Extrachromosomal Circular DNA Research Articles

Spotlight on the function and trends on Extrachromosomal circular DNA (eccDNA) : A bibliometric analysis from 2008-2023.

Extrachromosomal circular DNA (eccDNA),a type of circular DNA that has a nucleosomal structure, is widely distributed in eukaryotic chromosomes and has been found to modulate genome instability and plasticity, playing a role in regulating gene expression and genome evolution. To comprehensively outline the stages of eccDNA research development, including author collaborations, research topics and hotspots, and their temporal evolution trends, we conducted a bibliometric analysis of 242 publications related to eccDNA research published from 2008-2023 in the Web of Science Core Collection. The bibliometric analysis was performed using CiteSpace, the R package Bibliometrix, and VOSviewer. The USA, the University of California system, and Turner Km were found to be the most influential nation, organization, and author in this field, respectively. The exploration of Characterization and Diagnosis, Heterochromatin,Circ-Seq and Cancer Drug Resistance on eccDNA are the most concerned hotspots. EccDNA research has become a rapidly growing hotspot, receiving extensive attention from scholars in recent years. This study is the first to investigate the development and current challenges of eccDNA research through bibliometric analysis.The research on eccDNA has advanced from disorder to more intricate molecular functions. At present, the rapid growth of eccDNA studies in cancer has not been accompanied by an intuitive analysis of its evolutionary patterns. This review provides an overview of eccDNA's biological characteristics and functions, with a focus on its role in cancer research.

Confirmation of glyphosate-resistant Palmer amaranth (Amaranthus palmeri) populations in New York and responses to alternative chemistries

Abstract Palmer amaranth (Amaranthus palmeri S. Watson, AMAPA) is one of the most troublesome weeds in North America due to its rapid growth rate, substantial seed production, competitiveness and the evolution of herbicide-resistant populations. Though frequently encountered in the South, Midwest, and Mid-Atlantic regions of the United States, A. palmeri was recently identified in soybean [Glycine max (L.) Merr.] fields in Genesee, Orange, and Steuben counties, NY, where glyphosate was the primary herbicide for in-crop weed control. This research, conducted in 2023, aimed to (1) describe the dose response of three putative resistant NY A. palmeri populations to glyphosate, (2) determine their mechanisms of resistance, and (3) assess their sensitivity to other postemergence herbicides commonly used in NY crop production systems. Based on the effective dose necessary to reduce aboveground biomass by 50% (ED50), the NY populations were 42 to 67 times more resistant to glyphosate compared with a glyphosate-susceptible population. Additionally, the NY populations had elevated EPSPS gene copy numbers ranging from 25 to 135 located within extrachromosomal circular DNA (eccDNA). Label rate applications of Weed Science Society of America (WSSA) Group 2 herbicides killed up to 42% of the NY populations of A. palmeri. Some variability was observed among populations in response to WSSA Group 5 and 27 herbicides. All populations were effectively controlled by labeled rates of herbicides belonging to WSSA Groups 4, 10, 14, and 22. Additional research is warranted to confirm whether NY populations have evolved multiple resistance to herbicides within other WSSA groups and to develop effective A. palmeri management strategies suitable for NY crop production.

Comparative analysis of methodologies for detecting extrachromosomal circular DNA

Extrachromosomal circular DNA (eccDNA) is crucial in oncogene amplification, gene transcription regulation, and intratumor heterogeneity. While various analysis pipelines and experimental methods have been developed for eccDNA identification, their detection efficiencies have not been systematically assessed. To address this, we evaluate the performance of 7 analysis pipelines using seven simulated datasets, in terms of accuracy, identity, duplication rate, and computational resource consumption. We also compare the eccDNA detection efficiency of 7 experimental methods through twenty-one real sequencing datasets. Here, we show that Circle-Map and Circle_finder (bwa-mem-samblaster) outperform the other short-read pipelines. However, Circle_finder (bwa-mem-samblaster) exhibits notable redundancy in its outcomes. CReSIL is the most effective pipeline for eccDNA detection in long-read sequencing data at depths higher than 10X. Moreover, long-read sequencing-based Circle-Seq shows superior efficiency in detecting copy number-amplified eccDNA over 10 kb in length. These results offer valuable insights for researchers in choosing the suitable methods for eccDNA research.

