Showing papers on "Nuclear DNA published in 2023"

Mitochondrial H2O2 release does not directly cause genomic DNA damage

Abstract: Reactive Oxygen Species (ROS) derived from mitochondrial respiration are frequently cited as a major source of genomic DNA damage and subsequent mutations that contribute to cancer development and aging. However, experimental evidence showing that ROS released by mitochondrial can directly damage nuclear DNA under (patho)physiological conditions has been largely lacking. In this study we modeled the effects of mitochondrial H2O2 release and compared this to H2O2 production at the nucleosomes in an untransformed human cell line. We used a chemogenetic approach to produce localized H2O2 and combined it with a new method we developed to directly quantify the amount of H2O2 produced. This enabled us to precisely investigate to what extent DNA damage occurs downstream of near- and supraphysiological amounts of localized H2O2 generation. Nuclear H2O2 production gives rise to DNA strand breaks, subsequent activation of the DNA damage response, cell cycle arrest and eventually senescence. Release of H2O2 from mitochondria on the other hand shows none of these effects, even at levels that are orders of magnitude higher than what mitochondria normally produce. Artificially high levels of mitochondrial H2O2 release do result in DNA strand breaks, but in parallel invariably cause ferroptosis-mediated cell death, preventing propagation of DNA damage-induced mutations. This study shows that H2O2 released from mitochondria is unlikely to directly damage genomic DNA, limiting its contribution to oncogenic transformation and aging.

Nucleophagy contributes to genome stability through degradation of type II topoisomerases A and B and nucleolar components.

Abstract: The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

Somatic nuclear blebbing in Caenorhabditis elegans is not a feature of organismal aging but a potential indicator of germline proliferation in early adulthood

Abstract: Abstract Abnormal nuclear morphology is suggested to be a hallmark of aging and one such abnormality is nuclear blebbing. However, little is known about whether and how nuclear blebbing participates in animal aging, and what regulates it. In this study, we show that the frequency of nuclear blebbing in the hypodermis increases during aging in wild-type C. elegans. These nuclear blebs are enveloped by the nuclear lamina, the inner and the outer nuclear membrane, and 42% of them contain chromatin. Although nuclear blebbing could lead to DNA loss if chromatin-containing blebs detach and fuse with lysosomes, we find by time-lapse imaging that nuclear blebs rarely detach, and the estimated lifetime of a nuclear bleb is 772 h or 32 days. The amount of DNA lost through nuclear blebbing is estimated to be about 0.1% of the total DNA loss by adult Day 11. Furthermore, the frequency of nuclear blebbing does not correlate with the rate of aging in C. elegans. Old age does not necessarily induce nuclear blebbing, neither does starvation, heat stress, or oxidative stress. Intriguingly, we find that proliferation of germ cells promotes nuclear blebbing.

Mature Red Blood Cells Contain Long DNA Fragments and Could Acquire DNA from Lung Cancer Tissue

Abstract: Red blood cells (RBC) are commonly known as cells with no nucleus or mitochondria and are assumed to be a transportation vehicle. This study confirms that RBC contain long DNA fragments inside with stain by both microscope and flow cytometry, which covers most nuclear and mitochondrial genome regions by next‐generation sequencing (NGS). Such characteristics demonstrate a significant difference compared with A549 cell line or paired peripheral blood mononuclear cell as nucleated cells. To further explore the characteristics of RNA DNA, DNA from 20 RBC samples is sequenced by NGS. Interestingly, several gaps and multiple regions with copy number variation are observed significantly different between different samples, which could be used to distinguish samples with different health status accurately. Using an in vitro co‐culture system, it is shown that RBC could absorb DNA‐bearing tumorigenic mutations from cancer cell lines but requires cell‐to‐cell contact. Finally, based on a small scale clinical trial, it is confirmed that common genetic mutations of cancer tissues could be detected in RBC from patients with early‐stage non‐small‐cell lung cancer. This study highlights a new biological phenomenon involving RBC and its translational potential as a novel liquid biopsy technology platform for early cancer screening and diagnosis of malignancy.

