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.

Current Issues in Molecular Biology doi: 10.3390/cimb48070649

Authors: Olivia Tania Hernández-Hernández Dora María Velázquez-Hernández Ignacio Camacho-Arroyo

Endometriosis is a long-term gynecological condition marked by the growth of endometrial-like tissue outside the uterus, which undergoes proliferation, bleeding, and regeneration. This disease is associated with disrupted steroid hormone signaling, notably progesterone (P4) resistance and estradiol (E2) dominance. P4 resistance has been associated with impaired activation of the progesterone receptor (PR) and reduced transcription of P4 target genes, while elevated E2 levels induce estrogen receptor (ER)-mediated signaling, enhancing estrogen-dependent lesion growth. This hormonal imbalance contributes to a pro-inflammatory microenvironment, chronic pelvic pain, infertility, and enhanced neuroangiogenesis. Emerging evidence indicates that the coordinated regulation of neurotrophins and sex hormones promotes nerve fibers and blood vessel growth and invasion within endometriotic lesions. P4 and E2 have been shown to modulate the expression of key neurotrophins, including nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). This review presents current evidence on the interplay between neurotrophins and ovarian steroids in endometriosis, with a specific focus on their contribution to neuroangiogenesis and pain pathophysiology. The review includes articles in English containing the Medical Subject Headings (MeSH) terms: “endometriosis”, “neurotrophins”, “nerve growth factor”, “brain-derived neurotrophic factor”, “neuroangiogenesis”, “progesterone”, and “estradiol”, found in the PubMed database published between 2000 and 24 May 2026. This review included a range of original research articles, systematic reviews, meta-analyses, prospective observational studies, case–control studies, and review papers, for a total of 122 articles.

Current Issues in Molecular Biology doi: 10.3390/cimb48070648

Authors: Gülistan Öncü Murat Türkoğlu Ali Türkan Hakan Sevinç

Current treatment options for hair loss remain limited. Therefore, this study compared a botanical extract derived from multiple plants with the pharmaceutical agent minoxidil for topical application. The evaluated parameters included inflammatory cytokines (IL-1β, IL-6, TNF-α), growth factors (TGF-β, VEGF, KGF), and 5α-reductase type II (SRD5A2) expression in the human keratinocyte cell line HaCaT, as measured by ELISA. Both the botanical extract and minoxidil reduced IL-6 levels by 21% and 35%, and TNF-α levels by 13% and 35%, respectively. Treatment with the botanical extract and minoxidil increased VEGF expression by 50% and 85%, and KGF by 16% and 31%, respectively, while reducing SRD5A2 expression by 21% and 28%, respectively. Overall, the results of this in vitro study suggest that the botanical extract exhibits a response pattern similar to that of minoxidil, characterized by the suppression of pro-inflammatory cytokines and SRD5A2, along with enhanced expression of growth factors VEGF and KGF in HaCaT cells. These results provide a promising basis for further in vivo studies.

Current Issues in Molecular Biology doi: 10.3390/cimb48070647

Authors: Carolina Rivera Santiago Andrés Quintanar Stephano Hugo A. Barrera Saldaña

Background: Human growth hormone (hGH) deficiency (GHD) is typically treated with daily injections of recombinant human growth hormone (rhGH), which do not fully replicate physiological secretion patterns. This study evaluates a novel approach using synthetic mRNA encoding hGH encapsulated in lipid nanoparticles (LNPs) and designated VTRC-01 to enable endogenous hormone production. Methods: VTRC-01 was administered intramuscularly to hypophysectomized (Hypox) prepubertal Wistar rats, and its efficacy was compared with rhGH. A cohort of healthy rats was included to assess anabolic effects and safety. Results: VTRC-01 stimulated longitudinal growth in both Hypox and healthy rats, achieving effects comparable to rhGH. Treatment induced a significant anabolic response that exceeded the basal growth rate of healthy controls. Conclusions: These findings provide proof-of-concept for hGH mRNA-based therapy as a promising alternative to rhGH. Further improvements in mRNA and LNP technologies are expected to enhance safe hormone production. These promising results underscore the potential of reprogramming via therapeutic mRNA the synthesis of key endocrine regulators (such as hGH) directly within the organism, offering for the first time a powerful pathway for the potential treatment for endocrine therapies targeting growth hormone deficiency.

Current Issues in Molecular Biology doi: 10.3390/cimb48070646

Authors: Kristina Stavrakeva Elisaveta Apostolova Vesela Kokova Ivica Dimov Mariya Choneva Delyan Delev Ilia Kostadinov Ilia Bivolarski Maria Koleva Rumen Mladenov Plamen Stoyanov Anelia Bivolarska

Plant-derived compounds have recently attracted considerable scientific attention due to their potential therapeutic applications, which are largely attributed to their antioxidant properties. Tert-butyl hydroperoxide (t-BHP) is a potent inducer of intracellular oxidative stress, generating reactive free radicals, which significantly contribute to hepatic and renal damage. Micromeria frivaldszkyana (M. frivaldszkyana), a Bulgarian endemic species, contains high levels of phenolic compounds, including linarin, rosmarinic acid (RA), chlorogenic acid, rutin, quercetin, naringenin, and apigenin. In this study, male Wistar rats received oral treatment for 5 days comprising saline, 250, 400, or 500 mg/kg of M. frivaldszkyana methanolic extract, 100 mg/kg RA, or 125 mg/kg silymarin. On the final day, 0.5 mmol/kg of t-BHP was injected intraperitoneally, and blood and liver tissue samples were collected 18 h later for biochemical and histological analysis. Liver and kidney function was evaluated using biochemical markers (alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea, creatinine (Cr), uric acid (UA)), indicators of oxidative stress (malondialdehyde (MDA), 8-hydroxy-2′-deoxyguanosine (8-OHdG), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT)), and histopathology. Exposure to t-BHP resulted in significant hepatic and renal damage, including elevated serum markers, increased lipid peroxidation, and deoxyribonucleic acid (DNA) damage. Administration of 500 mg/kg M. frivaldszkyana markedly lowered elevated serum ALT and AST levels. The extract also significantly mitigated t-BHP-induced increases in serum Cr and UA. However, no significant increase in the levels of the antioxidant enzymes SOD and CAT or in GSH was observed at all tested doses. Malondialdehyde and 8-OHdG levels increased markedly following t-BHP exposure, whereas pretreatment with M. frivaldszkyana at all tested doses significantly ameliorated these oxidative alterations. These findings suggest that the methanolic extract of M. frivaldszkyana confers protective effects against t-BHP-induced toxicity, potentially through stabilisation of cell membranes, inhibition of lipid peroxidation, and reduction in DNA damage. The extract may therefore serve as a potential natural therapeutic agent against injuries caused by oxidative stress.

Current Issues in Molecular Biology doi: 10.3390/cimb48070645

Authors: Xiaobing Zhang Huiyu Li Jiaxin Zhao Jiawen Tang Xiaoqing Li Pengjing Li Qingyun Zhao Qi Wang Wei Zou

The second most prevalent neurodegenerative illness in the world, Parkinson’s disease (PD), currently has no viable treatments. Although it is yet unknown if mitochondrial dysfunction is an initial event or evolves as a result of neurodegeneration, it is thought to be a crucial component of Parkinson’s disease etiology. From the perspective of mitochondrial quality control (MQC), which includes PINK1/Parkin-mediated mitophagy, mitochondrial dynamics, and mitochondrial proteostasis, this article examines mitochondrial dysfunction. Together, these processes preserve mitochondrial homeostasis and prevent the buildup of damaged mitochondria. Dysfunctional mitochondria gradually build up and cause oxidative stress and aberrant cellular signaling when mitochondrial quality control is compromised. According to available data, mitochondrial reactive oxygen species (mtROS) primarily worsen pre-existing mitochondrial damage by encouraging α-synuclein aggregation, cardiolipin remodeling, and dopamine oxidation. In addition, innate immune pathways like cGAS–STING and TLR9 signaling can be triggered by mitochondrial damage-associated molecular patterns (mtDAMPs), especially mitochondrial DNA, which can lead to long-term neuroinflammatory reactions in PD. While new research suggests that m6A RNA modification may be involved in the regulation of mitochondrial stress, the PINK1/Parkin pathway is crucial for maintaining mitochondrial homeostasis. Therapeutic approaches that target mitophagy augmentation, neuroinflammatory signaling, and mitochondrial protection have garnered increasing attention. In an attempt to improve mitochondrial function and lessen persistent neuroinflammatory activation, future research will probably need to concentrate on combination treatment techniques.

Current Issues in Molecular Biology doi: 10.3390/cimb48070644

Authors: Anton V. Tyurin Bulat I. Yalaev Karina E. Akhiiarova Ilmira I. Galina Jie Li Rita I. Khusainova

Osteoarthritis (OA) and osteoporosis (OP) are prevalent conditions with a complex relationship, yet their shared epigenetic mechanisms remain poorly understood. While genes like DKK1, RHOJ, and SOX6 have been implicated in both diseases, the specific role of individual CpG sites has not been fully characterized. We investigated CpG methylation in these genes using bisulfite pyrosequencing of peripheral blood DNA from n = 96 postmenopausal women: n = 24 with comorbid OA and OP, n = 34 with OA, and n = 38 healthy controls. Methylation differences were analyzed using statistical tests and logistic regression. Comorbid patients showed significant hypermethylation at two DKK1 CpG sites compared to the OA-only group (padj = 0.0007 and padj = 0.042). Conversely, one DKK1 site was hypomethylated in the OA-only group relative to controls (padj = 0.03). A regression model combining three DKK1 sites and one SOX6 site demonstrated predictive value for comorbid disease, with an AUC of 0.696. These findings identify site-specific methylation of DKK1 and SOX6 as a molecular signature associated with comorbid OA and OP, offering new insights into their shared etiology.

Current Issues in Molecular Biology doi: 10.3390/cimb48060643

Authors: Siwen Tang Yongwei Li Xi Yu Ying Xiao Tian Zhong

To explore the associations between protein-level ratios (rQLTs) and duodenal ulcer (DU) risk using Mendelian randomization (MR), colocalization, and pathway analysis approaches. A bidirectional MR approach was used to identify molecular targets linking rQLTs with DU, employing the inverse variance weighted (IVW) method for causal estimation. Colocalization analysis ensured the reliability of inferred causal relationships. Gene interaction networks were constructed via STRING, and key regulatory hub-genes were identified through Cytoscape analysis. Significant inverse associations were found between rQLT-ACE2/GGT1 (Angiotensin-converting enzyme 2/γ-glutamyl transpeptidase 1) (IVW, OR (95% CI) = 0.754 (0.674–0.843), adjusted PIVW = 0.0005), and DU risk in the East Asian (Japanese) population. No statistically significant associations were observed in the European population. The findings indicate a genetic inverse association between rQLT-ACE2/GGT1 and DU risk in the East Asian (Japanese) population, while no corresponding association was observed in Europeans. These results provide genetic evidence consistent with a potential association rather than causal inference or biomarker validation. This study does not support conclusions regarding diagnostic or therapeutic utility at this stage.

Current Issues in Molecular Biology doi: 10.3390/cimb48060642

Authors: Yunfeng Shi Sheng Xia

Metformin, widely prescribed for type 2 diabetes mellitus (T2D), has emerged as a systemic immunomodulator with effects that extend far beyond glycemic control. Recent advances in immunometabolism reveal that metformin modulates innate immune responses through coordinated cellular metabolic reprogramming and epigenetic modification, which collectively modulate the functional phenotype of innate immune cells. This narrative review summarizes current evidence regarding the immunomodulatory effects of metformin on the innate immune system, with a focus on immunometabolism and epigenetic regulation. It explores how metformin modulates innate immunity by altering cellular energy sensing, mitochondrial function, and nutrient utilization. Such metabolic changes and alterations further reshape chromatin structure and architecture, as well as transcriptional profiles and programs. Through the regulation of glycolysis, fatty acid oxidation, and histone modification landscapes, metformin regulates the phenotypes of innate immune cells, which can be pro-inflammatory, tolerogenic, or homeostatic. This conceptual framework presents a new understanding of metformin. As well as acting as an anti-inflammatory agent, it may regulate immune memory.

Current Issues in Molecular Biology doi: 10.3390/cimb48060641

Authors: Christos Panagiotis Rigopoulos Ilias Georgakopoulos-Soares Apostolos Zaravinos

Immunotherapy has transformed cancer treatment; however, clinical benefit remains limited to a subset of patients, underscoring the need for robust biomarkers that capture tumor-immune interactions across cancer types. In this study, we performed a comprehensive pan-cancer, multi-omics characterization of the immune checkpoint–related molecules CD70, CD80, and TIGIT to evaluate their diagnostic, prognostic, and immunological relevance. Using integrative analyses of transcriptomic, epigenomic, genomic, pharmacogenomic, and single-cell RNA-sequencing data from The Cancer Genome Atlas and complementary resources, we assessed expression patterns, DNA methylation, somatic mutations, copy number alterations, immune infiltration, tumor stemness, and drug sensitivity. CD70, CD80, and TIGIT were broadly dysregulated across multiple malignancies, with coordinated overexpression particularly evident in kidney renal clear-cell carcinoma. Elevated expression of these immune checkpoints was associated with advanced tumor stage, aggressive molecular subtypes, and unfavorable survival outcomes in selected cancers, including uveal melanoma and renal malignancies. Functional analyses revealed significant associations between checkpoint expression and key oncogenic pathways, including epithelial–mesenchymal transition, apoptosis, and hormone receptor signaling, suggesting links with tumor progression and immune activation states. Immune deconvolution analyses indicated that TIGIT expression is associated with a T-cell–inflamed microenvironment and reduced neutrophil infiltration, while CD80 exhibited methylation-dependent associations with immune cell composition. Genomic and epigenetic alterations were found to correlate with checkpoint expression patterns and immune phenotypes across tumor types. Pharmacogenomic profiling identified associations between checkpoint expression and sensitivity to multiple anticancer agents; however, these findings are based on cell line datasets and should be considered predictive. Single-cell transcriptomic analyses further resolved cell-type–specific expression patterns, distinguishing tumor-intrinsic from immune-restricted expression profiles. Collectively, our findings establish CD70, CD80, and TIGIT as integrative biomarkers of tumor progression, immune contexture, and therapeutic response, providing a rationale for their clinical exploitation in precision immuno-oncology.

Current Issues in Molecular Biology doi: 10.3390/cimb48060640

Authors: Ekaterina A. Istomina Marina P. Slezina Tatyana I. Odintsova

Pathogenesis-related (PR) proteins are crucial for plant defense against pathogen infection. However, the specific role of thaumatin-like proteins (TLPs), which comprise the PR-5 family, in plant immune responses has not been thoroughly investigated. Filipendula ulmaria is a medicinal plant with valuable pharmacological properties, including antimicrobial, anti-inflammatory, gastroprotective, immunomodulatory, and anticancer activities. The structure of the TLP family and its role in the immune system of meadowsweet have not been studied so far. The goal of this study was to analyze in detail the TLP gene family in meadowsweet and explore its response to fungal infection. In the meadowsweet genome, we identified 27 putative TLP genes, examined their structure and location on chromosomes, analyzed cis-regulatory elements in the promoter regions, predicted the structure and physicochemical characteristics of the encoded proteins, and performed a phylogenetic analysis. We also studied the differential expression of TLP genes under Bipolaris sorokiniana infection. Of six differentially expressed genes, three genes were up-regulated 48 h post-infection, suggesting their involvement in defense response to the fungus. The results obtained shed light on the role of the TLP gene family in the immune system of F. ulmaria and form the foundation for the creation of disease-resistant crops in agriculture and the development of bio-based antimicrobials in medicine.

Current Issues in Molecular Biology doi: 10.3390/cimb48060639

Authors: Chris M. Njagi James J. Kelley Nikita Gulati Naman S. Sijwali Andrey Grigoriev

Transposable elements (TEs) are inserted into the genome and may change its properties; those occurring in or near regulatory regions may also alter gene expression. Given the challenges of detecting insertions in short-read sequencing, we analyzed structural variants in polar and brown bear genomes by a reciprocal alignment of one species’ sample genomes to a reference sequence of the other species, thus inferring TE insertion as the other genome’s “deletions”. With this approach, we detected short interspersed elements (SINEs) belonging to the CAN SINE family as dominant fixed TEs. We observed a non-random distribution of CAN SINE insertion positions near both protein- and RNA-coding genes, where TEs often overlap UTRs or occur in their vicinity. In contrast, SINEs avoid coding sequences, suggesting TE insertions that would disrupt such sequences are under purifying selection. We used black bear as an outgroup and determined that most of the CAN SINE insertions in the polar bear genome were derived, since they are not present in black or brown bear, while there is no dominant trend for CAN SINE insertions in brown bear relative to the outgroup. Many of the genes with UTRs affected by CAN SINEs are potentially relevant to the differences between the species (body shape, size, etc.) or to Arctic-adaptation phenotypes such as fur color, metabolism, and the immune system. This supports a model that CAN SINEs have contributed to regulatory evolution in bears and provides further evidence of such events across carnivore genomes in the animal kingdom.

Current Issues in Molecular Biology doi: 10.3390/cimb48060638

Authors: Jessica Walshe Sushanta Kumar Saha

Cyanobacteria are increasingly recognised as photosynthetic chassis for sustainable metabolic engineering because oxygenic photosynthesis generates ATP and NADPH via the photosynthetic electron transport chain, which drive CO2 fixation through the Calvin–Benson–Bassham cycle into carbon intermediates that can be redirected toward engineered heterologous pathways. Their genetic tractability, CO2-fixing capacity, ecological adaptability, and relatively simple cellular organisation make them attractive platforms for developing low-carbon biotechnological processes. This review explores recent progress in engineering cyanobacteria for heterologous pathway construction, critically evaluating genetic tools including transformation methods, genome integration strategies, promoter systems, and CRISPR-based editing, with specific emphasis on challenges of direct relevance to phototrophic chassis: host–pathway metabolic compatibility, precursor supply, cofactor balancing between photosynthetic output and heterologous pathway demand, and achieving genetic stability in polyploid cyanobacterial genomes. The review also addresses key limitations with mechanistic context: metabolic burden from multi-gene pathway expression reduces growth rate and selects against producing cells; polyploidy delays complete chromosomal segregation of engineered constructs; slow photoautotrophic growth constrains volumetric productivity; native regulatory networks resist carbon flux redirection; and cultivation constraints—including light attenuation in dense cultures and mismatches between photosynthetic ATP/NADPH supply and heterologous pathway demand—further limit achievable yields.

Current Issues in Molecular Biology doi: 10.3390/cimb48060637

Authors: Dmitry Sukhov Tatyana Petrova Daria Kruglova Inna Kholoshenko German Romanenko Yuri Utkin Pavel Pantyushenko Dmitry Trofimov Vladimir Korotkiy Ekaterina Barsova Yulia Kirillova

A chimeric RNase A inhibitor (SUMO-RI) was produced by fusing a SUMO domain to the N-terminus of the murine Rnh1 protein. Functional assays demonstrated that SUMO-RI effectively protects RNA from RNase A-mediated degradation under conditions mimicking real-time RT-PCR, with performance comparable to that of commercial RNase inhibitors. The primary advantage of the chimeric design is its improved technological suitability: SUMO-RI exhibits markedly enhanced storage stability relative to the recombinant Rnh1 inhibitor. However, this benefit comes with a trade-off—SUMO fusion reduces thermostability at temperatures above approximately 47 °C. Together, these findings establish SUMO fusion as a rational engineering strategy for RNase inhibitors, offering improved practical handling at the expense of thermal resilience.

