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18 pages, 3955 KB  
Systematic Review
Liquid Biopsy for Cerebral Aneurysms: Circulating RNA as Diagnostic and Prognostic Tools—A Systematic Review of Current Evidence and Perspectives
by Matteo Palermo, Alessandro Olivi and Carmelo Lucio Sturiale
Cells 2025, 14(19), 1525; https://doi.org/10.3390/cells14191525 - 30 Sep 2025
Abstract
Intracranial aneurysms (IAs) are potentially devastating cerebrovascular lesions, and predicting rupture risk remains a major clinical challenge. Conventional radiological and clinical scores offer only partial risk stratification, highlighting the need for complementary approaches. Liquid biopsy represents a promising non-invasive strategy to identify circulating [...] Read more.
Intracranial aneurysms (IAs) are potentially devastating cerebrovascular lesions, and predicting rupture risk remains a major clinical challenge. Conventional radiological and clinical scores offer only partial risk stratification, highlighting the need for complementary approaches. Liquid biopsy represents a promising non-invasive strategy to identify circulating biomarkers that reflect aneurysm biology and instability. We conducted a systematic review according to PRISMA 2020 guidelines, screening PubMed, Scopus, and Web of Science up to August 2025. Forty-eight eligible studies, encompassing 3515 IA patients, evaluated circulating RNA species, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) in serum, plasma, blood, or cerebrospinal fluid. Multiple candidates emerged as consistently dysregulated: upregulation of miR-21, miR-126, and miR-200a-3p, and downregulation of miR-143 and let-7b-5p were recurrently observed across independent cohorts. LncRNAs, such as MALAT1 and MIAT, and circRNAs, including circ_0000690 and circ_0021001, demonstrated diagnostic and prognostic potential, with some correlating with rupture status and clinical severity indices. Despite encouraging findings, heterogeneity in study design, sample handling, and analytic methods limits reproducibility. Large-scale, multicenter validation studies are essential to translate these biomarkers into clinical practice. Full article
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2 pages, 138 KB  
Correction
Correction: Zhang et al. Circular Nucleic Acids Act as an Oncogenic MicroRNA Sponge to Inhibit Hepatocellular Carcinoma Progression. Biomedicines 2025, 13, 1171
by Qianyi Zhang, Pengcheng Sun, Guang Hu, Xuanyao Yu, Wen Zhang, Xuan Feng, Lan Yu and Pengfei Zhang
Biomedicines 2025, 13(10), 2394; https://doi.org/10.3390/biomedicines13102394 - 29 Sep 2025
Abstract
There was an error in the original publication [...] Full article
(This article belongs to the Special Issue MicroRNA and Its Role in Human Health, 2nd Edition)
22 pages, 3777 KB  
Article
Comparative Transcriptomics Reveals Novel and Differential Circular RNA Responses Underlying Interferon-Mediated Antiviral Regulation in Porcine Alveolar Macrophages
by Jiuyi Li, Oluwaseun Adeyemi, Laura C. Miller and Yongming Sang
Viruses 2025, 17(10), 1307; https://doi.org/10.3390/v17101307 - 27 Sep 2025
Abstract
Porcine Reproductive and Respiratory Syndrome (PRRS) causes significant economic losses in the swine industry. Circular RNAs (circRNAs), a class of stable non-coding RNAs, are increasingly recognized as regulators in immune responses and host–virus interactions. This study investigated the genome-wide circRNA responses in porcine [...] Read more.
