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Abstract Background The dominant hypothesis about the pathogenesis of Alzheimer’s disease (AD) is the “amyloid cascade” concept and modulating the expression of proteins involved in the metabolism of amyloid-beta (Aβ) is proposed as an effective strategy for the prevention and therapy of AD. Recently, we found that an antibiotic ceftriaxone (CEF), which possesses neuroprotective activity, reduced cognitive deficits and neurodegenerative changes in OXYS rats, a model of sporadic AD. The molecular mechanisms of this effect are not completely clear, we suggested that the drug might serve as the regulator of the expression of the genes involved in the metabolism of Aβ and the pathogenesis of AD. The study was aimed to determine the effects of CEF on mRNA levels of Bace1 (encoding β-secretase BACE1 involved in Aβ production), Mme, Ide, Ece1, Ace2 (encoding enzymes involved in Aβ degradation), Epo (encoding erythropoietin related to endothelial function and clearance of Aβ across the blood brain barrier) in the frontal cortex, hippocampus, striatum, hypothalamus, and amygdala of OXYS and Wistar (control strain) male rats. Starting from the age of 14 weeks, animals received CEF (100 mg/kg/day, i.p., 36 days) or saline. mRNA levels were evaluated with RT-qPCR method. Biochemical parameters of plasma were measured for control of system effects of the treatment. Results To better understand strain variations studied here, we compared the gene expression between untreated OXYS and Wistar rats. This comparison showed a significant decrease in mRNA levels of Ace2 in the frontal cortex and hypothalamus, and of Actb in the amygdala of untreated OXYS rats. Analysis of potential effects of CEF revealed its novel targets. In the compound-treated OXYS cohort, CEF diminished mRNA levels of Bace1 and Ace2 in the hypothalamus, and Aktb in the frontal cortex. Furthermore, CEF augmented Mme, Ide, and Epo mRNA levels in the amygdala as well as the levels of Ece1 and Aktb in the striatum. Finally, CEF also attenuated the activity of ALT and AST in plasma of OXYS rats. Conclusion Those findings disclosed novel targets for CEF action that might be involved into neuroprotective mechanisms at early, pre-plaque stages of AD-like pathology development.

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., Competing Interests: None declared. As an Editorial Board Member of Precision Clinical Medicine, the corresponding author Alain R. Thierry was blinded from reviewing and making decisions on this manuscript., (© The Author(s) 2024. Published by Oxford University Press on behalf of the West China School of Medicine & West China Hospital of Sichuan University.)

We examined from a large exploratory study cohort of COVID-19 patients ( N  = 549) a validated panel of neutrophil extracellular traps (NETs) markers in different categories of disease severity. Neutrophil elastase (NE), myeloperoxidase (MPO), and circulating nuclear DNA (cir-nDNA) levels in plasma were seen to gradually and significantly ( p  < 0.0001) increase with the disease severity: mild (3.7, 48.9, and 15.8 ng/mL, respectively); moderate (9.8, 77.5, and 27.7 ng/mL, respectively); severe (11.7, 99.5, and 29.0 ng/mL, respectively); and critical (13.1, 110.2, and 46.0 ng/mL, respectively); and are also statistically different with healthy individuals (N = 140; p  < 0.0001). All observations made in relation to the Delta variant-infected patients are in line with Omicron-infected patients. We unexpectedly observed significantly higher levels of NETs in asymptomatic individuals as compared to healthy subjects ( p  < 0.0001). Moreover, the balance of cir-nDNA and circulating mitochondrial DNA level was affected in COVID-19 infected patients attesting to mitochondrial dysfunction., Competing Interests: A.R.T., E.P., and B.P. are author of a patent: Thierry A.R., Pisareva E., and B. Pastor NEW METHOD TO DIAGNOSE INFLAMMATORY DISEASES 11194720 PCT application number PCT/EP2022/072147, Date of receipt 05 August 2022., (© 2024 Published by Elsevier Inc.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

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., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pisareva, Roch, Sanchez, Pastor, Mirandola, Diab-Assaf, Mazard, Prévostel, Al Amir Dache and Thierry.)