Current Issues in Molecular Biology doi: 10.3390/cimb48060636

Authors: Petra Fodor Barbara Zsebik Ferenc Fenyvesi Zsuzsanna Szabó Anna Vass Gábor Halmos

Background: Uveal melanoma is the most common intraocular malignancy in adults with a poor prognosis. Although local therapies are effective, treatment options for advanced disease remain limited. Combination strategies using chemotherapeutic agents and natural compounds, such as quercetin, are in focus for their potential to enhance antitumor efficiency and overcome resistance. Methods: The effects of doxorubicin, quercetin, and their combination were investigated in uveal melanoma cell lines. Cell viability was determined by an MTT assay, and apoptosis and cell cycle distribution by flow cytometry. Invasion assays were performed to evaluate metastatic potential, while modifications in signaling pathways were analyzed by Western blotting and qPCR. Results: Both doxorubicin and quercetin significantly reduced cell viability and induced apoptotic and necrotic cell death. The combination treatment demonstrated additional inhibitory effects in both cell lines, shown by increased SubG1 populations, reduced invasive capacity, and modulation of signaling pathways. Cell cycle analysis indicated treatment-induced growth inhibition. Notably, pathway modifications varied between cell lines, suggesting heterogeneous responses. Conclusions: Quercetin may potentiate certain antitumor effects of doxorubicin in uveal melanoma, particularly by reducing post-treatment invasiveness and modulating certain PI3K/AKT pathway-related proteins. These results support the possibility of quercetin-based combination therapies, although further molecular and in vivo studies are required.

Current Issues in Molecular Biology doi: 10.3390/cimb48060635

Authors: Jai-Sing Yang Kun-Ching Cheng Yu-Hsiu Chuang Ping-Chung Kuo Tian-Shung Wu

An undescribed seco-kaurane diterpenoid, benincaside A (BA), was isolated from the seeds of Benincasa hispida. The seeds of B. hispida have been traditionally used in folk medicine and previous studies have reported anti-tumor potential in B. hispida seed extracts. Accordingly, we investigated the cytotoxicity and underlying mechanisms of BA in colorectal cancer cells. BA inhibited growth in HT29, Colo205, HCT116, and CT26 colorectal cancer cells, as determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while showing no toxicity toward normal human umbilical vein endothelial cells (HUVEC) and human fibroblast WS-1 cells. In HCT116 cells, BA-induced deoxyribonucleic acid (DNA) damage and apoptosis, as evidenced by morphological changes, 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and assays of caspase-8 and caspase-3 activities. BA triggered apoptotic cell death via the extrinsic pathway, as indicated by elevated caspase-8 and caspase-3 activities. Intracellular reactive oxygen species (ROS) generation was observed in BA-treated HCT116 cells. The growth-inhibitory effects were significantly attenuated by pretreatment with N-acetylcysteine (NAC, an antioxidant), caffeine (an ATM kinase inhibitor), z-VAD-fmk (pan-caspase inhibitor), or z-IETD-fmk (caspase-8-specific inhibitor). Colorimetric assays confirmed increased caspase-8 and caspase-3 activities in BA-treated cells. This study is the first to report ROS-dependent signaling as a key mechanism underlying BA-induced cell death in HCT116 human colorectal cancer cells.

Current Issues in Molecular Biology doi: 10.3390/cimb48060634

Authors: Razvan Vlad Opris Dan Alexandru Toc Alina Mihaela Baciu Ioana Alina Colosi Vlad Sever Neculicioiu Anca Onaciu Cristian-Silviu Moldovan Ana-Maria Vlase Carmen Costache Adrian Florea

Invasive candidiasis caused by drug-resistant Candida species represents a critical global health challenge, with few novel therapeutic scaffolds under development. Here, silver nanoparticles were synthesized using a 21-day fermented Chun Mee kombucha tea extract (K-AgNPs) and characterized by UV-Vis spectroscopy, transmission electron microscopy, nanoparticle tracking analysis, and Fourier-transform infrared spectroscopy. LC-MS/MS profiling of the kombucha substrate documented a phytochemical landscape dominated by epigallocatechin (up to 122,631 µg/mL) and epigallocatechin gallate (up to 415 µg/mL), with a progressive ~80% decline in epicatechin and concomitant increases in gallic acid and chlorogenic acid across the 21-day fermentation. K-AgNPs obtained were spherical, 19.4 nm (±7.9 nm SD) in diameter, with a surface plasmon resonance peak at 415 nm. FTIR confirmed phenolic, carboxylate, and glycosidic surface capping. Antifungal susceptibility testing against eight Candida species, including the WHO critical–priority pathogen Candidozyma auris, showed concordant minimum inhibitory and minimum fungicidal concentrations of 0.80–1.60 µg/mL, confirming fungicidal activity. In vivo evaluation in Galleria mellonella larvae across six infection models demonstrated that K-AgNP treatment at the species-specific MIC significantly improved larval survival versus untreated infected controls (p < 0.01–0.001), while nanoparticle-only groups maintained ≥98% survival, indicating negligible toxicity. Co-treatment amplified total hemocyte mobilization, and K-AgNP-only larvae maintained hemocyte viability above 96% at all time points, indistinguishable from negative controls. Together, these findings demonstrate antifungal activity of K-AgNPs across the genus Candida in standardized in vitro and in vivo settings and provide justification for further investigation, including head-to-head comparison against licensed antifungals and physicochemical validation of nanoparticle stability under assay conditions.

Current Issues in Molecular Biology doi: 10.3390/cimb48060633

Authors: Rosalinda Di Gerlando Francesca Dragoni Evelyne Minucchi Maria Garofalo Giulia Perini Alfredo Costa Antonio Pisani Carlo Morasso Matteo Cotta Ramusino Stella Gagliardi

Alzheimer’s disease (AD) and Frontotemporal Dementia (FTD) are complex neurodegenerative disorders, often sharing overlapping symptoms. Non-coding RNAs may be involved in pathological processes in these conditions, hence the study of miRNAs isolated from plasma-derived extracellular vesicles (EVs) could provide exploratory insights into the molecular background. The main aim of this work was to identify shared deregulated miRNAs presenting different expression patterns in the two pathologies. A selection of the identified deregulated miRNAs was further studied with the purpose of identifying their mRNA targets and generating hypotheses on their potential pathological involvement. A total of 340 and 291 differentially expressed miRNAs were found in FTD and AD, respectively. Among the commonly deregulated miRNAs with opposite expression patterns between the two conditions, miR-638 emerged as a candidate of interest, showing consistent patterns across our experimental analyses. Nevertheless, these findings are preliminary and intended to be interpreted cautiously, requiring validation in larger cohorts. In addition, the expression of two of its predicted targets in peripheral blood mononuclear cells (PBMCs) appeared to align with miR-638 expression in the same cell type and may reflect potential differences in underlying brain pathological states.

Current Issues in Molecular Biology doi: 10.3390/cimb48060632

Authors: Priscila Mendes Joana Pinto Carole Mateus Inês Guerra Vanessa Mateus

Background: Ischemic stroke remains a leading cause of mortality and disability worldwide. Despite extensive preclinical research, most neuroprotective strategies have failed to translate into clinical benefit, partly due to methodological variability. The transient intraluminal filament middle cerebral artery occlusion (tifMCAO) model is widely used, yet its implementation lacks consistency. This review aimed to characterize tifMCAO methodologies in adult rats and examine how experimental variability relates to reported outcomes. Methods: A systematic review was conducted following PRISMA guidelines. Studies using tifMCAO in adult rats were included. MEDLINE (PubMed), Web of Science, and Scopus were searched up to March 2025. Risk of bias was assessed using the SYRCLE tool and reporting quality using the ARRIVE checklist. The protocol was registered in PROSPERO (CRD420251140869). Results were synthesized narratively. Results: A total of 125 studies were included. A commonly used framework involved male Sprague–Dawley rats (6–12 weeks), silicone-coated monofilaments, occlusion durations of 60–120 min (most frequently 90 min), and isoflurane anesthesia, although this reflects methodological convergence rather than true standardization. Substantial variability was observed across methodological parameters. Variations in ischemia duration, filament properties, and anesthesia were associated with differences in infarct size, blood–brain barrier disruption, and functional outcomes. Conclusions: The tifMCAO model shows partial methodological convergence alongside significant variability influencing outcomes. Improved standardization and reporting are essential to enhance reproducibility and translational relevance.

Current Issues in Molecular Biology doi: 10.3390/cimb48060631

Authors: Iasmin V. Costa Ana Cecília R. Cruz Carlos Alberto M. Carvalho

Dengue is an arboviral disease of global significance caused by Orthoflavivirus denguei (DENV), which has four antigenically distinct serotypes. The envelope (E) protein plays a critical role in viral entry and eliciting immune responses. This study aimed to compare the biochemical and immunological properties of the E protein across the four DENV serotypes using in silico approaches. E protein reference sequences were retrieved from RefSeq and analyzed with various bioinformatics tools. Sequence alignment revealed identities ranging from 63.08% to 77.69%. Biochemical analysis showed minimal variation in molecular weight and isoelectric point; however, the net charge of DENV-3 E protein was notably lower. Secondary structure predictions indicated a predominance of alpha-helices in DENVs-1/2, while DENVs-3/4 featured more beta-sheets. Post-translational modification analysis revealed mostly casein kinase II phosphorylation sites across all serotypes, with DENV-4 uniquely presenting also tyrosine kinase sites. Amino acids W231/D341 in DENV-1, Q86 in DENVs-2/4, and D87/D339 in DENV-3 showed maximum antigenicity scores in B cell recognition, while the human leukocyte antigen (HLA) alleles B*08:01/B*39:01 and DRB4*01:01, recognized by T cells, presented the highest number of predicted epitopes for the different DENV serotypes. Conservation analysis showed that the major antigenic regions highlighted in this study are highly conserved among contemporary DENV isolates despite the genetic variability observed within each serotype. These findings suggest that subtle structural differences in the E protein may contribute to distinct immunogenic profiles, highlighting candidate regions for future investigation.

Current Issues in Molecular Biology doi: 10.3390/cimb48060630

Authors: Yağmur Dilber Erhan Dilber Kübra Yıldız Domaniç

Methyl methacrylate (MMA)-based materials are widely used in temporary and permanent prosthetic dentistry; the prolonged presence of these materials in the oral cavity and potential residual monomer release can affect local biological responses. This study aimed to evaluate the biocompatibility and toxicity profiles of MMA, the monomeric unit of polymethyl methacrylate (PMMA), a key component of dental materials used in temporary prosthetic restorations. Molecular docking simulations were performed using CB-Dock2 and Autodock vina, while protein–protein interaction (PPI) analysis was performed using STRING and Cytoscape. In addition, Swiss ADME Target Prediction, toxicity prediction, and enrichment analyses were used to characterize the biological significance of selected targets in more detail. Molecular docking studies revealed promising interactions of MMA with valuable biomolecular targets relevant to biocompatibility. The toxicity profile revealed aspects of MMA that could be improved. Pharmacophore modeling, highlighting the importance of carbonyl and hydroxyl groups as pharmacophoric properties, revealed compounds with suitable biocompatibility profiles. Consequently, it emphasizes the interactions of MMA with biomolecules and safety considerations. It can guide the design and optimization of biocompatible materials as an exploratory avenue for future developments in dental biomaterials.

Current Issues in Molecular Biology doi: 10.3390/cimb48060629

Authors: Amelia Tero-Vescan Ruxandra Ștefănescu Amalia Pușcaș Mădălina Buț Bianca-Eugenia Ősz Mark Slevin

Inflammaging is defined as chronic low-grade inflammation associated with aging and is increasingly recognized as a dynamic and mechanistically driven biological process rather than a state adequately described by circulating biomarkers alone. Traditional inflammatory markers alone, including interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and C-reactive Protein (CRP), fail to capture the complexity, tissue specificity, and causal architecture of inflammaging. Recent experimental evidence has demonstrated that diverse upstream drivers, including immunosenescence, gut microbiome dysbiosis, metabolic dysfunction, and cellular senescence, converge on a limited number of central inflammatory hubs, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, GMP–AMP synthase–stimulator of interferon genes (cGAS–STING), Janus kinase/signal transducer and activator of transcription (JAK/STAT), and p38 mitogen-activated protein kinase (p38 MAPK) signaling. These mechanistic nodes represent promising therapeutic targets, potentially modifiable biological processes, and support the emerging concept of ‘druggable inflammaging’, whereby senotherapeutics, inflammasome inhibitors, innate immune modulators, and metabolic interventions may actively modify aging-associated inflammatory biology rather than simply monitor it through biomarkers. This review highlights a paradigm shift from biomarker-based assessment toward mechanism-based intervention, where inflammaging can be characterized as a modifiable biological process and a central target for precision pharmacological strategies in aging-related diseases.

Current Issues in Molecular Biology doi: 10.3390/cimb48060627

Authors: David Aebisher Klaudia Dynarowicz Rostyslav Marunych Izabela Rudy Kacper Rogóż Aleksandra Kawczyk-Krupka Piotr Oleś Dorota Bartusik-Aebisher

Glioblastoma (GBM) remains one of the most aggressive malignancies, characterized by profound heterogeneity and a dismal prognosis. While genomic and transcriptomic profiling have provided structural insights, they often fail to capture the dynamic interactions within the tumor microenvironment (TME). Secretome analysis—the study of proteins actively secreted by tumor cells—offers a functional readout of these interactions and a reservoir for potential biomarkers. In this review, we critically evaluate the role of MALDI-TOF Mass Spectrometry as a strategic tool for GBM secretome profiling. While Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) remains the gold standard for deep protein discovery, we argue that MALDI-TOF’s speed, cost-effectiveness, and high-throughput capabilities position it as an ideal platform for clinical screening and “spectral phenotyping.” We discuss the technical hurdles, such as ion suppression and the “leakome” (intracellular contamination), and highlight how integrating MALDI with Extracellular Vesicle (EV) enrichment and Artificial Intelligence (AI) can bridge the gap between in vitro discovery and clinical application.

Current Issues in Molecular Biology doi: 10.3390/cimb48060628

Authors: Javier Azúa-Romeo Maria Cabetas Irene Rodriguez Bárbara Angulo Arantxa Andueza

Background: Lung cancer is a highly heterogeneous disease in which molecular characterization has become essential for guiding personalized therapies. The implementation of next-generation sequencing (NGS) allows for the simultaneous detection of multiple genomic alterations, improving tumor profiling and therapeutic decision-making. This study aimed to characterize the molecular landscape of lung cancer using NGS and to evaluate its association with histological subtypes and programmed death-ligand 1 (PD-L1) expression. Methods: A retrospective observational study was conducted on 96 patients diagnosed with lung cancer between 2023 and 2025. Molecular profiling was performed using the Action OncoKitDx panel. Associations between genetic alterations, histological subtypes, and PD-L1 expression were analyzed using Fisher’s exact test, with p < 0.05 considered statistically significant. Results: Adenocarcinoma was the most common histological subtype (67.7%), followed by squamous cell carcinoma (26%). The most common mutations were KRAS (34.4%), TP53 (29.2%), and EGFR (11.5%). KRAS mutations were significantly associated with adenocarcinoma (p = 0.001), while squamous cell carcinoma showed a higher frequency of cases without molecular alterations detected by the NGS panel (p = 0.002). Co-mutations were identified in 22.9% of cases, with KRAS–TP53 being the most common combination. Tumors harboring EGFR mutations showed a significantly lower frequency of co-mutations (p = 0.012). No significant associations were found between PD-L1 expression and either histological subtypes or the analyzed genetic alterations. Conclusions: Lung cancer exhibits marked molecular heterogeneity, with a predominance of KRAS mutations in adenocarcinoma. The low frequency of co-mutations in EGFR-mutated tumors supports their role as dominant driver alterations. The lack of association between PD-L1 expression and genomic alterations highlights the complexity of its regulation and suggests the involvement of multiple biological factors. These findings reinforce the clinical value of NGS in comprehensive tumor profiling and in the development of precision medicine strategies.

Current Issues in Molecular Biology doi: 10.3390/cimb48060626

Authors: Violeta Popovici Emma Adriana Ozon Manuela Apetrei Rodica Boca Cerasela Elena Gîrd

Human papillomavirus (HPV) has become a leading cause of oropharyngeal cancers, alongside well-known risk factors such as tobacco and alcohol use. Currently, HPV-positive oropharyngeal squamous cell carcinoma (HPV+ OPSCC) has increased significantly in developed countries, with HPV-16 being the most common high-risk subtype. Clinically, HPV+ OPSCC shows clear differences in prognosis compared to HPV-negative tumors, particularly regarding survival rates and treatment responses. Patients with HPV+ OPSCC tend to have notably better survival outcomes and a more favorable outlook. Strong evidence indicates that HPV-related oropharyngeal cancers represent a distinct epidemiological, clinical, and molecular group, setting them apart from non-HPV-related cancers. As a result, treatment strategies for these subtypes should follow specific clinical protocols to optimize outcomes. Additionally, the viral oncoproteins E6 and E7, which systematically disrupt host tumor-suppressor networks, provide strong reasons for targeted phytotherapeutic interventions. Therefore, there is increasing interest in exploring plant bioactive compounds with promising anti-HPV and anticancer effects that target key oncogenic pathways. This review aims to compile the latest data on bioactive phytochemicals with mechanistic evidence in HPV+ OPSCC, highlight their molecular interactions across oncogenic signaling pathways, and discuss evidence-based findings focusing on research published from 2000 to 2025.

Current Issues in Molecular Biology doi: 10.3390/cimb48060625

Authors: Massimo Iorizzo

Microbial α-L-rhamnosidases are increasingly recognised as selective biocatalysts in food biotechnology, nutraceutical production, and health-related applications. These glycoside hydrolases catalyse the hydrolysis of terminal alpha-L-rhamnose residues from flavonoids, terpenoids, saponins, and other glycosylated natural products, thereby modulating sensory properties, solubility, intestinal absorption, and biological activity. While their traditional uses include debittering citrus juice and enhancing wine aroma, recent evidence demonstrates their wider value in selective flavonoid biotransformation, production of rare mono-glycosylated derivatives, probiotic fermentations, and microbiome-associated metabolism. This review summarises microbial sources, catalytic mechanisms, CAZy classification, substrate specificity, structure–function relationships, analytical methods, industrial process engineering, and emerging applications in functional foods and targeted nutraceutical applications. Particular attention is given to the distinction between alpha-(1→2)- and alpha-(1→6)-linked substrates, the production of isoquercitrin and prunin, recombinant enzyme platforms, immobilised biocatalysts, and potential future opportunities arising from metagenomics, synthetic biology, and AI-assisted protein engineering.

Current Issues in Molecular Biology doi: 10.3390/cimb48060624

Authors: Bruna Freitas Marchi Vittoria de Lima Camandona Athirson Moraes Chanavat Gustavo Roncoli Reigado Carla Roberta de Oliveira Carvalho Felipe Santiago Chambergo Viviane Abreu Nunes

Type 2 diabetes mellitus (T2D) is associated with dyslipidemia, characterized by elevated plasmatic triglycerides and free fatty acids, particularly palmitate (PA), which may cause lipotoxicity in skeletal muscle cells. This leads to inflammation, activation of the unfolded protein response (UPR), insulin resistance, and cell death. Herbal medicines such as Uncaria tomentosa (UT) have shown potential as complementary treatments for T2D due to their protective effects. Purpose and study design: This study investigates the effect of UT aqueous extract on UPR and insulin resistance induced by PA in C2C12 myotubes. C2C12 myoblasts were grown in DMEM medium supplemented with 10% fetal bovine serum and differentiated into myotubes with 3.5% horse serum. The myotubes were incubated with 100 or 500 μM PA, 2–100 µM thapsigargin (Tg) or tunicamycin (Tn), in the presence or absence of 250 μg/mL UT extract or 100 µM TUDCA, for 2 or 6 h. The myotubes treated with UT extract for 6 h, after the incubation with 20 µM Tg, Tn or 500 µM PA, presented reduction in the expression of UPR-related genes ATF4 and CHOP by approximately 1.5-fold, and increased by 3-fold the expression of IRS-1, an insulin-signaling protein, when compared to myotubes incubated with only 20 µM Tg, Tn or 500 µM PA. These findings suggest that UT extract may serve as a modulator against skeletal muscle dyslipidemia by downregulating ATF4 and CHOP, reducing cell stress and death, while enhancing IRS-1 expression, which supports the use of the UT extract in managing insulin resistance and T2D.

