increased SOD and GSH-Px, decreased MDA), and modulated neurotransmitter balance (reduced Glu, increased GABA) in brain tissues. YPGE also upregulated P-glycoprotein expression and reduced neuronal apoptosis in the hippocampal CA1 region by upregulating Bcl-2 and downregulating Bax. These findings suggest that YPGE exerts multi-target antiepileptic effects through synergistic anti-inflammatory, antioxidant, and anti-apoptotic actions, providing experimental evidence for the development of novel antiepileptic therapies based on processed G. elata.

Current Issues in Molecular Biology doi: 10.3390/cimb48070687

Authors: Ioannis Tsomidis Angeliki Tsakou Argyro Voumvouraki Elias Kouroumalis

Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection remain a world health problem leading to fibrosis and cirrhosis. Liver damage is primarily mediated by the innate and adaptive immune responses since HBV and HCV are not directly cytotoxic. Kupffer cells and liver-recruited macrophages are heavily implicated in both viral elimination and progression of the disease. HBV and HCV proteins polarize macrophages into either an M1 pro-inflammatory phenotype, promoting hepatocyte damage or into an M2 immunosuppressive phenotype, leading to viral persistence and fibrogenesis via cytokines such as interleukin-10 (IL-10) and transforming growth factor-beta (TGF-β). In this review a brief overview of the heterogeneity of liver macrophages in health and during chronic viral infection is presented. Recognition of viruses by macrophages and the modulation of macrophages by viral proteins in the pathogenesis of liver inflammation and injury are discussed in detail. Most importantly, the mechanisms that HBV and HCV are using to manipulate macrophages and escape elimination are also presented. The role of macrophages in the evolution of acute-on-chronic liver failure is analyzed. Finally, a concise presentation of the emerging, but not yet clinically used, therapeutic strategies targeting macrophages to control chronic HBV infection and restore the dysregulated immune response is discussed. In conclusion, this integrated review of liver macrophage implication summarizes the pathophysiology and pathogenesis of HBV and HCV including acute-on-chronic- liver failure and viral cirrhosis.

Current Issues in Molecular Biology doi: 10.3390/cimb48070686

Authors: Diana Carolina Zambrano Ríos Miguel Ángel Gómez Juan Manuel Gómez Felipe Alberto Polo Betty Oviedo Sarria Julián Andrés Rivera Andrés Jenuer Matta

Obesity and metabolic dysregulation are closely associated with chronic low-grade inflammation, in which interleukin-6 (IL-6) plays a key regulatory role. Genetic variation in the IL-6 gene may influence inflammatory responses and metabolic homeostasis. To identify single-nucleotide variants (SNVs) in the IL-6 gene in a cohort of Colombian high-performance athletes and to evaluate their potential functional and structural consequences using bioinformatic prediction and protein-modeling approaches. A descriptive observational study was conducted in a cohort of 23 high-performance Colombian athletes from Valle del Cauca representing cycling, karate, and weightlifting disciplines. Genomic Deoxyribonucleic Acid (DNA) extracted from peripheral blood samples was analyzed using Next-Generation Sequencing (NGS). Identified variants were evaluated using several in silico prediction tools, including Basic Local Alignment Search Tool (BLAST version 2.16.0), Expert Protein Analysis System (ExPASy version 4.0), Open Reading Frame Finder (ORFfinder version 0.4.3), and population databases such as Genome Aggregation Database (gnomAD version 4.0). Structural modeling was used to explore the potential impact of amino-acid substitutions on IL-6 protein stability. Eight single-nucleotide variants were identified in the IL-6 gene. Among them, the rs1524107 variant generated a missense substitution predicted to modify the amino-acid sequence of the IL-6 protein. Structural modeling suggested a potential alteration in protein stability associated with this variant. The rs1524107 variant may influence IL-6 protein structure according to computational predictions. These findings provide preliminary hypothesis-generating evidence regarding the potential role of IL-6 genetic variation in inflammatory regulation; however, functional validation and larger cohort studies are required to determine their biological significance.

Current Issues in Molecular Biology doi: 10.3390/cimb48070685

Authors: Ru-Xi Yuan Xiao-Yang Ma Yang Lv Hong-Bin Si

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Current Issues in Molecular Biology doi: 10.3390/cimb48070684

Authors: Peiyang He Hanyang Cai

Soil salinization represents one of the most severe abiotic constraints on global forest productivity. Populus, the most widely cultivated fast-growing timber tree and a premier model woody plant, exhibits striking intrageneric variation in salt tolerance—from the extremely halophytic Populus euphratica to highly salt-sensitive cultivated clones. Understanding the molecular basis of this variation has profound implications for saline–alkali land reclamation and salt-tolerant variety breeding. This review systematically synthesizes current knowledge on Populus salt-stress responses, covering three primary injury mechanisms (osmotic stress, ionic toxicity, and oxidative damage) and the corresponding physiological countermeasures. We further survey functional genes across four major categories: ion transporters, osmotic-adjustment enzymes, antioxidant-defense components, and transcription factors. Crucially, we extend beyond the herbaceous-plant paradigm by examining salt-tolerance strategies that are specific to the woody architecture of Populus: long-distance radial and axial Na+ transport through tall stems, salt sequestration in senescent bark and wood parenchyma, and deep-root ion exclusion strategies. Comparative insights from other woody genera are incorporated to highlight convergent and divergent mechanisms. On this basis, we propose an integrated multi-level regulatory model in which Na+ compartmentalization/efflux serves as the core, ROS homeostasis as the key regulatory axis, and osmotic adjustment as the auxiliary strategy. Outstanding challenges—including unresolved primary salt-signal perception, insufficient pathway integration, and limited in planta gene-function verification—are critically assessed, and future research priorities encompassing multi-omics integration, CRISPR-based gene editing, and natural-population genomics are outlined.

Current Issues in Molecular Biology doi: 10.3390/cimb48070683

Authors: Gianluca Paventi Mariantonietta Succi Katia Maglieri Catello Di Martino Maria Virginia Soldovieri Massimo Iorizzo

Lactiplantibacillus plantarum is a highly versatile lactic acid bacterium, widely distributed across diverse ecological niches. Although often described as a nomadic species, increasing evidence suggests that strains from different habitats may retain niche-associated functional traits. This study investigated the phenotypic biodiversity of forty L. plantarum strains isolated from four ecologically distinct environments: wine, honeybee gut, trout intestine, and pre-weaning infant feces. Growth performance at different temperatures and on various carbon sources, acidification capacity, and β-glucosidase activity were evaluated and integrated using multivariate statistical analyses. Significant differences in β-glucosidase activity were observed among ecological groups (Kruskal–Wallis, p = 0.001), with wine-associated strains exhibiting the highest enzymatic activities and trout-derived isolates the lowest. Growth and acidification traits showed more limited variation among habitats, indicating that these physiological characteristics are largely conserved within the species. Heatmap visualization, principal component analysis (PCA), and hierarchical clustering revealed substantial phenotypic heterogeneity among strains. PCA indicated that growth performance and acidification traits contributed primarily to the first principal component, whereas β-glucosidase activity and differential fructose utilization were major contributors to the second component. Permutational multivariate analysis of variance (PERMANOVA) confirmed a significant effect of ecological origin on the overall phenotypic structure (p = 0.006), although habitat explained only 15.3% of the total variance (R2 = 0.153). Overall, the results show that ecological origin contributes to the phenotypic diversification of L. plantarum populations while preserving the extensive functional versatility characteristic of this species. β-Glucosidase activity emerged as the most discriminating phenotypic trait among ecological groups and represented the principal niche-associated functional signature identified in this study.

