Circulating DNA reveals a specific and higher fragmentation of the Y chromosome.

Chromosome stability is a key point in genome evolution, particularly that of the Y chromosome. The Y chromosome loss in blood and tumor cells is well established. Through processes that are common to other chromosomes too, the Y chromosome undergoes degradation and fragmentation in the blood stream before elimination. This process gives rise to circulating DNA (cirDNA) fragments, whose examination may provide potential insight into the role of DNA fragmentation in blood for the Y chromosome elimination. In this study, we employed shallow whole genome sequencing (sWGS) to comprehensively assess the total cirDNA and the individual chromosome fragment size profiles in the plasma of healthy male individuals. Here, we show that (i) the fragment size profiles of total circulating DNA (cirDNA) and DNA fragments originating from autosomes and the X chromosome in blood plasma are homogeneous, and have a remarkably low variability (mean CV = 7%) among healthy individuals, (ii) the Y chromosome has a distinct fragment size profile with the accumulation of the fragment < 145 bp and depletion of the dinucleosome-associated fragments (290-390 bp), and its fragment fraction in blood decreases with age. These results indicate a higher fragmentation of the Y chromosome compared to other chromosomes and this in turn might be due to its increased susceptibility to degradation. Our findings pave the way for an elucidation of the impact of chromosomal origin on DNA degradation and the Y chromosome biology.

Persistence of neutrophil extracellular traps and anticardiolipin auto-antibodies in post-acute phase COVID-19 patients.

In the early phase of the pandemic, we were among the first to postulate that neutrophil extracellular traps (NETs) play a key role in COVID-19 pathogenesis. This exploratory prospective study based on 279 individuals showed that plasma levels of neutrophil elastase, myeloperoxidase and circulating DNA of nuclear and mitochondrial origins in nonsevere (NS), severe (S) and postacute phase (PAP) COVID-19 patients were statistically different as compared to the levels in healthy individuals, and revealed the high diagnostic power of these NETs markers in respect to the disease severity. The diagnostic power of NE, MPO, and cir-nDNA as determined by the Area Under Receiver Operating Curves (AUROC) was 0.95, 097, and 0.64; 0.99, 1.0, and 0.82; and 0.94, 1.0, and 0.93, in NS, S, and PAP patient subgroups, respectively. In addition, a significant fraction of NS, S as well as of PAP patients exhibited aCL IgM/IgG and anti-B2GP IgM/IgG positivity. We first demonstrate persistence of these NETs markers in PAP patients and consequently of sustained innate immune response imbalance, and a prolonged low-level pro-thrombotic potential activity highlighting the need to monitor these markers in all COVID-19 PAP individuals, to investigate postacute COVID-19 pathogenesis following intensive care, and to better identify which medical resources will ensure complete patient recovery.

Towards systematic nomenclature for cell-free DNA.

Cell-free DNA (cfDNA) has become widely recognized as a promising candidate biomarker for minimally invasive characterization of various genomic disorders and other clinical scenarios. However, among the obstacles that currently challenge the general progression of the research field, there remains an unmet need for unambiguous universal cfDNA nomenclature. To address this shortcoming, we classify in this report the different types of cfDNA molecules that occur in the human body based on its origin, genetic traits, and locality. We proceed by assigning existing terms to each of these cfDNA subtypes, while proposing new terms and abbreviations where clarity is lacking and more precise stratification would be beneficial. We then suggest the proper usage of these terms within different contexts and scenarios, focusing mainly on the nomenclature as it relates to the domains of oncology, prenatal testing, and post-transplant surgery surveillance. We hope that these recommendations will serve as useful considerations towards the establishment of universal cfDNA nomenclature in the future. In addition, it is conceivable that many of these recommendations can be transposed to cell-free RNA nomenclature by simply exchanging "DNA" with "RNA" in each acronym/abbreviation. Similarly, when describing DNA and RNA collectively, the suffix can be replaced with "NAs" to indicate nucleic acids.

Blood contains circulating cell-free respiratory competent mitochondria.

Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.

Hypoxia differently modulates the release of mitochondrial and nuclear DNA.

BACKGROUND: We investigated the influence of hypoxia on the concentration of mitochondrial and nuclear cell-free DNA (McfDNA and NcfDNA, respectively). METHOD: By an ultra-sensitive quantitative PCR-based assay, McfDNA and NcfDNA were measured in the supernatants of different colorectal cell lines, and in the plasma of C57/Bl6 mice engrafted with TC1 tumour cells, in normoxic or hypoxic conditions. RESULTS: Our data when setting cell culture conditions highlighted the higher stability of McfDNA as compared to NcfDNA and revealed that cancer cells released amounts of nuclear DNA equivalent to the mass of a chromosome over a 6-h duration of incubation. In cell model, hypoxia induced a great increase in NcfDNA and McfDNA concentrations within the first 24 h. After this period, cfDNA total concentrations remained stable in hypoxia consecutive to a decrease of nuclear DNA release, and noteworthy, to a complete inhibition of daily mitochondrial DNA release. In TC1-engrafted mice submitted to intermittent hypoxia, plasma NcfDNA levels are much higher than in mice bred in normoxia, unlike plasma McfDNA concentration that is not impacted by hypoxia. CONCLUSION: This study suggests that hypoxia negatively modulates nuclear and, particularly, mitochondrial DNA releases in long-term hypoxia, and revealed that the underlying mechanisms are differently regulated.