Current Issues in Molecular Biology doi: 10.3390/cimb48060623

Authors: Ahmet Şengül Dilek Kaan Hatice Güler Hüseyin Yiğit

The study aimed to investigate the individual therapeutic effects of melatonin and umbilical cord-derived mesenchymal stem cell exosomes (UC-MSC-Exo) separately on BE(2)-C neuroblastoma cells. Melatonin is recognized for its anti-cancer, antioxidant, and apoptosis-inducing properties, and its ability to cross the blood–brain barrier. UC-MSC-Exos are nanovesicles from mesenchymal stem cells that can also cross the blood–brain barrier and transport biologically active molecules. The potential therapeutic benefits of each independent agent in treating BE(2)-C neuroblastoma cells were investigated. Melatonin and UC-MSC-Exos were examined on BE(2)-C neuroblastoma cells at varying concentrations and time intervals to evaluate cell viability and apoptosis. Both melatonin and UC-MSC-Exo independently reduced cell viability and induced apoptosis in a manner that depended on the dosage and duration of exposure. Melatonin had an IC50 of 2.68 mM after 24 h, while UC-MSC-Exo showed an IC50 of 25.3 μg/mL after 48 h, with no cytotoxic effects observed at 24 h. Specifically, individual concentrations of 2.5 mM and 5 mM of melatonin, as well as 50 µg/mL and 100 µg/mL of UC-MSC-Exo, led to significant levels of apoptotic and necrotic cells at 48 and 72 h (p < 0.001). Our findings suggest that the individual administration of melatonin and UC-MSC-Exo may hold therapeutic potential for neuroblastoma cells, particularly given their ability to cross the blood–brain barrier. Further in vivo research is required to evaluate their clinical utility.

Current Issues in Molecular Biology doi: 10.3390/cimb48060622

Authors: Ashwag Altayyar Li Liao

Disease comorbidity—the co-occurrence of two or more diseases in the same individual—has gained growing attention due to its association with adverse clinical outcomes and increased treatment complexity. Recent subgraph-based approaches for disease comorbidity prediction model disease modules as subgraphs induced by disease-associated genes in the protein–protein interaction (PPI) network and learn disease representations from subgraph topology. However, these approaches are constrained by incomplete disease–gene annotations, which may obscure important molecular relationships between diseases. Accordingly, disease comorbidity may also be influenced by molecular events beyond annotated disease genes, such as gene fusion events that have emerged as important contributors to disease mechanisms. Motivated by the role of gene fusions in disease development, we introduce Gene Fusion-Initiated Path Propagation for Disease Comorbidity Prediction (FuDiCo), a framework that models comorbidity through influence propagation over the PPI network. FuDiCo represents fusion-associated genes as localized perturbation sources and learns how their influence propagates along interaction paths toward disease subgraphs, thereby capturing propagation patterns that link related diseases and contribute to their comorbidity. Experiments on a benchmark disease comorbidity dataset show that FuDiCo outperforms state-of-the-art methods, achieving statistically significant improvements. These results shed light on the importance of gene fusion events in understanding disease relationships.

Current Issues in Molecular Biology doi: 10.3390/cimb48060621

Authors: Faris Alrumaihi

Ubiquitination, a crucial cellular protein regulation process, is linked to various diseases, including cancer. Deubiquitinases (DUBs) can reverse ubiquitination, offering a therapeutic strategy. USP7, a DUB, is a key target in oncology due to its role in destabilizing p53, and small-molecule inhibitors could restore p53 activity and combat tumor growth. In this study, we integrated a machine learning (ML)-based screening approach with molecular docking and molecular dynamics (MD) simulations in order to identify potential small-molecule inhibitors of USP7. ML-based screening identified 22 active molecules from a library of 2301 natural compounds. Among the 22 active compounds, only fifteen compounds fulfilled the drug-likeness criteria. Subsequently, molecular docking found three compounds, PubChem 162957515, 114917, and 442879 as potential inhibitors based on binding affinity and interactions. Further, MD simulations and MM-PBSA analyses were performed to evaluate the stability and dynamic behavior of the complexes. Binding energy calculations Molecular Mechanics Poisson–Boltzmann Surface Area (MM-PBSA) revealed that compounds PubChem 114917 and 162957515 exhibited strong binding affinities of −20.98 kcal/mol and −18.68 kcal/mol, respectively, implying that these compounds could serve as promising inhibitors for the development of anticancer therapeutics.

Current Issues in Molecular Biology doi: 10.3390/cimb48060620

Authors: Satoyasu Ito Eri Manabe Toshiyuki Shikata Kojiro Takamoto Shuhei Kobuchi

Hypertension remains a major driver of multi-organ damage, including cardiac remodeling and hepatic complications. The ketone body β-hydroxybutyrate (BHB) has emerged as a potential metabolic signaling molecule with anti-inflammatory properties. This study investigated whether BHB attenuates cardiac stress and hepatic injury in a salt-sensitive hypertensive model. Dahl salt-sensitive (DS) rats were fed a high-salt (HS) diet combined with a choline-deficient diet to induce cardiac inflammation and hepatic fibrosis. Rats received either BHB or a control vehicle. We found that BHB significantly suppressed hepatic lipid accumulation and fibrotic markers, including TGF-β and collagen III mRNA, even under severe dietary stress. In the heart, BHB attenuated the expression of inflammatory markers (TNF-α and ANP) despite the persistence of high systolic blood pressure. These results demonstrate that BHB exerts direct organ-protective effects through anti-inflammatory and anti-fibrotic actions that are independent of robust blood pressure reduction. Our findings suggest that BHB could be a promising metabolic intervention for managing multi-organ complications in hypertensive patients with metabolic comorbidities.

Current Issues in Molecular Biology doi: 10.3390/cimb48060619

Authors: Menghui Yang Xiaochuan Ma Konglin Zhou Sheng Lin Jianming Chen Zhenyue Lin

The genus Orthogalumna (Oribatida: Galumnidae) has been recognized for its phytophagous associations with aquatic plants, particularly its potential role in the biocontrol of the invasive weed Water hyacinth (Eichhornia crassipes). Despite its ecological significance, this genus remains poorly studied in terms of its micromorphological architecture, phylogenetics, and genomic resources. In this study, we report Orthogalumna cf. terebrantis from China, providing the first comprehensive characterization of an Orthogalumna species by integrating morphology, phylogeny, and transcriptomics. This record represents the first documented occurrence of O. cf. terebrantis in China, pending confirmation by voucher-based morphological comparison and molecular data. This work provides critical microstructural evidence to complement traditional morphological identification and establishes a foundational molecular dataset for future studies on the systematics, comparative genomics, and environmental adaptation of oribatid mites.

Current Issues in Molecular Biology doi: 10.3390/cimb48060618

Authors: Awal Prasetyo Dora Maftikhati Levina Athaya Anarizta Nazhira Ghina Setyawan Anindha Waradita Putri Yuwono Maria Meutia Saleha Farahdita Ramadhanti Annisa Mukti Hermawan Istiadi Udadi Sadhana Fathur Nur Kholis

This study compared the temporal effects of traditional cigarettes and e-cigarettes on lung health in male Rattus norvegicus over 8- and 12-week periods. Thirty rats were evaluated for tracheal/alveolar histopathology and systemic markers (IL-6, TNF-α, SOD-3, MDA). Chronic cigarette exposure (12 weeks) and nicotine aerosol (8 weeks) significantly suppressed weight gain, while ascorbic acid aerosol caused less growth inhibition. At 8 weeks, cigarette exposure (K3) induced adaptive tracheal mucosal thickening (66.88 ± 17.92 µm vs. 52.40 ± 2.63 µm in control K1), increased goblet cells (4.2 ± 2.44 N/mm), elevated SOD-3 (12.75 ± 1.10 pg/mL), and initiated emphysematous alveolar expansion (469.77 ± 91.31 µm vs. 202.03 ± 29.38 µm in K1 in K1). Conversely, 12-week cigarette smoke (K4) triggered epithelial exhaustion, significantly thinning the tracheal mucosa (34.65 ± 6.55 µm) and elevating systemic IL-6 (11.45 ± 1.17 pg/mL vs. 8.43 ± 0.88 pg/mL in control K2). Notably, chronic electronic ascorbic acid aerosolization (K6) preserved localized alveolar structural layouts and limited septal thickening compared with nicotine groups. However, it failed to suppress systemic inflammation, as evidenced by elevated IL-6 levels. In conclusion, while ascorbic acid aerosols moderate localized parenchymal destruction compared to nicotine, chronic aerosol exposure accelerates systemic immune activation.

Current Issues in Molecular Biology doi: 10.3390/cimb48060617

Authors: Zhi-Qiang Mu Xiao Zhang Jing-Jing Yan Bao-Ping Chen Hong-Wei Wang

To investigate the codon usage bias (CUB) and its influencing factors in the chloroplast genome of Styphnolobium japonicum f. oligophyllum, we sequenced, assembled and annotated the genome using Illumina high-throughput sequencing, and systematically analyzed 52 protein-coding sequences. The chloroplast genome is 158,739 bp with a typical quadripartite structure, containing 129 functional genes. It presents a mean GC3 content of 28.26% and a mean ENC value of 45.40, indicating weak CUB and low gene expression. Among 31 preferred codons (RSCU > 1), 29 (93.5%) end with A/U. Neutrality plot, ENC-plot and PR2-plot analyses reveal that natural selection is the primary regulator of CUB. A total of 19 optimal codons were identified. These results provide fundamental reference data that may facilitate future genetic engineering efforts in S. japonicum f. oligophyllum.

Current Issues in Molecular Biology doi: 10.3390/cimb48060616

Authors: Yue Sun Zhaorui Xu Yanxia Wu Mingxu Zhang Xuejing Lu

Photoreceptor (PR) degeneration is a shared pathological feature of multiple blinding retinal diseases. This narrative review examines the mechanisms underlying PR vulnerability to ferroptosis-associated lipid-peroxidation injury, with emphasis on three interconnected features: the marked enrichment of docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) in PR outer-segment disc membranes; the chronically high metabolic demand of PRs and the specialized spatial organization of their mitochondria; and retinal pigment epithelium (RPE)–PR metabolic coupling, including outer-segment renewal and phagocytic turnover, glucose transport and lactate shuttling, and visual-cycle–related all-trans-retinal (atRAL) clearance and bisretinoid accumulation. We also summarize antioxidant defense systems centered on the cystine/glutamate antiporter (xCT)–glutathione (GSH)–glutathione peroxidase 4 (GPX4) axis and mitochondrial GPX4 (mtGPX4), which restricts iron-dependent lipid peroxidation in PRs. We propose that highly oxidizable membrane phospholipid substrates, mitochondrial homeostatic imbalance, and impaired RPE–PR metabolic coupling may collectively shape PR susceptibility to ferroptosis-associated injury. From a therapeutic perspective, this framework supports multitarget strategies designed to interrupt lipid-peroxidation propagation, stabilize mitochondrial redox homeostasis and quality-control mechanisms, and restore RPE–PR metabolic support and local iron-buffering capacity.

Current Issues in Molecular Biology doi: 10.3390/cimb48060615

Authors: Dexian Wei Yuan Zhang Yanlin Wu Liqun Ren Qing He

Triple-negative breast cancer (TNBC), characterized by the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression, is a highly aggressive molecular subtype with high recurrence and metastasis rates. Due to the absence of reliable molecular targets, surgery combined with chemotherapy remains the mainstay of clinical treatment. In recent years, immunotherapy has provided new strategies for TNBC management. Immune checkpoints are key regulatory molecules that maintain immune homeostasis, and blocking these checkpoints can restore T cell activity and enhance tumor cell killing. Immune checkpoint inhibitors (ICIs) have demonstrated clinical benefit, particularly in combination with chemotherapy for patients with locally advanced or metastatic TNBC. This review focuses on immune checkpoint–based immunotherapy in TNBC, providing an overview from mechanistic insights to clinical applications and emerging therapeutic strategies. In addition to ICIs, we discuss alternative approaches, such as bispecific antibodies, antibody–drug conjugates (ADCs), chimeric antigen receptor T cell (CAR-T) therapy, tumor vaccines, and oncolytic viruses (OVs), highlighting their current research progress and clinical applications in TNBC treatment.

Current Issues in Molecular Biology doi: 10.3390/cimb48060614

Authors: Ming Lei Cui Li Jing Wang Mei Qin Li-Rong Huang Xia-Lian Ou Liang Kang Han Liu Zhan-Jiang Zhang

Corydalis ophiocarpa is a medicinally valuable plant, noted for its abundant alkaloid content. Despite its significance, the mitochondrial (mt) genome of this plant has not been characterized, which impedes both the phylogenetic understanding within the Corydalis genus and the comprehension of its full genetic potential. In this research, we successfully assembled the complete mitogenome of C. ophiocarpa by employing a hybrid method that integrates Oxford Nanopore long reads with Illumina short reads. The assembled genome forms a circular structure of 600,064 bp, with a GC content of 46.49%, and includes 63 genes, comprising 40 unique protein-coding genes (PCGs), 20 tRNAs, and three rRNAs. Through assembly and coverage analysis, we identified a 6383 bp forward repeat associated with a contig having approximately double the depth, indicating a repeat-mediated multipartite structure where the main circle may coexist with two smaller subgenomic forms. We discovered 775 C-to-U RNA editing sites across the 40 PCGs, with 95.4% being non-synonymous and favoring hydrophobic amino acid substitutions, particularly in Complex I subunits. Furthermore, we identified sixteen mt plastid DNA fragments constituting 2.43% of the mitogenome, a proportion more than double that found in the closely related C. saxicola. Phylogenetic analysis confirms that C. ophiocarpa is most closely related to C. saxicola, with C. pauciovulata as another close relative. This study presents the first complete mitogenome of C. ophiocarpa, providing a genomic basis for investigating the relationships between mt genome structure, post-transcriptional regulation, and specialized metabolism in the Corydalis genus.

Current Issues in Molecular Biology doi: 10.3390/cimb48060613

Authors: Tamara Drljača Vladimir Perović Nevena Veljković Branislava Gemović

Background: The population of Serbia faces a significant burden from cardiovascular diseases (CVDs). This study aimed to computationally investigate genetic factors that contribute to the prevalence of these diseases by examining the possible involvement of common variants on lipid metabolism. Methods: We examined how a variant prevalent in the Serbian population, chr7:g.56019730G>A in the PSPH gene, affects the phosphoserine phosphatase (PSP) protein interaction network, particularly involved in lipid metabolism. The Informational Spectrum Method (ISM), method for the analysis of protein sequence based on amplitude changes, was applied to single out the top 10 affected interactors. Their further functional annotation identified the pathways in which they jointly participate with PSP. An additional strategy encompassed the investigation of variant combinations in all analyzed genes and potential relevance of prevalent variant combinations on lipid metabolism. Results: The PSP interactions affected by the R49W variant, such as SHMT1/2, were primarily in pathways associated with serine, glycine, and sphingolipid metabolism, highly relevant for CVD etiology. Further, we identified frequent variant combinations within the LRCH1, CEP126, PIK3CG, and PIKFYVE genes in the Serbian cohort. Conclusions: This study underscores the importance of investigating genetic variant combinations in complex diseases, and provides a hypothesis generating foundation for future research into the relationship between these genes and cardiovascular diseases.

Current Issues in Molecular Biology doi: 10.3390/cimb48060612

Authors: Feliciana Menna Stefano Lupo Laura De Luca Antonio Baldascino Enzo Maria Vingolo Alessandro Meduri

Mitochondrial dysfunction and oxidative stress are increasingly recognized as key contributors to the development and progression of retinal degenerative diseases, including age-related macular degeneration and inherited retinal dystrophies. Growing evidence suggests that alterations in mitochondrial function, excessive production of reactive oxygen species, defective mitophagy, and chronic inflammatory responses are closely interconnected processes that contribute to retinal cell damage and degeneration. This review provides an overview of the current understanding of the molecular mechanisms linking mitochondrial dysfunction to retinal degeneration, with particular emphasis on the impact of oxidative stress, mitochondrial quality-control pathways, and inflammatory signaling. Available evidence indicates that mitochondrial DNA damage, impaired bioenergetics, and dysregulated mitochondrial dynamics play a crucial role in the degeneration of photoreceptors and retinal pigment epithelium cells. In turn, oxidative stress further exacerbates mitochondrial impairment, creating a self-sustaining cycle that promotes disease progression. Recent advances have also highlighted the therapeutic potential of targeting mitochondrial pathways. Although several mitochondria-directed strategies have shown encouraging results in experimental models, their translation into clinical practice remains at an early stage. Overall, the available data identify mitochondria as a promising therapeutic target and support the development of precision medicine approaches aimed at preserving retinal function and slowing disease progression in patients with retinal degenerative disorders.

Current Issues in Molecular Biology doi: 10.3390/cimb48060611

Authors: Maryam N. ALNasser Wayne G. Carter

Excitotoxicity caused by excessive activation of glutamate receptors, particularly N-methyl-D-aspartate receptors (NMDARs), significantly contributes to neuronal damage in neurodegenerative diseases (NDDs), such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. This systematic review aimed to evaluate the effects of plant extracts and phytochemicals on NMDAR-mediated excitotoxicity and to summarize their proposed neuroprotective mechanisms. The review protocol was registered in PROSPERO (CRD42024528160). A systematic search of Medline, Embase, Web of Science Core Collection, and PubMed identified 323 records, with an additional 7 records identified through manual searching that specifically considered in vitro and in vivo inhibitors of NMDAR excitotoxicity using plant extracts and isolated phytochemicals. Twenty-seven studies demonstrated that plant extracts and phytochemicals attenuate excitotoxicity through multiple mechanisms, including inhibition of NMDAR-induced currents, reduction of intracellular calcium influx, modulation of NMDAR expression, attenuation of oxidative stress, and mitochondrial dysfunction. However, the evidence base was largely dominated by in vitro and ex vivo studies, with limited in vivo validation, restricting translational relevance. Risk-of-bias assessment using an adapted version of the Office of Health Assessment and Translation (OHAT) Risk of Bias Tool indicated that 4 studies had a low overall risk of bias, 12 had low to moderate risk, and 11 were at moderate risk, with key limitations related to inadequate reporting of blinding, randomization, and allocation concealment. In contrast, exposure characterization, outcome assessment, and confounding control were generally strong across studies. Although the findings support the mechanistic neuroprotective potential of certain plant extracts and phytochemicals against NMDAR-mediated excitotoxicity, further well-designed in vivo and clinical studies are required to establish their therapeutic relevance for the treatment of neurodegenerative diseases.