Current Issues in Molecular Biology doi: 10.3390/cimb48070682

Authors: Wei Zhao Bao Yu Chengli Li Jingjing Li Haijun Huang Weiguo Cao

Cholestatic liver injury (CLI) is a complex condition for which current treatment options remain limited. Lysimachiae Herba (LH), a traditional Chinese medicine with hepatoprotective properties, has not yet been fully characterized in terms of its active constituents and underlying mechanisms in CLI. This study was designed to systematically determine the chemical composition of LH, characterize its absorbed constituents in vivo, and elucidate its therapeutic mechanisms against CLI. UPLC-Q-TOF-MS/MS was employed to analyze the chemical composition of LH and its absorbed components in rat serum. Key targets and signaling pathways were predicted using network pharmacology and molecular docking, followed by experimental validation in an ANIT-induced CLI mouse model and LCA-treated HepG2 cells through biochemical assays, histological examination, transcriptomic analysis, qRT-PCR, Western blotting, and immunofluorescence analysis. A total of 129 compounds were tentatively identified in LH, among which 26 were detected in the bloodstream. Network analysis and molecular docking suggested that LH regulates bile acid homeostasis predominantly by the FXR signaling pathway. Both in vivo and in vitro experiments provided convergent evidence that LH modulates the FXR-related bile acid regulatory network, enhances bile acid efflux transporter expression, and alleviates CLI. In conclusion, this study systematically elucidates the chemical composition, absorbed constituents, and pharmacological mechanisms of LH in CLI, highlighting the involvement of FXR-related bile acid regulation as an important mechanism and providing a scientific basis for the potential development of LH for cholestatic liver injury.

Current Issues in Molecular Biology doi: 10.3390/cimb48070681

Authors: Seongho Han Sung-Whan Kim

Ischemic vascular diseases remain a leading cause of morbidity and mortality worldwide and are frequently associated with irreversible tissue damage. Although stem cell-based therapies have shown promise for vascular regeneration, their clinical translation has been limited by poor survival, insufficient engraftment, functional heterogeneity, and immune rejection. Recent advances in genome-editing technologies, including CRISPR/Cas9, base editing, and prime editing, have provided powerful tools for overcoming these limitations through precise genetic modification of stem cells. Gene editing can enhance angiogenic potential, improve resistance to ischemic stress, augment paracrine activity, promote endothelial maturation, and reduce immunogenicity. In this review, we outline the current genome-editing toolbox and its application to stem cell engineering for vascular regeneration in ischemic disease. We also examine emerging therapeutic concepts, universal donor cell platforms, and key issues in safety and ethics, with a focus on translational pathways. Taken together, advances at the interface of genome editing and stem cell biology are likely to accelerate the development of regenerative therapies that deliver more durable vascular repair in ischemic vascular disease.

Current Issues in Molecular Biology doi: 10.3390/cimb48070680

Authors: Yuan Li Yao Liu Yuyi Cong Juan Liu Wen Pan Xiaobing Guan Jiaqi Wang

Oral squamous-cell carcinoma (OSCC) is a prevalent malignancy of the head and neck region. A delay in the diagnosis of OSCC often results in a high metastatic tendency, which is the main reason for the high patient mortality. Dynamic monitoring and management of the onset and progression of OSCC are critical for improving patient survival rates. Liquid biopsy technology—characterized by its non-invasive nature, procedural convenience, and capacity for longitudinal monitoring—is a promising adjunct to histopathological examination for the early diagnosis of OSCC. Epigenetic alterations, characterized by reversibility and long-term stability in physiological fluids, are critical enablers of liquid biopsy and its clinical utility. Advances in detection technologies, including quantitative polymerase chain reaction (qPCR), digital droplet PCR (ddPCR), next-generation sequencing (NGS), and electrochemical biosensors, have significantly facilitated the research and clinical translation of epigenetic biomarkers in oral liquid biopsies. However, translating epigenetic biomarkers from research discovery to clinical practice for OSCC remains hindered by several critical challenges: the scarcity of large-scale, rigorously designed cohort studies, limited multicenter validation, inconsistent preprocessing protocols, and a lack of harmonized analytical platforms. Finally, we propose a conceptual framework to outline potential clinical application models for these biomarkers.

Current Issues in Molecular Biology doi: 10.3390/cimb48070679

Authors: Ewa Nowak Emilia Morawiec Adam Pudełko Agnieszka Polak Mateusz Broncel Daria Matczyńska Dawid Zamojski Michał Czerwinski Anna Bednarska-Czerwińska

Efficient and reproducible lentiviral vector production and T-cell transduction remain important technical challenges in CAR-T (Chimeric Antigen Receptor T-cell) cell manufacturing. In this study, we optimized HEK293T transfection and primary T-cell transduction parameters for lentiviral CAR constructs targeting BCMA (B-cell maturation antigen) and GPRC5D (G-protein coupled receptor family C group 5 member D). Lipofectamine 3000 and TurboFectin 8.0 were compared across different seeding densities and reagent-to-DNA ratios, with vector yields quantified by qPCR (Quantitative Polymerase Chain Reaction) and p24 ELISA (Enzyme-linked Immunosorbent Assay). Lipofectamine 3000 consistently generated higher viral titers and transduction efficiencies, as reflected by a greater proportion of GFP-positive (Green Fluorescent Protein) cells than TurboFectin 8.0, reaching peak titers of 9.65 × 108 copies/mL for the anti-GPRC5D and 5.33 × 108 copies/mL for the anti-BCMA vectors. Under optimized conditions, transduction efficiencies reached 43.8% GFP+ cells for BCMA-CAR and approximately 13–14% GFP-positive transduced cells for the GPRC5D construct within the tested TU/mL range. Co-transduction experiments yielded approximately 62–66% GFP+ cells with detectable BCMA-binding and presumptive GPRC5D-CAR-expressing subpopulations identified based on GFP reporter expression. Immunophenotypic analysis demonstrated a relatively stable CD4/CD8 distribution (~65/35), enrichment of effector memory CD8+ cells, and expression of activation-associated markers. Collectively, these findings describe an optimized lentiviral transfection and transduction workflow that may support the further development of dual-targeting BCMA/GPRC5D CAR-T manufacturing strategies in research and early translational settings.

Current Issues in Molecular Biology doi: 10.3390/cimb48070678

Authors: Lianghua Huang Zhaoji Pan Meng Bai Jiyan Guo Jian Xiao Chenghai Gao

As a traditional element of Chinese medicine, Hippocampus erectus is well known for promoting adolescent growth, yet its active fractions and underlying molecular mechanisms remain unclear. In this study, the aqueous extract of H. erectus was subjected to in vitro simulated gastrointestinal digestion and ultrafiltration to separate three molecular weight fractions (<10 kDa, 10–30 kDa, >30 kDa). Their chemical profiles were characterized, and osteogenic activities were systematically evaluated using cell assays, a juvenile rat model, and integrated transcriptomics and data-independent acquisition (DIA) proteomics. Results revealed that chemical profiling showed the >30 kDa fraction was mainly composed of hemocyanin subunits, and the 10–30 kDa fraction was enriched in growth-related amino acids and steroid derivatives; functionally, the 10–30 kDa fraction promoted preosteoblast proliferation and early differentiation via enhanced alkaline phosphatase (ALP) activity, while the >30 kDa fraction dominated late osteoblast maturation and mineralization. Both fractions significantly increased rat body and bone length by expanding growth plate proliferative zones and elevating serum insulin-like growth factor-1 (IGF-1)/bone morphogenetic protein-2 (BMP-2) levels. Transcriptomic and proteomic analyses identified vascular endothelial growth factor (VEGF), Wingless-related integration site (Wnt), phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt), and extracellular matrix (ECM)–receptor interaction as potential core regulatory pathways. Integrated multi-omics analysis further confirmed Frizzled-related protein B (Frzb) and AKT1 substrate 1 (Akt1s1) as candidate key regulatory targets enriched in the Wnt and adenosine monophosphate-activated protein kinase (AMPK) signaling pathways. These findings elucidate the multi-fraction, multi-pathway mechanism of H. erectus in promoting skeletal development, providing scientific evidence for its traditional use and a theoretical basis for growth-promoting functional food development.