Long-read genome assembly of the insect model organism Tribolium castaneum reveals spread of satellite DNA in gene-rich regions by recurrent burst events.

Eukaryotic genomes are replete with satellite DNAs (satDNAs), large stretches of tandemly repeated sequences that are mostly underrepresented in genome assemblies. Here we combined nanopore long-read sequencing with a reference-guided assembly approach to generate an improved, high-quality genome assembly, TcasONT, of the model beetle Tribolium castaneum Enriched by 45 Mb in repetitive regions, the new assembly comprises almost the entire genome sequence. We use the enhanced assembly to conduct global and in-depth analyses of abundant euchromatic satDNAs. Unexpectedly, we show the extensive spread of satDNAs in gene-rich regions, including long arrays. The sequence similarity relationships between satDNA monomers and arrays indicate a recent exchange of satDNA arrays between different chromosomes. We propose a scenario of their genome dynamics characterized by repeated bursts of satDNAs spreading through euchromatin, followed by a process of elongation and homogenization of arrays. We find that suppressed recombination on the X Chromosome has no significant effect on the spread of satDNAs but the X rather tolerates the amplification of satDNAs into longer arrays. Analyses of arrays' neighboring regions show a tendency of one satDNA to be associated with transposable-like elements. Using 2D electrophoresis followed by Southern blotting, we prove Cast satDNAs' presence in the fraction of extrachromosomal circular DNA (eccDNA). We point to two mechanisms that enable this satDNA spread to occur: transposition by transposable elements and insertion mediated by eccDNA. The presence of such a large proportion of satDNA in gene-rich regions inevitably gives rise to speculation about their possible influence on gene expression.

Long-read sequencing of an advanced cancer cohort resolves rearrangements, unravels haplotypes, and reveals methylation landscapes

The Long-Read Personalized OncoGenomics (POG) dataset comprises a cohort of 189 patient tumors and 41 matched normal samples sequenced using the Oxford Nanopore Technologies PromethION platform. This dataset from the POG program and the Marathon of Hope Cancer Centres Network includes DNA and RNA short-read sequence data, analytics, and clinical information. We show the potential of long-read sequencing for resolving complex cancer-related structural variants, viral integrations, and extrachromosomal circular DNA. Long-range phasing facilitates the discovery of allelically differentially methylated regions (aDMRs) and allele-specific expression, including recurrent aDMRs in the cancer genes RET and CDKN2A. Germline promoter methylation in MLH1 can be directly observed in Lynch syndrome. Promoter methylation in BRCA1 and RAD51C is a likely driver behind homologous recombination deficiency where no coding driver mutation was found. This dataset demonstrates applications for long-read sequencing in precision medicine and is available as a resource for developing analytical approaches using this technology.

Protection of the genome and the central exome by peripheral non-coding DNA against DNA damage in health, ageing and age-related diseases.

DNA in eukaryotic genomes is under constant assault from both exogenous and endogenous sources, leading to DNA damage, which is considered a major molecular driver of ageing. Fortunately, the genome and the central exome are safeguarded against these attacks by abundant peripheral non-coding DNA. Non-coding DNA codes for small non-coding RNAs that inactivate foreign nucleic acids in the cytoplasm and physically blocks these attacks in the nucleus. Damage to non-coding DNA produced during such blockage is removed in the form of extrachromosomal circular DNA (eccDNA) through nucleic pore complexes. Consequently, non-coding DNA serves as a line of defence for the exome against DNA damage. The total amount of non-coding DNA/heterochromatin declines with age, resulting in a decrease in both physical blockage and eccDNA exclusion, and thus an increase in the accumulation of DNA damage in the nucleus during ageing and in age-related diseases. Here, we summarize recent evidence supporting a protective role of non-coding DNA in healthy and pathological states and argue that DNA damage is the proximate cause of ageing and age-related genetic diseases. Strategies aimed at strengthening the protective role of non-coding DNA/heterochromatin could potentially offer better systematic protection for the dynamic genome and the exome against diverse assaults, reduce the burden of DNA damage to the exome, and thus slow ageing, counteract age-related genetic diseases and promote a healthier life for individuals.

Extrachromosomal circular DNAs in prostate adenocarcinoma: global characterizations and a novel prediction model.

