Search Results (867)

The size of the mitogenome varies greatly in angiosperms from different species, but the causes of expansion remain unclear. Species from the Zingiberaceae family often carry exceptionally large mitogenomes, where this phenomenon can be readily studied. C. kwangsiensis is a medicinal plant that is native to Guangxi, China, and it is traditionally used to treat blood stasis and gynecological conditions. We assembled its complete mitogenome by using the Illumina and Nanopore reads obtained from its analysis. The genome comprises 12 circular contigs with a multi-branched structure, totaling 7.76 Mb. It contains 39 protein-coding genes, 30 tRNAs (transfer RNAs), three rRNAs (ribosomal RNAs), and 652 C-to-U RNA edited sites. Repeated analysis revealed that the dispersed repeats are the major contributors to genome expansion. Comparisons with two other Zingiberaceae mitogenomes suggested that the large genome arose in a common ancestor rather than from a whole-genome duplication, with little contribution from DNA transfers from the chloroplasts and nuclei. Thus, the exceptionally large mitogenome of Curcuma appears to result largely from repeated accumulation in the non-coding regions, such as nuclear genome expansion in angiosperms. This finding also highlights the conservation of core mitochondrial genes. Our work provides new insights into mitogenomic size variations and gene conservation in plant species, including Zingiberaceae. Full article

Background/Objectives: eccDNA is a promising cancer biomarker in liquid biopsy. However, the reliability and reproducibility of eccDNA studies rely on the standardization of pre-analytical handling and processing of eccDNA, as well as transparent methodological reporting. Although evidence-based guidelines for cell-free DNA handling provide clear recommendations for plasma/serum processing, the extent to which eccDNA studies report and adhere to these key procedures remains uncertain. Methods: We systematically reviewed 14 studies (2017–2025) assessing eccDNA in plasma or serum from cancer patients. Each study was evaluated against 22 checklist items summarized from the NCI Biospecimen Collection and Processing Guideline, categorized into biospecimen collection, blood processing, and eccDNA processing. Items were classified as “reported,” “not reported,” or “deviated from NCI guideline,” and missing/deviation rates were calculated. Results: Publication was largely post-guideline (13/14 after 2020). Reporting gaps were widespread. Items with 100% missing were venipuncture site (14/14), freeze–thaw cycles (14/14), and fitness for downstream analysis/pre-assay QC (14/14). Other frequently missing elements included blood volume (10/14; 71%), collection tube type (7/14; 50%), date/time from draw to processing (11/14; 79%), second centrifugation parameters (8/14; 57%), storage temperature before freezing (6/14; 43%), frozen storage duration (13/14; 93%), and post-thaw handling (13/14; 93%). By contrast, the extraction method was consistently reported (0% missing), and most reported quantification (21% missing). Deviations from the NCI guideline were uncommon when items were reported, such as a blood processing delay (2/14; 14%) and the use of circulating nucleic acid kits for eccDNA extraction (1/14; 7%). Conclusions: Inconsistent reporting of pre-analytical procedures limits the reproducibility, transparency, and clinical translation of eccDNA research. We propose a concise reporting checklist informed by the NCI guideline. By focusing on the most frequently underreported aspects of key pre-analytical eccDNA procedures, the checklist provides researchers with an efficient and succinct methodological reporting framework that will enhance transparency and promote standardization in future eccDNA studies. Full article

The pathogenic free-living amoeba Naegleria fowleri is the cause of primary amebic meningoencephalitis (PAM), a central nervous system infection that is almost always lethal. One of the unusual features of the amoebae is the presence of ~4000 copies of a nucleolar-localized closed circular extrachromosomal ribosomal DNA element (CERE) that encodes the cell’s ribosomal RNA repertoire. It has historically been challenging to purify large quantities of CERE, limiting our understanding of the nucleic acid. Here, we describe a methodology for CERE purification that improves yield, reduces processing times, and maintains the integrity of the plasmid. This approach will enable the study of this unique DNA architecture, advancing our understanding of the pathobiology of the organism. Full article