Current Issues in Molecular Biology doi: 10.3390/cimb48060610

Authors: Lillian Schneider Maria Ruano Camryn R. Mackey Kiersten Spiegel Renee A. Bouley Ruben C. Petreaca Ryan J. Yoder

Cannabinoids, including the psychoactive D9-tetrahydrocannabinol (THC) and the non-psychoactive cannabidiol (CBD), interact with receptors within the endocannabinoid system. The major receptors within this system are CNR1 (cannabinoid receptor 1) and CNR2 (cannabinoid receptor 2), which are both seven-transmembrane G-protein-coupled receptors. In this report, we used the Catalogue of Somatic Mutations in Cancers (COSMIC) to map and analyze mutations arising in CNR1 and CNR2. The goal was to determine if any trends or signatures could be identified. We identified several mutations in both CNR1 and CNR2. In silico 3D structure of proteins reveals that these mutations cluster on the intracellular regions of CNR1 and CNR2, and certain residues may be able to destabilize the interaction with the G-alpha protein due to their close proximity. mRNA expression showed that CNR1 and CNR2 are within normal expression levels in most cancer types except kidney, where there is a tendency towards over-expression. Neither CNR1 nor CNR2 is a driver gene, and our analysis shows that mutations in cancer cells are deactivating (e.g., loss of function).

Current Issues in Molecular Biology doi: 10.3390/cimb48060609

Authors: Abdulkadir Kankilic Ibrahim Basar Selim Karahan Ulas Alabalik Revsa Evin Canpolat Erkan Omer Karakoyun Ismail Yildiz Mehmet Yigit Akgun Ozkan Ates Meral Erdinc

This study investigated the effects of different doses of ranolazine in a middle cerebral artery occlusion/reperfusion (MCAO-I/R) model by evaluating histopathological changes and serum Sirtuin 1 (SIRT1), Apela peptide (APELA), and Apelin-13 (APL13) levels. A total of 47 male Sprague Dawley rats (250 ± 20 g) were randomly assigned to five groups: Sham (n = 7), MCAO (n = 10), MCAO+RAN10 (n = 10), MCAO+RAN30 (n = 10), and MCAO+RAN50 (n = 10). MCAO-I/R was induced by transient filament occlusion of the right middle cerebral artery for 90 min followed by reperfusion. Ranolazine was administered intraperitoneally once daily for 21 days in the treatment groups. Serum SIRT1, APELA, and APL13 levels were measured using enzyme-linked immunosorbent assay (ELISA), and brain tissues were evaluated histopathologically for neuronal degeneration and apoptotic cell counts. Histopathological analysis revealed significant neuronal degeneration and increased apoptosis in the MCAO group compared with the Sham group. Ranolazine treatment did not demonstrate significant histopathological improvement compared with the untreated MCAO group. Among the treatment groups, the MCAO+RAN50 group showed higher apoptotic cell counts and lower serum biomarker levels than the other ranolazine-treated groups. Serum SIRT1, APELA, and APL13 levels were lowest in the MCAO+RAN50 group, with selected pairwise differences reaching statistical significance. Under the present experimental conditions, clear evidence of neuroprotection could not be demonstrated. None of the ranolazine-treated groups showed significant histopathological improvement compared with the untreated MCAO group. These findings indicate that higher-dose ranolazine was not associated with neuroprotection under the conditions of this study. However, given the limited sample size, absence of infarct volume analysis, lack of neurological functional assessment, and absence of tissue-level molecular validation, further studies are required to clarify the biological significance and potential clinical relevance of the observed biomarker changes.

Current Issues in Molecular Biology doi: 10.3390/cimb48060608

Authors: Yuanyuan Bai Kangzhen Chen Taojie Yao Shengbo Shi Hongmei Duan Peng Hao Wen Zhao Yudan Gao Xiaoguang Li Zhaoyang Yang

Temporal lobe epilepsy (TLE) is a common drug-resistant epilepsy characterized by recurrent seizures, cognitive impairment, aberrant adult hippocampal neurogenesis, inhibitory circuit disruption, and persistent inflammatory remodeling. Current anti-seizure medications primarily offer symptomatic control and do not target the progressive structural and functional deterioration of epileptic hippocampal networks. Here, we investigated whether local nerve growth factor (NGF)-hydrogel delivery during the latent phase after status epilepticus could mitigate hippocampal pathological remodeling and improve long-term outcomes in a kainic acid (KA)-induced mouse model (utilizing C57BL/6J and Nestin-CreERT2 mice). Animals were randomly assigned to three groups: the saline control group, the untreated KA epilepsy group, and the KA + NGF-hydrogel treatment group. NGF-hydrogel was administered into hippocampal Cornu Ammonis 1 (CA1) beginning 3 days post-kainic acid and repeated every 15 days. Histological, immunofluorescence, circuit-tracing, electrophysiology, electroencephalography (EEG), and behavioral assessments were used to evaluate neurogenesis, microenvironment, circuit readouts, seizure burden, and cognition. NGF-hydrogel treatment was associated with preserved dentate gyrus neural stem cell populations, improved newborn granule cell localization and maturation, attenuated neuroinflammation and gliosis, and partial recovery of inhibitory interneuron markers. These changes were accompanied by improved hippocampal circuit readouts, reduced chronic spontaneous seizure burden, and enhanced recognition and spatial memory. Our findings indicate that local NGF-hydrogel delivery following status epilepticus is associated with improved hippocampal remodeling and functional outcomes, and suggest that biomaterial-based neurotrophic support may be a promising strategy for providing targeted neuroprotection and facilitating excitatory/inhibitory (E/I) balance reconstruction in the epileptic hippocampus.

Current Issues in Molecular Biology doi: 10.3390/cimb48060607

Authors: Zhiyun Cheng Ruyu Bai Yong Diao

Venous thromboembolism (VTE) is a major cause of morbidity and mortality, underscoring the need for new molecular markers to enable early detection and clarify underlying mechanisms. Iron metabolism is linked to oxidative stress, endothelial injury, and inflammation, all central to thrombosis, yet its transcriptomic contribution to VTE remains unclear. We analyzed gene expression profiles from GSE19151 and GSE48000 using differential expression and weighted gene co-expression network analysis (WGCNA), integrating results with an iron metabolism gene set. Three hub genes were identified, arachidonate 5-lipoxygenase (ALOX5), Rho GTPase activating protein 1 (ARHGAP1), and glucose-6-phosphate dehydrogenase (G6PD), all downregulated in VTE. Gene set enrichment indicated that ALOX5 is involved in endothelial regulation, lipid metabolism, and immune pathways. A three-gene signature showed high diagnostic accuracy (AUC = 0.924 in the discovery cohort; 0.705 in validation). Immune deconvolution revealed broad immune remodeling and associated ALOX5 with multiple immune cell subsets, especially M0 macrophages, and with regulators such as TGFB1 and IL6R. Western blot analysis further showed that ALOX5 protein expression was significantly increased in LPS-activated HUVECs, supporting its involvement in inflammatory endothelial injury. DrugBank screening identified 19 approved drugs targeting ALOX5, supporting its potential for mechanistic and clinical investigation.

Current Issues in Molecular Biology doi: 10.3390/cimb48060606

Authors: Ruoyu Wang Jie Li Yu Zhai Qin Song Pengyu Xia Bowen Jiang Minghang Chen Minghan Liu Changqing Li

Atrophic fracture nonunion is a clinically challenging form of failed bone repair, particularly under inflammatory conditions, but the cell-intrinsic programs that impair the function of skeletal stem/progenitor cells (SSPCs) remain incompletely defined. Here, we integrated public and in-house single-cell RNA sequencing datasets from mouse periosteum, normal fracture healing, and inflammation-associated fracture nonunion models to characterize stromal cell fate changes. Trajectory inference, transcription factor network analysis, and intercellular communication modeling were combined with in vitro and in vivo validation experiments. SSPCs in the nonunion microenvironment were arrested in an undifferentiated state and acquired a pro-inflammatory and pro-ferroptotic phenotype, with enrichment of ferroptosis-related genes including Acsl4. Computational analyses nominated IL-1β as a candidate upstream inflammatory signal, with neutrophils representing a potential source, and linked this signal to NF-κB activation and increased Epas1 activity in SSPCs. In primary SSPCs, IL-1β induced lipid peroxidation, intracellular ferrous iron accumulation, ferroptosis-related protein expression, and impaired osteochondrogenic differentiation. Ferroptosis inhibitor treatment further attenuated IL-1β-induced ferroptosis-related protein changes, supporting pathway specificity. Pharmacological inhibition of EPAS1 with PT2385 attenuated IL-1β-induced ferroptotic stress and restored SSPC differentiation in vitro, while also improving IL-1β-impaired fracture repair in vivo. Mendelian randomization analysis provided additional genetic evidence supporting potential links among IL-1β, EPAS1, and human nonunion risk. Together, these findings suggest that an IL-1β/EPAS1-associated ferroptotic program contributes to SSPC dysfunction during inflammation-associated fracture nonunion and may represent a potential targetable mechanism for improving impaired bone repair.

Current Issues in Molecular Biology doi: 10.3390/cimb48060605

Authors: Sonia Hussain Georg Jander

Bemisia tabaci (whitefly; Hemiptera: Aleyrodidae), a complex of morphologically similar but genetically distinct species, causes enormous agricultural damage worldwide. Farmers incur billions of dollars in losses each year from whiteflies, both through direct feeding damage and from the transmission of numerous plant viruses. Important crops that are heavily damaged by whiteflies include tomato, eggplant, cucumber, cotton, cucurbits, beans, and cassava. The global invasiveness and persistence of B. tabaci are largely attributed to its exceptional biological traits. Understanding these traits is essential for developing effective, long-term pest management strategies. This review describes in detail how the basic biology studies of B. tabaci provide a foundation for developing pest management strategies. Specifically, we discuss: (1) insights into the development of insecticide resistance can guide resistance management strategies; (2) knowledge of natural enemies supports the advancement of biological control approaches; and (3) understanding plant–insect interactions reveals molecular targets for innovative pest management solutions. We also examine emerging research trends and offer future perspectives on how ongoing studies may drive the development of next-generation control strategies (RNA interference, clustered regularly interspaced short palindromic repeats—CRISPR-associated protein 9 (CRISPR-Cas9), and horizontally transferred genes as targets).

Current Issues in Molecular Biology doi: 10.3390/cimb48060604

Authors: Tsutomu Arakawa

Proteins are extensively used for research, food processing and pharmaceuticals [...]

Current Issues in Molecular Biology doi: 10.3390/cimb48060603

Authors: Giorgia Francesca Saraceno Gessica Bonavota Emilia Furia Erika Cione Paola Tucci

A critical yet frequently overlooked factor is the tumor’s metabolic profile. Diabetes and chronic moderate hyperglycemia are known risk factors for triple-negative breast cancer (TNBC) that do not respond to hormonal therapy. So, identifying novel therapeutic targets and developing more effective treatments is needed. One of the key pathways involved in the aggressive nature of TNBC is the Toll-like receptor 4 (TLR4) signaling cascade. To this end, curcumin (CUR) has shown effects consistent with modulating inflammatory stress by inhibiting TLR4/MD-2. This study evaluated CUR at concentrations observed in the bloodstream (0.025–25 ng/mL) in MDA-MB-231 TNBC cells under different glucose conditions (normal, moderate, and severe hyperglycemia) and inflammatory states (LPS-induced), using cell viability assays and molecular docking. A zinc complex (Zn–CUR) was also used. Results were validated through cell viability assays. Under severe hyperglycemia, CUR unexpectedly increased cell viability in a dose-dependent manner, while Zn–CUR had no activity across all glucose levels. In LPS-induced inflammation, CUR exhibited a biphasic, dose-dependent response, being protective at mid-level doses but cytotoxic at higher doses, whereas Zn–CUR showed more consistent effects, consistent with modulation of inflammatory stress. Molecular docking suggests that Zn–CUR binds more stably within the MD-2 hydrophobic pocket than CUR, particularly when bound to LPS, with binding energies of −8.7 and −8.3 kcal/mol, respectively. However, better in silico affinity did not always translate into improved cellular effects. These findings indicate that metabolic context significantly influences CUR’s biological activity and that forming a zinc complex offers a safer, more reliable profile. This positions Zn–CUR as a candidate warranting further investigation for TNBC, particularly in the context of hyperglycemia.

Current Issues in Molecular Biology doi: 10.3390/cimb48060602

Authors: Shanyu Li Xinjie Zhang Leiming Cao Yang Zhou Ruitong Zhang Lisi Jiang Wei Fu

Glycerophosphate diester phosphodiesterase (GDPD) catalyzes the decomposition of glycerophosphate diester into sn-glycerol-3-phosphate and corresponding alcohols. In this study, six GDPD genes were identified in the cucumber genome, named CsGDPD1 to CsGDPD6, and distributed on chromosomes 1, 3, 4, 5, 6, and 7. All six proteins exhibited similar predicted three dimensional structures, suggesting conserved biochemical functions. Phylogenetic and dN/dS selection pressure analyses revealed that CsGDPD genes are evolutionarily close to their Arabidopsis homologs and have evolved under purifying selection, indicating functional conservation. Synteny analysis identified five collinear gene pairs between cucumber and Arabidopsis, but no synteny with rice. Promoter cis-acting element analysis showed the presence of multiple stress- and hormone-responsive elements. Tissue-specific expression profiling demonstrated that CsGDPD1, CsGDPD2, and CsGDPD6 are broadly expressed across tissues, whereas CsGDPD4 and CsGDPD5 show preferential expression in reproductive organs. qRT-PCR under drought and salt stress, with or without the plant growth promoting rhizobacterium GD17, revealed that drought alone upregulates all CsGDPD genes; PGPR-GD17 alone (+PGPR) suppresses their expression; and combined PGPR + Drought leads to synergistic suppression. Under salt stress, CsGDPD5 was dramatically upregulated (20-fold), and PGPR-GD17 partially reversed salt induced changes. These results provide a comprehensive foundation for understanding the evolutionary and functional roles of the GDPD gene family in cucumber stress responses.

Current Issues in Molecular Biology doi: 10.3390/cimb48060601

Authors: Maria-Alexandra Martu Stefan-Lucian Burlea Silvia Martu Sorina-Mihaela Solomon Ionut Luchian Liliana Pasarin Ioana Martu Mihaela Salceanu Elena-Odette Luca Diana-Maria Anton Diana Tatarciuc Irina-Georgeta Sufaru

Periodontitis and type 2 diabetes mellitus (T2DM) are linked through systemic inflammation and endothelial dysfunction, yet it remains uncertain whether periodontal inflammatory burden independently reflects early, multi-organ microvascular vulnerability beyond glycemic exposure. This study aimed to assess the independent association between periodontal inflammatory burden, measured by PISA, and retinal microvascular impairment on OCT-A, and to examine relationships with renal trajectories, small-fiber neuropathy, and inflammatory/endothelial biomarkers. This cross-sectional observational study included 285 never-smoking adults with T2DM. The primary outcome was a pre-specified OCT-A microvascular impairment composite. Secondary outcomes included eGFR slope and log(UACR) slope, corneal nerve fiber length (CNFL), and a multi-organ microvascular burden score. Biomarkers comprised hsCRP, IL-6, sICAM-1, sVCAM-1, sE-selectin, PAI-1, angiopoietin-2 (Ang-2), and vWF:Ag. Multivariable linear regression estimated associations per 1 SD higher PISA, adjusting for age, sex, diabetes duration, HbA1c, CGM time in range, CGM coefficient of variation, systolic blood pressure, LDL cholesterol, BMI, and medication classes (SGLT2 inhibitors, GLP-1 receptor agonists, ACEi/ARB, statins). False discovery rate (FDR) control (q = 0.10) was applied for secondary endpoints. Higher PISA was independently associated with worse OCT-A microvascular impairment (adjusted β = 0.138, 95% CI 0.061–0.216; p = 0.0005). Although statistically significant, the effect sizes were modest in magnitude, and their translation into clinically meaningful differences in microvascular outcomes warrants investigation in prospective settings. Higher PISA was also associated with greater multi-organ microvascular burden (β = 0.101, 95% CI 0.040–0.163; p = 0.0014; FDR q = 0.005) and lower CNFL (β = −0.224, 95% CI −0.397 to −0.052; p = 0.0113; FDR q = 0.023). PISA was associated with higher levels of inflammatory and endothelial activation/injury biomarkers (all FDR q < 0.10). In this cross-sectional study, periodontal inflammatory burden was independently associated with quantitative retinal microvascular impairment, lower corneal nerve fiber length, and a consistent pattern of endothelial activation biomarker elevations in never-smoking adults with T2DM. The clinical significance of the observed effect sizes requires further evaluation, and longitudinal studies are needed to establish temporality.

Current Issues in Molecular Biology doi: 10.3390/cimb48060600

Authors: Feifei Li Yue Zou Tong Li Lingling Xie Dandan Liu Kunhong Song Yanting Luo Dan Qin Youjin Hao Bo Li

Phylogenetic reconstruction is a multi-step process that typically involves sequence retrieval, alignment, trimming, and tree inference, often requiring the integration of multiple independent tools. This fragmented workflow increases technical complexity and limits reproducibility, particularly in large-scale analyses. Here, we present phyloPipeR, an R package that provides an integrated and automated framework for end-to-end phylogenetic analysis and tree comparison within a unified environment. The phyloPipeR enables complete workflows from ortholog retrieval to tree inference and quantitative comparison, while also supporting modular execution of individual steps. The package implements multiple phylogenetic inference methods and supports both concatenation and coalescent strategies for multi-gene analyses. By integrating tree reconstruction and quantitative comparison within a single framework, phyloPipeR improves reproducibility, reduces technical barriers, and provides a scalable solution for systematic and integrative evolutionary studies.

Current Issues in Molecular Biology doi: 10.3390/cimb48060598

Authors: Mabel Gethsemani Jaimes-Gonzalez Roberto Montes-de-Oca-Jimenez Martha Elba Ruiz-Riva-Palacio Gabriel Arteaga-Troncoso Jorge Pablo Acosta-Dibarrat Pilar Eliana Rivadeneira-Barreiro Pablo Cleomenes Zambrano-Rodriguez Dan Israel Zavala-Vargas Siomar de Castro Soares Victor Augusto Sallum Ceballos Pedro Sanchez-Aparicio Vasco Ariston de Carvalho Azevedo

Pathogenicity islands (PAIs) are regions of bacterial genomes that harbor genes encoding virulence factors. Identifying molecules that enhance pathogenicity is crucial for understanding the mechanisms pathogens employ to cause disease and their evolution. Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a pathogenic microorganism that causes caseous lymphadenitis (CLA) in sheep and goats. Despite its prevalence in Mexico, its genetic material has not been analyzed for virulence factors acquired through horizontal gene transfer. Therefore, the aim of this study was to characterize the complete genomes of Mexican C. pseudotuberculosis strains and identify virulence-related genes harbored with PAIs. Seventeen strains of C.pseudotuberculosis biovar ovis isolated from Mexico were whole-genome sequenced using illumina technology, assembled de novo with SPAdes, and annotated using Prokka. PAIs were predicted with GIPSy based on genomic signatures associated with horizontal gene transfer, including G + C deviation, codon usage, virulence factors, transposases, and tRNA-flanking regions. Positive selection was assessed using POTION v1.2 by identifying orthologous groups enriched in non-synonymous substitutions. This represents the first comprehensive PAI analysis of Mexican C. pseudotuberculosis strains, identifying 14 putative pathogenicity islands harboring 51 virulence-associated genes. Additionally, positive selection analysis identified five coding sequences, including radA and rpiB, that are undergoing adaptive evolutionary changes. These findings elucidate the pathogenic mechanisms and genomic plasticity of Mexican C. pseudotuberculosis strains. They also highlight novel genetic targets for vaccine and therapeutic development against CLA.