The role of focal amplifications and extrachromosomal circular DNA (eccDNA) is still uncertain in prostate adenocarcinoma (PRAD). Here, we first mapped the global characterizations of eccDNA and then investigate the characterization of eccDNA-amplified key differentially expressed encoded genes (eKDEGs) in the progression, immune response and immunotherapy of PRAD. Circular_seq was used in conjunction with the TCGA-PRAD transcriptome dataset to sequence, annotate, and filter for eccDNA-amplified differentially expressed coding genes (eDEGs) in PRAD and para-cancerous normal prostate tissues. Afterwards, risk models were created and eKDEGs linked to the PRAD prognosis were identified using Cox and Lasso regression analysis. The immune microenvironment of the risk model was quantified using a variety of immunological algorithms, which also identified its characteristics with regard to immunotherapy, immune response, and immune infiltration. In this research, there was no significant difference in the size, type, and chromosomal distribution of eccDNA in PRAD and para-cancerous normal prostate tissues. However, 4,290 differentially expressed eccDNAs were identified and 1,981 coding genes were amplified. Following that, 499 eDEGs were tested in conjunction with the transcriptome dataset from TCGA-PRAD. By using Cox and Lasso regression techniques, ZNF330 and PITPNM3 were identified as eKDEGs of PRAD, and a new PRAD risk model was conducted based on this. Survival analysis showed that the high-risk group of this model was associated with poor prognosis and validated in external data. Immune infiltration analysis showed that the model risks affected immune cell infiltration in PRAD, not only mediating changes in immune cell function, but also correlating with immunophenotyping. Furthermore, the high-risk group was negatively associated with anti-CTLA-4/anti-PD-1 response and mutational burden. In addition, Tumor Immune Dysfunction and Exclusion analyses showed that high-risk group was more prone to immune escape. Drug sensitivity analyses identified 10 drugs, which were instructive for PRAD treatment. ZNF330 and PITPNM are the eKDEGs for PRAD, which can be used as potential new prognostic markers. The two-factor combined risk model can effectively assess the survival and prognosis of PRAD patients, but also can predict the different responses of immunotherapy to PRAD patients, which may provide new ideas for PRAD immunotherapy.

Extrachromosomal circular DNA-related SPOCK1 contributes to development and enzalutamide resistance of prostate cancer by regulating epithelial mesenchymal transition

Prostate cancer is a significant contributor to cancer-related mortality, and the tumor typically develops into castration-resistant prostate cancer (CRPC). Hence, few effective clinical strategies are available to patients with advanced disease. Extrachromosomal circular DNA (eccDNA) is a type of circular DNA originating from the chromosomes but is likely independent of them. Because of its unique structural characteristics, eccDNA has extensive applications as a new biomarker for cancer prevention and treatment. Circle-seq obtains a comprehensive picture of the overall landscape of eccDNA sizes and content in cell populations. In this study, we used Circle-seq and studied the distribution pattern and expression level of eccDNA in prostate cancer. We confirmed that eccDNA is derived from every human chromosome and has sequences from all known types of genomic structures, revealing it is a common mutational element in prostate cancer. We also identified an eccDNA-related gene SPOCK1 that promotes drug resistance, proliferation, and metastasis of many cancers through the epithelial-mesenchymal transition (EMT) mechanism. The SPOCK1-associated eccDNA was highly upregulated in various groups of sequencing results, and SPOCK1 was highly expressed in prostate cancer tissues and cells. Therefore, SPOCK1 exists as eccDNA in prostate cancer and encourages its development and drug resistance via the EMT mechanism. Our results suggest that upregulated genes in the form of eccDNA are oncogenes in prostate cancer and play a pivotal role in carcinogenesis.

Identification and Characterization of Extrachromosomal Circular DNA in Slimming Grass Carp.