Porcine Reproductive and Respiratory Syndrome (PRRS) causes significant economic losses in the swine industry. Circular RNAs (circRNAs), a class of stable non-coding RNAs, are increasingly recognized as regulators in immune responses and host–virus interactions. This study investigated the genome-wide circRNA responses in porcine alveolar macrophages (PAMs), key cell targets of PRRSV, following treatment with a modified live virus (MLV) vaccine or two interferon (IFN) subtypes (IFN-α1, IFN-ω5). Using RNA sequencing, we identified over 1000 differentially expressed circRNAs across treatment groups, revealing both conserved and distinct expression profiles. Gene Ontology and KEGG pathway analyses indicated that circRNA-associated genes are significantly enriched in immune-related processes and pathways, including cytokine signaling and antiviral defense. Notably, IFN-ω5 treatment induced a pronounced circRNA response, aligning with its potent antiviral activity. We further explored the regulatory potential of these circRNAs by predicting miRNA binding sites, revealing complex circRNA-miRNA interaction networks. Additionally, we assessed the coding potential of differentially expressed circRNAs by identifying open reading frames (ORFs), internal ribosome entry sites (IRESs), and N6-methyladenosine (m6A) modification sites, suggesting a subset may undergo non-canonical translation. These findings provide a comprehensive landscape of circRNA expression in PAMs under different antiviral conditions, highlighting their potential roles as immune regulators and novel players in interferon-mediated antiviral responses, particularly downstream of IFN-ω5. This work contributes to understanding the non-coding RNA landscape in the PRRSV-swine model and suggests circRNAs as potential targets for future antiviral strategies. Full article
(This article belongs to the Special Issue Host Cell-Virus Interaction, 4th Edition)
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25 pages, 5106 KB  
Article
Circular Approach in Development of Microbial Biostimulants Using Winery Wastewater
by Tatjana Dujković, Ivana Danilov, Vanja Vlajkov, Marina Savić, Zdravko Šumić, Aleksandar Jokić and Jovana Grahovac
Agronomy 2025, 15(10), 2272; https://doi.org/10.3390/agronomy15102272 - 25 Sep 2025
Abstract
Sustainable development requires implementation of eco-friendly practices and a circular approach in both agricultural and industrial systems. This study evaluated winery flotation wastewater (WFW) as a cultivation substrate for Bacillus sp. 10/R isolated from grapevine rhizosphere for sustainable biostimulant production. The bacterial isolate [...] Read more.
Sustainable development requires implementation of eco-friendly practices and a circular approach in both agricultural and industrial systems. This study evaluated winery flotation wastewater (WFW) as a cultivation substrate for Bacillus sp. 10/R isolated from grapevine rhizosphere for sustainable biostimulant production. The bacterial isolate was characterized by 16S rRNA sequencing and biochemical tests, showing the highest similarity with Bacillus mojavensis and Bacillus halotolerans. Plant growth-promoting traits were assessed via assays for hydrolytic enzymes, ACC (1-aminocyclopropane-1-carboxylate) deaminase, and IAA (indole acetic acid) production, as well as for phosphate solubilization. The isolate was cultivated in WFW, including monitoring of biomass growth, enzymatic activity, and substrate composition changes. The resulting cultivation broths based on WFW (WFW-CB) and nutrient broth (NB-CB) were tested as barley seed treatment at five dosages, using sterile media and water as controls. The results have displayed strong pectinase (EAI–enzyme activity index 2.79) and cellulase activity (2.33), moderate xylanase (1.75) and ACC deaminase activity (growth zone 54.67 ± 0.58 mm), and moderate IAA production (9.66 µg/mL). Biomass content has increased by two log units within 48 h (up to 9.06 log CFU/mL), with stable pectinase activity (~2.2 U/mL). Germination assays revealed that 10% WFW-CB and 50% WFW enhanced germination indices and biomass, whereas undiluted WFW and WFW-CB inhibited germination. These results indicate that WFW is a suitable substrate for Bacillus sp. 10/R cultivation, linking industrial wastewater valorization with plant biostimulant production in a circular economy framework. Full article
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24 pages, 715 KB  
Review
Role of Non-Coding RNAs in Acute Myeloid Leukemia
by Shailendra S. Maurya, Sarita Maurya and Sumit K. Chaturvedi
Non-Coding RNA 2025, 11(5), 70; https://doi.org/10.3390/ncrna11050070 - 19 Sep 2025
Viewed by 223
Abstract
Acute myeloid leukemia (AML) is a highly heterogeneous disease, with significantly higher incidence and fatality rates in the elderly. Even with recent decades of research progress in AML, the exact etiology of this deadly disease is still not fully understood, with recent advancements [...] Read more.