Current Issues in Molecular Biology doi: 10.3390/cimb48060599

Authors: Tiago Azevedo Jessica Silva Maria J. Pires Mário Ginja Tiane C. Finimundy Maria J. Neuparth Manuela Matos Lillian Barros Paula A. Oliveira Ana I. Faustino-Rocha

Background: Cardiotoxicity remains a major concern in breast cancer management, with implications for prognosis and quality of life. Natural compounds have shown chemopreventive potential while preserving cardiac safety. This study evaluated the cardiac effects of a Santolina chamaecyparissus aqueous extract (SCE), characterized by high levels of 1,3-O-dicaffeoylquinic acid and myricetin-O-glucuronide, in female Wistar rats subjected to N-methyl-N-nitrosourea (MNU)-induced mammary carcinogenesis. Methods: Twenty-eight animals were assigned to four groups (n = 7/group): control (CTRL), MNU-induced (IND), SCE-supplemented (SCE), and MNU-induced SCE-supplemented (SCE+IND). SCE was administered in water (120 µg/mL) for 20 weeks, and MNU was injected intraperitoneally (50 mg/kg) at 50 days of age. At the end of the experiment, body and heart weights were recorded, creatine kinase MB (CK-MB) concentrations assessed, and echocardiography performed to evaluate cardiac structure and function. Results: Final body and relative heart weights did not differ among groups. CK-MB was lower in SCE-supplemented groups compared with CTRL and IND (p < 0.05). IND animals exhibited a hyperdynamic functional profile accompanied by impaired ventricular filling, which was attenuated in SCE+IND animals (p < 0.05). Cardiac structural parameters were largely preserved with SCE, despite an increased left ventricular mass (p < 0.05). Conclusions: SCE did not induce adverse cardiac effects and partially mitigated early carcinogenesis-associated cardiac alterations under the tested conditions, supporting its cardiac safety as a potential phenolic-rich chemopreventive strategy.

Current Issues in Molecular Biology doi: 10.3390/cimb48060597

Authors: Zijia Cui Hua Wu Zhicheng Yang Bohao Xu Fan Jiang Rien Lai Lu Han Ciding Lu Dandan Li Kehui Zheng

Tieguanyin (Camellia sinensis cv. Tieguanyin) is an important oolong tea cultivar in China, and heat stress has become a major environmental constraint affecting its growth and productivity. Calmodulin-binding transcription activators (CAMTAs) are important transcription factors involved in calcium/calmodulin-mediated signaling and plant responses to environmental stresses. However, systematic knowledge of the CAMTA gene family in Tieguanyin remains limited. In this study, 20 CsCAMTA genes were identified from the Tieguanyin genome and characterized based on their physicochemical properties, phylogenetic relationships, conserved motifs, gene structures, chromosomal distribution, collinearity, promoter cis-acting elements, and functional annotation. The 20 CsCAMTA genes were unevenly distributed across eight chromosomes, and collinearity analysis suggested that segmental duplication may have contributed to the expansion of this gene family. Conserved motif and domain analyses indicated that CsCAMTA proteins retained typical structural features of CAMTA transcription factors, including CG-1, ANKYR, TIG, and CaMBD/IQ-related regions. Promoter analysis showed that CsCAMTA genes harbored multiple cis-acting elements related to hormone responsiveness, stress response, light response, and growth regulation. Furthermore, qRT-PCR analysis of 18 representative CsCAMTA genes under 40 °C heat treatment revealed distinct temporal expression patterns, suggesting that different CsCAMTA members may respond to heat stress at different stages. Several genes, such as CsCAMTA2, CsCAMTA10, and CsCAMTA16, showed marked transcriptional changes and may represent candidate heat-responsive genes in Tieguanyin. These results provide a systematic overview of the CsCAMTA gene family and lay a foundation for further functional studies of heat stress responses in Tieguanyin.

Current Issues in Molecular Biology doi: 10.3390/cimb48060596

Authors: Quan Zhao Peian Zhang Yang Song Fayong Li Yingyao Liu Jun Chen Dongfeng Liu

Ougan (Citrus suavissima Hort. et Tanaka) is valued for its distinctive sweet–bitter flavor and nutritional properties; however, tissue-resolved metabolic differences between two cultivar forms (seeded and seedless) of Ougan (C. suavissima) remains poorly understood. In this study, a comprehensive UPLC-MS/MS-based metabolomic analysis was conducted on peel (SP and NP), pulp (SF and NF), segment membrane (SM and NM) and seed tissues (SS, from seeded fruit only) of seeded and seedless Ougan fruits. A total of 1333 metabolites were annotated, with flavonoids (48.53%) and phenolics (12.25%) representing the predominant compound classes. Tissues specificity was the primary determinant of metabolic variation, with peel and segment membrane tissue showing relatively high abundance (fold change ≥ 2, |Log2FC| ≥ 1) of phenylpropanoid- and flavonoid-derived metabolites. Comparative analysis between seeded and seedless tissues revealed significant modulation of phenylpropanoid biosynthesis, flavonoid biosynthesis, phenylalanine metabolism, and related secondary metabolite pathways. Seeded tissues showed a higher relative abundance of selected flavonol glycosides (6-hydroxykaempferol-3,6-O-diglucoside), hydroxycinnamic acid derivatives, and santhocyanin-related compounds, whereas seedless tissues showed higher relative abundance of selected flavanones and malonylated flavonoid glycosides. Seeds were characterized by high limonin content, consistent with limonoid-associated bitterness chemistry. Overall, our findings provide a tissue-resolved metabolomic framework for understanding quality-associated secondary metabolite variation in mature Ougan fruit.

Current Issues in Molecular Biology doi: 10.3390/cimb48060595

Authors: Xiaoxuan Pan Xin Chen Chunyuan Zhang Xin Ma Jieru Han

This review argues that protein lactylation—a lactate-driven posttranslational modification—serves as the long-sought molecular bridge that coordinates these two hallmarks in gastric cancer (GC). Far from being a passive metabolic byproduct, lactylation operates as a central molecular hub with a dual function: intracellularly, it directly drives malignant phenotypes by modifying key oncoproteins such as YAP and metabolic enzymes; extracellularly, it remodels the tumor immune microenvironment by polarizing tumor-associated macrophages toward an immunosuppressive M2 phenotype, upregulating PD-L1 expression, and impairing CD8+ T-cell function. We propose that these two arms constitute a self-reinforcing metabolic–epigenetic–immunological circuit, wherein lactylation both originates from and perpetuates the Warburg effect, creating a vicious cycle that sustains malignancy and immune evasion. This framework positions lactylation not merely as a mechanistic detail, but as a unifying principle that integrates metabolic reprogramming, epigenetic regulation, and immune suppression in GC. We critically evaluate the current landscape of lactylation “writers,” “erasers,” and “readers”; highlight the translational potential of targeting this pathway; and identify the conceptual and technical bottlenecks that must be overcome—including the lack of causality in current studies, the absence of specific research tools, and the unresolved heterogeneity of lactylation across cell types and disease stages. By reframing lactylation as an actionable hub rather than a downstream consequence, this review provides a roadmap for advancing lactylation-based precision medicine in GC.

Current Issues in Molecular Biology doi: 10.3390/cimb48060594

Authors: Fumihiro Nishimura Manabu Sasada Fumio Fukai Kyoko Imanaka-Yoshida Akihiro Sudo Masahiro Hasegawa

Osteoarthritis (OA) is highly prevalent worldwide. Fibronectin (FN) has been associated with OA pathology; however, its role remains unexplored. In this study, we hypothesized that FNIII14 induces chondrocyte apoptosis by inactivating β1 integrin and aimed to clarify the role of FNIII14 in OA pathology. Immunohistochemistry, immunofluorescence, flow cytometry, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay, a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, real-time quantitative polymerase chain reaction, and Western blotting were performed using cartilage obtained from patients who underwent total knee arthroplasty. In a mouse model, FNIII14 or phosphate-buffered saline was administered to the knees, and cartilage degeneration and synovitis were evaluated using the Mankin and Synovitis scores, respectively. Statistical significance was determined using the Mann–Whitney U or Kruskal–Wallis test (p < 0.05). FNIII14 was detected in highly degenerated OA cartilage, with decreased β1 integrin activity, suppressed cell proliferation, and induced apoptosis in chondrocytes. FNIII14 decreased the gene expression of cartilage-specific markers and anabolic factors and increased inflammatory cytokine gene expression and phosphorylation of extracellular signal-regulated protein kinase-1/2. FNIII14 induced cartilage degeneration in mouse knees with almost no synovitis. Regulation of FNIII14 production and action may play an important role in suppressing cartilage degeneration in human OA.

Current Issues in Molecular Biology doi: 10.3390/cimb48060593

Authors: Yihan Wang Mengchen Lv Ying Li

Cadmium (Cd) is one of the most toxic heavy metals in the environment, and it severely represses photosynthesis, growth, development and nutrient uptake in photosynthetic organisms. Excessive cadmium (Cd) taken up by plants seriously threatens global food security and human health. Therefore, designing an eco-friendly and sustainable strategy that can reduce the accumulation of Cd in plants is a major challenge. Phosphorus (P), as an essential nutrient for plant growth, has been shown to play a pivotal role in mediating Cd-induced stress response. However, the molecular mechanisms underlying the crosstalk between phosphate signaling and Cd stress response remain largely uncharacterized, especially the role of the core phosphate homeostasis regulator Phosphate Starvation Response 1 (PSR1). Here, we used the model green microalga Chlamydomonas reinhardtii to investigate the physiological and transcriptomic responses to Cd stress in wild type (WT, CC-125) and PSR1 loss-of-function mutant (Crpsr1, CC-4267). Our results showed that the Crpsr1 mutant exhibited significantly enhanced Cd tolerance compared with WT under P-sufficient conditions, with a better growth phenotype and a significantly lower Cd accumulation. Transcriptome analysis revealed distinct gene expression profiles between WT and the Crpsr1 mutant in response to Cd treatment. Gene Ontology (GO) enrichment analysis showed that differentially expressed genes (DEGs) were mainly involved in primary metabolism, protein kinase activity, ion binding and transmembrane transport, which are critical processes for mitigating Cd stress. Notably, key genes associated with iron uptake and homeostasis were significantly upregulated in the Crpsr1 mutant under Cd stress, indicating a potential regulatory link between PSR1, iron homeostasis and Cd tolerance. Taken together, our findings establish a functional association between the central phosphate signaling regulator PSR1 and Cd stress response in green microalgae, and provide novel candidate genes and regulatory networks for developing engineered microalgae with enhanced Cd phytoremediation capacity.

Current Issues in Molecular Biology doi: 10.3390/cimb48060592

Authors: Pei-Yi Chen Je-Wen Liou Ming-Jiuan Wu Jui-Hung Yen

Disruption of cholesterol homeostasis is closely associated with hypercholesterolemia, dyslipidemia, atherosclerotic cardiovascular disease (ASCVD), and metabolic disorders such as metabolic dysfunction-associated fatty liver disease (MAFLD). Intestinal and hepatic cholesterol absorption are central to maintaining systemic cholesterol balance, with Niemann–Pick C1-Like 1 (NPC1L1) acting as a key transporter that mediates cholesterol uptake in enterocytes and hepatocytes. Aberrant NPC1L1 expression or activity promotes excessive cholesterol accumulation in both plasma and liver, thereby contributing to dyslipidemia and hepatic steatosis. Consequently, NPC1L1 has emerged as an important therapeutic target for reducing cholesterol absorption and improving lipid homeostasis. Although ezetimibe is currently the only clinically approved NPC1L1 inhibitor, its limited efficacy as monotherapy highlights the need for alternative or complementary therapeutic strategies. Growing evidence indicates that natural phytochemicals, particularly polyphenols and flavonoids, can modulate NPC1L1 at both transcriptional and functional levels. These compounds not only suppress intestinal cholesterol absorption but also attenuate hepatic lipid accumulation, ultimately improving circulating lipid profiles. This review summarizes recent advances in understanding the role of NPC1L1 in cholesterol metabolism and highlights the emerging therapeutic potential of phytochemicals as novel complementary approaches for the prevention and treatment of lipid metabolic disorders.

Current Issues in Molecular Biology doi: 10.3390/cimb48060591

Authors: Saba Iordanishvili Nazibrola Chiradze Dodo Agladze Marine Kikvidze Revaz Solomonia Vincenzo Lagani

Blood lipid responses to diet vary substantially between individuals, limiting the effectiveness of uniform dietary recommendations, and genetic variation may contribute to this heterogeneity through gene–diet interactions. This systematic review and meta-analysis evaluated nutrigenetic interactions affecting blood lipid traits. Web of Science Core Collection and MEDLINE were searched in April 2026 to identify human studies testing interactions between dietary exposures—including macronutrient composition, fat quantity, fat type [polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), and saturated fatty acids (SFA)], carbohydrate, and protein—and lipid-related genes. Interaction p-values were synthesized using a weighted Stouffer’s Z method with Benjamini–Hochberg false discovery rate correction. Twenty studies (n = 20), comprising approximately 9800 participants, met the inclusion criteria. The most consistent evidence was observed for CETP, APOE, and APOB, particularly in relation to broader macronutrient composition and fat-related exposures, while ABCA1 and APOA5 showed significant but more limited evidence. PUFA was the most consistent specific dietary exposure. In contrast, ABCG5, ABCG8, and CYP7A1 lacked sufficient data for meta-analysis, highlighting major gaps in the current literature. Overall, the findings support the view that lipid responses to diet are partly genotype-dependent, while also underscoring the need for larger, better harmonized studies to clarify and extend the current evidence base.

Current Issues in Molecular Biology doi: 10.3390/cimb48060590

Authors: Li Wang Yaoxing Wang Mingkai Shao Tao Wang Wanbao Zheng Jun Cao Renwen Luo Youyan Tu Yiting Xia Yiming Wei Ning Liu Wenjie Lu Youzhi Xu

In the original publication [...]

Current Issues in Molecular Biology doi: 10.3390/cimb48060589

Authors: Arzu Ay Nevra Alkanli Gokay Taylan Esra Ergin

Chronic inflammation mediated by cytokines is central to the pathogenesis of coronary artery disease (CAD). This exploratory study aimed to investigate potential associations between functional gene variations of the cytokines interleukin 18 (IL-18) and interleukin 8 (IL-8) and the CAD susceptibility within a specific regional cohort, while accounting for common clinical comorbidities. Genotype distributions of IL-18 (−607 C/A, −137 G/C) and IL-8 (+781 C/T) were analyzed in a cohort of 102 patients with angiographically confirmed CAD and 102 healthy controls. Genotyping was performed using PCR, allele-specific PCR, and RFLP techniques. Multivariate logistic regression was utilized to assess potential independent associations, adjusting for age and traditional clinical risk factors. In this specific cohort, after adjusting for age, hypertension, diabetes, cholesterol, family history, and smoking, the IL-18 (−137 G/C) CC genotype was observed more frequently in CAD patients (adjusted odds ratio [AOR] = 6.15, 95% CI = 2.10–18.05, p = 0.001). An exploratory analysis of genotype combinations suggested that the IL-18 (−607/−137) CA-CC profile may be linked to an increased risk (AOR = 3.65, p = 0.028), while tentative protective trends were noted for certain IL-18/IL-8 combinations. Notably, a significant deviation from a Hardy–Weinberg equilibrium (HWE) was observed in the control group for the IL-18 loci, which represents a substantial methodological limitation that may influence these risk estimates. Our preliminary findings suggest that specific IL-18 and IL-8 variations may contribute to CAD susceptibility independently of traditional risk factors in the studied population. However, given the modest sample size and the observed HWE deviation, these associations should be regarded as suggestive rather than definitive. While these genetic variations underscore the importance of cytokine pathways in cardiovascular research, they do not currently support clinical implementation for risk stratification. Large-scale, multi-center prospective studies are necessary to validate these preliminary signals and evaluate their long-term scientific utility.

Current Issues in Molecular Biology doi: 10.3390/cimb48060588

Authors: Diana Basharova Ayazhan Bekbayeva Gulnara Svyatova Aizhan Darmeshova Elena Zholdybayeva

Background: Alport syndrome is a hereditary disorder caused by defects in the type IV collagen network. Although exon variants are primarily associated with Alport syndrome, the clinical significance of intronic variants remains incompletely characterized. The aim of this study was to characterize the clinical and molecular features of a familial case of Alport syndrome associated with the intronic variant c.1588-2A>G and to assess its impact using in silico tools. Case description: Two affected siblings presented with hematuria, proteinuria, and renal biopsy demonstrated focal global and segmental glomerulosclerosis, findings consistent with Alport syndrome. Whole-exome sequencing was subsequently performed in patients. The variant (NM_033380.2, c.1588-2A>G) in intron 23 of the COL4A5 gene was identified in both probands. SpliceAI analysis demonstrated a complete loss of the canonical acceptor site and a high probability of cryptic site activation. Conclusion: The evidence suggests a likely pathogenic role of the COL4A5 c.1588-2A>G variant in Alport syndrome.

Current Issues in Molecular Biology doi: 10.3390/cimb48060587

Authors: Quan Gu Wenyang Zhang Ziping Chen Na Li Shuwen Xu

Although previous studies have indicated that heme oxygenase 1 (HO1/HY1) regulates the flowering time via the photoperiod pathway, the specific mechanism is still elusive. Here, we found that the Arabidopsis hy1-100 mutant displayed early flowering, and the characteristic expression patterns of several master genes involved in the autonomous pathway were altered. Notably, the transcript levels of FLOWERING LOCUS C (AtFLC) gene declined developmentally in both wild-type and hy1-100 mutant, with a more pronounced fold reduction observed in the mutant. Genetic evidence further underlined that hy1-100/FLCOE plants partially reversed the early flowering phenomenon of hy1-100 mutant, suggesting that AtHO1 regulated flowering at least partially through the AtFLC-involved autonomous pathway, as supported by changes in FLOWERING LOCUS T (AtFT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (AtSOC1) transcripts. Further analysis of hy1-100/ft mutants revealed that hy1-100/ft and ft mutants displayed similar late flowering phenotypes, accompanied by downregulated APETALA1 (AtAP1) and AtSOC1, indicating that AtFT played a crucial role in AtHO1-regulated flowering. Two key conclusions are drawn: first, the loss of AtHO1 function promotes early flowering in Arabidopsis, which was genetically linked to the autonomous pathway regulating AtFLC expression; second, AtFT was an essential downstream factor mediating AtHO1-regulated flowering.

Current Issues in Molecular Biology doi: 10.3390/cimb48060586

Authors: Marko Mladenović Bojana Banović Đeri Ana Nikolić Dragana Dudić Slaven Prodanović Sanja Z. Perić Nikola Grčić

Knowledge of population structure and genetic relationship among inbred lines is essential for exploiting heterosis in maize breeding programs. This study evaluated the concordance between 25k Illumina® Infinum Maize SNP (Single Nucleotide Polymorphism) Array (25k SNP array)-derived and ribonucleic acid (RNA)-sequencing (seq)-derived markers in estimating genetic relatedness and population structure within the Maize Research Institute “Zemun Polje” (MRIZP) breeding program. A panel of 28 elite MRIZP maize inbred lines, along with two public lines, was analyzed. For the RNA-seq data, three alternative SNP datasets were generated based on heterozygous-site handling (ALL, HOM, and FINAL) to assess their impact on downstream genetic inference. These approaches had distinct effects on clustering resolution and genetic relationship structure. The FINAL dataset, in which heterozygous positions were recoded as missing values and re-filtered, was selected as the most balanced dataset for comparative analyses with 25k SNP array data. Only a limited number of overlapping SNP positions were identified between RNA-seq and 25k SNP array datasets (six in ALL, two in FINAL, and none in HOM), all located within coding regions. Despite this minimal overlap, distance-based analyses revealed partial concordance in genetic relationship patterns and population structure between platforms. Genetic distances estimated from 25k SNP array markers were consistent with pedigree records and provided more informative insights than pedigree data alone. Population structure inferred from 25k SNP array data showed high concordance with previously defined heterotic groups, correctly assigning 29 out of 30 lines (96.67%) to their expected clusters. In contrast, RNA-seq-derived SNPs showed moderate concordance with expected heterotic groupings (56.67%), indicating that transcriptome-derived markers capture part of the underlying breeding structure, but do not fully resolve heterotic group separation. Overall, these results support the 25k SNP array as a robust tool for assessing genetic relatedness and population structure in maize breeding programs, while RNA-seq-derived SNPs provide complementary but less reliable information for routine heterotic assignment.