Current Issues in Molecular Biology doi: 10.3390/cimb48070677

Authors: Wenxiu Yin Wenxin Zhang Quan Zhang Zhengping Ying Shan Wu Huizhen Yu Mingzhe Zhang

The rapid proliferation of genetically modified (GM) crops and the uncontrolled distribution of GM-based food and feed have become a growing global concern, posing new challenges for regulatory oversight and traceability. The traditional PCR detection method cannot simultaneously meet the needs of high-throughput, high-specificity and high-sensitivity detection of transgenic organisms. In this study, a multiplex fluorescence PCR-capillary electrophoresis platform was developed by labeling primers of endogenous and exogenous genes with different fluorescent groups. The system enabled the simultaneous detection of 27 GM-related genes and events in a single analytical workflow. The results demonstrated accurate identification of all seven GM maize events, with correct detection achieved for each individual strain. In addition, the method enabled precise discrimination of a mixed sample containing five GM maize varieties. The assay also achieved a detection sensitivity of 0.1% in gradient mixtures with different GM contents. Our platform integrates a larger number of targets into a single PCR reaction, thereby simplifying the detection workflow while maintaining high analytical performance. Furthermore, the combination of multicolor fluorescence labeling and capillary electrophoresis provides high-resolution fragment discrimination and robust multiplex detection capability. This platform provides a novel and effective tool for rapid detection in food safety of transgenic crops and related areas, and can be applied in import/export inspection, quarantine, and biosafety surveillance.

Current Issues in Molecular Biology doi: 10.3390/cimb48070676

Authors: Hamad Hussain Nooral Amin Imran Ali Abdul Wakeel Umar Naveed Ahmad

The continued rise in atmospheric carbon dioxide (CO2) concentrations presents a strategic opportunity to harness climate change variables within the framework of precision agriculture. Despite the well-established role of elevated CO2 (eCO2) in enhancing biomass accumulation, its largely underexplored potential to drive the biosynthesis of secondary metabolites represents a more significant and promising avenue of investigation. This review appraises the physiological and molecular mechanisms through which eCO2 enrichment redirects metabolic flux toward secondary metabolite biosynthesis, with far-reaching implications for plant productivity and resilience. Special emphasis is placed on critically evaluating the scientific literature to explore how CO2-mediated modulation of the carbon–nutrient balance (CNB) can be strategically leveraged to enhance secondary metabolite yields. Moving from observation to application, integrated strategies are proposed to exploit CO2 enrichment in advanced bioreactor systems and controlled-environment greenhouses as a means of maximizing bioactive compound production in ginseng. Pinpointing the regulatory sweet spots at which carbon saturation elicits maximum ginsenoside expression opens a promising avenue for engineering ginseng cultivation systems with sustainable potency and superior bioactivity. Though the full molecular architecture of these pathways in Panax awaits elucidation, converging evidence from related plant systems furnishes a credible mechanistic scaffold for future research.

Current Issues in Molecular Biology doi: 10.3390/cimb48070675

Authors: Roxana-Cristina Dobriceanu Ianis Kevyn Stefan Boboc Liliana Mititelu Tartau Andreea Daniela Meca Carmen Nicoleta Oancea Maria-Alexandra Paceana Marius-Mihai Pastiu Adina Turcu-Stiolica Maria Bogdan

Type 2 diabetes mellitus is associated with chronic low-grade inflammation contributing to endothelial dysfunction, metabolic imbalance, and cardiovascular complications. Although semaglutide (SEM) and tirzepatide (TIR) provide important metabolic and cardioprotective benefits, their early anti-inflammatory effects and potential sex-dependent differences remain incompletely understood. This study comparatively evaluated the effects of SEM and TIR on systemic inflammatory biomarkers in a murine model of streptozotocin-induced diabetes mellitus. Thirty BALB/c mice were allocated into six experimental groups according to sex and treatment: control, SEM, and TIR groups (n = 5/group). Diabetes was induced by intraperitoneal streptozotocin administration, followed by treatment with SEM or TIR. Circulating interleukin-1β (IL-1β) and pentraxin-3 (PTX-3) levels were measured at baseline, one week after streptozotocin administration, and after six weeks of treatment. Control groups exhibited progressive increases in IL-1β and PTX-3 levels, indicating sustained inflammatory activation. In contrast, SEM- and TIR-treated animals showed attenuated inflammatory responses characterized by transient or stabilized biomarker profiles. Differential inflammatory responses were observed between treatments and sexes. Male SEM and Male TIR groups demonstrated stable IL-1β levels, whereas female treated groups showed persistent elevations, particularly Female TIR animals. PTX-3 responses also displayed differential sex-dependent patterns, with Female SEM animals exhibiting the most stable inflammatory profile. These findings suggest differential early immunomodulatory effects of the two modern antidiabetic drugs, characterized by distinct biomarker responses according to sex and inflammatory marker profile. IL-1β and PTX-3 may represent complementary biomarkers for the assessment of early inflammatory activation associated with diabetes mellitus and its cardiometabolic complications.

Current Issues in Molecular Biology doi: 10.3390/cimb48070674

Authors: Danielle Cristina Honorio França Alan Cardec Barbosa Elton Brito Ribeiro Anibal Monteiro de Magalhães Neto Emanuelle Carolina Honorio França Maraisa Delmult Borges Patrícia Gelli Feres de Marchi Adenilda Cristina Honorio-França Eduardo Luzía França

Background: Type 2 diabetes mellitus is characterized by chronic low-grade inflammation, immune dysregulation, and metabolic impairment. This study investigated cytokine profiles associated with physical exercise and insulin therapy in patients with Type 2 diabetes mellitus. Methods: Blood samples were collected from 51 volunteers to evaluate metabolic parameters and cytokine concentrations. According to glycemic status and insulin use, participants were classified into non-diabetic, non-insulin-dependent Type 2 Diabetes Mellitus, and insulin-dependent Type 2 Diabetes Mellitus groups. Results: Physically active individuals with non-insulin-dependent Type 2 Diabetes Mellitus exhibited increased IL-4, IL-6, and IL-10 levels, suggesting enhanced immunoregulatory and anti-inflammatory responses. Physically active patients with insulin-dependent Type 2 Diabetes Mellitus showed elevated IL-17 concentrations. In contrast, sedentary individuals with insulin-dependent Type 2 Diabetes Mellitus exhibited higher TNF-α levels, indicating a more pronounced proinflammatory profile. IFN-γ concentrations were significantly higher in patients with insulin-dependent Type 2 Diabetes Mellitus, regardless of exercise status. Correlation analyses demonstrated distinct cytokinemetabolic interaction patterns according to metabolic condition and physical exercise. Conclusion: Cytokines can serve as molecular markers of immunometabolic responses associated with physical exercise and insulin therapy in Type 2 Diabetes Mellitus, reflecting alterations in systemic inflammatory regulation and immune–metabolic crosstalk related to glycemic adaptation.

Current Issues in Molecular Biology doi: 10.3390/cimb48070673

Authors: Anna Długaszek Jacek Kabut Małgorzata Domagała-Haduch Anita Gorzelak-Magiera Joanna Sadurska Maria-Laura Morawiec Aleksandra Mielczarek-Palacz Iwona Gisterek-Grocholska

Ovarian cancer, particularly high-grade serous ovarian cancer (HGSOC), remains one of the most lethal gynecological malignancies due to late diagnosis and the development of chemoresistance. The tumor microenvironment (TME) plays an important role in disease progression, with chemokines influencing cell recruitment, angiogenesis, metastasis, and immune modification. This review synthesizes current evidence on key chemokine axes in ovarian cancer, highlighting their dual roles as prognostic biomarkers and therapeutic targets. The most important axes include CXCL12/CXCR4 (which drives tumor proliferation, angiogenesis and chemoresistance via epithelial–mesenchymal transition), CCL2/CCR2 (promoting immunosuppressive tumor-associated macrophages and resistance), and CCL5/CCR5 (enhancing pro-oncogenic signaling and Treg/MDSC infiltration). Pro-angiogenic ELR+CXC chemokines like CXCL8 induce vascularization and inflammation. On the contrary, effector chemokines (CXCL9/10/11/13) correlate with “hot” immune subtypes and improved survival in several studies. High expression of immunosuppressive chemokines predicts poorer prognosis and therapy resistance, while immune-attracting profiles associate with better outcomes and chemotherapy responsiveness. Therapeutically, inhibitors like plerixafor (CXCR4), PF-04136309 (CCR2), and maraviroc (CCR5) show preclinical promise, synergizing with chemotherapy, anti-VEGF, and checkpoint inhibitors. Chemokines also represent actionable molecular targets to overcome ovarian cancer’s “cold” immune phenotype. Future research should validate multi-chemokine signatures for patient stratification and advanced clinical trials toward personalized therapies.

