Search Results (24)

Legionella pneumophila (L. pneumophila) infection is characterized by a wide spectrum of manifestations, from influenza-like illness to life-threatening atypical pneumonia with multiorgan failure. The aim of our study was the assessment of in vitro and ex vivo neutrophil activation in L. pneumophila infections, as well as the role of neutrophils’ peptides such as LL-37 in infection. The ability of neutrophils to form ex vivo extracellular traps (NETs) in response to bacterial infection was examined by immunofluorescence. In parallel, patients’ sera, as well as opsonized standard L. pneumophila strains, were used for in vitro activation of neutrophils from healthy individuals. The serum levels of interleukins were assessed using the LEGENDplexTM Multi-Analyte Flow Assay Kit. Furthermore, citrullinated cf-DNA as a marker of neutrophil extracellular traps (NETs) was detected in the serum of patients with acute infection. It was demonstrated that neutrophils released NETs in vitro and ex vivo upon L. pneumophila (interaction in an autophagy-independent manner. Notably, IL-1b was detected on NETs, but an antimicrobial peptide LL-37 was absent. The lack of antimicrobial activity failed to inhibit bacterial proliferation. In addition, in vitro and ex vivo NETs formation was observed during the Clarithromycin treatment. Those NETs were decorated with bioactive antimicrobial peptide LL-37, which inhibits bacterial proliferation. The findings provide evidence that neutrophils release NETs in vitro and ex vivo by expressing the IL1β protein in them. The lack of expression of the antimicrobial peptide LL-37 on the NETs demonstrates the inability of the cells to inhibit proliferation, and consequently the elimination of L. pneumophila. Clarithromycin plays a dual role in the elimination. Full article

Background: Preimplantation genetic testing for aneuploidy (PGT-A) is a popular approach in assisted reproductive technology that improves embryo selection and implantation rates. Traditional approaches rely on trophectoderm (TE) biopsy, which is an invasive procedure that might jeopardize embryo integrity and create technical constraints such as mosaicism-related misclassification. Non-invasive preimplantation genetic testing (niPGT) has emerged as a possible alternative, using embryonic cell-free DNA (cfDNA) extracted from wasted culture media or blastocoel fluid to assess chromosomal status without requiring direct embryo manipulation. Methods: This systematic study investigates the molecular mechanisms behind cfDNA release, its biological properties, and the technological concerns that influence its utilization in niPGT. We look at recent advances in next-generation sequencing (NGS), whole-genome amplification (WGA), and bioinformatic techniques that improve cfDNA-based aneuploidy detection. In addition, we compare the sensitivity, specificity, and concordance rates of niPGT to conventional TE biopsy, highlighting the major aspects impacting its diagnostic performance. Results: The release of cfDNA from embryos is influenced by apoptotic and necrotic processes, active DNA shedding, and extracellular vesicle secretion, which results in fragmented chromosomal material of different qualities and quantities. While niPGT has shown promise as a noninvasive screening approach, significant variability in cfDNA yield, maternal DNA contamination, and sequencing biases all have an impact on test accuracy. Studies show that niPGT and TE biopsies have moderate-to-high concordance, although there are still issues in detecting mosaicism, segmental aneuploidies, and DNA degradation artifacts. Conclusions: NiPGT is a safer and less intrusive alternative to TE biopsy, with potential clinical benefits. However, technical advancements are required to improve cfDNA collecting procedures, reduce contamination, and improve sequencing accuracy. Additional large-scale validation studies are needed to create standardized methodologies and ensure that niPGT achieves the diagnostic reliability requirements required for widespread clinical deployment in IVF programs. Full article

Type 2 diabetes mellitus (T2DM) significantly impairs fracture healing, with neutrophils playing a crucial role in this process. In T2DM, these immune cells are over-activated, leading to the excessive release of neutrophil extracellular traps (NETs), increasing inflammation and hindering recovery. Thus, a need for markers to assess patients in the risk group arises. This study demonstrates that circulating cell-free DNA (cfDNA) can be efficiently quantified from serum samples by a single-step qPCR and be used as a marker for NETosis. Our results revealed that trauma patients with T2DM have the highest cfDNA levels, followed by trauma patients, and the healthy group has the lowest. The method shows strong correlations between cfDNA and neutrophil-specific markers such as MPO, citH3, AZU1, and α-defensin, highlighting its potential as a rapid indicator of NETosis. This approach could allow the timely interference for high-risk patients, ultimately improving healing outcomes and reducing complications such as chronic inflammation, non-union fractures, and diabetic foot ulcers. Full article