Current Issues in Molecular Biology doi: 10.3390/cimb48060585

Authors: Kenta Hosomi Mitsuaki Ishida Kensuke Nakanishi Kohei Taniguchi Jun Arima Atsushi Tomioka Mitsuhiro Asakuma Sang-Woong Lee Ko Fujimori Yoshinobu Hirose

Acinar-to-ductal metaplasia (ADM)—a process involving the dedifferentiation or transdifferentiation of pancreatic acinar cells—is recognized as an initial event in pancreatic tumorigenesis. Studies in mouse models have revealed that tuft cells, which are chemosensory epithelial cells, appear in ADM following tissue injury, and tuft cell-produced prostaglandin (PG) D2 may suppress inflammation and tumorigenesis. However, the presence and role of tuft cells in the human pancreas remain unclear. Therefore, in this study, we investigated the presence of tuft cells and PGD2 production in human ADM. We analyzed ADM lesions from consecutive patients undergoing surgical resection for pancreatic tumors using dual immunohistochemical staining for POU domain class 2 transcription factor 3 (POU2F3) and hematopoietic PGD synthase (H-PGDS). All 29 patients (13 men and 16 women) with diagnoses including pancreatic ductal adenocarcinoma and intraductal papillary mucinous neoplasms exhibited ADM in regions of obstructive pancreatitis. Immunohistochemical analysis showed that 67.3% of ADM lesions contained POU2F3- and/or H-PGDS-positive cells. Among these, 85.5% of POU2F3-positive cells co-expressed H-PGDS, and 76.2% of H-PGDS-positive cells were POU2F3-positive. These findings indicate that tuft cells present in human ADM produce PGD2, suggesting a role in tissue repair. Tuft cells may represent a potential therapeutic target in pancreatitis, warranting further investigation into their functional role in ADM.

Current Issues in Molecular Biology doi: 10.3390/cimb48060583

Authors: Shifang Zhang Guo Hu Yuan Guo Xudong Chang Haijun Li Yingzhu Liu

The brown frog (Rana dybowskii) is an important cold-temperate amphibian species in northeastern China, with considerable ecological and resource value. Because its survival, metabolism, and reproduction are highly sensitive to environmental temperature, elucidating the molecular mechanism’s underlying temperature response is of great significance for understanding environmental adaptation and reproductive regulation in amphibians. In this study, R. dybowskii was used as the experimental model, and a multi-tissue transcriptomic analysis was conducted on the brain, liver, spleen, ovary, oviduct, and testis from both females and males under 2 °C and 12 °C conditions to characterize tissue- and sex-specific responses to temperature variation. The results showed that the global transcriptomic landscape of R. dybowskii was primarily driven by tissue type, whereas temperature effects were mainly manifested within individual tissues. The liver, brain, and spleen exhibited pronounced temperature responsiveness in both sexes, with the liver mainly showing metabolic reprogramming-related functional changes. Intersection analysis further revealed that temperature-responsive genes included not only conserved modules shared across tissues but also a large number of tissue-specific genes. Reproductive tissues displayed more pronounced functional divergence: the ovary showed relatively limited transcriptional changes and remained comparatively stable; the oviduct underwent marked transcriptional remodeling, with upregulated genes mainly involved in glycosylation and macromolecule processing, whereas downregulated genes were primarily associated with ribosomes, translation, and RNA splicing; the testis was likewise highly sensitive to temperature changes, with upregulated genes mainly enriched in amino acid metabolism, mitochondrial function, and DNA repair, while downregulated genes were mainly related to cellular structure and intercellular junctions. Collectively, these findings indicate that the molecular adaptation of R. dybowskii to temperature variation is characterized by strong tissue specificity and sex-biased responses in reproductive tissues. This study provides a transcriptomic basis for elucidating the molecular responses to short-term temperature variation is important for understanding environmental sensitivity and reproductive regulation in amphibians.

Current Issues in Molecular Biology doi: 10.3390/cimb48060584

Authors: Caiyun Sun Shi Tang Binlong Chen Mo-Zi-Li Adu

Fingered citron volatile oil is a volatile oil with a fresh and slightly floral aroma, extracted from fresh fingered citron. It is one of the natural perfume essential oils. At present, scholars at home and abroad mainly focus their research on fingered citron volatile oil on optimizing extraction processes, component analysis, pharmacological effects, and other aspects. With the continuous development and application of fingered citron volatile oil and related products, the research scope of fingered citron volatile oil is becoming increasingly extensive. As a narrative review, this paper summarizes and compares eight distinct extraction techniques for fingered citron volatile oil and clarifies the merits and drawbacks of each method. Steam distillation is one of the most employed approaches for volatile oil extraction in laboratory settings, whereas mechanical pressing is extensively applied and serves as the dominant and preferred industrial process for fingered citron volatile oil production. This paper also reviews its potential biological activities, including antidepressant, anti-anxiety, and sleep-improving effects, as well as the development and application of related products. Fingered citron volatile oil has been applied in products such as cigarettes and insecticides. Studying the chemical components of fingered citron volatile oil can not only help research its medicinal mechanisms but also promote the development of natural spices, providing a useful reference for the further utilization of fingered citron volatile oil in the future.

Current Issues in Molecular Biology doi: 10.3390/cimb48060582

Authors: Yanni Cao Xiaohui Li Jiangshan Liu Junyuan Zhang Kangcheng Xu Hao Lin Yuxian Liu

Background: Breast cancer (BRCA) is a common malignant tumor that seriously threatens women’s health. Studies have shown that histone modifications (HMs) play a vital role in the occurrence and development of BRCA. This study aims to explore the distribution patterns of HMs in the mammary epithelial cell line (HMEC) and breast cancer cell line (MCF-7), and their potential associations with gene expression, patient prognosis, and drug efficacy. Methods: First, the distribution of histone modification (HM) signals in HMEC and MCF-7 cell lines was analyzed. Multiple algorithms were then used to predict the effects of different HMs and their modified regions on gene expression in the two cell lines. Based on four key regions identified from this analysis, 268 HM-related immune-related genes (H_IRGs) were screened, followed by functional enrichment and pathway analysis. Subsequently, Cox and LASSO regression analyses were performed on the H_IRGs to construct a risk scoring model. Results: The random forest algorithm showed the best predictive performance (AUC = 0.92) and identified three key HMs (H3K4me2, H3K27me3, and H3K36me3) and four key regions that strongly influenced gene expression. A risk scoring model was constructed from 11 key IRGs (BCL2A1, PSME2, STC2, ESRRG, CRISP3, IL13RA1, LCN1, EED, CLEC10A, SLURP1, and FGF12). This model effectively predicted patients’ survival in both the training and validation cohorts. Conclusions: In summary, our research results provide a theoretical basis for the occurrence and development of BRCA, and the 11 key IRGs discovered are expected to become potential biomarkers for BRCA prognostic assessment and treatment response prediction.

Current Issues in Molecular Biology doi: 10.3390/cimb48060581

Authors: Dragana Zaklan Nikola Martić Bojana Andrejić Višnjić Milana Bosanac Snežana Hadžistević Aleksandar Rašković Nebojša Pavlović

Carnosine is widely recognized for its antioxidant and cytoprotective properties and is being increasingly used in dietary supplements. However, its effects in drug-induced liver injury remain insufficiently studied. This study aimed to investigate and compare the hepatoprotective and antioxidative effects of pure carnosine and an antioxidant-enriched commercial carnosine supplement in a murine model of paracetamol-induced hepatotoxicity. Adult male Swiss Webster mice were pretreated orally for seven days with carnosine or a commercial carnosine supplement prior to a single hepatotoxic dose of paracetamol. The serum biochemical parameters, hepatic oxidative stress markers, histopathology, and immunohistochemical expression of CYP2E1, COX-2, and Iba1 were evaluated 24 h after paracetamol administration. Paracetamol increased serum aminotransferases, lipid peroxidation, CYP2E1 expression, and histological liver injury. Pure carnosine pretreatment tended to exacerbate biochemical liver injury, whereas the commercial supplement attenuated lipid peroxidation, preserved bilirubin levels, and reduced histological damage. Both formulations decreased CYP2E1 expression and were associated with less necrosis and COX-2 immunoreactivity compared with paracetamol alone. Antioxidant enzyme activities and macrophage markers showed no consistent intergroup differences. These findings indicate that carnosine may not consistently exert hepatoprotective effects in acute drug-induced liver injury and that accompanying antioxidants may critically modify its biological actions.

Current Issues in Molecular Biology doi: 10.3390/cimb48060579

Authors: Marcos Antônio Denk Isabella Cristina Mendes Rossa Luize Kremer Gamba Anna Clara Faidiga Silva Julia Letícia de Bortolo Paulo Cesar Lock Silveira Camila da Costa Júlio Cesar Francisco Luiz César Guarita-Souza

Background/Objectives: Acute myocardial infarction (AMI) remains a major cause of global morbidity and mortality and is a leading factor in the development of heart failure. This study investigated the regenerative potential of decellularized human amniotic membrane (HAM) and Wharton’s jelly (WJ) in a rat model of left ventricular dysfunction induced by acute myocardial infarction (AMI). Methods: Twenty-three rats underwent left anterior descending coronary artery ligation and were randomized into three groups: control (saline), WJ (decellularized WJ), and HAM (decellularized HAM). Results: After 30 days, echocardiographic, histopathological, and immunohistochemical assessments were performed. No significant differences in ventricular function were observed among groups. However, the HAM-treated group showed a significant reduction in myocardial fibrosis compared with the control (p = 0.009), suggesting attenuation of post-infarction remodeling. Despite the absence of measurable functional recovery, HAM demonstrated potential to promote more favorable tissue organization. Study limitations include the lack of a sham-operated group, short follow-up period, and absence of quantitative decellularization validation. Conclusions: Overall, the results indicate that decellularized HAM may act as a structural modulator of myocardial remodeling, warranting further studies with longer follow-up and combination approaches, such as cell-based or growth factor-enhanced therapies.

Current Issues in Molecular Biology doi: 10.3390/cimb48060578

Authors: Bingpeng Yang Yanhui Wang Yufei Teng Yan Liu Xinjun Fan Qianrui Yang Na Li Binwen Shi Wanchao Zhu Shutu Xu Wenqian Mei Jiquan Xue

Plant height is a fundamental quantitative phenotypic trait affecting maize (Zea mays) planting density and is an important focus in the breeding of varieties suitable for mechanical harvesting. In this study, we found a natural extreme dwarf mutant designated as d25, whose dwarf phenotype is controlled by a single recessive gene. The phenotype was restored by spraying with gibberellic acid 3 (GA3), which indicated that the mutant phenotype of the d25 mutant resulted from mutations in genes involved in the gibberellin (GA) metabolic pathway. Additionally, performing bulked segregant analysis on 30 extreme phenotypic plants in the F2 population (d25 × P002), we located one major quantitative trait locus (QTL) at chromosome 3 from 9.3 to 11 Mb. In combination with transcriptome sequencing analysis of d25 and WT plants, we identified the cloned typical plant height-related gene D1, whose expression was significantly higher in d25 mutant plants than that in WT plants. Further analysis revealed that a 275 bp structural variant spanning exonic and intronic regions of the D1 gene accounted for the dwarf phenotype in the d25 mutant. Protein prediction revealed that this variant site alters the translated protein sequence of this region, thereby modifying its function and impairing the critical pathway responsible for hormone synthesis, resulting in reduced plant height. These findings indicate that the d25 mutant may be a novel mutant allele of the D1 gene affecting maize plant development.

Current Issues in Molecular Biology doi: 10.3390/cimb48060580

Authors: Xiao Han Xiaojie Zhang Rui Wang Lili Gu Wenxian Yang Yiqiang Ren Zhenwei Ren

The PIN-LIKES (PILS) gene family is crucial for regulating auxin homeostasis and stress adaptation in plants; nevertheless, a comprehensive study on this family in alfalfa (Medicago sativa) remains insufficient. This research found 46 MsPILS genes within the tetraploid alfalfa genome and categorized them into four subfamilies. The genes are irregularly allocated throughout 16 chromosomes, with tandem duplications acting as a primary catalyst for family expansion. Analysis indicated that all MsPILS proteins contain the conserved Mem_trans domain. The promoter study revealed that MsPILS genes had many cis-elements that respond to abiotic stressors and hormones. qRT-PCR research indicated that MsPILS genes exhibit variable expression across several tissues and respond to multiple abiotic stressors. Protein–protein interaction (PPI) research revealed PIN3, PIN5, and PIN6 as principal interacting partners of the MsPILS proteins. Subcellular localization studies indicated that MsPILS1c is in the nucleus, plasma membrane, and endoplasmic reticulum (ER). This research offers significant genetic resources and a theoretical framework for elucidating the activities of PILS genes and for molecular breeding aimed at improving stress tolerance in alfalfa.

Current Issues in Molecular Biology doi: 10.3390/cimb48060577

Authors: Jesús Alonso Gándara-Mireles Elio Aarón Reyes Espinoza Verónica Loera-Castañeda Lourdes Patricia Córdova Hurtado Antonio Emilio González Font Julio Cesar Grijalva Ávila Ignacio Villanueva Fierro Ismael Lares-Asseff Cynthia Mora Muñoz Gabriela Velasco Villa Hugo Payán Gándara Leslie Patrón-Romero Horacio Almanza-Reyes

Doxorubicin (Dox) is a cornerstone in the treatment of pediatric acute lymphoblastic leukemia (ALL), but its use is limited by dose-dependent cardiotoxicity. Oxidative stress, arising from mitochondrial dysfunction, enzymatic generation of reactive oxygen species, and cardiotoxic metabolites, has been implicated as a central mechanism, with interindividual variability partly influenced by genetic factors. This study evaluated oxidative DNA damage 8-hydroxy-2′-deoxyguanosine (8-OHdG) as an integrative marker of redox-related pathways in Dox-induced cardiotoxicity. In a prospective case–control study, 93 pediatric patients with ALL treated with Dox and 63 controls were included. Cardiotoxicity was assessed by serial echocardiography, and 8-OHdG levels were measured by ELISA. Genotyping of ABCC1 rs3743527, NCF4 rs1883112, and CBR3 rs1056892 was performed, and multivariable analyses were conducted. Dox-treated patients showed higher 8-OHdG levels than controls, and patients with cardiotoxicity (n = 11) had higher levels than those without. A higher frequency and severity of cardiotoxicity was observed in female patients, although this finding should be interpreted cautiously. Although allele frequencies did not reach statistical significance, distinct distribution patterns were observed between groups. These findings suggest that 8-OHdG may function as an integrative marker of redox dysfunction associated with Dox-induced cardiotoxicity.

Current Issues in Molecular Biology doi: 10.3390/cimb48060576

Authors: Bilian Hu Yuting Dai Can Cheng Jihua Zhou Fuan Niu Bin Sun Anpeng Zhang Liming Cao Huangwei Chu

Boro II (BT), the first cytoplasmic male sterility (CMS) system in rice, is widely used in three-line japonica hybrid rice production. Accurate detection of maintainer-seed contamination in BT-type CMS seed lots is critical for ensuring genetic purity and hybrid seed quality. In this study, we developed a SYBR Green-based quantitative real-time PCR (qPCR) assay for the detection and quantification of maintainer-seed contamination in BT-type CMS seed lots. Maintainer-specific primers targeting a mitochondrial sequence unique to the maintainer line, together with an endogenous reference targeting a conserved mitochondrial sequence present in both maintainer and CMS lines, were validated for specificity. A standard curve was constructed using defined CMS–maintainer seed mixtures (0.1–5% contamination), and ΔCt values were converted to relative abundance (2−ΔCt). The assay exhibited high specificity, reproducibility, and sensitivity, with a strong linear relationship between 2−ΔCt values and actual contamination levels (R2 > 0.99). Performance testing using simulated contamination samples (0.2–3.13%) demonstrated accurate quantification with acceptable recovery rates. This method provides a rapid, robust, and reliable tool for routine genetic purity testing and quality control in BT-type CMS hybrid rice seed production.

Current Issues in Molecular Biology doi: 10.3390/cimb48060575

Authors: Eirini Ilia Dimitrios Papoutsis Vasiliki Michou Aikaterini Itziou

Background: Long-term exposure to ambient air pollution during pregnancy has been strongly associated with oxidative-stress-mediated adverse maternal and fetal outcomes. Aim: The present study aimed to evaluate systemic oxidative stress and antioxidant capacity in pregnant women residing in a highly polluted area (Kozani) compared with a less polluted region (Grevena) in Western Macedonia, Greece. Methods: Oxidative stress was assessed using derivatives of reactive oxygen metabolites (d-ROMs), while antioxidant capacity was evaluated through biological antioxidant potential (BAP). Results: The findings of the study demonstrated that pregnant women in the polluted area exhibited elevated d-ROMs levels and significantly reduced BAP levels compared with controls. Although unadjusted oxidative stress differences were not statistically significant, adjusted analyses revealed significantly higher oxidative stress in the exposed group. These results suggest that air pollution exposure is associated with systemic redox homeostasis, primarily through depletion of antioxidant defenses. Conclusions: This study provides novel biomonitoring evidence linking environmental exposure to redox imbalance during pregnancy. As Western Macedonia transitions to a post-lignite era, the decrease in air pollution is anticipated to lead to significant improvements in public health, while these findings establish an important baseline for evaluating the effectiveness of environmental and public health interventions.

Current Issues in Molecular Biology doi: 10.3390/cimb48060574

Authors: Nada Tawfig Hashim Rasha Babiker Muhammed Mustahsen Rahman Riham Mohammed Vivek Padmanabhan Md Sofiqul Islam Nallan C. S. K. Chaitanya Bakri Gobara Shadi El Bahra

Periodontal disease is a chronic inflammatory condition driven by polymicrobial biofilms whose interaction with the host immune response drives the destruction of tooth-supporting tissues. Within these communities, bacterial cell–cell communication—particularly quorum sensing (QS)—coordinates virulence factor expression, biofilm maturation, and interspecies behaviour, allowing pathogens to mount population-dependent attacks on the host. Disrupting these signals has therefore drawn growing attention as an anti-virulence strategy for biofilm-associated oral infection. Quorum quenching (QQ)—the inhibition or disruption of QS pathways—prevents bacteria from coordinating these virulence-related activities. The candidate inhibitors investigated to date fall into three broad classes: conventional antibiotics used at sub-inhibitory concentrations, plant-derived natural compounds, and synthetic molecules designed to interfere with signal synthesis, signal reception, or signal transduction. In experimental work on periodontal pathogens, agents from each class reduce biofilm formation, suppress virulence factor production, and disrupt microbial communication within polymicrobial biofilms. Clinical translation, however, lags behind the laboratory evidence. Most data still come from in vitro systems and animal models, and the ecological complexity of the oral biofilm makes therapeutic targeting difficult: signals that drive virulence in pathogens also support cooperation among commensals. Toxicity profiles, pharmacokinetics, and well-powered clinical trials are needed before quorum-quenching agents can be considered for routine periodontal care. Even with these caveats, targeting bacterial communication offers a different therapeutic logic from conventional antimicrobials: attenuating virulence rather than killing cells, and so exerting weaker selective pressure for resistance. Further dissection of QS networks in oral biofilms—and the rational design of quenching agents that act on pathogenic rather than commensal signalling—may yield useful adjuncts to current periodontal therapy.