A new DNA extraction method (HV-CTAB-PCI) for amplification of nuclear markers from open ocean-retrieved faeces of an herbivorous marine mammal, the dugong

Abstract: Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new ‘High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol’ (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of single nucleotide polymorphism (SNP) markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.

Museomics reveals the phylogenetic position of the extinct Moroccan trout Salmo pallaryi.

Abstract: We used museomics to reconstruct the mitochondrial genome from two individuals of the Moroccan, endemic and extinct trout, Salmo pallaryi. We further obtained partial data from 21 nuclear genes previously used for trout phylogenetic analyses. Phylogenetic analyses including publicly available data from the mitochondrial control region and the cytochrome b gene, and the 21 nuclear genes place S. pallaryi among other North African trouts. Mitochondrial DNA places S. pallaryi close to Salmo macrostigma within a single North African clade. Although the nuclear coverage of the genome was low, both specimens were independently positioned as sisters to one of two distantly related North African clades, viz. the Atlas clade with the Dades trout, Salmo multipunctatus as sister. Phylogenetic discordance between mitochondrial DNA and nuclear DNA phylogenies is briefly discussed. As several specimens that were extracted failed to produce DNA of sufficient quality, we discuss potential reasons for that. We suggest that museum specimens in poor physical condition may be better for DNA extraction compared to better-preserved ones, possibly related to the innovation of formalin as a fixative before ethanol storage in the early 20th century. This article is protected by copyright. All rights reserved.

Reassessment of species delimitation using nuclear markers in three lentic-breeding salamanders from the Chugoku District of Japan (Amphibia: Caudata: Hynobiidae)

Abstract: Hynobius akiensis sensu lato has recently been split into three species based on short sequence analyses of cyt-b gene of mtDNA and without data of nuclear DNA, and strange sympatric distribution in some areas has been indicated in two species. We analyzed nuclear DNA marker (SNPs) and complete sequence of cyt-b in H. akiensis sensu lato to reassess species delimitation and genetic introgression among species. As a result, we found two lineages with discordant mitochondrial and nuclear DNA in some areas. Of H. akiensis sensu lato, each of the two contains the type locality of two species recently reported (H. sumidai and H. geiyoensis), and the use of these names has been previously advocated. However, their sympatric distribution was rejected based on nuclear DNA data, which we consider is more reliable than mtDNA. We thus clarify geographic boundary of these two species and revise the species delimitations.

Abstract 4819: Mechanism of disturbance of nuclear size homeostasis in cancer cells

Abstract: Abnormal increase in nuclear size is one of the criteria for distinguishing cancer cells. The main causes and functional consequences of nuclear size increase in cancer cells are not well known. In this study, we analyzed genome-wide RNAi library screen data to discover genes involved in the maintenance of nuclear size homeostasis. Importantly, many of the screen hits mapped to the functional categories of replication stress and DNA damage repair. We confirmed that depletion of genes required to prevent replication fork collapse causes a significant increase in nuclear size. Excessive replication stress and defects in the DNA repair system are common in many cancers. Therefore, disturbances of nuclear size homeostasis may be due to excessive replication stress and enhanced DNA damage response. To identify effectors that link replication stress and nuclear size change, we investigated candidates such as cell cycle progression, the mTOR pathway, and the cytoskeleton. As previously known, cell cycle arrest at G2 phase increased nuclear size, but replication fork collapse increased nuclear size even more excessively, suggesting the existence of other mechanisms. Inhibition of the mTOR pathway did not block replication stress-induced nuclear enlargement. Interestingly, we found that actin polymerization inside the nucleus plays a role in determining nuclear size in replication stress condition. Inhibition of nuclear actin polymerization reversed nuclear enlargement due to replication fork collapses. The Increase in nuclear size appeared to reduce the invasive ability of cancer cells. In addition, the enlarged nuclei resisted mechanical stress-mediated nuclear envelope rupture. We also observed a change in the abundance of the tri-methylation of lysine 27 on histone H3. These results suggest that nuclear size homeostasis is perturbed as a result of excessive replication stress, and changes in nuclear size affect the invasive capacity and gene expression of cancer cells. However, it is not clear whether nuclear enlargement act as a strong barrier to metastasis, as nuclear size can readily change through changes in nuclear actin dynamics. Further studies are needed to understand the impact of nuclear size changes on cancer initiation and progression. Citation Format: Changgon Kim, Joon Kim. Mechanism of disturbance of nuclear size homeostasis in cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4819.