Acute myeloid leukemia (AML) is a highly heterogeneous disease, with significantly higher incidence and fatality rates in the elderly. Even with recent decades of research progress in AML, the exact etiology of this deadly disease is still not fully understood, with recent advancements in sequencing technologies highlighting the role of a growing number of non-coding RNAs (ncRNAs) that are intimately associated with AML leukemogenesis. These ncRNAs have been found to have a significant role in leukemia-related cellular processes such as cell division, proliferation, and death. A few of these non-coding RNAs exhibit potential as prognostic biomarkers. The three main groups of ncRNAs that contribute unique activities, especially in cancer, are microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Their existence or altered expression levels frequently offer vital information on the diagnosis, course of treatment, and follow-up of cancer patients. The identification of ncRNAs has opened up new avenues for the diagnosis, prognosis, and therapy of acute myeloid leukemia. In order to provide a clear understanding of the significant influence that lncRNAs have on prognostic predictions and diagnostic accuracy in AML, this review aims to provide a comprehensive and insightful understanding of how these molecules actively participate in the complex landscape of the disease. Full article
(This article belongs to the Section Long Non-Coding RNA)
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20 pages, 4824 KB  
Article
Assembly and Analysis of the Complete Mitochondrial Genome of Eryngium foetidum L. (Apiaceae)
by Lihong Zhang, Wenhu Zhang, Yongjian Luo, Jun Liu, Qing Li and Qiongheng Liu
Biology 2025, 14(9), 1296; https://doi.org/10.3390/biology14091296 - 19 Sep 2025
Viewed by 346
Abstract
Eryngium foetidum L. belongs to the Apiaceae family and is a perennial herb. The entire plant is rich in essential oils, which have a distinctive aroma similar to cilantro. This plant exhibits significant biological activity and possesses characteristics such as disease resistance and [...] Read more.
Eryngium foetidum L. belongs to the Apiaceae family and is a perennial herb. The entire plant is rich in essential oils, which have a distinctive aroma similar to cilantro. This plant exhibits significant biological activity and possesses characteristics such as disease resistance and antimicrobial properties, showing great potential in medical and food applications. Additionally, its essential oil has substantial commercial value. Mitochondria play a crucial role as organelles within plant cells; however, the mitochondrial genome of E. foetidum remains underexplored. To fill this research gap, we conducted sequencing and assembly of the mitochondrial genome of E. foetidum, aiming to uncover its genetic mechanisms and evolutionary trajectories. Our investigation reveals that the mitochondrial genome of E. foetidum is a circular structure, similar to that of other species, with a length of 241,660 bp and a GC content of 45.35%, which is within the range observed in other organisms. This genome encodes 59 genes, comprising 37 protein-coding sequences, 18 tRNA genes, and 4 rRNA genes. Comparative analysis highlighted 16 homologous regions between the mitochondrial and chloroplast genomes, with the longest segment spanning 992 bp. By analyzing 37 protein-coding genes (PCGs), we identified 479 potential RNA editing sites, which induce the formation of stop codons in the nad3 and atp6 genes, as well as start codons in the ccmFC, atp8, nad4L, cox2, cox1, and nad7 genes. Meanwhile, the genome shows a preference for A/T bases and A/T-ending codons, with 32 codons having a relative synonymous codon usage (RSCU) value greater than 1. The codon usage bias is relatively weak and mainly influenced by natural selection. Most PCGs are under purifying selection (Ka/Ks < 1), while only a few genes, such as rps7 and matR, may be under positive selection. Phylogenetic analysis of mitochondrial PCGs from 21 species showed E. foetidum at the basal node of Apiaceae, consistent with the latest APG angiosperm classification and chloroplast genome-based phylogenetic relationships. In summary, our comprehensive characterization of the E. foetidum mitochondrial genome not only provides novel insights into its evolutionary history and genetic regulation but also establishes a critical genomic resource for future molecular breeding efforts targeting mitochondrial-associated traits in this economically important species. Full article
(This article belongs to the Section Genetics and Genomics)
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12 pages, 1507 KB  
Article
Identification of Riboflavin Metabolism Pathway in HepG2 Cells Expressing Genotype IV Swine Hepatitis E Virus ORF3 Protein
by Jing Tu, Shengping Wu, Lingjie Wang, Chi Meng, Gengxu Zhou, Jianhua Guo, Jixiang Li, Liting Cao, Zhenhui Song and Hanwei Jiao
Vet. Sci. 2025, 12(9), 912; https://doi.org/10.3390/vetsci12090912 - 19 Sep 2025
Viewed by 256
Abstract
(1) Background: Hepatitis E (HE) is a novel zoonotic disease caused by hepatitis E virus (HEV). In particular, swine hepatitis E virus (SHEV) genotype IV is one of the main genotypes that infect humans. Open reading frame 3 (ORF3) is an important virulence [...] Read more.