Oxford Nanopore technology enables cost-effective, portable, long-read analyses of pathogen genomes. Accurate detection and interpretation of small circular viral genomes, including Anelloviridae, remain challenging due to limited base-level error quantification in rolling-circle amplification (RCA)-derived datasets. Here, we characterized read-level sequencing error profiles using M13mp18, a 7.2 kb circular phage genome, subjected to 1X and 3X shearing during library preparation. M13mp18 DNA was serially diluted into pooled anellovirus-positive plasma DNA extracts. Using custom error-analysis pipelines, we quantified mismatch, insertion, and deletion rates and evaluated consensus reconstruction accuracy across simulated sequencing depths. Since metagenomic viromes contain mixtures of related genomes and uneven coverage across taxa, depth-normalized subsampling was used to assess the precision of read-level error estimates under heterogeneous coverage. Across four benchmarked datasets, per-base error rates ranged from 0.018 to 0.022 errors per aligned base. Complete M13mp18 reference reconstruction was achieved at input levels ≥ 4.6 log₁₀ copies, and consensus sequences reached 100% identity at depths ≥ 15X when sufficient reads were available. Below 4.6 log₁₀ input copies, recovery was inconsistent. These findings provide a controlled empirical characterization of read-level error behavior in RCA-derived nanopore sequencing and support the interpretation of circular DNA virome data generated in complex metagenomic backgrounds. Full article

Chronic hepatitis B virus (HBV) infection remains one of the main causes of hepatocellular carcinoma (HCC), even though vaccination and long-term viral suppression have reduced new infections and circulating viral replication. This residual cancer risk suggests that serum HBV DNA alone does not capture the full biology of HBV-related carcinogenesis. Hepatitis B virus X protein (HBx) is a relevant entry point because it maintains the transcriptional competence of covalently closed circular DNA (cccDNA), engages host chromatin regulators, and may persist in tumors as cccDNA-derived, integration-derived, full-length, truncated, or fusion forms. This review focuses on a specific question: does the available literature support HBx-associated reactivation of the IGF2 locus in chronic HBV infection and HBV-related hepatocarcinogenesis, and, if so, at which regulatory layer is the claim defensible? The most direct evidence remains promoter-proximal. Classic mechanistic work shows acute HBx-dependent activation of IGF2 promoter P4 through Sp1- and PKC/ERK-dependent signaling. Human tissue and cell-based studies also support a broader fetal-promoter compartment, including P3/P4 transcript enrichment, local promoter hypomethylation, MBD2-HBx-CBP/p300 recruitment, and increased histone H3/H4 acetylation. These observations do not, however, establish HBV exclusivity, uniform loss of imprinting, or direct HBx-mediated rewiring of the human IGF2/H19 topological domain. Recent integration-aware and long-read studies further argue against treating tumor-stage HBx as a single biological variable. In the present evidence framework, HBx-associated IGF2 locus reactivation is therefore more appropriately viewed as a stage-aware, promoter-resolved, biomarker-oriented hypothesis than as a universal mechanism or a treatment algorithm for HBV-related HCC. Full article

Current 3D tumor models for aggressive breast cancers inadequately recapitulate the native tumor microenvironment (TME), leading to poor translational potential. There is a critical need for models capable of mimicking the unique biochemical signals present in the TME. To address this gap, breast tissue and a patient-derived xenograft tumor were decellularized and processed to produce breast tissue- and tumor-specific decellularized extracellular matrices (dECM). Histology confirmed complete cellular removal while maintaining the ECM. Further, DNA content was significantly reduced while ECM composition (POSTN, COLI, FN1) was retained. Breast dECM was incorporated (0, 5, 10, 20, and 50 µg/mL) with triple-negative breast cancer cell lines to generate spheroids. Imaging and histology demonstrated that cells in low dECM (5 and 10 µg/mL) formed compact singular spheres, while higher dECM concentrations (20 and 50 µg/mL) resulted in cells concentrated on the outer edge of the sphere and irregular sphere circularity. RNA-sequencing of MDA-MB-231 dECM spheres demonstrated that gene changes were mediated by both the inclusion of dECM and its composition. High-density tumor dECM upregulated genes associated with metastasis, while high-density breast dECM enhanced tumor suppressors and anti-metastasis genes. These findings indicate that dECM provides physiological cues in 3D tumor models by incorporating TME. Full article