Slimming grass carp is a commercial variety with good body form and meat quality, which is cultured by starving common grass carp in a clean flowing water environment. Compared to common grass carp, slimming grass carp has a far higher economic value. Until now, no molecular study has concentrated on the regulation mechanism of the muscle characteristics of slimming grass carp. This study first reported the gene expression profile of the muscle characteristics of slimming grass carp based on the level of extrachromosomal circular DNAs (eccDNAs). EccDNAs are double-stranded circular DNAs derived from genomic DNAs and play crucial roles in the functional regulation of a wide range of biological processes, none of which have been shown to occur in fish. Here, muscle eccDNAs from slimming grass carp and common grass carp were both generally sequenced, and the information, as well as the expression profile of eccDNAs, were compared and analysed. The findings reveal that 82,238 and 25,857 eccDNAs were detected from slimming grass carp and common grass carp, respectively. The length distribution of eccDNAs was in the range of 1~1000 bp, with two peaks at about 200 bp and 400 bp. When the expression profiles of eccDNAs between slimming grass carp and common grass carp were compared, 3523 up-regulated and 175 down-regulated eccDNAs were found. Enrichment analysis showed that these eccDNA genes were correlated with cellular structure and response, cell immunology, enzyme activity, etc. Certain differentially expressed eccDNAs involved in muscle characteristics were detected, which include myosin heavy chain, myosin light chain, muscle segment homeobox C, calsequestrin, calmodulin, etc., among which the majority of genes were linked to muscle structure and contraction. This indicates that during the process of cultivating from common grass carp to slimming grass carp, the treatment primarily affected muscle structure and contraction, making the meat quality of slimming grass carp different from that of common grass carp. This result provides molecular evidence and new insights by which to elucidate the regulating mechanism of muscle phenotypic characterisation in slimming grass carp and other fish.

Genome-wide characterization of extrachromosomal circular DNA in SLE and functional analysis reveal their association with apoptosis

Extrachromosomal circular DNA (eccDNA) derived from linear chromosomes, are showed typical nucleosomal ladder pattern in agarose gel which as a known feature of apoptosis and demonstrated to be immunogenicity. In systemic lupus erythematosus (SLE) patients, elevated levels of cell-free DNA (cfDNA) can be found in either linear forms or circular forms, while circular ones are much less common and harder to detect. The molecular characteristics and function of circular forms in plasma SLE patients remains elusive. Herein, we characterized the hallmarks of plasma eccDNA in SLE patients, including the lower normalized number and GC content of eccDNA in SLE plasma than in the healthy, and SLE eccDNA number positively correlated with C3 and negatively with anti-dsDNA antibodies. The differential eccGenes (eccDNAs carrying the protein coding gene sequence) of SLE was significantly enriched in apoptosis-related pathways. The artificially synthesized eccDNA with sequences of the PRF1 exon region could promote transcriptional expression of PRF1, IFNA and IFIT3 and inhibit early-stage apoptosis. Plasma eccDNA can serve as a novel autoantigen in the pathogenesis of SLE.

Predicting gestational diabetes mellitus risk at 11–13 weeks’ gestation: the role of extrachromosomal circular DNA

BackgroundGestational diabetes mellitus (GDM) significantly impacts maternal and infant health both immediately and over the long term, yet effective early diagnostic biomarkers are currently lacking. Thus, it is essential to identify early diagnostic biomarkers for GDM risk screening. Extrachromosomal circular DNA (eccDNA), being more stable than linear DNA and involved in disease pathologies, is a viable biomarker candidate for diverse conditions. In this study, eccDNA biomarkers identified for early diagnosis and assessment of GDM risk were explored.MethodsUsing Circle-seq, we identified plasma eccDNA profiles in five pregnant women who later developed GDM and five matched healthy controls at 11–13 weeks of gestation. These profiles were subsequently analyzed through bioinformatics and validated through outward PCR combined with Sanger sequencing. Furthermore, candidate eccDNA was validated by quantitative PCR (qPCR) in a larger cohort of 70 women who developed GDM and 70 normal glucose-tolerant (NGT) subjects. A ROC curve assessed the eccDNA’s diagnostic potential for GDM.Results2217 eccDNAs were differentially detected between future GDM patients and controls, with 1289 increased and 928 decreased in abundance. KEGG analysis linked eccDNA genes mainly to GDM-related pathways such as Rap1, MAPK, and PI3K-Akt, and Insulin resistance, among others. Validation confirmed a significant decrease in eccDNA PRDM16circle in the plasma of 70 women who developed GDM compared to 70 NGT women, consistent with the eccDNA-seq results. PRDM16circle showed significant diagnostic value in 11–13 weeks of gestation (AUC = 0.941, p < 0.001).ConclusionsOur study first demonstrats that eccDNAs are aberrantly produced in women who develop GDM, including PRDM16circle, which can predict GDM at an early stage of pregnancy, indicating its potential as a biomarker.Trial registration ChiCTR2300075971, http://www.chictr.org.cn. Registered 20 September 2023.