SARS-CoV2 may evade innate immune response, causing uncontrolled neutrophil extracellular traps formation and multi-organ failure.

We demonstrate that the general clinical conditions, risk factors and numerous pathological and biological features of COVID-19 are analogous with various disorders caused by the uncontrolled formation of neutrophil extracellular traps and their by-products. Given the rapid evolution of this disease's symptoms and its lethality, we hypothesize that SARS-CoV2 evades innate immune response causing COVID-19 progresses under just such an amplifier loop, leading to a massive, uncontrolled inflammation process. This work allows us to propose new strategies for treating the pandemic.

Guidelines for the Preanalytical Conditions for Analyzing Circulating Cell-Free DNA.

Circulating cell-free DNA (cfDNA) isolated from blood has been identified as a potential biomarker in numerous fields, and has been the object of intensive research over the past decade, although its original discovery dates back 60 years. While it is already used routinely in commercial and clinical practice in oncology and prenatal testing, other potential applications have emerged, including for diabetes, cardiovascular diseases, organ transplantation, autoimmune diseases, sepsis, trauma, and sport management. As with the discovery and development of any biomarker, preanalytical requirements and documentation are as important as analytical requirements. Except for the case of noninvasive prenatal testing and prenatal diagnosis, the implementation of cfDNA in a clinical setting remains limited because of the lack of standardization of cfDNA analysis. In particular, only a few attempts have been made to collect and pool scientific data on the relevant preanalytical factors, and no standard operating procedure has yet been set. For this report, we have performed a thorough and systematic search via MEDLINE® for relevant preanalytical variables and patient factors. These form the basis of the guidelines we propose for analyzing nuclear cfDNA.

Association of the immediate perioperative dynamics of circulating DNA levels and neutrophil extracellular traps formation in cancer patients.

Objectives: Elevated circulating DNA (cirDNA) concentrations were found to be associated with trauma or tissue damage which suggests involvement of inflammation or cell death in post-operative cirDNA release. We carried out the first prospective, multicenter study of the dynamics of cirDNA and neutrophil extracellular trap (NETs) markers during the perioperative period from 24 h before surgery up to 72 h after curative surgery in cancer patients. Methods: We examined the plasma levels of two NETs protein markers [myeloperoxidase (MPO) and neutrophil elastase (NE)], as well as levels of cirDNA of nuclear (cir-nDNA) and mitochondrial (cir-mtDNA) origin in 29 colon, prostate, and breast cancer patients and in 114 healthy individuals (HI). Results: The synergistic analytical information provided by these markers revealed that: (i) NETs formation contributes to post-surgery conditions; (ii) post-surgery cir-nDNA levels were highly associated with NE and MPO in colon cancer [r = 0.60 (P < 0.001) and r = 0.53 (P < 0.01), respectively], but not in prostate and breast cancer; (iii) each tumor type shows a specific pattern of cir-nDNA and NETs marker dynamics, but overall the pre- and post-surgery median values of cir-nDNA, NE, and MPO were significantly higher in cancer patients than in HI. Conclusion: Taken as a whole, our work reveals the association of NETs formation with the elevated cir-nDNA release during a cancer patient's perioperative period, depending on surgical procedure or cancer type. By contrast, cir-mtDNA is poorly associated with NETs formation in the studied perioperative period, which would appear to indicate a different mechanism of release or suggest mitochondrial dysfunction.

Corrigendum: What is the role of the neutrophil extracellular traps in the cardiovascular disease burden associated with hemodialysis bioincompatibility?

[This corrects the article DOI: 10.3389/fmed.2023.1268748.].

Impact of platelet activation on the release of cell-free mitochondria and circulating mitochondrial DNA.

BACKGROUND: Research on circulating mitochondrial DNA (cir-mtDNA) based diagnostic is insufficient, as to its function, origin, structural features, and particularly its standardization of isolation. To date, plasma preparation performed in previous studies do not take into consideration the potential bias resulting from the release of mitochondria by activated platelets. METHODS: To tackle this, we compared the mtDNA amount determined by a standard plasma preparation method or a method optimally avoiding platelet activation. MtDNA extracted from the plasma of seven healthy individuals was quantified by Q-PCR in the course of the process of both methods submitted to filtration, freezing or differential centrifugation. RESULTS: 98.7 to 99.4% of plasma mtDNA corresponded to extracellular mitochondria, either free or into large extracellular vesicles. Without platelet activation, the proportion of both types of entities remained preponderant (76-80%), but the amount of detected mtDNA decreased 67-fold. CONCLUSION: We show the high capacity of platelets to release free mitochondria in "in vitro" conditions. This represents a potent confounding factor when extracting mtDNA for cir-mtDNA investigation. Platelet activation during pre-analytical conditions should therefore be avoided when studying cir-mtDNA. Our findings lead to a profound revision of the assumptions previously made by most works in this field. Overall, our data suggest the need to characterize or isolate mtDNA associated various structural forms, as well as to standardize plasma preparation, to better circumscribe cir-mtDNA's diagnostic capacity.