Background/Objectives: Cervical cancer is mainly driven by persistent infection with high-risk human papillomaviruses (HPV), particularly HPV16 and HPV18. Despite advances in cytology, HPV-DNA testing and vaccination, challenges remain in the triage of HPV-positive individuals, prognostic stratification and prediction of treatment response. Non-coding RNAs (ncRNAs), including microRNAs, long non-coding RNAs and circular RNAs, together with host genetic factors influencing ncRNA expression and emerging lncRNA-encoded peptides, are increasingly recognized as regulators of HPV-associated carcinogenesis. This review summarizes their biological and potential clinical relevance. Methods: A structured literature search was conducted in PubMed and Scopus. Eligible studies included experimental, clinical, observational, genomic and translational investigations on ncRNA dysregulation, circulating or exosomal ncRNAs, treatment-response signatures, host genetic variation and lncRNA-encoded peptides in HPV-associated cervical precancer and cancer. Results: HPV oncoproteins can reshape host ncRNA networks through transcriptional and epigenetic mechanisms. Several miRNAs, lncRNAs and circRNAs are involved in cell-cycle control, apoptosis, senescence, epithelial–mesenchymal transition, immune regulation, DNA repair and treatment resistance. Circulating, exosomal and urinary ncRNA signatures have shown diagnostic or prognostic potential in exploratory cohorts. Specific lncRNAs, including ENSG00000267838/lnc-LENG9-5 and lncRNA-EME1, have been associated with chemoradiotherapy response and radioresistance. The lncRNA-encoded peptide TUBORF represents a novel preclinical therapeutic candidate, while genetic variation may further modulate lncRNA function in HPV-related cervical cancer. Conclusions: ncRNAs are promising candidates for risk stratification, non-invasive diagnosis, treatment-response prediction and therapeutic development in HPV-associated cervical disease. However, evidence remains exploratory, requiring prospective multicentre validation and standardized workflows before clinical implementation. Full article

Sustainable agriculture increasingly relies on organic amendments that integrate circular economy principles. Municipal Solid Waste (MSW)-derived compost (MSW-compost) represents a promising candidate as soil amendment in viticulture, yet its impact on soil microbiota remains poorly investigated. This study assessed the effects of MSW-compost application on the bacterial microbiota of a Mediterranean vineyard soil over a twelve-month period, comparing two application methods (surface mulching and tillage incorporation). Soil DNA was analyzed by 16S rRNA gene metabarcoding, complemented by functional prediction (Picrust2) and the Tea Bag Index to assess soil decomposition activity. MSW-compost significantly increased alpha-diversity and affected beta-diversity (p = 0.001) of the microbiota, regardless of the application method, with significant effects persisting throughout the entire observation period despite a clearly diminishing trend. Devosia emerged as the hub taxon of the co-occurrence network and was increased by compost addition. MSW-compost application mode remarkably affected the microbial network, with mulched treatment leading to a more complex, denser, and more interconnected network. While a similar number of taxa were increased or decreased, functional prediction revealed a notable enrichment of metabolic pathways, both synthetic and degradative, in the MSW-compost amended samples; this finding was supported by the enhanced red tea decomposition data (p = 0.007). Our results indicate that MSW-compost acts as a beneficial soil amendment, simultaneously enhancing microbial diversity and soil decomposition activity. This study provides novel evidence supporting the use of MSW-compost as a sustainable tool for improving soil microbiological quality in productive vineyards. Full article