Reconstructing extrachromosomal DNA structural heterogeneity from long-read sequencing data using Decoil.

Circular extrachromosomal DNA (ecDNA) is a form of oncogene amplification found across cancer types and associated with poor outcome in patients. ecDNA can be structurally complex and can contain rearranged DNA sequences derived from multiple chromosome locations. As the structure of ecDNA can impact oncogene regulation and may indicate mechanisms of its formation, disentangling it at high resolution from sequencing data is essential. Even though methods have been developed to identify and reconstruct ecDNA in cancer genome sequencing, it remains challenging to resolve complex ecDNA structures, in particular amplicons with shared genomic footprints. We here introduce Decoil, a computational method that combines a breakpoint-graph approach with LASSO regression to reconstruct complex ecDNA and deconvolve co-occurring ecDNA elements with overlapping genomic footprints from long-read nanopore sequencing. Decoil outperforms de novo assembly and alignment-based methods in simulated long-read sequencing data for both simple and complex ecDNAs. Applying Decoil on whole-genome sequencing data uncovered different ecDNA topologies and explored ecDNA structure heterogeneity in neuroblastoma tumors and cell lines, indicating that this method may improve ecDNA structural analyses in cancer.

Beyond the Chromosome: Recent Developments in Decoding the Significance of Extrachromosomal Circular DNA (eccDNA) in Human Malignancies.

Extrachromosomal circular DNA (eccDNA) is a form of a circular double-stranded DNA that exists independently of conventional chromosomes. eccDNA exhibits a broad and random distribution across eukaryotic cells and has been associated with tumor-related properties due to its ability to harbor the complete gene information of oncogenes. The complex and multifaceted mechanisms underlying eccDNA formation include pathways such as DNA damage repair, breakage-fusion-bridge (BFB) mechanisms, chromothripsis, and cell apoptosis. Of note, eccDNA plays a pivotal role in tumor development, genetic heterogeneity, and therapeutic resistance. The high copy number and transcriptional activity of oncogenes carried by eccDNA contribute to the accelerated growth of tumors. Notably, the amplification of oncogenes on eccDNA is implicated in the malignant progression of cancer cells. The improvement of high-throughput sequencing techniques has greatly enhanced our knowledge of eccDNA by allowing for a detailed examination of its genetic structures and functions. However, we still lack a comprehensive and efficient annotation for eccDNA, while challenges persist in the study and understanding of the functional role of eccDNA, emphasizing the need for the development of robust methodologies. The potential clinical applications of eccDNA, such as its role as a measurable biomarker or therapeutic target in diseases, particularly within the spectrum of human malignancies, is a promising field for future research. In conclusion, eccDNA represents a quite dynamic and multifunctional genetic entity with far-reaching implications in cancer pathogenesis and beyond. Further research is essential to unravel the molecular pathways of eccDNA formation, elucidate its functional roles, and explore its clinical applications. Addressing these aspects is crucial for advancing our understanding of genomic instability and developing novel strategies for tailored therapeutics, especially in cancer.

Integrated analyses highlight interactions between the three-dimensional genome and DNA, RNA and epigenomic alterations in metastatic prostate cancer

The impact of variations in the three-dimensional structure of the genome has been recognized, but solid cancer tissue studies are limited. Here, we performed integrated deep Hi-C sequencing with matched whole-genome sequencing, whole-genome bisulfite sequencing, 5-hydroxymethylcytosine (5hmC) sequencing and RNA sequencing across a cohort of 80 biopsy samples from patients with metastatic castration-resistant prostate cancer. Dramatic differences were present in gene expression, 5-methylcytosine/5hmC methylation and in structural variation versus mutation rate between A and B (open and closed) chromatin compartments. A subset of tumors exhibited depleted regional chromatin contacts at the AR locus, linked to extrachromosomal circular DNA (ecDNA) and worse response to AR signaling inhibitors. We also identified topological subtypes associated with stark differences in methylation structure, gene expression and prognosis. Our data suggested that DNA interactions may predispose to structural variant formation, exemplified by the recurrent TMPRSS2–ERG fusion. This comprehensive integrated sequencing effort represents a unique clinical tumor resource.