Background: The clinical relevance of circulating cell-free DNA (cfDNA) in oncology has gained significant attention, with its potential as a biomarker for cancer diagnosis and monitoring. However, its precise role in cancer biology and progression remains unclear. cfDNA in cancer patients’ blood has been shown to activate signaling pathways, such as those mediated by toll-like receptors (TLRs), suggesting its involvement in cancer cell adaptation to the tumor microenvironment. Methods: This impact of cfDNA released from MDA-MB-231, a triple-negative breast cancer (TNBC) cell line was assessed, focusing on glucose availability and culture duration. The impact of cfDNA on the proliferation of MDA-MB-231 cells was investigated using proliferation curves, while cellular migration was evaluated through wound healing assays. The metabolic alterations induced by distinct cfDNA variants in MDA-MB-231 cells were investigated through nuclear magnetic resonance (NMR) spectroscopy, and their effect on cisplatin resistance was evaluated using flow cytometry. Furthermore, the expression levels of DNA-sensitive Toll-like receptor 9 (TLR9) were quantified via immunofluorescence, alongside its colocalization with lysosome-associated membrane protein 1 (LAMP1). Results: This study indicates that cfDNA facilitates metabolic adaptation, particularly under metabolic stress, by modulating glucose and glutamine consumption, key pathways in tumor cell metabolism. Exposure to cfDNA induced distinct metabolic shifts, favoring energy production through oxidative phosphorylation. The anti-cancer activity of cfDNA isolated from conditioned media of cells cultured under stressful conditions is influenced by the culture duration, emphasizing the importance of adaptation and se-lection in releasing cfDNA that can drive pro-tumoral processes. Additionally, cfDNA exposure influenced cell proliferation, quiescence, and migration, processes linked to metastasis and treatment resistance. These findings underscore cfDNA as a key mediator of metabolic reprogramming and adaptive responses in cancer cells, contributing to tumor progression and therapy resistance. Furthermore, the activation of TLR9 signaling suggests a mechanistic basis for cfDNA-induced phenotypic changes. Conclusions: Overall, cfDNA serves as a crucial signaling molecule in the tumor microenvironment, orchestrating adaptive processes that enhance cancer cell survival and progression. Full article

Background: Cell-free DNA (cfDNA) is present in healthy individuals but is elevated in those undergoing physical exertion, trauma, sepsis, and certain cancers. Maintaining cfDNA concentrations is vital for immune homeostasis and preventing inflammatory responses. Understanding cfDNA release and clearance is essential for using cfDNA as a biomarker in clinical diagnostics. We focused on the fragment size of cfDNA and investigated cfDNA dynamics and half-life, particularly the 100–250 base pair fragments. Methods: Healthy, adult men (n = 5; age 40 ± 4.1 years) were subjected to a 30 min treadmill exercise. Blood samples were collected at 0, 5, 10, 15, 30, and 60 min post-exercise using PAXgene^® Blood ccfDNA tubes to stabilize and prevent nuclease-mediated cfDNA degradation and minimize genomic DNA contamination risk. The cfDNA concentration was measured using an electrophoresis-based technique (4150 TapeStation system) to quantify the concentration based on cfDNA fragment size. Results: The results showed a cfDNA half-life of 24.2 min, with a transient increase in 100–250 base pair cfDNA fragments post-exercise, likely due to nuclease activity. These levels rapidly reverted to the baseline within an hour. Conclusions: The rapid clearance of cfDNA underscores its potential as a biomarker for real-time disease monitoring and the evaluation of treatment efficacy. This study is expected to standardize cfDNA investigations, enhancing diagnosis and treatment monitoring across various disease conditions. Full article

The aim of this study was to examine the components of the cell-free supernatant (CFS) derived from a novel strain of psychrophilic Lactobacillus, Dellaglioa algida, and to further elucidate the impact of this CFS on various cellular processes. Specifically, we sought to understand its effects on the cell membrane, protein and DNA release, protease activity, and metabolites of Pseudomonas fluorescens and Pseudomonas fragi, thereby clarifying the antibacterial mechanism involved. The CFS components were analyzed using Gas Chromatography–Mass Spectrometry (GC-MS), the Coomassie Brilliant Blue method, and the phenol–sulfuric acid method. The inhibitory effect of the CFS on Pseudomonas fluorescens and Pseudomonas fragi was assessed using the ethidium bromide (EB) assay, Oxford cup assay, and ultramicroassay. Additionally, we analyzed the metabolites produced by Pseudomonas fluorescens and Pseudomonas fragi when treated with the CFS. The findings reveal that the CFS of Dellaglioa algida contains 94 volatile components, with protein and sugar concentrations of 32.857 ± 0.9705 mg/mL and 98.250 ± 4.210 mg/L, respectively. The CFS induces varying degrees of damage to the cell membranes of both Pseudomonas fluorescens and Pseudomonas fragi, leading to the release of intracellular proteins and DNA. Furthermore, the CFS reduced the protease activity and metabolic capacity of Pseudomonas fluorescens and Pseudomonas fragi. These results enhance our understanding of the mechanism by which psychrophilic Dellaglioa algida inhibits Pseudomonas fluorescens and Pseudomonas fragi, confirming that its inhibitory effect predominantly occurs through damage to the biological cell membranes of Pseudomonas. Dellaglioa algida is a newly identified cold-adapted inhibitor of Pseudomonas, indicating that its CFS is an effective microbial inhibitor in cold environments. This discovery suggests potential applications in inhibiting the growth and reproduction of Pseudomonas fluorescens and Pseudomonas fragi in food, pharmaceuticals, perfumes, and other chemicals, providing a valuable new reference for industrial preservation. Full article