The vertebrate mitochondrial genome (mitogenome) is a small, circular DNA molecule typically ~16–17 kb in length, encoding 37 genes that are essential for the electron transport chain, the mechanism that drives mostly all the ATP synthesis in cells. Owing to its central role in energy metabolism, its structure is highly conserved across vertebrate lineages in both the number and relative position of each gene in the genome. Nevertheless, different variations have been found in several teleost lineages, including antarctic fishes (Nototheniidae), gadiforms, hatchetfishes (Sternoptychidae), and Batrachoidiformes. The explanation for these phenomena remains unknown yet may reflect shifts in functional constraints and can provide insights into lineage-specific and/or coevolutionary processes. This raises the possibility that mitogenome structure is related to habitat selection, potentially reflecting environmental influences on energetic regulation. To further test this hypothesis, we studied more than 400 teleost species across all major teleost lineages. The mitogenome sequences were downloaded from NCBI and annotated using two independent algorithms (MITOZ and MITOS) and then compared with a reference (Danio rerio) to find any deviation from the standard structure. Similarly, ecological data was downloaded from FishBase using the R Package “rfishbase” 5.0.3. Two independent ancestral reconstruction analyses were carried out for both traits, “Mitogenome” and “Habitat”, using a reference evolutionary tree for teleosts to unravel both evolutionary histories. The possible association between mitogenome and habitat was then assessed using a suite of phylogenetic comparative methods, including Pagel’s correlation test (corHMM) to evaluate whether both traits evolved in a correlated fashion, branch-level co-transition analysis to identify lineages where structural changes and habitat shifts co-occurred, and node-by-node comparisons of ancestral state probabilities across the phylogeny. Preliminary results suggest a correlation between some deep-sea environments and a modified mitogenome structure, with structural deviations tending to cluster in lineages inhabiting greater depths. These exploratory findings raise the possibility that changes in mitogenome architecture may be linked to adaptations in energetic metabolism required for life in extreme low-energy environments. Further analyses are underway to clarify the functional significance of these genomic changes and their relationship to ecological and metabolic pressures in teleost evolution. Full article

Background: Extrachromosomal DNA (ecDNA) amplification represents a distinct mechanism of genomic instability in cancer, increasingly recognized for its role in aggressive disease progression. This review examines how ecDNA drives tumour evolution and assesses its potential as both a prognostic marker and therapeutic target. Methods: The authors integrate findings from multiple detection platforms—including FISH, whole-genome sequencing, and specialized reconstruction algorithms—and present data across diverse cancer types; no preregistration is noted, and no animal studies are included. Results: ecDNA consists of circular, acentric DNA elements carrying high-copy oncogene amplifications (such as EGFR, MYC, MDM2, and CDK4). Unlike chromosomal DNA, ecDNA segregates unevenly during cell division, generating intratumoral heterogeneity, accelerating adaptation to selective pressures, and promoting resistance to therapy. Pan-cancer surveys summarized here reveal ecDNA in a significant subset of tumours, with particularly high frequencies in liposarcoma, glioblastoma, and HER2-positive breast cancer, and consistent associations with worse clinical outcomes. Conclusions: The authors conclude that ecDNA amplification serves as a credible adverse prognostic indicator and holds promise for refining risk stratification and guiding treatment strategies. However, they stress that clinical adoption remains constrained by the absence of standardized, scalable, and reproducible detection. Full article

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. Full article

Mono- and bis-guanyl hydrazone-functionalized tricyclic compounds were here designed and investigated as putative G-quadruplex ligands in the context of anticancer drug development. The G-quadruplex on Controlled Pore Glass (G4-CPG) assay, a fast and easy screening method based on affinity chromatography for identifying potential G-quadruplex binders, together with biophysical techniques such as circular dichroism and fluorescence spectroscopy, demonstrated a higher selectivity of mono- with respect to disubstituted derivatives in recognizing G-quadruplexes from telomeric and oncogenic DNA regions vs. duplexes. Among the mono-substituted compounds, higher G-quadruplex selectivity was found for those containing the pyrido [3,4-b]indole and dibenzofuran scaffolds compared to the 9H-fluorene, 9H-carbazole, and dibenzothiophene ones. Molecular docking studies suggested that the investigated ligands bound the hybrid telomeric G-quadruplex model by adopting a coplanar arrangement of the core and guanyl hydrazone moieties, both stacked on the 5′-G-quartet, while in the interaction with the parallel oncogenic G-quadruplex model the guanyl hydrazone moieties pointed towards the grooves/loops. Finally, biological assays highlighted the higher potential of mono-guanyl hydrazone-derivatized tricyclic compounds as selective anticancer agents, showing higher anticancer activity and selectivity of action than the bis-guanyl hydrazone derivatives. Full article