EccDNA in plant-stress and biotechnological solutions in agriculture

Extrachromosomal circular DNA (eccDNA) is genetic material that exists outside of chromosomes and holds potential for next-generation genetic engineering in plant biology. By improving plant resilience, growth, and productivity, eccDNA offers a promising solution to global challenges in food security and environmental sustainability, making this a transformative era in agricultural biotechnology.

Transfection of a Molecular Clone of Naegleria gruberi rDNA into N. gruberi Trophozoites.

All ribosomal genes of Naegleria trophozoites are maintained in a closed circular extrachromosomal ribosomal DNA (rDNA) containing element (CERE). While little is known about the CERE, a complete genome sequence analysis of three Naegleria species clearly demonstrates that there are no rDNA cistrons in the nuclear genome. Furthermore, a single DNA origin of replication has been mapped in the N. gruberi CERE, supporting the hypothesis that CERE replicates independently of the nuclear genome. This CERE characteristic suggests that it may be possible to use engineered CERE to introduce foreign proteins into Naegleria trophozoites. As the first step in exploring the use of a CERE as a vector in Naegleria, we developed a protocol to transfect N. gruberi with a molecular clone of the N. gruberi CERE cloned into pGEM7zf+ (pGRUB). Following transfection, pGRUB was readily detected in N. gruberi trophozoites for at least seven passages, as well as through encystment and excystment. As a control, trophozoites were transfected with the backbone vector, pGEM7zf+, without the N. gruberi sequences (pGEM). pGEM was not detected after the first passage following transfection into N. gruberi, indicating its inability to replicate in a eukaryotic organism. These studies describe a transfection protocol for Naegleria trophozoites and demonstrate that the bacterial plasmid sequence in pGRUB does not inhibit successful transfection and replication of the transfected CERE clone. Furthermore, this transfection protocol will be critical in understanding the minimal sequence of the CERE that drives its replication in trophozoites, as well as identifying regulatory regions in the non-ribosomal sequence (NRS).

METB-08. CIRCULAR EXTRACHROMOSOMAL DNA IS ASSOCIATED WITH POOR SURVIVAL IN CHILDHOOD CANCER TYPES

Abstract BACKGROUND Circular extrachromosomal DNA (ecDNA) is a common form of oncogene amplification in aggressive cancers and a promising target for improved cancer therapy. The frequency and diversity of ecDNA has been described in different types of adult cancers and in selected childhood cancer types such as neuroblastoma and medulloblastoma. However, the frequency of ecDNA and its association with survival has not been comprehensively analyzed across the spectrum of pediatric cancers. METHODS We performed a pediatric pan-cancer study of ecDNA in which we mined large pediatric cancer genomics repositories and reconstructed ecDNA sequence structures from whole genome sequencing data. RESULTS We analyzed a retrospective cohort of 1684 tumor biopsies from 1506 different patients, covering all major childhood cancers, many of which occur exclusively in children, including rare embryonal tumor types. The fraction of patients with ecDNA-positive tumors was 10%, varying significantly by tumor type. Aggressive tumor types had particularly high incidences of ecDNA, including embryonal tumors with multilayered rosettes (n = 6, 67%), osteosarcoma (n = 55, 47%), rhabdomyosarcoma (n = 38, 39%), and neuroblastoma (n = 111, 30%). Survival analysis revealed that ecDNA amplifications are significantly associated with poor outcome. Furthermore, we catalogued known and potentially novel oncogenes that are recurrently amplified on ecDNA in pediatric cancers and showed that ecDNA often evolves during disease progression. CONCLUSIONS These results reveal childhood tumor types in which ecDNA is widely prevalent and highlight patient populations that could potentially benefit from future therapies targeting mechanisms of ecDNA biogenesis and function.

Extrachromosomal circular DNA (eccDNA) characteristics in the bile and plasma of advanced perihilar cholangiocarcinoma patients and the construction of an eccDNA-related gene prognosis model.