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., (© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: As circulating DNA (cirDNA) is mainly detected as mononucleosome-associated circulating DNA (mono-N cirDNA) in blood, apoptosis has until now been considered as the main source of cirDNA. The mechanism of cirDNA release into the circulation, however, is still not fully understood. This work addresses that knowledge gap, working from the postulate that neutrophil extracellular traps (NET) may be a source of cirDNA, and by investigating whether NET may directly produce mono-N cirDNA., Methods: We studied (1) the in vitro kinetics of cell derived genomic high molecular weight (gHMW) DNA degradation in serum; (2) the production of extracellular DNA and NET markers such as neutrophil elastase (NE) and myeloperoxidase (MPO) by ex vivo activated neutrophils; and (3) the in vitro NET degradation in serum; for this, we exploited the synergistic analytical information provided by specifically quantifying DNA by qPCR, and used shallow WGS and capillary electrophoresis to perform fragment size analysis. We also performed an in vivo study in knockout mice, and an in vitro study of gHMW DNA degradation, to elucidate the role of NE and MPO in effecting DNA degradation and fragmentation. We then compared the NET-associated markers and fragmentation size profiles of cirDNA in plasma obtained from patients with inflammatory diseases found to be associated with NET formation and high levels of cirDNA (COVID-19, N = 28; systemic lupus erythematosus, N = 10; metastatic colorectal cancer, N = 10; and from healthy individuals, N = 114)., Results: Our studies reveal that gHMW DNA degradation in serum results in the accumulation of mono-N DNA (81.3% of the remaining DNA following 24 h incubation in serum corresponded to mono-N DNA); "ex vivo" NET formation, as demonstrated by a concurrent 5-, 5-, and 35-fold increase of NE, MPO, and cell-free DNA (cfDNA) concentration in PMA-activated neutrophil culture supernatant, leads to the release of high molecular weight DNA that degrades down to mono-N in serum; NET mainly in the form of gHMW DNA generate mono-N cirDNA (2 and 41% of the remaining DNA after 2 h in serum corresponded to 1-10 kbp fragments and mono-N, respectively) independent of any cellular process when degraded in serum; NE and MPO may contribute synergistically to NET autocatabolism, resulting in a 25-fold decrease in total DNA concentration and a DNA fragment size profile similar to that observed from cirDNA following 8 h incubation with both NE and MPO; the cirDNA size profile of NE KO mice significantly differed from that of the WT, suggesting NE involvement in DNA degradation; and a significant increase in the levels of NE, MPO, and cirDNA was detected in plasma samples from lupus, COVID-19, and mCRC, showing a high correlation with these inflammatory diseases, while no correlation of NE and MPO with cirDNA was found in HI., Conclusions: Our work describes the mechanisms by which NET and cirDNA are linked. In doing so, we demonstrate that NET are a major source of mono-N cirDNA independent of apoptosis and establish a new paradigm of the mechanisms of cirDNA release in normal and pathological conditions. We also demonstrate a link between immune response and cirDNA., (© 2022. The Author(s).)

We postulate that a significant part of circulating DNA (cirDNA) originates in the degradation of neutrophil extracellular traps (NETs). In this study, we examined the plasma level of two markers of NETs (myeloperoxidase (MPO) and neutrophil elastase (NE)), as well as cirDNA levels in 219 patients with a metastatic colorectal cancer (mCRC), and in 114 healthy individuals (HI). We found that in patients with mCRC the content of these analytes was (i) highly correlated, and (ii) all statistically different ( p  < 0.0001) than in HI (N = 114). These three NETs markers may readily distinguish between patients with mCRC from HI, (0.88, 0.86, 0.84, and 0.95 AUC values for NE, MPO, cirDNA, and NE + MPO + cirDNA, respectively). Concomitant analysis of anti-phospholipid (anti-cardiolipin), NE, MPO, and cirDNA plasma concentrations in patients with mCRC might have value for thrombosis prevention, and suggested that NETosis may be a critical factor in the immunological response/phenomena linked to tumor progression., Competing Interests: TM reported receiving grants from Amgen SAS; nonfinancial support from Servier and MSD; and personal fees from Merck Serono, Bristol Myers Squibb, Sanofi Genzyme, Sandoz, and Bayer outside the submitted work. AA has reported a consulting/advisory role and/or receiving honoraria from Bristol-Myers Squibb, MSD Oncology, and Servier; and has received research funding from Bayer Pharmaceuticals. ART is a DiaDx stockholder. BP, JDA, EP, CS, AK, LM, RT, VP, MY, and AM declare no competing interests., (© 2022 The Authors.)