(1) Background: Hepatitis E (HE) is a novel zoonotic disease caused by hepatitis E virus (HEV). In particular, swine hepatitis E virus (SHEV) genotype IV is one of the main genotypes that infect humans. Open reading frame 3 (ORF3) is an important virulence protein of SHEV, which is involved in virus assembly, release, and regulation of host cell signaling pathways. Circular RNAs (circRNAs), as a type of competitive endogenous RNA (ceRNA), have a closed-loop structure and are special non-coding RNA molecules. They participates in the regulation of multiple biological processes by adsorbing microRNAs (miRNAs). Riboflavin, also known as vitamin B2, is a component of the coenzyme of flavoenzymes in the body. When there is a deficiency of riboflavin, it will affect the biological oxidation process of the host, leading to metabolic disorders. In addition, riboflavin can also affect the synthesis, transportation and decomposition of lipids in the body. It mainly maintains the normal transportation process of fat in the liver. Therefore, the deficiency of riboflavin will lead to the disorder of lipid metabolism in the body. Thus, viral hepatitis is closely related to riboflavin metabolism. However, there are very few reports on SHEV ORF3 affecting the riboflavin metabolism of target cells and thereby influencing viral infection. Therefore, this study investigates this highly significant scientific issue. (2) Methods: In the previous research of our group, adenovirus was used to mediate the overexpression of SHEV ORF3 genotype IV in HepG2 cells. Total RNA was extracted for high-throughput sequencing of circRNAs and transcriptome. KEGG functional enrichment analysis was performed on the data to identify the differentially expressed circRNAs and miRNAs after SHEV infection, and the relevant circRNA-miRNA network in the riboflavin metabolism pathway in HepG2 cells was found. (3) Results: We identified 4 circRNAs in the riboflavin metabolism pathway of HepG2 cells expressing the ORF3 protein of SHEV genotype IV and successfully found 26 relevant circRNA-miRNA networks. (4) Conclusion: We successfully screened and identified circRNAs related to riboflavin metabolism, further identifying the circRNA-miRNA network and its functional targets. For the first time, we investigated the key mechanism by which ORF3 protein influences riboflavin metabolic pathways in target cells through circRNAs, preliminarily revealing that ariboflavinosis can lead to lipid metabolic disorder in the organism. This indicates a close association between viral HE and riboflavin metabolism. Full article
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11 pages, 1779 KB  
Article
The First Complete Chloroplast Genome Sequence of the Cyrtomium hemionitis Fern
by Junxi Zhao, Panpan Shi, Xiaoxuan Wang, Shuosheng Zhang and Haixian Zhan
Curr. Issues Mol. Biol. 2025, 47(9), 771; https://doi.org/10.3390/cimb47090771 - 18 Sep 2025
Viewed by 200
Abstract
Cyrtomium hemionitis is a Cyrtomium fern with potential medicinal value; however, the lack of chloroplast genome data for this species limits its utilization and exploitation. In this study, the Illumina NovoSeq 6000 platform and SPAdes v3.14.1 were used to sequence and assemble the [...] Read more.