Extrachromosomal DNAs (eccDNAs) frequently carry amplified oncogenes. This investigation aimed to examine the occurrence and role of eccDNAs in individuals diagnosed with advanced perihilar cholangiocarcinoma (pCCA) who exhibited distinct prognostic outcomes. Five patients with poor survival outcomes and five with better outcomes were selected among patients who received first-line hepatic arterial infusion chemotherapy from June 2021 to June 2022. The extracted eccDNAs were amplified for high-throughput sequencing. Genes associated with the differentially expressed eccDNAs were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The differentially expressed bile eccDNA-related genes were used to construct a prognostic model. Across all 10 patients, a total of 19,024 and 3,048 eccDNAs were identified in bile and plasma, respectively. The concentration of eccDNA detected in the bile was 9-fold higher than that in plasma. The chromosome distribution of the eccDNAs were similar between bile and matched plasma. GO and KEGG pathway analyses showed enrichment in the mitogen-activated protein kinase (MAPK) and Wnt/β-catenin pathways in patients with poor survival outcomes. According to the prognostic model constructed by eccDNA-related genes, the high-risk group of cholangiocarcinoma patients displayed significantly shorter overall survival (p < 0.001). Moreover, the degree of infiltration of immunosuppressive cells was higher in patients in the high-risk group. In conclusion, EccDNA could be detected in bile and plasma of pCCA patients, with a higher concentration. A prognostic model based on eccDNA-related genes showed the potential to predict the survival and immune microenvironment of patients with cholangiocarcinoma.

Landscape of extrachromosomal circular DNAs, transcriptome, and proteome analysis reveals insights into alcoholic liver cirrhosis

Alcoholic liver cirrhosis (ALC) is a result of excessive and chronic alcohol consumption. Because alchol can cause DNA damage, extrachromosomal circular DNA (eccDNA) was investigated in ALC liver due to it can be a result of DNA damage. Considering eccDNA has ability to lead to genomic instability as an enhancer of gene transcription, we utilized Circle-Seq to identify differences in eccDNA profiles and gene expression patterns in liver samples obtained from ALC patients (n = 3) and healthy controls (n = 3) to investigate the role of eccDNA in the development of ALC. The abundance of eccDNA in ALC (mean = 13,349) were higher than the healthy control (mean = 11,557) without significant difference (pvalue = 0.6530). We observed 1,032 eccDNA containing genes showed higher expression in ALC patients compared to healthy controls (p < 0.05, log2FC > 1). Notably, we discovered seven genes that exhibited a significant positive correlation between eccDNA abundance and gene expression levels. These genes include A disintegrin and metalloproteinase with thrombospondin motifs 2 (ADAMTS2), Voltage-dependent L-type calcium channel subunit alpha-1C (CACNA1C), Protein TANC1 (TANC1), Integrin alpha-2 (ITGA2), EH domain-containing protein 4 (EHD4), Phosphofurin acidic cluster sorting protein 1 (PACS1), and Neuron navigator 2 (NAV2). Through mass spectrometry proteomics, ITGA2 were found to have significantly higher abbudance in ALC. Integrins are a family of proteins plays key roles in the fibrosis development of liver. Thus, our study opens a new perspective for liver fibrosis development.

Exploring the extrachromosomal plasmid rDNA of Naegleria fowleri AY27 genotype II: A human brain-eating amoeba via high-throughput sequencing

Naegleria fowleri, also known as brain-earing amoeba, causes severe and rapidly fatal CNS infection in humans called primary amebic meningoencephalitis (PAM). The DNA from the N. fowleri clinical isolate was sequenced for circular extrachromosomal ribosomal DNA (CERE - rDNA). The CERE contains 18 S, 5.8 S, and 28 S ribosomal subunits separated by internal transcribed spacers, 5 open reading frames (ORFs), and mostly repeat elements comprising 7268 bp out of 15,786 bp (46%). A wide variety of variations and recombination events were observed. Finally, the ORFs that comprised only 4 hypothetical proteins were modeled and screened against Zinc drug-like compounds. Two compounds [ZINC77564275 (ethyl 2-(((4-isopropyl-4 H-1,2,4-triazol-3-yl) methyl) (methyl)amino) oxazole-4-carboxylate) and ZINC15022129 (5-(2-methoxyphenoxy)-[2,2’-bipyrimidine]-4,6(1 H,5 H)-dione)] were finalized as potential druggable compounds based on ADME toxicity analysis. We propose that the compounds showing the least toxicity would be potential drug candidates after laboratory experimental validation is performed.