Current Issues in Molecular Biology doi: 10.3390/cimb48070672

Authors: Yi Wu Guimei Yang Yixian Li Yunjing Ruan Qianmei Yang

Dendrobium officinale polysaccharide (DOP) shows efficacy against type 2 diabetes (T2D), but its mechanisms remain unclear. The present investigation aimed to identify potential biomarkers associated with DOP-mediated therapeutic interventions in T2D. Datasets related to T2D were excavated from the Gene Expression Omnibus (GEO) database. Candidate genes were acquired from the intersection of genes obtained from weighted gene co-expression network analysis (WGCNA) and differential analysis. Subsequently, Mendelian randomization (MR) identified causal biomarkers, validated by Receiver Operating Characteristic (ROC) curves and expression profiling. Then, a nomogram, immune infiltration, single-cell analysis, and molecular docking were performed. Among the 12 candidate genes, 7 with available eQTL instruments were included in MR analysis, while 5 lacking genome-wide significant IVs (p < 5 × 10−8) were excluded. Three genes demonstrated significant MR associations with T2D, and biomarkers GLI1 and LGALS9 showed strong diagnostic performance and were upregulated in T2D. The nomogram had good predictive value. Seventeen immune cells differed significantly between T2D and controls, with GLI1 and LGALS9 positively correlating with most and primarily expressed in stellate cells. Finally, D-Galacturonic acid, D-Mannose, and L-rhamnose monohydrate were compounds showing predicted binding potential with candidate biomarkers GLI1 and LGALS9 emerged as promising potential molecular candidates associated with DOP-mediated T2D regulation, offering novel mechanistic perspectives on DOP’s anti-diabetic properties.

Current Issues in Molecular Biology doi: 10.3390/cimb48070671

Authors: Joanna Szmydtka Maja Ziemian Rafał Banaszak Martyna Ciesielska Jagoda Ładosz Urszula Maliszewska Adrian Nowakowski Martyna Paszek Gabriela Suproń Zuzanna Świętochowska Agata Tkaczuk Szymon Wojciechowski Grzegorz Węgrzyn

Mucopolysaccharidoses (MPS) are severe, inherited metabolic diseases, classified among lysosomal storage diseases (LSDs). The presence of pathological variants of genes coding for enzymes involved in the degradation of glycosaminoglycans (GAGs) is a primary cause of each MPS type, and accumulation of these compounds is a characteristic feature of MPS. Depending on the kind of defective enzyme and the type of stored GAG(s), 12 classical types are distinguished, and a few other related diseases, whose classification is unclear. Although there is no fully effective cure for MPS, several kinds of therapeutic approaches have been proposed to treat these diseases, and some of them have been introduced into clinical practice. In this review article, we present and discuss very recent advances in developing various therapies for MPS, also indicating problems and limitations. This paper focuses on enzyme replacement therapy (ERT), cell- and gene-based therapies (including hematopoietic stem cell transplantation and gene therapy), inhibition of GAG synthesis, and some other newly developed therapeutic approaches. Perspectives on MPS therapies are also discussed.

Current Issues in Molecular Biology doi: 10.3390/cimb48070670

Authors: Prawej Ansari Alexa D. Reberio Asif Ali Md Hamza Naquib Sandeep Kumar Dhivya C Md Abeduzzaman Anon Hajera Khatun Md Ferdos Ahamed Peter R. Flatt Yasser H. A. Abdel-Wahab

Pomegranate (Punica granatum), a shrub belonging to the Lythraceae family, has long been recognized for its diverse pharmacological benefits, including potential roles in managing inflammation and diabetes. The present study explored the insulin-secretory and β-cell proliferative properties of the ethanol extract of P. granatum fruit peel (EEPG) and assessed its influence on glucose regulation in high-fat-fed diet-induced obese mice (HFDi-OM) through in vivo and in silico studies. In vitro, EEPG was found to activate cAMP-dependent pathways and regulate KATP channels, thereby enhancing glucose-stimulated insulin secretion from BRIN-BD11 β-cells, with partial reliance on extracellular calcium. EEPG promoted β-cell proliferation, as indicated by an increase in Ki-67 positive cells, and displayed inhibitory effects on glucose diffusion and starch hydrolysis, suggesting a capacity to delay carbohydrate digestion and absorption. Furthermore, EEPG demonstrated antioxidant activity by neutralizing free radicals. In an acute test, EEPG (at doses of 150 and 250 mg/5 mL/kg) improved oral glucose tolerance and elevated plasma insulin levels. Long-term oral treatment for 21 days to HFDi-OM led to a significant reduction in fasting blood glucose, body weight, and food and fluid intake. It also enhanced gastrointestinal motility and improved lipid profiles by increasing HDL and lowering total cholesterol, LDL, and triglycerides. The therapeutic properties of EEPG are likely attributed to its rich bioactive components, including flavonoids (quercetin, kaempferol, catechin, and epicatechin) and phenolic acids (ellagic acid), which exhibited strong multi-target binding affinities in in silico molecular docking studies toward SUR1, PDE4, PI3K, and α-amylase, thereby supporting enhanced insulin secretion, β-cell function and glucose homeostasis.

Current Issues in Molecular Biology doi: 10.3390/cimb48070669

Authors: Ye Ryeong Kim Ayun Seol Su Jin Lee Ji Eun Kim Hee Jin Song Su Jeong Lim Su Ha Wang Ye Eun Ryu Young Whan Choi Sun Il Choi Dae Youn Hwang

Although oxidative stress plays a critical role in age-related macular degeneration (AMD) progression, natural product–derived single compounds against AMD remain largely unexplored. We investigated the protective effects and underlying mechanism of α-iso-cubebene against AMD-like retinal injury. Alterations in key phenotypes for AMD were analyzed in AMD-mimicking models using ARPE-19 cells co-treated with blue light (BL) and N-retinylidene-N-retinylethanolamine (A2E), as well as BL-exposed BALB/c mice. In BL+A2E-treated ARPE-19 cells, α-iso-cubebene reduced intracellular reactive oxygen species (ROS) and nitric oxide (NO) production and restored superoxide dismutase (SOD) activity and nuclear factor erythroid 2–related factor 2 (Nrf2), suggesting enhancement of the antioxidant defense system. Furthermore, α-iso-cubebene improved cell viability, reduced apoptotic cell populations, and regulated apoptosis-related signaling pathways under oxidative stress conditions. It also attenuated cyclooxygenase-2 (COX-2)-mediated inducible nitric oxide synthase (iNOS) signaling and was associated with reduced inflammasome-related signaling. Importantly, these protective effects were consistently observed regarding the protection of histopathological structure and normalization of inflammatory cytokines in the retina of BL-exposed BALB/c mice. Collectively, our results demonstrate that α-iso-cubebene, as a potential therapeutic candidate, alleviates AMD-like retinal injury and was associated with enhanced antioxidant responses and reduced inflammatory and apoptotic signaling markers.