An evaluation of plant ploidy is an important task in breeding and biotechnology. Current methods of ploidy assessment (flow cytofluorometry and microscopy) are time-consuming and costly, and not applicable to real-world agricultural conditions. We developed an automated method for ploidy assessment based on fluorescence microscopy, which aims to accelerate and reduce the cost of plant ploidy analysis. The method is based on the automated selection of plant nuclei in fluorescence micrographs, followed by analysis of nuclear area, fluorescence intensity of the Hoechst DNA-binding probe, and nuclear geometry (circularity, roundness, solidity). The study was conducted on monocotyledonous and dicotyledonous plants with known genome sizes. Triticum aestivum Wt (6n, hexaploid) and Temp (4n, tetraploid) are monocotyledonous, and Capsella bursa-pastoris (4n, tetraploid) and Capsella rubella (2n, dT/iploid) are dicotyledonous. A simple fluorescent staining protocol combined with automated analysis using our ImageJ macro enables reliable separation of both monocotyledonous and dicotyledonous plants by genome size with an accuracy close (for dicots) or comparable (for monocots) to flow cytofluorometry. For ploidy separation in monocots, the most sensitive parameters are fluorescence intensity, nucleus area, and circularity. For ploidy separation in dicots, the most sensitive parameters are nucleus area, fluorescence intensity, and circularity. Full article

The Ros/MucR family is constituted by proteins controlling the expression of genes crucial for the interaction with eukaryotic hosts. Ros/MucR family members were classified as H-NS-like proteins in α-proteobacteria, as they share fundamental features with H-NS proteins playing a pivotal role in controlling gene expression by structuring the bacterial genome. Here, we identified two new Ros/MucR family members in Sinorhizobium meliloti. They differ from classical MucR homologs since MucR2 lacks the circular oligomeric structure typical of other family members and MucR3 shows a concentration-dependent oligomerization ability with a low propensity to form circular particles, as shown by cryogenic electron microscopy. Moreover, MucR2 and MucR3 present a new zinc coordination sphere. The newly identified MucRs bind DNA, but lack the DNA bridging activity, which is crucial for structuring the bacterial genome. Using mass spectrometry, light scattering, NMR, EMSA and bridging assay, our study reports the first identification and characterization of two new MucRs and indicates that Ros/MucR family members control gene expression through distinct mechanisms. These results provide an important framework for future studies aimed at dissecting the interplay among MucR proteins and understanding how they can jointly orchestrate condition-dependent gene expression in bacterial species expressing multiple mucR homologous genes. Full article

Digestive tract structure is a key indicator of fish health and environmental adaptation, while seasonal dynamics of the gut microbiota reflect host responses to environmental changes. In this study, the digestive tract microstructure of Hypomesus nipponensis from Bosten Lake was characterized using H&E staining and scanning electron microscopy, followed by 16S rDNA gene V3-V4 region sequencing and analysis of the gut microbiota in spring, summer, and autumn. The results showed that the esophageal mucosa of H. nipponensis is a stratified columnar epithelium, with abundant gastric glands, and the circular muscle layer of the stomach caeca is significantly thickened (244.84 ± 49.01 μm). The pyloric caeca resemble the gut in structure; both are covered with dense microvilli on the luminal surface. Collectively, these features constitute the structural basis for its carnivorous diet. Microbiota analysis revealed that the diversity of gut microbiota fluctuated significantly with season: the Chao, Ace, and Sob indices in spring (144.63 ± 30.27) were significantly higher than in summer (82.13 ± 21.45) and autumn (83.25 ± 15.30) (p < 0.001), with no significant difference between summer and autumn (p > 0.05). The dominant marker genera of H. nipponensis in spring, summer, and autumn were Bacillus (31.60%), Clostridium (32.20%), and Sarcina (29.32%), respectively. This study describes the adaptive characteristics of the digestive tract structure and feeding habits of H. nipponensis and reveals the seasonal changes in its gut microbiota. Importantly, since the digestive tract structure data were collected only in summer, the direct relationship between the structure and seasonal microbial dynamics cannot be determined, and multi-season histological sampling is needed for further investigation. Nevertheless, these findings provide preliminary morphological and microbiological references for the ecological adaptation of this species in Bosten Lake and offer a scientific basis for water resource management in this area. Full article