Current Issues in Molecular Biology doi: 10.3390/cimb48060573

Authors: Gian Carlos Nascimento Melina Laura Moretti Pinheiro Brenda Veridiane Dias Raphael Ocelli Pinheiro Maria Aparecida Vasconcelos Paiva Brito Afonso Henrique de Oliveira Júnior Lara Louzada Aguiar Rodinei Augusti Julio Onesio-Ferreira Melo Rafael Bastos Teixeira Ana Cardoso Clemente Filha Ferreira de Paula

Medicinal plants have attracted increasing scientific interest due to the diversity of bioactive compounds reported across different species. They may represent complementary sources of bioactive compounds alongside conventional antimicrobials, which may pose risks to animal health and compromise treatment efficacy. Considering the importance of alternative compounds, we aimed to evaluate the antimicrobial activity in vitro of medicinal plants Stryphnodendron adstringens (Mart.) Coville, known as barbatimão, Baccharis crispa Spreng, known as carqueja and Azadirachta indica, known as neem. S. adstringens (Mart.) Coville and B. crispa Spreng were used as extract and obtained from plants collected in the municipality of Bambuí, state of Minas Gerais, Brazil. A. indica was evaluated as extract and oil, and the crushed leaves and oil were purchased from a commercial company. Antimicrobial activity was determined by the minimum bactericidal concentration (MBC) test-against Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Escherichia coli, and Salmonella spp., isolated from bovine mastitis. The bacteria were submitted to the MBC test at concentrations of 100, 50, 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19 and 0.09 mg/mL. The bacteria evaluated were sensitive to most plant extracts for at least one of the concentrations evaluated, except for Gram-negative bacteria, Escherichia coli, and Salmonella spp. There was no activity of B. crispa Spreng extract and A. indica against E. coli and neither of B. crispa Spreng extract against Salmonella spp. even at the highest concentration evaluated. S. adstringens (Mart.) Coville was considered the extract with the highest activity against the bacteria evaluated and S. uberis the most susceptible to antimicrobial action. The results indicate detectable antimicrobial activity of the evaluated extracts and oil, suggesting their potential relevance as complementary sources of bioactive compounds for further investigation, rather than as direct alternatives to conventional antibiotic therapies. Paper spray mass spectrometry (PS-MS) was employed as an exploratory phytochemical screening approach, and all metabolite assignments reported herein should be regarded as tentative or putative annotations under the analytical conditions used, consistent with MSI Level 3 confidence.

Current Issues in Molecular Biology doi: 10.3390/cimb48060572

Authors: Mark Okot Aneesa Ahmed Colin W. Wright Md Talat Nasim

Pulmonary arterial hypertension (PAH) is a progressive, fatal disease of the pulmonary vasculature characterized by obliterative remodeling of small pulmonary arteries, leading to sustained elevation of pulmonary vascular resistance, right ventricular failure, and premature death. The diagnostic gold standard remains right heart catheterization, requiring a mean pulmonary artery pressure greater than 20 mmHg at rest, a pulmonary arterial wedge pressure of 15 mmHg or below, and a pulmonary vascular resistance exceeding 2 Wood units. PAH is an autosomal dominant disorder with markedly incomplete penetrance of approximately 20–30%, indicating that germline mutations alone are insufficient to cause disease. Disease manifestation requires additional “second hits”, including chronic hypoxia, systemic inflammation, hemodynamic stress, hormonal influences, and common genetic modifiers such as single-nucleotide polymorphisms (SNPs). This genetic and environmental complexity underpins the broad clinical heterogeneity observed across PAH subtypes, which include idiopathic PAH, heritable PAH, and disease associated with connective tissue disorders, HIV infection, portal hypertension, congenital heart disease, schistosomiasis, and drug or toxin exposure. This review provides a comprehensive and critical appraisal of the molecular-genetic architecture of PAH. Thirty genes have now been implicated in disease pathogenesis, spanning seven functional categories: receptors of the TGF-β/BMP signaling family (BMPR2, ACVRL1, ENG, BMPR1B); circulating BMP ligands (GDF2, BMP10); transcription factors (TBX4, SOX17, KLF4, FOXF1, SMAD1, SMAD4, SMAD9); membrane and polyamine transporters (ATP13A3, AQP1); potassium channel regulators (KCNA5, KCNK3, ABCC8); metabolic and mitochondrial genes (EIF2AK4, NFU1, GGCX); signaling receptors and structural proteins (NOTCH3, KDR, CAV1, PLEKHH2); vasoactive and extracellular matrix regulators (KLK1, CBLN2, CD248); and epigenetic regulators (TET2, TOPBP1). Among these, BMPR2 is the dominant contributor, accounting for 53–86% of heritable PAH and 14–35% of idiopathic cases. The remaining genes each account for fewer than 5% of cases individually, collectively reflecting a broad landscape of rare and ultra-rare genetic contributions. For each gene, we critically evaluate the strength of genetic evidence, pathogenic mechanisms, degree of mechanistic resolution, and clinical relevance. We further discuss the contribution of emerging technologies, including whole-genome sequencing, single-cell and spatial transcriptomics, multi-omics integration, iPSC-derived vascular models, and artificial intelligence, to expanding the PAH genetic architecture beyond single-gene discovery. A key theme across this landscape is convergence: despite mechanistic diversity at the gene level, most PAH-associated variants ultimately impair endothelial quiescence, promote smooth muscle proliferation, and drive apoptosis resistance through disruption of BMP signaling amplitude, transcriptional stability, ion channel homeostasis, metabolic integrity, or epigenetic regulation. This convergence supports both a unified therapeutic rationale and a precision medicine framework for genotype-stratified intervention in PAH.

Current Issues in Molecular Biology doi: 10.3390/cimb48060570

Authors: Changli Yang Qi Jia Yongxia Zhao Lin Qin Daopeng Tan Yuqi He

Dendrobium nobile Lindl. is a valuable medicinal orchid in traditional Chinese medicine. It abounds in alkaloids with extensive pharmacological properties, holding promising prospects for pharmaceutical development. In this review, the relevant literature was systematically retrieved from PubMed, Web of Science and CNKI using the keywords Dendrobium nobile Lindl., alkaloids, dendrobine, pharmacological activities and biosynthetic pathways. We comprehensively summarize the chemical composition, pharmacological effects, biosynthetic routes, clinical application potential and research prospects of alkaloids derived from D. nobile. The main alkaloid components including dendrobine, dendramine and nobilonine are classified into dendrobine, dendroxine and nobiline structural types. This paper further elaborates the anti-inflammatory, antioxidant and neuroprotective effects of these alkaloids, as well as their regulatory mechanisms on apoptosis-associated proteins and signaling cascades. Key enzymes and regulatory genes participating in the mevalonate pathway-mediated biosynthesis of sesquiterpenoid alkaloids are also discussed. Collectively, this review provides a theoretical basis and reference for subsequent basic research and therapeutic development of D. nobile alkaloids.

Current Issues in Molecular Biology doi: 10.3390/cimb48060571

Authors: Andria Kotsoni Evelina Ioannou Evangelia Barmparousi Lefteris C. Zacharia

β2-adrenergic receptor (β2-AR) agonists have been implicated in neuroprotection, yet their mechanisms remain obscure. We examined whether salbutamol (SA, short-acting) or formoterol (FO, long-acting) protect PC-12 cells from okadaic acid (OA), and evaluated receptor dependence, antioxidant capacity, and apoptotic signaling. Viability was quantified with crystal violet and MTT assays. OA reduced viability to approximately 60%, and SA or FO (0.1–10 µM) improved survival, which reached 76–83% at 10 µM (p < 0.05). β2-AR blockage with ICI-118,551, and ADRB2 mRNA knockdown did not abolish protection by FO or SA, suggesting a possible β2-AR-independent protective component. However, as knockdown was not confirmed at the protein level and signaling was not directly assessed, the evidence remains provisional. FO, but not SA, exhibited direct antioxidant activity in the DPPH assay, but both at 50 μΜ lowered H2O2-induced intracellular reactive oxygen species (from 167.9% to baseline, p < 0.05). Both compounds reduced the OA-induced expression of selected pro-apoptotic transcripts, although these mRNA data do not establish the functional inhibition of apoptosis. FO reduced fold change relative to untreated control from 5.8 to 2.6 for Bax, and 6.4 to 3.4 for Bak, whereas SA achieved a significant reduction only for Bax, from 5.8 to 4.4. Taken together, SA and FO offer a partial protection to PC-12 cells from OA cytotoxicity through pathways suggesting a β2-AR-independent protective component, with FO showing additional antioxidant properties and a reduced expression of selected pro-apoptotic transcripts. These findings provide preliminary evidence that select β2 agonists may exert cytoprotective effects that are consistent with, but do not establish, a receptor-independent component. These findings warrant further protein-level and functional validation.

Current Issues in Molecular Biology doi: 10.3390/cimb48060569

Authors: Zhijun Fan Yi Li Shuting Geng Yu Si Yuerong Yang Huali Yu Xue Hu Jianhai Jiang

N-acetylneuraminate pyruvate lyase (NPL) is a key enzyme in sialic acid catabolism that links sialylation to cellular metabolism, but its role in cancer cell metabolic adaptation is poorly defined. In particular, it remains unclear whether NPL contributes to ATP maintenance in hepatocellular carcinoma (HCC) under nutrient stress through its role in sialic acid catabolism. HCC is a highly lethal malignancy characterized by extensive metabolic reprogramming. Here, we investigated whether NPL links sialic acid catabolism to ATP maintenance in HCC cells under glucose deprivation. Glucose deprivation induced NPL expression in Huh7 and PLC/PRF/5 cells, which is consistent with an adaptive response to energetic stress. Stable NPL knockdown reduced intracellular ATP levels. Consistently, cell growth was significantly reduced, as assessed by Cell Counting Kit-8 (CCK-8) and colony formation assays. These effects were more pronounced in the absence of glucose. Exogenous pyruvate partially restored ATP levels and the growth inhibition caused by NPL knockdown, particularly in the absence of glucose. This rescue further suggests that NPL may support ATP maintenance under glucose deprivation partly through pyruvate metabolism. Together, these findings indicate that NPL contributes to maintaining intracellular ATP levels during glucose deprivation, thereby supporting HCC cell adaptation to metabolic stress. To extend these biological findings to potential therapeutic exploration, we performed virtual screening and molecular docking using a U.S. Food and Drug Administration (FDA)-approved drug library. Candidate compounds predicted to bind NPL were identified, providing a basis for further validation and optimization.

Current Issues in Molecular Biology doi: 10.3390/cimb48060568

Authors: Ya Zhu Xiaochun Zhang Chao Jin Heying Jin

Inflammatory bowel disease (IBD) is associated with skeletal muscle loss and mitochondrial dysfunction, which may compromise therapeutic responsiveness. We investigated whether progressive resistance training could improve muscle oxidative phosphorylation gene expression and enhance the efficacy of anti-TNF-α therapy (infliximab) in a dextran sulfate sodium (DSS)-induced colitis mouse model. Male C57BL/6 mice with subacute DSS-induced colitis were assigned to control, DSS, DSS + resistance training, DSS + anti-TNF-α, and DSS + resistance training + anti-TNF-α groups (n = 6 each). Resistance training consisted of 8 weeks of progressive ladder climbing; anti-TNF-α was administered at weeks 0, 2, and 6. Outcomes included body weight, disease activity, muscle function, histology, serum cytokines, muscle transcriptomics, and qPCR. DSS caused weight loss, functional decline, elevated proinflammatory cytokines, suppressed mitochondrial gene expression, and muscle inflammation. Resistance training or anti-TNF-α alone partially restored performance and mitochondrial gene expression while reducing inflammation. Their combination yielded superior effects (p < 0.01), normalizing histology. These findings suggest that resistance training may improve muscle metabolic status and attenuate systemic inflammation, thereby contributing to enhanced anti-TNF-α therapy outcomes in this preclinical model. Further studies are warranted to elucidate the underlying mechanisms and evaluate the translational potential.

Current Issues in Molecular Biology doi: 10.3390/cimb48060567

Authors: Claudia Moriello Nicola Alessio Rosario Finamore Chiara De Rosa Chiara Schiraldi Caterina Manna Stefania D’Angelo Pasquale Perrone

Oxidative stress plays a central role in the development of cardiovascular diseases (CVDs) and is strongly influenced by environmental toxicants such as heavy metals. Mercury (Hg) is a well-known pro-oxidant agent capable of disrupting cellular redox homeostasis, promoting reactive oxygen species (ROS) generation, and inducing structural and functional alterations in circulating blood cells. In particular, erythrocytes (RBC) are highly susceptible to oxidative damage due to their continuous exposure to oxygen and limited antioxidant repair mechanisms. In this study, we investigated the protective effects of polyphenolic extracts obtained from two tomato (Solanum lycopersicum) cultivars, Piccadilly and Piennolo, against HgCl2-induced oxidative stress in human RBC. Chemical characterization revealed that Piennolo extracts contained higher levels of total polyphenols, flavonoids, and ortho-diphenols compared with Piccadilly samples. Despite these differences, both extracts showed comparable antioxidant capacity. In RBC exposed to HgCl2, tomato extracts significantly reduced ROS production, lipid peroxidation (TBARS), methemoglobin formation, and sulfhydryl depletion, while restoring intracellular glutathione levels. Importantly, pretreatment with the extracts markedly decreased phosphatidylserine externalization, indicating preservation of membrane phospholipid asymmetry and a reduction in the procoagulant phenotype induced by oxidative stress. Overall, these findings suggest that bioactive compounds present in polyphenol-rich tomato extracts exert protective effects on RBC through antioxidant and membrane-stabilizing mechanisms.

Current Issues in Molecular Biology doi: 10.3390/cimb48060566

Authors: Camille Jacques Flora Marchand Mathias Chatelais Elías Hurtado-Gaitán Joana M. Buades Ilaria Floris

The micro-immunotherapy medicine (MIM) 2LEID-N® was developed to sustain immune response, notably in the framework of respiratory infections. This pilot study investigated the potential effects of one capsule of this MIM, referred to as 2LEID-N-9 throughout the manuscript, containing IL-12 (5 CH), IFN-γ (6 CH), and TNF-α (5 CH). Phagocytosis and surface marker expression were assessed using flow cytometry, and cytokine secretion was assessed by ELISA. Cellular models included human monocyte-derived macrophages, peripheral blood mononuclear cells (PBMCs) from healthy donors, and THP-1 cells. Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS) was used to detect the actives. Compared with vehicle control, 2LEID-N-9 showed a trend towards the enhanced phagocytic activity of macrophages. In PBMCs, 2LEID-N-9 upregulated the secretion of several cytokines, including IL-2, IL-4, IL-13, IFN-γ, and TNF-α in both basal and CD3/CD28-stimulated conditions. Notably, a tendency towards increased secretion of TNF-α was found in LPS-stimulated THP-1 cells. The presence of the three actives, as assessed by LC-HRMS/MS, combined with the functional data, provide promising exploratory evidence of immunomodulatory effects and tendencies towards the stimulation of innate and adaptive immune cells, warranting further investigation.

Current Issues in Molecular Biology doi: 10.3390/cimb48060565

Authors: Ahmet Ali Berber Cansu Akbulut Esra Yıldız Sinem Öztürk Şefika Nur Demir Nurcan Berber

This study evaluates the cytotoxic and genotoxic-like potential of an ipfencarbazone-based herbicide formulation (IPF-BH; commercial product Hokuto, containing 250 g/L of the triazolinone herbicide ipfencarbazone) in human peripheral lymphocytes in vitro across a concentration range of 62.5–1000 µg/mL. Cytotoxicity was monitored via the mitotic index (MI), while cytogenetic damage was assessed using the cytokinesis-block micronucleus (MN) assay and the alkaline comet assays. Comparisons were performed using one-way ANOVA, followed by Dunnett’s post hoc test, against the negative control. Results indicated a concentration-dependent cytotoxic effect, with a marked reduction in MI observed at all tested concentrations (p < 0.001). MN frequency was significantly elevated at concentrations ≥125 µg/mL, whereas the 62.5 µg/mL concentration did not induce significant micronuclei formation. The comet assay revealed increased DNA damage parameters (tail length, tail intensity (%), and tail moment) across the tested concentration range, albeit with a non-monotonic profile for tail length and tail intensity. These findings suggest that IPF-BH exposure is associated with marked cytotoxicity and a genotoxic response in this in vitro model at concentrations within the OECD 487-acceptable cytotoxicity window, together with cytotoxicity-associated genotoxic-like effects at strongly cytotoxic concentrations in human peripheral lymphocytes under in vitro conditions. Because IPF-BH is a commercial formulation, and no direct mechanistic endpoints (e.g., reactive oxygen species, mitochondrial transmembrane potential, lipid peroxidation, glutathione) were measured, and because the present design was performed without exogenous metabolic activation (no S9 supplementation), the observed effects cannot be unambiguously attributed to ipfencarbazone alone or to a defined mechanism of action; extrapolation to in vivo genotoxicity requires complementary +S9 and rodent in vivo follow-up studies.

Current Issues in Molecular Biology doi: 10.3390/cimb48060564

Authors: Luyao Qi Jiale Feng Xinyi Tan Meng Yang Lilian Ji Weicheng Hu

Sebacic acid (SA), a ten-carbon medium-chain dicarboxylic acid, has emerged as a multifunctional bioactive metabolite with potential applications in metabolic regulation and regenerative medicine. Evidence indicates that SA exerts anti-inflammatory effects by modulating nuclear factor kappa B (NF-kB), mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription (STAT) pathways, improves glucose homeostasis by enhancing mitochondrial function and suppressing hepatic gluconeogenesis, and contributes to lipid metabolism via peroxisomal and mitochondrial β-oxidation. Beyond metabolic regulation, SA promotes bone repair by stimulating osteoblast differentiation and inhibiting osteoclast activity, and supports muscle regeneration by enhancing energy supply, cell proliferation, and the microenvironment. SA also serves as a monomer for poly (glycerol sebacate) (PGS), enabling its use in biodegradable tissue engineering scaffolds. This review synthesizes current experimental and preclinical findings on the biological functions of SA, elucidates the underlying molecular mechanisms, and highlights its translational potential for the treatment of metabolic disorders and tissue regeneration.