Cyrtomium hemionitis is a Cyrtomium fern with potential medicinal value; however, the lack of chloroplast genome data for this species limits its utilization and exploitation. In this study, the Illumina NovoSeq 6000 platform and SPAdes v3.14.1 were used to sequence and assemble the chloroplast genome of C. hemionitis. The chloroplast genome was 151,295 bp in length and exhibited a typical circular, double-stranded, quadripartite plastome architecture, with a GC content of 42.43%. Additionally, it included 30 high-frequency codons, 26 of which ended with A or U. In total, we annotated 130 coding genes, which included 88 protein-coding genes, 8 rRNA genes, and 34 tRNA genes. The IR (inverted repeat) boundaries of the genus Cyrtomium differed from those of common plants, with differences discovered in the JLB (large single-copy, inverted repeat b) and JLA (large single-copy, inverted repeat a) boundaries in this genus. Additionally, the phylogeny of this genus showed that C. hemionitis was more closely related to C. falcatum, whereas Dryopteris crassirhizoma was closely related to the genus Cyrtomium. These findings have significant implications for future research and can serve as a reference for the molecular evolution, systematic development, and utilization of C. hemionitis. Full article
(This article belongs to the Section Molecular Plant Sciences)
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48 pages, 557 KB  
Review
Molecular Signatures of Obesity-Associated Infertility in Polycystic Ovary Syndrome: The Emerging Role of Exosomal microRNAs and Non-Coding RNAs
by Charalampos Voros, Georgios Papadimas, Despoina Mavrogianni, Aristotelis-Marios Koulakmanidis, Diamantis Athanasiou, Kyriakos Bananis, Antonia Athanasiou, Aikaterini Athanasiou, Ioannis Papapanagiotou, Dimitrios Vaitsis, Charalampos Tsimpoukelis, Maria Anastasia Daskalaki, Vasileios Topalis, Marianna Theodora, Nikolaos Thomakos, Fotios Chatzinikolaou, Panagiotis Antsaklis, Dimitrios Loutradis, Evangelos Menenakos and Georgios Daskalakis
Genes 2025, 16(9), 1101; https://doi.org/10.3390/genes16091101 - 17 Sep 2025
Viewed by 328
Abstract
Polycystic ovarian syndrome (PCOS) is one of the most common endocrine and metabolic conditions affecting women of reproductive age. This condition affects around 20% of this demographic and is characterized by polycystic ovarian morphology, hyperandrogenism, and chronic anovulation. Obesity, impacting 40–85% of women [...] Read more.
Polycystic ovarian syndrome (PCOS) is one of the most common endocrine and metabolic conditions affecting women of reproductive age. This condition affects around 20% of this demographic and is characterized by polycystic ovarian morphology, hyperandrogenism, and chronic anovulation. Obesity, impacting 40–85% of women with PCOS, exacerbates insulin resistance, increases insulin levels, and intensifies low-grade inflammation. This exacerbates the reproductive and metabolic complications associated with the condition. Recent advancements in molecular biology have underscored the significance of non-coding RNAs, including as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), as crucial regulators of gene expression and prospective biomarkers for PCOS. Exosome-derived microRNAs (ex-miRNAs) have emerged as compelling candidates due to their stability in body fluids and their capacity to promote intercellular communication among adipose tissue, the ovary, and the endometrium. Research, encompassing both experimental and clinical studies, has shown that ex-miRNAs display differing expression levels in women with obesity-related PCOS. Several of these ex-miRNAs are associated with networks that govern inflammation, glucose metabolism, steroidogenesis, and folliculogenesis. Moreover, the encapsulation of these chemicals within exosomes safeguards them from enzymatic breakdown, hence augmenting their potential as non-invasive biomarkers for diagnosis, prognosis, and treatment monitoring. Despite the initial results being encouraging, challenges remain in standardising exosome separation, quantifying miRNA, and analyzing functional data within the complex pathophysiology of PCOS. This narrative review consolidates existing evidence regarding the molecular signatures of obesity-related infertility in PCOS, emphasising the growing significance of exosomal miRNAs and other non-coding RNAs, while examining their translational potential for early diagnosis and personalised therapeutic approaches. Full article
(This article belongs to the Section Genetic Diagnosis)
35 pages, 1232 KB  
Review
Non-Coding RNAs in Health and Disease: From Biomarkers to Therapeutic Targets
by Marios A. Diamantopoulos, Michaela A. Boti, Triantafyllia Sarri and Andreas Scorilas
LabMed 2025, 2(3), 17; https://doi.org/10.3390/labmed2030017 - 17 Sep 2025
Viewed by 295
Abstract
Non-coding RNAs (ncRNAs) are critical regulators of gene expression, taking part in the modulation of multiple biological functions across a range of cell types. Initially dismissed as transcriptional noise, ncRNAs are now recognized for their significant roles in key cellular mechanisms, including differentiation, [...] Read more.