A New Paradigm of the Origins of Circulating DNA in Patients with Cancer.

SUMMARY: By shedding light on the cellular origins of circulating DNA (cirDNA), this research provides important insights into the mechanisms of cirDNA production in cancer. Contrary to expectations, the increased cirDNA in patients with cancer was not derived predominantly from neoplastic cells or surrounding nonneoplastic epithelial cells; rather, the excess cirDNA originated primarily from leukocytes, implying a systemic impact of cancer on cell turnover or DNA clearance. See related article by Mattox et al., p. 2166 (1).

Mitochondria-derived cell-to-cell communication.

In addition to their intracellular mobility, mitochondria and their components can exist outside the cells from which they originate. As a result, they are capable of acting on non-parental distant cells and mediate intercellular communication in physiological conditions and in a variety of pathologies. It has recently been demonstrated that this horizontal transfer governs a wide range of biological processes, such as tissue homeostasis, the rescue of injured recipient cells, and tumorigenesis. In addition, due to mitochondria's bacterial ancestry, they and their components can be recognized as damage-associated molecular patterns (DAMPs) by the immune cells, leading to inflammation. Here, we provide an overview of the most current and significant findings concerning the different structures of extracellular mitochondria and their by-products and their functions in the physiological and pathological context. This account illustrates the ongoing expansion of our understanding of mitochondria's biological role and functions in mammalian organisms.

What is the role of the neutrophil extracellular traps in the cardiovascular disease burden associated with hemodialysis bioincompatibility?

Despite significant progress in dialysis modalities, intermittent renal replacement therapy remains an "unphysiological" treatment that imperfectly corrects uremic disorders and may lead to low-grade chronic inflammation, neutrophil activation, and oxidative stress due to repetitive blood/membrane interactions contributing to the "remaining uremic syndrome" and cardiovascular disease burden of hemodialysis patients. Understanding dialysis bioincompatibility pathways still remains a clinical and biochemical challenge. Indeed, surrogate biomarkers of inflammation including C-reactive protein could not discriminate between all components involved in these complex pathways. A few examples may serve to illustrate the case. Cytokine release during dialysis sessions may be underestimated due to their removal using high-flux dialysis or hemodiafiltration modalities. Complement activation is recognized as a key event of bioincompatibility. However, it appears as an early and transient event with anaphylatoxin level normalization at the end of the dialysis session. Complement activation is generally assumed to trigger leukocyte stimulation leading to proinflammatory mediators' secretion and oxidative burst. In addition to being part of the innate immune response involved in eliminating physically and enzymatically microbes, the formation of Neutrophil Extracellular Traps (NETs), known as NETosis, has been recently identified as a major harmful component in a wide range of pathologies associated with inflammatory processes. NETs result from the neutrophil degranulation induced by reactive oxygen species overproduction via NADPH oxidase and consist of modified chromatin decorated with serine proteases, elastase, bactericidal proteins, and myeloperoxidase (MPO) that produces hypochlorite anion. Currently, NETosis remains poorly investigated as a sensitive and integrated marker of bioincompatibility in dialysis. Only scarce data could be found in the literature. Oxidative burst and NADPH oxidase activation are well-known events in the bioincompatibility phenomenon. NET byproducts such as elastase, MPO, and circulating DNA have been reported to be increased in dialysis patients more specifically during dialysis sessions, and were identified as predictors of poor outcomes. As NETs and MPO could be taken up by endothelium, NETs could be considered as a vascular memory of intermittent bioincompatibility phenomenon. In this working hypothesis article, we summarized the puzzle pieces showing the involvement of NET formation during hemodialysis and postulated that NETosis may act as a disease modifier and may contribute to the comorbid burden associated with dialysis bioincompatibility.

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

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.

A straightforward method to quantify circulating mRNAs as biomarkers of colorectal cancer.

Optimizing the biomarker combination to be analyzed in liquid biopsies should improve personalized medicine. We developed a method to purify circulating cell-free mRNAs from plasma samples and to quantify them by RT-qPCR. We selected three candidate colorectal cancer biomarkers (B2M, TIMP-1, and CLU). Their mRNA levels were significantly higher in plasma of patients with metastatic colorectal cancer patients (mCRC) (n = 107) than in healthy individuals (HI) (n = 53). To increase the discriminating performance of our method, we analyzed the sum of the three mRNA levels (BTC index). The area under the ROC curve (AUC) to estimate the BTC index capacity to discriminate between mCRC and HI plasma was 0.903. We also determined the optimal BTC index cut-off to distinguish between plasma samples, with 82% of sensitivity and 93% of specificity. By using mRNA as a novel liquid biopsy analytical parameter, our method has the potential to facilitate rapid screening of CRCm.