Current Issues in Molecular Biology doi: 10.3390/cimb48060563

Authors: Yu Liu Hao Liang Xiaxiang Zhang Qiang Zhang Nanqing Liu

The fructose 1,6-bisphosphate aldolase (FBA) gene family plays crucial roles in plant energy metabolism, growth, development, and abiotic stress responses, as it modulates antioxidant synthesis and soluble sugar accumulation to enhance plant cadmium tolerance. Seashore paspalum (Paspalum vaginatum Sw.), a halophytic perennial C4 turfgrass renowned for its exceptional cadmium tolerance, is ideal for phytoremediation of cadmium-contaminated soil. FBA family genes have been identified in several grass species, such as maize, rice, and wheat, but systematic investigations into FBA family genes and their functions in seashore paspalum remain scarce. In this study, seven class I FBAs (named as PvFBA1–PvFBA7) and one class II FBA (named as PvFBA8) in seashore paspalum were identified. The physicochemical properties, evolutionary relationships, gene structures, conserved domains, protein structures, cis-acting regulatory elements, chromosomal localizations, and collinearity relationships of eight PvFBAs were analyzed. These analyses suggested that PvFBA genes had highly conserved domains and belonged to ultra-conserved core genes. Expression pattern analysis indicated that the PvFBA gene family was dynamically responsive to cadmium stress. PvFBA6 and PvFBA7 were highly expressed in leaves, whereas PvFBA1 and PvFBA3 showed almost no expression. The RT-qPCR results suggested that the expression levels of PvFBA5 and PvFBA6 were highly consistent with the FPKM value trends analyzed in the transcriptomic data. Collectively, this study not only provides a theoretical foundation for the understanding of the evolution of the PvFBA gene family but also offers potential candidate genes for enhancing cadmium stress tolerance in plants.

Current Issues in Molecular Biology doi: 10.3390/cimb48060562

Authors: Xiaoyan He Ying Liang Chunli Wang Xinghao Li Shuangshuang Qin Linxuan Li Guili Wei Danfeng Tang Zhanjiang Zhang Fan Wei

Sophora tonkinensis Gagnep. is an important medicinal shrub native to the karst regions of southwestern China, where long-term overharvesting and habitat fragmentation have markedly reduced wild resources. Although recent phytochemical, transcriptomic, and chloroplast genomic studies have improved understanding of this species, its maternally inherited population structure has remained unclear. To address this gap, we developed nine novel chloroplast simple sequence repeat (cpSSR) markers and used them to genotype 274 individuals from eighteen wild populations. A total of 41 alleles were detected, with 2–10 alleles per locus, indicating moderate to high polymorphism at the species level. By combining the nine cpSSR loci, we further identified 25 chlorotypes, including 19 private chlorotypes. Within-population chloroplast diversity was generally low, and five populations were monomorphic, whereas HJSE and LYNG retained comparatively high chlorotype diversity. Genetic differentiation among populations was extremely strong (mean FST = 0.808), whereas historical gene flow was very limited (Nm = 0.112), and AMOVA showed that 85% of total chloroplast variation occurred among populations. Taken together, chlorotype network analysis, chlorotype geographic distribution, UPGMA, PCoA, and exploratory STRUCTURE analysis supported three geographically structured chloroplast groups, indicating long-term restriction of seed-mediated dispersal across the fragmented karst landscape. These newly developed cpSSR markers and the derived chlorotype framework provide a practical basis for tracing maternal lineages, prioritizing conservation units, guiding ex situ germplasm sampling, and informing future breeding of this nationally protected species. Overall, the present results describe chloroplast-based maternal structure rather than total genome-wide diversity in S. tonkinensis.

Current Issues in Molecular Biology doi: 10.3390/cimb48060561

Authors: Mónica Vázquez-Del Mercado Beatriz Teresita Martín-Márquez Erika Aurora Martínez-García Marcelo Heron Petri

The prostaglandin D2 receptor (PTGDR) -441 C/T polymorphism has been previously associated with inflammatory diseases such as asthma. The goal of this study was to explore the association of this polymorphism with idiopathic inflammatory myopathies (IIM) and their clinical features. Seventy-two healthy subjects (HS) and forty-eight patients with IIM from Guadalajara and Mexico were recruited over three years. Clinical features and enzymes used for diagnostics and follow-up, such as creatine phosphokinase (CPK), lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine aminotransferase (ALT), were collected at the recruitment point and from the chart at the diagnostic point. PCR-ARMS was used to define genotypes. The Chi-square test was used to compare genotype and allele frequencies and clinical features between IIM patients and healthy subjects. A one-way ANOVA on ranks was performed to compare enzymatic levels. The allele distribution was not in Hardy–Weinberg equilibrium in the controls. There was no difference in age or gender distribution between the groups. The polymorphic (T) allele was more common in the IIM group than in the HS group. At the same time, the wild-type (CC) genotype presented more clinical features, such as heliotrope rash, fever, dyspnea, and weight loss, than the TT genotype. No significant differences were found regarding the enzyme levels. To further understand the role of this polymorphism in IIM, a bigger sample size is required along with mechanistical studies. Nevertheless, the polymorphic allele of the PTGDR -441 C/T polymorphism suggests susceptibility to IIM, whereas the wild-type CC genotype is associated with more clinical features.

Current Issues in Molecular Biology doi: 10.3390/cimb48060560

Authors: Ivo Nikolaev Sirakov Kalina Shishkova Raina Gergova Stefan Dimitrov Gergov Elena Tasheva-Terzieva

Type 2 diabetes is a multifactorial metabolic disease characterized by chronic hyperglycemia, insulin resistance, and persistent low-grade inflammation. All of these factors lead to dysregulation of the immune system. Of particular interest is the interaction between immune dysregulation in type 2 diabetes and oncogenic viruses such as Human papillomavirus (HPV) and Epstein–Barr virus (EBV), which play an essential role in the etiology of Head and neck cancer on the one hand and have mechanisms for escaping the immune response on the other. The aim of the present study is to perform an analysis of patients with head and neck cancer divided into two groups, with and without diabetes, aimed at studying the relationship between type 2 diabetes and the established viral status. It was found that for all viruses proven by us, the frequency of positive tests for them was higher in the group with type 2 diabetes compared to the group of patients without diabetes. The study provides new insights and suggestions for a significant association between type 2 diabetes mellitus, increased prevalence of EBV, and some low-risk HPV genotypes in patients with head and neck tumors. Continuing from our previous study, the association between EBV and HPV44, after strict statistical adjustment, highlights their potential biological and clinical significance within the oncogenic environment in the presence of type 2 diabetes.

Current Issues in Molecular Biology doi: 10.3390/cimb48060559

Authors: Eun-Ji You Boyong Kim

Fermentation is widely used to enhance the bioactivity of herbal phytochemicals through microbial bioconversion. Rehmannia glutinosa contains catalpol, an iridoid glycoside with metabolic and immunomodulatory potential; however, its efficacy in the unfermented form is limited. This study investigated whether fermentation enhances catalpol production and improves metabolic and immune-regulating functions via glucagon-like peptide-1 receptor (GLP-1R) signaling. Rehmannia glutinosa extract was fermented under optimized conditions, and catalpol and iridoid precursor levels were quantified to assess bioconversion efficiency. Biological effects were evaluated in intestinal epithelial cells, macrophages, and an Artemia model, focusing on glucose transport, GLP-1 secretion, dipeptidyl peptidase-4 (DPP-4) expression, mucosal defense, and GLP-1R/protein kinase A/cAMP response element-binding protein (PKA/CREB) signaling. Fermentation significantly increased catalpol content while reducing iridoid precursors. The fermented extract suppressed intestinal glucose absorption by downregulating sodium–glucose cotransporter 1 (SGLT1) and glucose transporter 2 (GLUT2). It also enhanced GLP-1 secretion and reduced DPP-4 expression, leading to activation of GLP-1R/PKA/CREB signaling. This activation increased mucin 2 (MUC2) expression and promoted anti-inflammatory.

Current Issues in Molecular Biology doi: 10.3390/cimb48060558

Authors: Jirong Liu Nanyan Mao Yaru Cui Yiyao Bao Chao Tang

Multisystem inflammatory syndrome in children (MIS-C) is a severe systemic inflammatory complication triggered by prior SARS-CoV-2 infection. It predominantly affects the cardiovascular system, and coronary artery injury, myocardial dysfunction, and myocardial ischemia are closely associated with disease severity and clinical outcomes. This article reviews the immunopathological characteristics and clinical manifestations of MIS-C-related coronary artery lesions, including coronary artery dilation and aneurysm formation, as well as the key pathophysiological mechanisms leading to myocardial ischemia. Based on recent clinical and translational research, we summarize current approaches to diagnosis, risk stratification, acute medical management, and long-term follow-up strategies. By synthesizing updated evidence, this review aims to provide theoretical support and practical clinical guidance for the early identification, timely intervention, and optimized management of affected children, ultimately improving their long-term cardiovascular prognosis.

Current Issues in Molecular Biology doi: 10.3390/cimb48060557

Authors: Michail Varras Fani-Niki Varra Viktoria-Konstantina Varra Panagiotis Theodosis-Nobelos

Autoimmune diseases are characterized by chronic inflammation, immune dysregulation, and excessive oxidative stress, which collectively contribute to a progressive tissue damage and organ dysfunction. Although conventional immunosuppressive and anti-inflammatory therapies remain the main therapeutic approach, their clinical efficacy is often limited by poor pharmacokinetic properties, low tissue selectivity, systemic toxicity, and adverse effects following long-term administration. In this context, antioxidant-based nanoformulations have emerged as promising multi-target therapeutic strategies for the modulation of oxidative and inflammatory pathways involved in autoimmune disorders. This review focuses on polymeric and non-polymeric nanoformulations designed to improve the solubility, stability, bioavailability, controlled release, and targeted delivery of antioxidant and anti-inflammatory agents for autoimmune disease treatment. Recent advances in nanocarrier systems applications, including nanogels, poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polymethacrylate, chitosan, hyaluronic acid, hydroxyapatite (HAP), lipid-based and ROS-responsive nanosystems, are discussed. The therapeutic potential of nanoencapsulated steroidal and non-steroidal anti-inflammatory drugs, antioxidant compounds, enzymes, inorganic elements, and nucleic acid-binding systems is evaluated through preclinical and limited clinical evidence. Many of these reported nanoformulations exhibit enhanced therapeutic efficacy, improved tissue targeting, reduced systemic toxicity, and the ability to simultaneously modulate oxidative stress and inflammatory signaling pathways. Despite the encouraging findings, important challenges remain regarding clinical translation, long-term safety, reproducibility, and large-scale production. In overall, antioxidant nanoformulations represent a promising and evolving platform for the development of more effective and targeted therapies against autoimmune diseases.

Current Issues in Molecular Biology doi: 10.3390/cimb48060556

Authors: Jintana Sirivarasai Sorsia Muttrarak Prapimporn Chattranukulchai Shantavasinkul Sittiruk Roytrakul Waraporn Malilas Pachara Panpunuan Piyamitr Sritara

Coffee consumption has been associated with metabolic and cardiovascular health, but the molecular mechanisms underlying these associations remain unclear. This study investigated the association between coffee intake and circulating proteomic profiles across metabolic conditions using a pooled-serum, exploratory design. Participants were classified into four groups: normal weight (NW), normal weight with coffee intake (NWC), obese with hypertension (OBHT), and obese with hypertension with coffee intake (OBHTC). Differentially expressed proteins (DEPs) were identified using volcano plot criteria (|log2FC| ≥ 1, FDR < 0.05), followed by Reactome pathway enrichment, Gene Ontology (GO) molecular function, and Enrichr-derived protein–protein interaction (PPI) analyses. Results: In NW vs. NWC, coffee intake was associated with proteins enriched in receptor-mediated signaling and phosphoinositide pathways. In OBHT vs. OBHTC, DEPs were linked to mitochondrial respiration and oxidoreductase activity. The NW vs. OBHT comparison showed downregulation of metabolic and signaling proteins with enrichment of mitochondrial and stress-response functions. In NWC vs. OBHTC, proteins related to cytokine signaling and vascular function were reduced, while redox-associated regulators were increased. PPI networks highlighted interconnected hubs integrating signaling, metabolism, and immune responses. Conclusion: These findings suggest context-dependent proteomic patterns associated with coffee intake. Given the pooled design and small sample size, results are hypothesis-generating and require validation.

Current Issues in Molecular Biology doi: 10.3390/cimb48060555

Authors: Zitong Pan Yi Wang Jieya Zhang Xiaoran Bian Huaxue Zhang Jiahao Pan Xinyu Wang Dadong Guo

Regulatory T cells (Tregs, CD4+ CD25+ Foxp3+) play a crucial role as a core cell subset in maintaining immune homeostasis in the ocular immune-privileged microenvironment. This review systematically summarizes the stage-specific regulatory mechanisms of Treg cells in common inflammatory diseases such as keratitis, uveitis, and dry eye syndrome, including intercellular interactions, signal pathway mediation, and cytokine network regulation, as well as key experimental evidence (animal/cell models and clinical sample data) and research progress in targeted therapy. Studies have shown that Treg cells maintain ocular immune balance by secreting anti-inflammatory cytokines (such as IL-10 and TGF-β), regulating signaling pathways (STAT, PI3K/AKT, SIRT1, etc.), and interacting with immune cells (macrophages, dendritic cells). Their functions are regulated by multiple factors such as cytokine networks, epigenetic modifications, and delivery vectors. Targeted interventions based on Treg cells (cell therapy, drug intervention, and signaling pathway regulation) and combined treatment strategies have shown good anti-inflammatory potential. This article, in light of current research limitations (such as insufficient analysis of cell heterogeneity and the disconnect between basic and clinical research), proposes future research directions, providing a theoretical basis for the understanding of the pathogenesis of inflammatory eye diseases and the development of new immunomodulatory therapies, and establishing a complete research framework of “mechanism–evidence–treatment”.

Current Issues in Molecular Biology doi: 10.3390/cimb48060554

Authors: Krastena Nikolova Ivan Antonov Victoria Ilieva Valentina Gavazova Daniela Virovska Denitsa Nencheva Silviya Abarova

In this study, we investigated the interaction of a dapagliflozin-containing medicinal product (the commercial drug Forxiga®) with human serum albumin (HSA) at different temperatures using steady-state fluorescence spectroscopy, competitive displacement assays, UV–Vis absorption spectroscopy, and thermodynamic analysis. Increasing concentrations of Forxiga induced a gradual, concentration-dependent quenching of the intrinsic fluorescence of HSA (λex=284 nm; λemmax≈334–339 nm), indicating perturbation of the microenvironment surrounding Trp-214 located in subdomain IIA. Stern–Volmer analysis showed that the quenching constants were temperature-dependent. Meanwhile, the high apparent bimolecular quenching constants suggested a predominantly static quenching mechanism associated with ground-state complex formation. By performing a modified Scatchard-type double-logarithmic analysis, we identified a primary binding site, particularly at lower temperatures. Van’t Hoff analysis revealed negative enthalpy and entropy changes. This indicates that the interaction was spontaneous and exothermic, mainly driven by hydrogen bonding and van der Waals forces. The competitive displacement assays confirmed preferential binding at Sudlow’s site I, in proximity to Trp-214. Additionally, the UV–Vis spectroscopy, supported by ligand-induced perturbation of aromatic residues, confirmed the absence of significant inner-filter effects. Differential scanning calorimetry suggested partial thermal stabilization of HSA upon ligand binding. This finding is consistent with the formation of a stabilized protein–ligand complex. These results suggest that Forxiga forms a relatively stable ground-state complex with HSA, primarily at Sudlow’s site I, and that the interaction is influenced by temperature-dependent conformational changes in the protein.

Current Issues in Molecular Biology doi: 10.3390/cimb48060553

Authors: Rogelio Frank Jiménez-Ortega Rosa M. Salgado Berenice Rivera-Paredez Nelly Patiño Tania Hilario-Huerta Silvia Arenas-Díaz Rafael Velázquez-Cruz Alberto Hidalgo-Bravo

Osteosarcoma is the most common primary bone tumor in children, adolescents, and young adults, and metastasis remains the main determinant for a poor outcome. We conducted an exploratory retrospective cohort study using formalin-fixed, paraffin-embedded tissue from 97 patients with histopathologically confirmed osteosarcoma treated between 2005 and 2019 to evaluate telomere maintenance mechanisms. To assess alternative lengthening of telomeres (ALT), colocalization of telomeric DNA and promyelocytic leukemia protein (TEL–PML) was evaluated as a tissue-based proxy. TEL–PML colocalization was assessed using combined PML immunofluorescence and telomere DNA PNA-FISH. Furthermore, telomerase reverse transcriptase (TERT) expression was evaluated by immunohistochemistry in relation to metastasis and disease progression. Logistic regression models were adjusted for age, sex, and smoking. TEL–PML was evaluable in 45/97 cases, including 10 positive and 35 negative tumors; the remaining samples were non-evaluable because of non-determinable signal or predominant necrosis. TERT immunohistochemistry was scorable in 58/97 cases, of which 33 were positive. TEL–PML evaluability was associated with amputation specimens, whereas TERT positivity was associated with non-osteoblastic histology and was inversely associated with age. Neither TEL–PML nor TERT was significantly associated with metastasis, recurrence, or death. Exploratory time-to-metastasis curves suggested an earlier increase of metastatic events among TEL–PML-positive cases. However, the small number of positive tumors precludes definitive prognostic interpretation. These hypothesis-generating findings indicate that TEL–PML assessment is feasible in osteosarcoma, but it is strongly influenced by tissue adequacy. On the other hand, TERT immunohistochemistry appears to reflect subtype- and age-related heterogeneity rather than providing robust outcome stratification in this cohort.

Current Issues in Molecular Biology doi: 10.3390/cimb48060552

Authors: Tian Xia Changhe Wei Xiaofan Chen

Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as the most transformative cellular immunotherapy modality, with its evolutionary trajectory intrinsically coupled to advances in immune receptor structure–function paradigms. Recent technological breakthroughs have yielded unprecedented mechanistic insights into immune receptors. Cryo-electron microscopy, single-cell omics, and structural biology have revealed the molecular architecture and functional dynamics of key receptors, including T-cell receptors (TCRs) and B-cell receptors (BCRs). This comprehensive review systematically integrates the latest discoveries in immune receptor structure–function relationships, emphasizing the mechanistic underpinnings of receptor diversity generation, signal transduction networks, and their direct translational impact on CAR-T therapeutic optimization. We critically examine the innovative design principles governing fourth-generation CAR-T cells, delineate breakthrough strategies for overcoming solid tumor immunoresistance, and analyze the synergistic potential of CAR-T and TCR-T technological convergence. Particular attention is devoted to elucidating how fundamental immune receptor research can be harnessed to address the tripartite challenges of safety, efficacy, and persistence that currently constrain CAR-T clinical applications. This review establishes a mechanistic framework for developing next-generation CAR-T technologies grounded in immune receptor biology and provides strategic insights for accelerating cellular immunotherapy clinical translation.

Current Issues in Molecular Biology doi: 10.3390/cimb48060551

Authors: Lucía Ceballos-Romero Soraya Herrera-Espejo Daniel Atassi Pilar Sánchez-Suero Jerónimo Pachón José Miguel Cisneros María Eugenia Pachón-Ibáñez

Rapid detection of Pseudomonas aeruginosa and its virulence factor ExoU is essential for improving patient outcomes. In this study, a CRISPR–Cas13a-based diagnostic assay combined with recombinase polymerase amplification (RPA) was developed to detect P. aeruginosa and the exoU gene in blood samples. The assay demonstrated robust amplification, with detection limits of 6 log10 and 8 log10 CFU/mL in Luria–Bertani medium and blood, respectively, and a 100% specificity, without cross-reactivity against four Gram-negative bacilli and Staphylococcus aureus reference strains. The utilisation of a fluorescence-based readout facilitated unambiguous discrimination between P. aeruginosa and P. aeruginosa/exoU+ isolates vs. negative controls. In conclusion, these results support the potential of RPA/CRISPR-Cas13a diagnostics for the rapid identification of P. aeruginosa and its ExoU virulence factor. Further optimisation and clinical validation are required to confirm its utility as a bedside diagnostic test, where its application would speed up clinical decisions in the treatment of these infections.