Importance: The COVID-19 pandemic has been associated with substantial reduction in screening, case identification, and hospital referrals among patients with cancer. However, no study has quantitatively examined the implications of this correlation for cancer patient management., Objective: To evaluate the association of the COVID-19 pandemic lockdown with the tumor burden of patients who were diagnosed with metastatic colorectal cancer (mCRC) before vs after lockdown., Design, Setting, and Participants: This cohort study analyzed participants in the screening procedure of the PANIRINOX (Phase II Randomized Study Comparing FOLFIRINOX + Panitumumab vs FOLFOX + Panitumumab in Metastatic Colorectal Cancer Patients Stratified by RAS Status from Circulating DNA Analysis) phase 2 randomized clinical trial. These newly diagnosed patients received care at 1 of 18 different clinical centers in France and were recruited before or after the lockdown was enacted in France in the spring of 2020. Patients underwent a blood-sampling screening procedure to identify their RAS and BRAF tumor status., Exposures: mCRC., Main Outcomes and Measures: Circulating tumor DNA (ctDNA) analysis was used to identify RAS and BRAF status. Tumor burden was evaluated by the total plasma ctDNA concentration. The median ctDNA concentration was compared in patients who underwent screening before (November 11, 2019, to March 9, 2020) vs after (May 14 to September 3, 2020) lockdown and in patients who were included from the start of the PANIRINOX study., Results: A total of 80 patients were included, of whom 40 underwent screening before and 40 others underwent screening after the first COVID-19 lockdown in France. These patients included 48 men (60.0%) and 32 women (40.0%) and had a median (range) age of 62 (37-77) years. The median ctDNA concentration was statistically higher in patients who were newly diagnosed after lockdown compared with those who were diagnosed before lockdown (119.2 ng/mL vs 17.3 ng/mL; P < .001). Patients with mCRC and high ctDNA concentration had lower median survival compared with those with lower concentration (14.7 [95% CI, 8.8-18.0] months vs 20.0 [95% CI, 14.1-32.0] months). This finding points to the potential adverse consequences of the COVID-19 pandemic and related lockdown., Conclusions and Relevance: This cohort study found that tumor burden differed between patients who received an mCRC diagnosis before vs after the first COVID-19 lockdown in France. The findings of this study suggest that CRC is a major area for intervention to minimize pandemic-associated delays in screening, diagnosis, and treatment.

To unequivocally address their unresolved intimate structures in blood, we scrutinized the size distribution of circulating cell-free DNA (cfDNA) using whole-genome sequencing (WGS) from both double- and single-strand DNA library preparations (DSP and SSP, n = 7) and using quantitative PCR (Q-PCR, n = 116). The size profile in healthy individuals was remarkably homogenous when using DSP sequencing or SSP sequencing. CfDNA size profile had a characteristic nucleosome fragmentation pattern. Overall, our data indicate that the proportion of cfDNA inserted in mono-nucleosomes, di-nucleosomes, and chromatin of higher molecular size (>1000 bp) can be estimated as 67.5% to 80%, 9.4% to 11.5%, and 8.5% to 21.0%, respectively. Although DNA on single chromatosomes or mono-nucleosomes is detectable, our data revealed that cfDNA is highly nicked (97%-98%) on those structures, which appear to be subjected to continuous nuclease activity in the bloodstream. Fragments analysis allows the distinction of cfDNA of different origins: first, cfDNA size profile analysis may be useful in cfDNA extract quality control; second, subtle but reliable differences between metastatic colorectal cancer patients and healthy individuals vary with the proportion of malignant cell-derived cfDNA in plasma extracts, pointing to a higher degree of cfDNA fragmentation and nuclease activity in samples with high malignant cell cfDNA content.

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., (© 2019 American Association for Clinical Chemistry.)

Mutations at BRAF codon V600 are used as predictive biomarkers for targeted therapy of skin melanoma. Here, a simple sensitive test to detect mutations of BRAF-V600 was developed using real-time PCR with allele-specific primers and TaqMan probes. Two versions of the test using sense and antisense allele-specific primers were designed and evaluated. The test detected 1% mutant allele V600E/K in 10 ng DNA standard made from wild-type human DNA spiked with BRAF-V600E or the V600K plasmid. The test was validated on clinical formalin-fixed paraffin-embedded samples of skin melanoma using pyrosequencing as a reference method. In the clinical samples, we detected the common mutation V600E, as well as the rare mutations V600K, V600E2 (codon GAA), V600E2 K601del, V600D-K601del, and V600R. In comparison with pyrosequencing, both versions of the test had 100% specificity with sensitivities of 97 and 86% for sense and antisense allele-specific primers, respectively. Using the PCR test with sense allele-specific primers, mutations in V600 were found in 33 of 51 Russian patients (64.7%) with cutaneous melanoma. This closed-tube real-time PCR test can be used as a simple and sensitive assay for mutations of BRAF-V600 in cutaneous melanoma.