Determination of Nuclear DNA Content and Chromosome Number of Verbascum scamandri Murb. (Scrophulariaceae)

Abstract: The aim of the study was to determine the chromosome number and nuclear DNA content of an endemic plant, V. scamandri, using 14-week-old plants germinated from seed and to determine the genetic stability based on flow cytometry analyses in callus tissue induced MS medium containing 1 mg/L Kin + 1 mg/L 2,4-D and 9-week-old propagated plants, induced and developed on MS medium containing 2 mg/L Kin + 0.1 mg/L 2,4-D. In the mitotic chromosome counts showed V. scamandri had 2n = 16. Flow cytometric analysis revealed that 14-week-old in vitro grown plants has 0.73 pg/2C ± 0.01, callus tissue has 0.76 pg/2C ± 0.02 and propagated plantlets has as 0.79 pg/2C ± 0.01. The results proved that propagated plants had similar DNA content to the seed-derived plants which showed analysed plants were genetically stable.

Supplementary Data from <i>Helicobacter pylori</i> Infection Induces Genetic Instability of Nuclear and Mitochondrial DNA in Gastric Cells

Abstract: Supplementary Data from <i>Helicobacter pylori</i> Infection Induces Genetic Instability of Nuclear and Mitochondrial DNA in Gastric Cells

Nuclear DNA Content Estimation of Seaweed by Fluorimetry Analysis.

Abstract: Fluorimetry analysis of nuclear DNA content allows identification of genome size and ploidy levels of different life phases, tissues, and populations in seaweed species. It is an easy method that saves time and resources compared to more complex techniques. Here we describe the methodology for measuring nuclear DNA content in seaweed species by DAPI fluorochrome staining and its comparison with the standard Gallus gallus erythrocytes nuclear content, one of the preferred internal standards. With this methodology, up to a thousand nuclei can be measured in a single staining session, allowing for a quick analysis of the studied species.

QIAamp DNA Extraction Protocol v1

Abstract: Non-invasively collected faecal samples are an alternative source of DNA to tissue samples, that may be used in genetic studies of wildlife when direct sampling of animals is difficult. Although several faecal DNA extraction methods exist, their efficacy varies between species. Previous attempts to amplify mitochondrial DNA (mtDNA) markers from faeces of wild dugongs (Dugong dugon) have met with limited success and nuclear markers (microsatellites) have been unsuccessful. This study aimed to establish a tool for sampling both mtDNA and nuclear DNA (nDNA) from dugong faeces by modifying approaches used in studies of other large herbivores. First, a streamlined, cost-effective DNA extraction method that enabled the amplification of both mitochondrial and nuclear markers from large quantities of dugong faeces was developed. Faecal DNA extracted using a new ‘High Volume- Cetyltrimethyl Ammonium Bromide- Phenol-Chloroform-Isoamyl Alcohol’ (HV-CTAB-PCI) method was found to achieve comparable amplification results to extraction of DNA from dugong skin. As most prevailing practices advocate sampling from the outer surface of a stool to maximise capture of sloughed intestinal cells, this study compared amplification success of mtDNA between the outer and inner layers of faeces, but no difference in amplification was found. Assessment of the impacts of faecal age or degradation on extraction, however, demonstrated that fresher faeces with shorter duration of environmental (seawater) exposure amplified both markers better than eroded scats. Using the HV-CTAB-PCI method, nuclear markers were successfully amplified for the first time from dugong faeces. The successful amplification of SNP markers represents a proof-of-concept showing that DNA from dugong faeces can potentially be utilised in population genetic studies. This novel DNA extraction protocol offers a new tool that will facilitate genetic studies of dugongs and other large and cryptic marine herbivores in remote locations.