Current Issues in Molecular Biology doi: 10.3390/cimb48070668

Authors: Şerife Efsun Antmen Hasan Öz Cem Yalaza Necmiye Canacankatan

This study aims identify plant-derived compounds that can inhibit glutathione peroxidase 4 (GPX4) enzyme and evaluate them through molecular docking, dynamics simulations, and ADMET analyses. The 3D structure of the GPX4 protein (PDB ID: 2OBI) was obtained from the Protein Data Bank. The plant-derived ligand library was compiled from the PubChem database and screened for compliance with Lipinski’s rules using ADMETLAB 2.0. Molecular docking simulations were performed using Autodock Vina. Molecular dynamics simulations of 100 nanoseconds were performed for the selected ligand–protein complexes using AMBER Tools and OpenMM software. The ADMET properties of the ligands were evaluated using the pKCSM web server. Compared to the reference inhibitor RSL3 (−7.2 kcal/mol), five plant compounds showed stronger binding affinity: withaferin A (−8.0 kcal/mol), mahanine (−7.9 kcal/mol), pseudobufarenogin (−7.8 kcal/mol), cucurbitacin I (−7.6 kcal/mol), and liquiritin (−7.5 kcal/mol). Molecular dynamics simulations showed that the complexes of withaferin A, mahanine, and liquiritin exhibited superior structural stability. ADMET analysis revealed that the compounds generally possess acceptable pharmacokinetic profiles but require some bioavailability optimization. The identified plant-derived compounds can be considered as potential therapeutic agents in cancer treatment by inducing ferroptosis via GPX4 inhibition. These findings provide an important basis for natural product-derived drug discovery studies.

Current Issues in Molecular Biology doi: 10.3390/cimb48070667

Authors: Sylia Ardache Shu Tang Endong Bao

Cisplatin (CP) chemotherapy is limited by nephrotoxicity, primarily involving tubular epithelial cell apoptosis. αB-crystallin (CryAB) is a small heat shock protein that plays a cytoprotective role in stressed kidneys but can also promote tumor progression. Its precise role and molecular mechanisms in CP-induced kidney injury remain largely unclear. This study highlighted the function of CryAB and its regulatory pathways in CP nephrotoxicity by employing in vitro models of rat renal tubular epithelial cells (NRK-52E) with CryAB gene knockdown/overexpression, and in vivo models of CryAB knockout/wild-type mice, followed by CP treatment. Apoptosis and key signaling pathways (NF-κB, MAPK, AKT) were evaluated in this study. The results indicated that CP treatment (20 µM) significantly upregulated CryAB expression in renal cells (p < 0.01) and triggered both apoptosis and MAPK activation. CryAB deficiency sensitized cells and mice to CP, exacerbating renal dysfunction, tubular injury, and apoptosis, as evidenced by increased Bax, cyt c release, and caspase-3 cleavage. Conversely, CryAB overexpression attenuated these effects. Furthermore, our findings suggest that the lack of CryAB favors the cytoplasmic retention of NF-κB, and that CryAB status can influence MAPK signaling, pointing to a potential regulatory loop. Additionally, CP-induced AKT phosphorylation was diminished in CryAB-deficient models. Therefore, CryAB may exert a cytoprotective role in CP nephrotoxicity, potentially mitigating tubular apoptosis by modulating the mitochondrial apoptotic pathway, supporting NF-κB-mediated survival signaling, and cross-talking with MAPK and AKT pathways. Our findings suggest that CryAB serves as an important regulator of renal cell fate and a potential therapeutic target for mitigating CP-induced kidney injury.

Current Issues in Molecular Biology doi: 10.3390/cimb48070666

Authors: Qingyu Cao Mengyuan Zhang Dan Wang Kaixuan He Yuanyuan Mei Ning Ning Wang

Monoubiquitination is a significant post-translational modification that plays a pivotal role in various biological processes. Chemical monoubiquitination holds significant value in investigating the functional implications of site-specific ubiquitination on target proteins. Despite all progress made in this area, conventional enzymatic methods so far rely largely on high yields of substrate proteins and the removal of tags to prevent non-specific ubiquitin binding, which poses substantial challenges for low-solubility proteins. Here, an optimized chemical monoubiquitination system that facilitates precise, site-specific ubiquitination of low-solubility target protein was developed using SSPP as an example. A cysteine-free GST tag (GST4CS) was engineered, and a flexible (GGGGS)3 linker was incorporated to mitigate steric hindrance and enhance the solubility of GST-SSPP fusion protein, resulting in a 2.5-fold increase in purification yield. Successful monoubiquitination of SSPP at the position of lysine 305 was achieved using disulfide-mediated conjugation, as proven via SDS-PAGE and Western blotting. Moreover, the phosphatase assay showed that monoubiquitination at residue C305 of the mutated SSPP significantly decreased its phosphatase activity. This system eliminates tag interference and enhances compatibility with low-solubility targets, providing a robust platform for functional studies of plant protein ubiquitination.

Current Issues in Molecular Biology doi: 10.3390/cimb48070665

Authors: Anatoliy Ivashchenko Anna Pyrkova Raigul Niyazova Saltanat Orazova

Londin et al. discovered a novel group of miRNAs, referred to as LmiRNAs, whose properties had been studied little for unknown reasons. In this study, we examine fully complementary interactions between LmiRNAs and mRNAs of human genes. Using the MirTarget program, we identified a significant number of target genes showing unique interaction with LmiRNAs. Among the 3707 LmiRNAs, fully complementary binding sites (BSs) were found in the 5′UTR of 75 target genes, with their interactions exhibiting high free energy. Fully complementary LmiRNA binding sites were located within the CDS of 81 target genes, while only seven LmiRNAs were found to bind to the 3′UTR of target genes. The KIFC3, PHF15, RPL15, and SNX11 genes were found to encode both LmiRNA-5p and LmiRNA-3p, which actively bind to their respective mRNAs. While the mRNA of most genes was targeted by only a single LmiRNA, the BMP8B, FGFRL1, and SDC3 genes included mRNAs bound by the specific pair ID00121.5p and ID02992.5p. These results expand our understanding of LmiRNAs and support their potential as diagnostic and therapeutic agents for various diseases.

Current Issues in Molecular Biology doi: 10.3390/cimb48070664

Authors: Margarida Queirós Rute S. Moura Eduardo J. Gudiña

The prevalence of diabetes has increased considerably in recent decades, representing a global health problem. Although several pharmacological approaches for the treatment of diabetes are available, it is pertinent to explore more effective alternatives with reduced adverse effects. In this work, the hypoglycemic activity of rhamnolipids was studied using the chicken embryo (in ovo) model. The results obtained demonstrated that rhamnolipids produced by Pseudomonas aeruginosa #112 and commercial rhamnolipids (RL-90) significantly reduced blood glucose levels of chicken embryos on embryonic day 11 (from 135 ± 11 mg/dL to 94–107 mg/dL). These values were similar to those achieved with human insulin (104 ± 20 mg/dL) and the rapid-acting human insulin analog FIASP® (95 ± 18 mg/dL). It was also verified that rhamnolipids did not have a negative effect on chicken embryo development at the concentrations tested. Regarding the molecular mechanisms involved in the decrease in blood glucose levels, for both insulins, a reduction in the expression of genes encoding the glucose transporter 2 (glut2) and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase 2 and fructose-1,6-biphosphatase 1 was observed. However, in the case of rhamnolipids, only a reduction in the expression of glut2 was observed. According to the results obtained, rhamnolipids are potential candidates for further studies on the development of new alternative treatments for diabetes symptoms.