Non-coding RNAs (ncRNAs) are critical regulators of gene expression, taking part in the modulation of multiple biological functions across a range of cell types. Initially dismissed as transcriptional noise, ncRNAs are now recognized for their significant roles in key cellular mechanisms, including differentiation, apoptosis, and proliferation, as well as their profound implications for the pathogenesis of numerous human diseases. Due to their remarkable stability, tissue-specific expression patterns, and abundance in body fluids, ncRNAs hold significant promise as non-invasive biomarkers for diagnosis, prognosis, and therapeutic monitoring. Furthermore, advances in RNA-targeted therapeutics have introduced novel strategies to modulate ncRNA activity, although challenges related to delivery efficiency, specificity, and clinical validation remain. This review comprehensively summarizes the classification, biogenesis, and molecular functions of ncRNAs, elucidates their involvement in health and disease, and evaluates their potential as clinical biomarkers and therapeutic targets. Additionally, it discusses the emerging technologies for RNA manipulation, including CRISPR-based RNA editing, that can advance ncRNA research and revolutionize ncRNA-based therapeutics. Full article
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16 pages, 3557 KB  
Article
Mechanisms of Variation in Abdominal Adipose Color Among Male Kazakh Horses Through Non-Coding RNA Sequencing
by Yuhe Zhou, Xinkui Yao, Jun Meng, Jianwen Wang, Yaqi Zeng, Linling Li and Wanlu Ren
Biology 2025, 14(9), 1285; https://doi.org/10.3390/biology14091285 - 17 Sep 2025
Viewed by 253
Abstract
The Kazakh horse is a highly valuable indigenous Chinese breed known for its use in both milk and meat production. However, the mechanisms underlying color variation in the abdominal adipose tissue of this breed remain poorly understood. In this study, the sequencing of [...] Read more.
The Kazakh horse is a highly valuable indigenous Chinese breed known for its use in both milk and meat production. However, the mechanisms underlying color variation in the abdominal adipose tissue of this breed remain poorly understood. In this study, the sequencing of non-coding RNAs (ncRNAs) was conducted on abdominal adipose tissue of different colors from Kazakh horses, with the aim of investigating the molecular mechanisms responsible for this variation. A total of 205 differentially expressed long non-coding RNAs (DELncRNAs) including ENSECAG00000003836, ENSECAG00000017858, and ENSECAG00000035167; 52 differentially expressed microRNAs (DEmiRNAs) including miR-200-y and eca-miR-9a; and 559 differentially expressed circular RNAs (DEcircRNAs) including ZNF226 and ITPKC, were identified between Group W and Group Y. GO annotation and KEGG enrichment analyses of the DEGs revealed that these genes were primarily involved in biological processes such as chemical homeostasis (biological process, BP), intracellular components (cellular component, CC), and iron-sulfur cluster binding (molecular function, MF) as well as in metabolic pathways related to lipid biosynthesis and metabolism including vitamin B6 metabolism, tryptophan metabolism, and glycerolipid metabolism. The sequencing accuracy was further validated using reverse transcription quantitative PCR (RT-qPCR). This study identified key DEGs and signaling pathways associated with the color variation in adipose tissue of Kazakh horses and sheds light on the regulatory genes and biological processes involved. These findings provide a theoretical basis and research foundation for future studies on color variations in the adipose tissue of equine species. Full article
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18 pages, 2168 KB  
Article
Effective Reduction in Nuclear DNA Contamination Allows Sensitive Mitochondrial DNA Methylation Determination by LC-MS/MS
by Lin Liang, Luis Alfonso González Molina, Pytrick G. Jellema, Martijn van Faassen, Laura T. A. Otten, Kevin P. Mennega, Ingrid H. Hof, D. A. Janneke Dijck-Brouwer, Amalia M. Dolga, Marianne G. Rots and Klary E. Niezen-Koning
Int. J. Mol. Sci. 2025, 26(18), 8864; https://doi.org/10.3390/ijms26188864 - 11 Sep 2025
Viewed by 442
Abstract
Mitochondria are essential organelles for cellular energy production, playing a central role in driving metabolic processes and supporting critical intracellular functions. Neurometabolic disorders encompass a wide variety of conditions characterized by mitochondrial dysfunction. Owing to their bacterial ancestry, mitochondria possess an independent genome [...] Read more.