Assembly‐free quantification of vagrant DNA inserts

Abstract: Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low‐coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re‐estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re‐analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low‐coverage high‐throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont‐derived sequences. We provide an R implementation of our methods called “vagrantDNA” and code to simulate test data sets.

Nuclear and mitochondrial DNA alterations in pheochromocytomas and paragangliomas, and their potential treatment

Abstract: Mitochondrial DNA (mtDNA) alterations have been reported in different types of cancers and are suggested to play important roles in cancer development and metastasis. However, there is little information about its involvement in pheochromocytomas and paragangliomas (PCCs/PGLs) formation. PCCs and PGLs are rare endocrine tumors of the chromaffin cells in the adrenal medulla and extra-adrenal paraganglia that can synthesize and secrete catecholamines. Over the last 3 decades, the genetic background of about 60% of PCCs/PGLs involving nuclear DNA alterations has been determined. Recently, a study showed that mitochondrial alterations can be found in around 17% of the remaining PCCs/PGLs. In this review, we summarize recent knowledge regarding both nuclear and mitochondrial alterations and their involvement in PCCs/PGLs. We also provide brief insights into the genetics and the molecular pathways associated with PCCs/PGLs and potential therapeutical targets.

Assembly-free quantification of vagrant DNA inserts

Abstract: Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence, and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1x) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extra-nuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris , exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics dataset of the parrot Psephotellus varius , obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called “vagrantDNA” and code to simulate test datasets.

Nuclear genetic control of mtDNA copy number and heteroplasmy in humans

Abstract: Human mitochondria contain a high copy number, maternally transmitted genome (mtDNA) that encodes 13 proteins required for oxidative phosphorylation. Heteroplasmy arises when multiple mtDNA variants co-exist in an individual and can exhibit complex dynamics in disease and in aging. As all proteins involved in mtDNA replication and maintenance are nuclear-encoded, heteroplasmy levels can, in principle, be under nuclear genetic control, however this has never been shown in humans. Here, we develop algorithms to quantify mtDNA copy number (mtCN) and heteroplasmy levels using blood-derived whole genome sequences from 274,832 individuals of diverse ancestry and perform GWAS to identify nuclear loci controlling these traits. After careful correction for blood cell composition, we observe that mtCN declines linearly with age and is associated with 92 independent nuclear genetic loci. We find that nearly every individual carries heteroplasmic variants that obey two key patterns: (1) heteroplasmic single nucleotide variants are somatic mutations that accumulate sharply after age 70, while (2) heteroplasmic indels are maternally transmitted as mtDNA mixtures with resulting levels influenced by 42 independent nuclear loci involved in mtDNA replication, maintenance, and novel pathways. These nuclear loci do not appear to act by mtDNA mutagenesis, but rather, likely act by conferring a replicative advantage to specific mtDNA molecules. As an illustrative example, the most common heteroplasmy we identify is a length variant carried by >50% of humans at position m.302 within a G-quadruplex known to serve as a replication switch. We find that this heteroplasmic variant exerts cis-acting genetic control over mtDNA abundance and is itself under trans-acting genetic control of nuclear loci encoding protein components of this regulatory switch. Our study showcases how nuclear haplotype can privilege the replication of specific mtDNA molecules to shape mtCN and heteroplasmy dynamics in the human population.

p53-dependent DNA repair during the DNA damage response requires actin nucleation by JMY