Current Issues in Molecular Biology doi: 10.3390/cimb48070663

Authors: Yunjiao Shen Yuanyuan Yao Zhihui Liu Yi Li Shijie Cao Xinchi Feng

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by glycolipid dysregulation and hepatic steatosis. Centella asiatica (CA) and its triterpenoid constituents exert metabolic benefits. In addition, previous metabolomics study found that asiatic acid regulated pyrimidine metabolism in obese mice, while the key target and pathway were undefined. This study investigated the regulatory effects of CA and its active constituents on T2DM-related glycolipid disorders, focusing on the pyrimidine metabolism pathway. T2DM mice were established using a high-fat diet combined with streptozotocin (STZ) and treated with Centella asiatica ethanolic extract or asiatic acid (AA), with glibenclamide as a positive control. Then, glycolipid metabolism, hepatic function, pyrimidine metabolites, and related mechanisms were assessed using biochemical assays, LC–MS/MS, cellular experiments, molecular analyses, and molecular docking. CAE and AA significantly reduced FBG (decreased by 51.01% and 53.01%), improved glucose intolerance, corrected dyslipidemia, alleviated hepatic steatosis, and attenuated insulin resistance in T2DM mice. They elevated hepatic uridine, cytidine, and UDP-glucose (UDPG) levels, promoted glycogen synthesis, inhibited uridine phosphorylase 1 (UPP1) activity, upregulated UDPG synthesis genes (PGM1, UGP2), and downregulated lipogenic genes (ACACA, Fasn, SREBP1/2). Molecular docking indicated specific binding of AA and asiaticoside to UPP1. This work distinguishes from our prior research by identifying UPP1 as a functional target and elucidating the detailed molecular mechanism. CA improves T2DM-associated glycolipid disorders and hepatic injury by modulating the pyrimidine metabolism-UDPG-glycogen synthesis pathway and targeting UPP1, highlighting its therapeutic potential for metabolic diseases.

Current Issues in Molecular Biology doi: 10.3390/cimb48070662

Authors: Galiya Gimalova Irina Gilyazova Elza Khusnutdinova Valentin Pavlov

This review summarizes current data on the role of small nucleolar RNAs (snoRNAs) and their host genes (SNHGs) in the development of bladder cancer (BC). It examines snoRNA biogenesis, classical functions (rRNA modification), and non-canonical oncogenic mechanisms, including microRNA sponging, sdRNA production, and protein interactions (EZH2, DNMT3A, hnRNPK). The factors involved in the deregulation of snoRNA/SNHG expression during tumour transformation are described, such as amplifications, epigenetic changes, and transcriptional control (c-Myc, p53). Studies have shown that in BC, the majority of snoRNAs/SNHGs (SNHG1, SNHG3, SNHG6, SNHG13, SCARNA12) act as oncogenes, activating the PI3K/AKT, Wnt/β-catenin, NF-κB, and c-Myc pathways, thereby enhancing proliferation, EMT, invasion, and metastasis. Suppressor molecules (SNHG2/GAS5) are also discussed. The clinical potential of snoRNAs as prognostic signatures (SNORS), diagnostic biomarkers (SNHG1 in urine), and therapeutic targets (e.g., SNHG3) is analyzed. Thus, snoRNAs and SNHGs represent a promising class of molecules for the development of new diagnostic and therapeutic approaches for BC, although further investigation in prospective studies is required.

Current Issues in Molecular Biology doi: 10.3390/cimb48070661

Authors: Xin-Yue Tang Ke-Jin Lu Rui Zhu Yue Gao Dong-Yan Wei Xi-Yu Zhang Yi-Cheng Ma Fei-Fei Wang Cheng-Gang Zou

Nicotinamide (NAM), a precursor of nicotinamide adenine dinucleotide (NAD+), and NAD+ are integral to a variety of cellular processes. NAM supplementation has been shown to have benefits for cellular senescence. However, the mechanism by which NAM improves skin photoaging remains unclear. In this study, the multi-omics analysis revealed that insufficient nicotinamide metabolism may be associated with a decrease in NAD+ synthesis during skin aging. Importantly, we found that NAM has an ameliorating effect on the skin photoaging in mice. Supplementation with NAM restored the expression of the salvage-pathway enzymes and NAD+ consumers. In addition, the supplementation with NAM was shown to restore the expression of skin barrier-related proteins (ZO1 and E-cadherin) and collagen I, while reducing the expression of senescence markers (γ-H2AX, p53, and p21). Furthermore, we found that NAM effectively suppresses the senescence-associated secretory phenotype (SASP) factors’ expression in skin photoaging. Our research reveals the dual role of NAM in attenuating skin photoaging, acting not only to delay cellular senescence but also to suppress the SASP.

Current Issues in Molecular Biology doi: 10.3390/cimb48070660

Authors: Maria Erodotou Alkistis Kapelouzou Konstantinos S. Mylonas Ioanna Soukouli John N. Boletis Gerasimos Tsourouflis Theodore Liakakos Dimitrios Schizas

Sepsis is a life-threatening condition characterized by a dysregulated host response to infection, leading to multi-organ dysfunction. Toll-like receptor signaling via MYD88- and TRIF-dependent pathways plays a central role in this process; however, its temporal and tissue-specific dynamics remain incompletely understood. The aim of this study was to investigate time-dependent transcriptional changes in MYD88- and TRIF-dependent signaling pathways across multiple organs in a murine model of sepsis. mRNA expression of MYD88, IRAK1, IRAK4, NF-kB, CCL4, CCL20, CCR2, IFN-β, IFN-γ, TNF-α, IL-1β, IL-2, IL-4, IL-8, IL-10, IL-18, Klotho, KLF4, HOXA5, NANOG and HIF1α was quantified using qRT-PCR in intestinal, kidney, liver and lung tissues at 24, 48, and 72 h following cecal ligation and puncture-induced sepsis in male C57BL/6J mice. Significant upregulation of innate immune signaling molecules, cytokines, chemokines, and interferon-related genes was observed in all tissues compared with controls. Genes associated with hypoxia and cellular regulation were also increased. These responses were tissue-specific and progressively intensified over time. Sepsis represents a dynamic, time-dependent, and tissue-specific process characterized by sustained activation of immune and hypoxic pathways, providing potential targets for time-stratified therapeutic strategies.

Current Issues in Molecular Biology doi: 10.3390/cimb48070659

Authors: Klaudia Dynarowicz Joanna Katarzyna Strzelczyk Dorota Bartusik-Aebisher Wiktoria Mytych Alina Pietryszyn-Bilińska Aleksandra Kawczyk-Krupka Dorota Hudy Oliwia Trzaskoś Jacek Tabarkiewicz David Aebisher

Glioblastoma multiforme (GBM) remains one of the most aggressive primary brain tumors with poor prognosis despite multimodal therapy. Photodynamic therapy (PDT) using indocyanine green (ICG) is an emerging adjuvant approach aimed at eliminating residual tumor cells after resection. While ICG-PDT exerts cytotoxic effects, its impact on molecular pathways regulating programmed cell death in glioma cells is not fully understood. In this study, T98G and U-118MG glioblastoma cells were divided into four groups: untreated control, light-only (10 min broadband irradiation), ICG-only (15 min incubation), and ICG-PDT (15 min ICG + 10 min broadband irradiation). Relative mRNA expression of apoptosis-related genes (BAX, BCL2, CASP3, FAS) and ferroptosis-related genes (GPX4, ACSL4, SLC7A11, GCH1) was quantified 24 h post-treatment by RT-qPCR using the 2−ΔΔCt method. ICG-PDT significantly reduced cell viability to 67.79% ± 3.39% (vs. 86.66% ± 4.33% in control), confirming effective phototoxicity. No statistically significant differences in mRNA levels were observed for any of the investigated genes across the groups (one-way ANOVA and Kruskal–Wallis, all p > 0.05). The largest non-significant deviation was a mild decrease in GPX4 (fold change 0.87) in the ICG-PDT group. Fluctuations in GCH1 were accompanied by high variance, likely reflecting technical noise rather than a true biological trend. The mRNA BAX/BCL2 ratio remained stable (~30) across all conditions. In contrast, the U-118MG line showed greater transcriptional sensitivity, with statistically significant decreases in CASP3 (p = 0.012) and ACSL4 (p = 0.031) expression, along with downward trends in BCL2 and GPX4 following ICG-PDT. ICG-PDT does not induce significant transcriptional changes in the analyzed genes T98G at the 24 h time point under the applied experimental conditions. In U-118MG cells, moderate transcriptional engagement of both apoptotic and ferroptotic routes was observed. Further studies at the protein and functional levels, across multiple time points and models, are warranted to fully elucidate the mechanisms of ICG-PDT in glioblastoma.