Mitochondria are essential organelles for cellular energy production, playing a central role in driving metabolic processes and supporting critical intracellular functions. Neurometabolic disorders encompass a wide variety of conditions characterized by mitochondrial dysfunction. Owing to their bacterial ancestry, mitochondria possess an independent genome consisting of a circular DNA molecule (mtDNA), which has been reported to be subject to methylation. However, the technical challenges in the detection of mtDNA methylation have led to debates on its existence. One of the concerns is that the compactness of mtDNA can lead to suboptimal bisulfite conversion, thereby causing mtDNA methylation overestimation. To address this, liquid chromatography tandem mass spectrometry (LC-MS/MS) offers a bisulfite-independent readout; however, this method requires mtDNA samples devoid of nuclear DNA (nDNA) contamination. To diminish nDNA contamination, we isolated mtDNA from the TRIzol RNA phase. Importantly, pyrosequencing showed no significant difference in the methylation levels of mtDNA isolated from the TRIzol RNA phase compared to those from the TRIzol DNA phase, or isolated via total genomic DNA (gDNA). Across different human cell lines, LC-MS/MS detected significantly lower global methylation levels for DNA isolated from the TRIzol RNA phase than those from the TRIzol DNA or gDNA isolation. Moreover, using mtDNA isolated from the TRIzol RNA phase, LC-MS/MS validated the enhanced mtDNA methylation in HepG2 transgenic cell lines expressing mitochondrial-targeted DNA methyltransferases (means of 2.89% and 2.03% for MCviPI and MSssI transgenic cell lines, respectively), compared to two negative control cell lines (1.36 and 1.39%). When applying it to clinically relevant material, LC-MS/MS demonstrated a significantly lower global methylation level for platelet DNA isolated from the TRIzol RNA phase (mean of 1.98%) compared to gDNA isolations (mean of 4.32%). Similar findings were confirmed in mouse brain tissue, in which a significantly lower methylation level was detected in DNA isolated from the TRIzol RNA phase (1.79%) compared to that from gDNA isolation (5.12%). In conclusion, isolating mtDNA from the TRIzol RNA phase holds significant potential in future studies, particularly for the quantification of mtDNA global methylation by LC-MS/MS, a technique that is independent of bisulfite conversion and bioinformatic analysis. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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16 pages, 1153 KB  
Article
Guanidino-Aryl Derivatives: Binding to DNA, RNA and G-Quadruplex Structure and Antimetabolic Activity
by Davor Margetić, Petra Jadrijević-Mladar, Anamaria Brozovic and Lidija-Marija Tumir
Molecules 2025, 30(18), 3682; https://doi.org/10.3390/molecules30183682 - 10 Sep 2025
Viewed by 385
Abstract
A series of novel guanidino-aryl (GA) compounds containing phenanthrene, fluoranthene, fluorene, and naphthalene aromatic cores were synthesized to investigate their interactions with DNA, RNA, and G-quadruplex structures. Among the novel compounds, the phenanthrene-guanidino compound demonstrated the highest micromolar affinity for AT-DNA, [...] Read more.
A series of novel guanidino-aryl (GA) compounds containing phenanthrene, fluoranthene, fluorene, and naphthalene aromatic cores were synthesized to investigate their interactions with DNA, RNA, and G-quadruplex structures. Among the novel compounds, the phenanthrene-guanidino compound demonstrated the highest micromolar affinity for AT-DNA, possibly due to partial phenanthrene intercalation in addition to hydrogen bonding and electrostatic interactions of guanidine cation. All new guanidino-aryl GA compounds bind strongly to the Tel22 G-quadruplex structure with similar affinities regardless of aromatic core size. The 1:1 stoichiometric complex is stabilised by π-π stacking interactions with the top or bottom G-tetrad, together with strong electrostatic interactions of the guanidino cation. The guanidino-porphyrin PoGU displayed distinct binding stoichiometry, indicating possible sandwiching between two G-quadruplex structures. Within the GA compounds tested, guanidino-fluorene exhibited moderate antimetabolic activity against the HeLa cell line, without selectivity against the healthy cell line. Full article
(This article belongs to the Special Issue Design, Synthesis and Applications of Bioactive Compounds)
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18 pages, 4805 KB  
Article
Circ-06958 Is Involved in Meat Quality by Regulating Cell Proliferation Through miR-31-5p/AK4 Axis in Pigs
by Xiaohan Zhang, Rongru Zhu, Xiaoxu Wu, Minghang Chang, Yuanlu Sun, Liang Wang, Ming Tian, Dongjie Zhang, Di Liu and Xiuqin Yang
Cells 2025, 14(18), 1416; https://doi.org/10.3390/cells14181416 - 10 Sep 2025
Viewed by 324
Abstract
Circular RNA (CircRNA) can regulate gene expression through acting as a competitive endogenous RNA (ceRNA), thus becoming involved in various biological processes. However, little was known about the role of circRNA in the formation of meat quality in pigs. Here, circRNAs were first [...] Read more.