Abstract: The tumour suppressor p53 is a nuclear transcription factor with key roles during DNA damage to enable a variety of cellular responses including cell cycle arrest, apoptosis and DNA repair. JMY is an actin nucleator and DNA damage-responsive protein whose sub-cellular localisation is responsive to stress and during DNA damage JMY undergoes nuclear accumulation. To gain an understanding of the wider role for nuclear JMY in transcriptional regulation, we performed transcriptomics to identify JMY-mediated changes in gene expression during the DNA damage response. We show that JMY is required for effective regulation of key p53 target genes involved in DNA repair, including XPC, XRCC5 (Ku80) and TP53I3 (PIG3). Moreover, JMY depletion or knockout leads to increased DNA damage and nuclear JMY requires its Arp2/3-dependent actin nucleation function to promote the clearance of DNA lesions. In human patient samples a lack of JMY is associated with increased tumour mutation count and in cells results in reduced cell survival and increased sensitivity to DNA damage response kinase inhibition. Collectively, we demonstrate that JMY enables p53-dependent DNA repair under genotoxic stress and suggest a role for actin in JMY nuclear activity during the DNA damage response.

Cardiomyocyte Nuclear Remodeling after Mechanical Unloading.

Abstract: Cardiomyocytes increase DNA content in response to stress in humans. DNA content is reported to decrease in association with increased markers of proliferation in cardiomyocytes following LVAD unloading. However, cardiac recovery resulting in LVAD explant is rare. Thus, we sought to test the hypothesis that changes in DNA content with mechanical unloading occurs independent of cardiomyocyte proliferation by quantifying cardiomyocyte nuclear number, cell size, DNA content and the frequency of cell cycling markers using a novel imaging flow cytometry methodology comparing human subjects undergoing LVAD implantation or primary transplantation. We found that cardiomyocyte size was 15 percent smaller in unloaded versus loaded samples without differences in the percentage of mono-, bi, or multi-nuclear cells. DNA content per nucleus was significantly decreased in unloaded hearts versus loaded controls. Cell cycle markers, Ki67 and phospho-histon3 (H3P) were not increased in unloaded samples. In conclusion, unloading of failing hearts is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell cycle markers, they may represent a regression of hypertrophic nuclear remodeling and not proliferation.

Data from Mitochondrial Genomic Backgrounds Affect Nuclear DNA Methylation and Gene Expression

Abstract: <div>Abstract<p>Mitochondrial DNA (mtDNA) mutations and polymorphisms contribute to many complex diseases, including cancer. Using a unique mouse model that contains nDNA from one mouse strain and homoplasmic mitochondrial haplotypes from different mouse strain(s)—designated Mitochondrial Nuclear Exchange (MNX)—we showed that mtDNA could alter mammary tumor metastasis. Because retrograde and anterograde communication exists between the nuclear and mitochondrial genomes, we hypothesized that there are differential mtDNA-driven changes in nuclear (n)DNA expression and DNA methylation. Genome-wide nDNA methylation and gene expression were measured in harvested brain tissue from paired wild-type and MNX mice. Selective differential DNA methylation and gene expression were observed between strains having identical nDNA, but different mtDNA. These observations provide insights into how mtDNA could be altering epigenetic regulation and thereby contribute to the pathogenesis of metastasis. <i>Cancer Res; 77(22); 6202–14. ©2017 AACR</i>.</p></div>

Comparison of the structures and topologies of plasma extracted circulating nuclear and mitochondrial cell-free DNA