Current Issues in Molecular Biology doi: 10.3390/cimb48070658

Authors: Jae Heon Kim Miho Song Yun Seob Song

Progressive bladder fibrosis and impaired detrusor function represent converging pathological endpoints across diverse bladder disorders, including bladder outlet obstruction (BOO) associated with benign prostatic hyperplasia, spinal cord injury (SCI)-induced neurogenic bladder, radiation cystitis, and interstitial cystitis/bladder pain syndrome. Conventional therapies primarily manage symptoms and rarely reverse established fibrosis or restore durable bladder homeostasis. Mesenchymal stem/stromal cells (MSCs) have attracted considerable interest as therapeutic agents owing to their antifibrotic, immunomodulatory, angiogenic, and trophic paracrine activities. This review synthesises six key studies from our group and places them within the broader international literature on bladder regenerative medicine: (i) feasibility of superparamagnetic iron oxide (SPIO)-based molecular MRI tracking of transplanted human MSCs (hMSCs) in the bladder; (ii) SPIO-hMSC therapy for BOO-associated fibrosis with concurrent MRI monitoring; (iii) hepatocyte growth factor (HGF)-overexpressing engineered hMSC (B10.HGF) therapy in BOO; (iv) hMSC transplantation into the SCI-injured bladder wall monitored by MRI; (v) systematic review and meta-analysis of stem cell therapy effects on urodynamic outcomes in SCI models; and (vi) HGF-overexpressing hMSC therapy for BOO-induced underactive bladder. These six key studies are contextualised within the broader literature addressing cell sources, biomaterial-assisted delivery platforms, mechanistic pathways, emerging clinical evidence, and the evolving regulatory landscape for cell-based advanced therapy medicinal products. Key translational challenges include product standardisation, long-term durability, and mechanism-linked potency assay development.

Current Issues in Molecular Biology doi: 10.3390/cimb48070657

Authors: Yuexinzi Jin Yuan Mu Li Wang Bingfeng Zhang Suli Ge Huaguo Xu Jian Xu Jiexin Zhang

Accurate measurement of serum uric acid (UA) is critical for disease assessment and therapeutic monitoring; however, numerous factors can compromise the accuracy of UA detection. This study describes a novel circulating immunoglobulin M (IgM)-involved protein complex that interferes with the uricase method and reduces serum UA measurement accuracy. A total of 24 serum samples were collected from 18 patients, and complete clinical information and laboratory data were obtained. Samples were divided into three groups according to their UA reaction curves. Optical density values were extracted to analyze differential insoluble properties, serum viscosity was measured, coimmunoprecipitation was performed for IgM complex detection, and three additional clinical methodologies were used for UA measurement and comparison. All samples exhibiting unique reaction curves showed simultaneous elevations in IgM and complement component 4 (C4) levels. Circulating IgM formed a protein complex with C4d without significantly increasing serum viscosity. The insolubility of the IgM–C4d complex was attributable to the particular alkaline component of the uricase reagent. Inaccurate UA measurements could only be corrected by mass spectrometry. This study represents the first report of the circulating IgM–C4d protein complex. Given that serum UA serves as a crucial therapeutic indicator for specific patient populations, mass spectrometry is the preferred analytical method for accurate UA quantification in these individuals.

Current Issues in Molecular Biology doi: 10.3390/cimb48070656

Authors: Elica Valkova Vasil Atanasov Kiril Kirilov Kristian Yakimov Yordan Kutsarov

The aim of the present study was to evaluate the bioavailability and associated bioactivity of p-coumaric (p-COA), caffeic (CA), and ferulic (FA) hydroxycinnamic acids (HCAs) isolated from an aqueous extract of plant material. An aqueous extract is more applicable in practice because this form is the most commonly used for oral administration. The p-COA, CA, and FA acids were evaluated for their behavior in the processes of absorption, distribution, metabolism, and excretion (ADME) using modern methods for assessing their functional groups according to Lipinski’s Rule of Five and the Rule of Nines. Given the available data on extensive metabolism of hydroxycinnamic acids during the first pass through the liver, it is necessary to consider an alternative route of administration, namely the sublingual route. Sublingual delivery of exogenous molecules obtained from plant material by extraction may represent a preferable alternative to oral administration, as first-pass hepatic metabolism is bypassed when dosage forms are administered sublingually.

Current Issues in Molecular Biology doi: 10.3390/cimb48070655

Authors: Menglong Xu Yongchao Li Wenqiang Sun Haocheng Guan Tinghui Wu Shuwei Li

Acetaminophen (APAP) overdose is a major cause of drug-induced liver injury and remains a widely used model of xenobiotic-induced hepatotoxicity. Oxidative stress, mitochondrial dysfunction, and ferroptosis are key events in APAP-mediated liver damage. In this study, we investigated whether L-menthol pretreatment protects against APAP-induced acute liver injury and explored the underlying mechanisms in vivo and in vitro. Male C57BL/6 mice were pretreated with L-menthol (100 mg/kg/day) for 7 days before APAP challenge (300 mg/kg). L-menthol markedly attenuated hepatic necrosis, inflammatory infiltration, and hepatocyte injury, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, suppressed IL-1β, IL-6, and TNF-α production, restored hepatic glutathione and superoxide dismutase levels, and decreased malondialdehyde accumulation. Transcriptomic analysis revealed significant enrichment of differentially expressed genes in reactive oxygen species- and ferroptosis-related pathways. In APAP-challenged HepG2 cells, L-menthol improved cell viability, preserved mitochondrial ultrastructure, reduced ferrous iron accumulation, was associated with upregulation of Keap1/Nrf2/HO-1/NQO1 pathway-related proteins, and restored GPX4 expression. Collectively, these findings indicate that L-menthol pretreatment attenuates APAP-induced hepatotoxicity, possibly through enhancement of antioxidant defenses and attenuation of ferroptosis-associated changes, supporting its potential as a preventive hepatoprotective small molecule against xenobiotic-induced liver injury.

Current Issues in Molecular Biology doi: 10.3390/cimb48070654

Authors: Dante L. Adorada Encarnación E. Adorada Niroshini Gunasinghe

Charcoal rot, caused by the pathogen Macrophomina, is becoming an increasing challenge in Australia’s northern cropping systems, with few effective management options available. The use of non-indigenous biocontrol agents raises ecological and regulatory concerns, which highlights the need to identify locally adapted microbial antagonists. In this study, indigenous Trichoderma isolates were collected from rhizosphere soils across Queensland and northern New South Wales and characterised using multilocus sequencing (ITS, tef-1α, rpb2) coupled with phylogenetic analysis. Twenty-six isolates were resolved into six species, dominated by T. azevedoi and T. afroharzianum. Dual-culture assays revealed substantial variation in antagonistic capacity, with several isolates achieving >70% inhibition of Macrophomina growth and maintaining consistent performance across pathogen genotypes. Functional screening indicated that enzyme-associated antibiosis was widespread, whereas volatile-mediated inhibition was restricted to a small subset of isolates. These findings demonstrate that biocontrol potential in indigenous Trichoderma populations is highly strain-dependent rather than species-driven. By integrating multilocus identification with functional screening, this study provides a practical framework for selecting locally adapted biocontrol candidates. This work establishes a foundation for developing region-specific biological control strategies and supports a shift toward targeted, strain-level selection for effective management of charcoal rot.