Circular RNA (CircRNA) can regulate gene expression through acting as a competitive endogenous RNA (ceRNA), thus becoming involved in various biological processes. However, little was known about the role of circRNA in the formation of meat quality in pigs. Here, circRNAs were first characterized in muscles with differential meat quality and myofiber composition, longissimus thoracis, and semitendinosus muscles, with RNA-sequencing (RNA-seq). A total of 1126 differentially expressed circRNAs were identified. Among them, Circ-06958 is highly expressed in both muscles. Circ-06958 originated from Long-chain acyl-CoA synthetase 1 (ACSL1), a gene involved in muscle development. Circ-06958 was then characterized experimentally for the first time. Next, it was revealed that Circ-06958 increased proliferation of muscle cells, including porcine skeletal muscle satellite cells (PMSCs) and C2C12 myoblasts, by promoting cell cycle progression. Circ-06958 was mainly localized in cytoplasm, indicating it can function as a ceRNA. A regulatory axis Circ-06958/miR-31-5p/Adenylate Kinase 4 (AK4) axis was constructed with molecular biology techniques. Afterward, it was shown that miR-31-5p inhibited cell proliferation by affecting cell cycle progression in the two cells, while AK4 increased it. We made it clear that Circ-06958 promoted muscle cell proliferation via the miR-31-5p/AK4 axis. The results will contribute to further revealing the mechanisms through which meat quality generates. Full article
(This article belongs to the Special Issue Gene and Cellular Signaling Related to Muscle)
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18 pages, 10923 KB  
Article
PAFAH1B3 Exists in Linear Chromosomal and Extrachromosomal Circular DNA and Promotes HCC Progression via EMT
by Dandan Li, Huishan Sun, Yingjie Wang, Yicong Yin, Ying Zhu, Xia Qian, Shanshan Wang, Longhao Zhang, Haitao Zhao and Ling Qiu
Int. J. Mol. Sci. 2025, 26(18), 8801; https://doi.org/10.3390/ijms26188801 - 10 Sep 2025
Viewed by 266
Abstract
Recent evidence highlights the role of extrachromosomal circular DNAs (eccDNAs) in cancers. However, reports regarding its role in hepatocellular carcinoma (HCC) are infrequent. The abundance of eccDNAs from five HCC/adjacent tissue pairs was explored using Circle-Sequencing. eccDNA PAFAH1B3 was selected as one of [...] Read more.
Recent evidence highlights the role of extrachromosomal circular DNAs (eccDNAs) in cancers. However, reports regarding its role in hepatocellular carcinoma (HCC) are infrequent. The abundance of eccDNAs from five HCC/adjacent tissue pairs was explored using Circle-Sequencing. eccDNA PAFAH1B3 was selected as one of the objects. The effect of eccDNA PAFAH1B3 on HCC progression was determined using EdU, Transwell, and apoptosis assays. Additionally, the expressions of eccDNA PAFAH1B3, mRNA PAFAH1B3, and epithelial–mesenchymal transition (EMT)-related markers were determined using RT-PCR and WB. A xenograft tumor model was established to explore the function of PAFAH1B3 in vivo, and EMT-related markers were detected using RT-PCR and IHC analyses. The abundance of eccDNA PAFAH1B3 was significantly increased in HCC cell lines after transfection with eccDNA PAFAH1B3, and promoted the proliferation, migration, and invasion of liver cells while inhibiting apoptosis. The levels of mRNA PAFAH1B3 were also upregulated. Furthermore, intratumoral injection of PAFAH1B3 inhibitor suppressed tumor growth, and PAFAH1B3 knockdown increased and decreased the levels of the E-cadherin and N-cadherin, respectively. Our study findings reveal that eccDNA PAFAH1B3 may promote the occurrence and development of HCC by enhancing the expression of PAFAH1B3 and regulating EMT. Full article
(This article belongs to the Section Molecular Genetics and Genomics)
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