Abstract: Introduction: The function, origin and structural features of circulating nuclear DNA (cir-nDNA) and mitochondrial DNA (cir-mtDNA) are poorly known, even though they have been investigated in numerous clinical studies, and are involved in a number of routine clinical applications. Based on our previous report disproving the conventional plasma isolation used for cirDNA analysis, this work enables a direct topological comparison of the circulating structures associated with nuclear DNA and mitochondrial cell-free DNA. Materials and methods: We used a Q-PCR and low-pass whole genome sequencing (LP-WGS) combination approach of cir-nDNA and cir-mtDNA, extracted using a procedure that eliminates platelet activation during the plasma isolation process to prevent mitochondria release in the extracellular milieu. Various physical procedures, such as filtration and differential centrifugation, were employed to infer their circulating structures. Results: DSP-S cir-mtDNA mean size profiles distributed on a slightly shorter range than SSP-S. SSP-S detected 40-fold more low-sized cir-mtDNA fragments (<90 bp/nt) and three-fold less long-sized fragments (>200 bp/nt) than DSP-S. The ratio of the fragment number below 90 bp over the fragment number above 200 bp was very homogenous among both DSP-S and SSP-S profiles, being 134-fold lower with DSP-S than with SSP-S. Cir-mtDNA and cir-nDNA DSP-S and SSP-S mean size profiles of healthy individuals ranged in different intervals with periodic sub-peaks only detectable with cir-nDNA. The very low amount of cir-mtDNA fragments of short size observed suggested that most of the cir-mtDNA is poorly fragmented and appearing longer than ∼1,000 bp, the readout limit of this LP-WGS method. Data suggested that cir-nDNA is, among DNA extracted in plasma, associated with ∼8.6% of large structures (apoptotic bodies, large extracellular vesicles (EVs), cell debris…), ∼27.7% in chromatin and small EVs and ∼63.7% mainly in oligo- and mono-nucleosomes. By contrast, cir-mtDNA appeared to be preponderantly (75.7%) associated with extracellular mitochondria, either in its free form or with large EVs; to a lesser extent, it was also associated with other structures: small EVs (∼18.4%), and exosomes or protein complexes (∼5.9%). Conclusion: This is the first study to directly compare the structural features of cir-nDNA and cir-mtDNA. The significant differences revealed between both are due to the DNA topological structure contained in the nucleus (chromatin) and in the mitochondria (plasmid) that determine their biological stability in blood. Although cir-nDNA and cir-mtDNA are principally associated with mono-nucleosomes and cell-free mitochondria, our study highlights the diversity of the circulating structures associated with cell-free DNA. They consequently have different pharmacokinetics as well as physiological functions. Thus, any accurate evaluation of their biological or diagnostic individual properties must relies on appropriate pre-analytics, and optimally on the isolation or enrichment of one category of their cirDNA associated structures.

Preliminary assessment of nuclear DNA content in Chondrilla (Asteraceae) plants of European Russia and Western Kazakhstan using flow cytometry

Abstract: Preliminary data on the genome size of representatives of the genus Chondrilla (Asteraceae) of European Russia and Western Kazakhstan were obtained using flow cytometry. Among all studied specimens of the genus, for specimens of C. ambigua and C. pauciflora, a direct dependence of the genome size on the number of chromosomes was established. In our study, the DNA content of the diploid C. ambigua was 2С = 1.69 pg, and that of the triploid C. pauciflora was 2С = 2.65 pg. 2С values are within 2.29–2.69 pg in most specimens of the genus Chondrilla (typical for 13 out of 23 specimens) belonging to the following taxa: C. paucifora, C. laticoronata, C. brevirostris, C. canescens, C. graminea, C. latifolia, C. juncea. Most likely, they are triploids, and in many cases with a number of chromosomes deviating from a multiple of the main number of chromosomes. The C. latifolia sample from the population of the Kamyshinsky district of the Volgograd region showed two peaks on the histogram of the relative fluorescence intensity, corresponding to two values of the relative DNA content, 1.68 and 2.58 pg, i.e. it turned out to be a mixoploid with two levels of ploidy (2n = 2x = 10 and 2n = 3x = 15). The diversity of genome sizes within the genus can be explained by the previously established high variability in the number of chromosomes associated with aneu- and mixoploidy.

Mitochondrial DNA

Abstract: Mitochondrial DNA (mtDNA) is maternally inherited and present in higher copy number in the cell than nuclear DNA (nDNA). Therefore, its main forensic applications involve samples that do not contain enough nDNA or that may derive from maternally related individuals. The past decade witnessed major technological changes and improvements in mtDNA typing including diverse Massively Parallel Sequencing (MPS) approaches. MPS-based mtDNA data are more complex and more informative than conventional Sanger-based electrophoretic sequence data. This can be used to better discern genuine signals from artefacts, heteroplasmies from other damage patterns, and nuclear elements of mtDNA form extraneous mixture contributions.