Current Issues in Molecular Biology doi: 10.3390/cimb48070653

Authors: Hideaki Hasegawa Aruma Watanabe Yasuhiro Katahira Izuru Mizoguchi Tatsuo Maeda Junya Mizugami Isao Naguro Hidenori Ichijo Kazutoshi Harada Yukari Okubo Takayuki Yoshimoto

Imiquimod-induced skin inflammation is the most widely used psoriasis mouse model. Although p38 mitogen-activated protein kinase reportedly plays a role in the pathogenesis of psoriatic inflammation, the purpose of one of its upstream activators, apoptosis signal-regulating kinase 1 (ASK1), remains unclear. This study investigated the role of ASK1 and its molecular mechanism in the imiquimod-induced psoriasis model. Compared to wild-type mice, the ASK1 knockout (KO) mouse skin lesion showed a higher clinical score and a thicker epidermis. The mRNA expression of pro-inflammatory cytokines, such as IL-17 and TNF-α, was also higher. Notably, the expression of aryl hydrocarbon receptor (AhR), a sensor for xenobiotic chemicals that is expressed in the skin to strengthen the skin barrier and accelerate terminal differentiation of the epidermis—as well as its downstream molecule CYP1A1, but not NRF2—was increased in the ASK1 KO psoriatic skin lesion. Immunoprecipitation analysis, followed by Western blotting, revealed that ASK1 interacts with AhR in cells transfected with their respective expression vectors, potentially leading to reduced AhR expression. These results suggest that ASK1 negatively regulates the development of the imiquimod-induced mouse psoriasis model by interacting with AhR and presumably antagonizing the AhR-CYP1A1 axis.

Current Issues in Molecular Biology doi: 10.3390/cimb48070652

Authors: Xingxin Cao Xiangkai Cai Mingxue Li Weihua Jin Fengmei Yang Suqin Duan Yanyan Li Zhanlong He

Bisphenol A (BPA) is a common industrial chemical primarily used in the manufacture of plastics, and it has been found in more than 90% of people worldwide. As an endocrine disruptor, BPA can impair reproduction, development, immunity, metabolism, and cognition; it also disturbs immune balance and thus fosters chronic inflammation. A number of population-based studies have indicated a link between environmental BPA exposure and atopic dermatitis (AD). Nevertheless, the detailed molecular pathways connecting BPA to AD remain poorly understood. AD is the leading chronic recurrent inflammatory skin disorder, characterized by severe itching and repeated eczema-like lesions. Its prevalence is roughly 13% among children and 5% among adults, and its global incidence continues to rise, imposing heavy health and economic burdens on societies. To clarify whether and how BPA may promote or worsen AD, we carried out a comprehensive computational study that integrated network toxicology, transcriptomic data, machine learning, molecular docking, and molecular dynamics simulations. From the CTD, ChEMBL, and SwissTargetPrediction databases, we collected 5701 potential BPA targets; from GeneCards and OMIM, we obtained 3270 genes linked to AD. The overlap between these two gene sets gave a group of common candidate genes. Enrichment analyses using GO and KEGG showed that these common genes were significantly overrepresented in the PI3K-Akt signaling pathway, Th17 cell differentiation, and the JAK-STAT signaling pathway—all central to immune and inflammatory regulation. We then built a protein–protein interaction (PPI) network by submitting the common genes to the STRING database and employed Cytoscape to extract hub genes from that network. By integrating human AD transcriptomic profiles with the hub genes and applying two machine learning techniques (LASSO and SVM), we identified six core toxic targets of BPA in AD: TIGIT, JAK3, IL22, S100A8, CCL2, and FCER1G. These six targets fall into two main functional categories: immune dysregulation and inflammatory cell infiltration. Subsequent molecular docking and molecular dynamics simulation experiments confirmed that BPA binds well to all six targets and can form stable complexes with them. Collectively, our findings offer a preliminary experimental foundation for future investigations into the pathogenesis of BPA-induced AD and provide important molecular evidence for understanding how environment–gene interactions contribute to complex inflammatory skin diseases such as AD.

Current Issues in Molecular Biology doi: 10.3390/cimb48070651

Authors: Bernard Kordas Jan Banach Judyta K. Juranek

Type 2 diabetes (T2D) is often accompanied by chronic low-grade inflammation, with altered cytokine balance. Although cytokine profiles have often been compared between patients with T2D and healthy individuals, less is known about how they differ among patients with varying disease duration. The aim of this exploratory study was to compare selected pro-inflammatory and anti-inflammatory cytokines in patients with shorter and longer duration of clinically diagnosed T2D. Anonymized surplus serum samples from 18 patients with T2D were analyzed. Patients were divided into two groups according to disease duration: 1–7 years and 8–16 years post-T2D diagnosis. Serum concentrations of six pro-inflammatory cytokines (IL-1β, IL-5, IL-6, IL-8, TNF-α and IFN-γ), three cytokines with anti-inflammatory or immunoregulatory functions (IL-2, IL-4, IL-10), and pro- and anti-inflammatory ratios were measured. All tests were performed using MAGLUMI X8 (Snibe Diagnostics, Shenzhen, China) high-sensitivity chemiluminescent immunoassay according to the manufacturer’s guidelines. Statistical analysis of the data obtained was performed using GraphPad Prism (Boston, MA, USA). The longer-duration T2D group showed higher median concentrations of several pro-inflammatory cytokines, particularly IL-6, IL-8, TNF-α, and IFN-γ, compared with the shorter-duration group. Several values in the longer-duration group exceeded the assay-specific reference intervals provided by the diagnostic platform. Anti-inflammatory and immunoregulatory cytokines showed less consistent differences between groups. Correlation analysis indicated stronger correlations among pro-inflammatory cytokines than among anti-inflammatory or immunoregulatory cytokines. This cross-sectional study suggests that cytokine profiles may differ between patients with shorter and longer durations of T2D, with a pattern consistent with a more pro-inflammatory profile in the longer-duration group. Because of the small sample size, absence of healthy controls, and limited availability of clinical covariates, these findings should be interpreted as descriptive rather than confirmatory and require validation in larger, longitudinal studies with detailed metabolic characterization.

Current Issues in Molecular Biology doi: 10.3390/cimb48070650

Authors: Muhammad Imran Nosheen Mushtaq Safdar Hussain

Lead (Pb) induces oxidative stress, inflammation, and hepatorenal injury. We evaluated whether fenugreek (Trigonella foenum-graecum) seed powder (200 mg/kg) protects against subchronic Pb-acetate exposure in male albino mice. Sixty mice were randomized to six groups (n = 10): control (G1), fenugreek-only (G2), Pb 150 mg/kg (G3), and three co-exposure groups receiving fenugreek with Pb at 50, 100, and 150 mg/kg (G4–G6), gavaged daily for 8 weeks. LC–DAD–ESI–MS/MS of the seed batch tentatively identified 32 metabolites, dominated by flavonoid C-glycosides, luteolin dihydrogalloyl-glucosyl-pentosyl glucoside (15.90%), vicenin-3 (14.46%), vicenin-2 (9.66%), vicenin-1 (8.80%), kaempferol 7-O-rhamnosyl-glucoside (8.71%), with additional acylated phenolic conjugates. Pb exposure (G3) significantly reduced growth and intake, elevated serum ALT, AST, ALP, urea, and creatinine, raised blood Pb, and produced hepatic necrosis, vacuolation, and inflammation. Molecularly, Pb upregulated Nrf2, HO-1, SCD-1, TNF-α, and IL-6 and suppressed SOD-3. Fenugreek co-treatment attenuated all these changes across the three Pb doses, with greatest effect at the lowest Pb load (G4). Notably, fenugreek co-treatment reduced rather than further increased Nrf2 and HO-1 expression relative to Pb alone, a pattern most consistent with lowering the upstream oxidative stimulus rather than direct induction of these pathways. The seed’s polyphenolic profile—rich in vicenin-type C-glycosides and luteolin and kaempferol derivatives—offers a plausible chemical basis for the antioxidant, anti-inflammatory, and modest Pb-lowering effects observed; however, because whole seed powder was administered and metabolite identifications are tentative, these structure–activity relationships are presented as hypotheses for future bioactivity-guided fractionation rather than as demonstrated mechanisms. These preclinical findings support further investigation of fenugreek as a candidate dietary adjunct against environmental Pb exposure, contingent on protein-level validation, pharmacokinetic characterization, benchmarking against a standard chelator, and bioactivity-guided fractionation.