Chimeric antigen receptor T-cell (CAR-T) therapy is a novel anticancer therapy using autologous or allogeneic T-cells. To date, six CAR-T therapies for specific B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin lymphomas (NHL), and multiple myeloma (MM) have been approved by the Food and Drug Administration (FDA). Significant barriers to the effectiveness of CAR-T therapy include cytokine release syndrome (CRS), neurotoxicity in the case of Allogeneic Stem Cell Transplantation (Allo-SCT) graft-versus-host-disease (GVHD), antigen escape, modest antitumor activity, restricted trafficking, limited persistence, the immunosuppressive microenvironment, and senescence and exhaustion of CAR-Ts. Furthermore, cancer drug resistance remains a major problem in clinical practice. CAR-T therapy, in combination with checkpoint blockades and bispecific T-cell engagers (BiTEs) or other drugs, appears to be an appealing anticancer strategy. Many of these agents have shown impressive results, combining efficacy with tolerability. Biomarkers like extracellular vesicles (EVs), cell-free DNA (cfDNA), circulating tumor (ctDNA) and miRNAs may play an important role in toxicity, relapse assessment, and efficacy prediction, and can be implicated in clinical applications of CAR-T therapy and in establishing safe and efficacious personalized medicine. However, further research is required to fully comprehend the particular side effects of immunomodulation, to ascertain the best order and combination of this medication with conventional chemotherapy and targeted therapies, and to find reliable predictive biomarkers. Full article

Irritable bowel syndrome (IBS) involves low-grade mucosal inflammation. Among the various approaches capable of managing the symptoms, physical activity is still under investigation. Despite its benefits, it promotes oxidative stress and inflammation. Mitochondria impacts gut disorders by releasing damage-associated molecular patterns, such as cell-free mtDNA (cf-mtDNA), which support inflammation. This study evaluated the effects of a 12-week walking program on the cf-mtDNA and DNase in 26 IBS and 17 non-IBS subjects. Pro- and anti-inflammatory cytokines were evaluated by ELISA. Digital droplet PCR was used to quantify cf-mtDNA; DNase activity was assessed using a single radial enzyme diffusion assay. PCR-RFLP was used to genotype DNASE1 rs1053874 SNP. Significantly lower IL-10 levels were found in IBS than in non-IBS individuals. Exercise reduced cf-mtDNA in non-IBS subjects but not in IBS patients. DNase activity did not correlate with the cf-mtDNA levels in IBS patients post-exercise, indicating imbalanced cf-mtDNA clearance. Different rs1053874 SNP frequencies were not found between groups. The study confirms the positive effects of regular moderate-intensity physical activity in healthy subjects and its role in cf-mtDNA release and clearance. Walking alone might not sufficiently reduce subclinical inflammation in IBS, based on imbalanced pro- and anti-inflammatory molecules. Prolonged programs are necessary to investigate their effects on inflammatory markers in IBS. Full article

Cell-free DNA molecules are released into the plasma via apoptotic or necrotic events and active release mechanisms, which carry the genetic and epigenetic information of its origin tissues. However, cfDNA is the mixture of various cell fragments, and the efficient enrichment of cfDNA fragments with diagnostic value remains a great challenge for application in the clinical setting. Evidence from recent years shows that cfDNA fragmentomics’ characteristics differ in normal and diseased individuals without the need to distinguish the source of the cfDNA fragments, which makes it a promising novel biomarker. Moreover, cfDNA fragmentomics can identify tissue origins by inferring epigenetic information. Thus, further insights into the fragmentomics of plasma cfDNA shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In this review, we focus on the cfDNA fragment characteristics and its potential application, such as fragment length, end motifs, jagged ends, preferred end coordinates, as well as nucleosome footprints, open chromatin region, and gene expression inferred by the cfDNA fragmentation pattern across the genome. Furthermore, we summarize the methods for deducing the tissue of origin by cfDNA fragmentomics. Full article

Primary graft dysfunction (PGD) is characterized by alveolar epithelial and vascular endothelial damage and inflammation, lung edema and hypoxemia. Up to one-third of recipients develop the most severe form of PGD (Grade 3; PGD3). Animal studies suggest that neutrophils contribute to the inflammatory process through neutrophil extracellular traps (NETs) release (NETosis). NETs are composed of DNA filaments decorated with granular proteins contributing to vascular occlusion associated with PGD. The main objective was to correlate NETosis in PGD3 (n = 9) versus non-PGD3 (n = 27) recipients in an exploratory study. Clinical data and blood samples were collected from donors and recipients pre-, intra- and postoperatively (up to 72 h). Inflammatory inducers of NETs’ release (IL-8, IL-6 and C-reactive protein [CRP]) and components (myeloperoxidase [MPO], MPO-DNA complexes and cell-free DNA [cfDNA]) were quantified by ELISA. When available, histology, immunohistochemistry and immunofluorescence techniques were performed on lung biopsies from donor grafts collected during the surgery to evaluate the presence of activated neutrophils and NETs. Lung biopsies from donor grafts collected during transplantation presented various degrees of vascular occlusion including neutrophils undergoing NETosis. Additionally, in recipients intra- and postoperatively, circulating inflammatory (IL-6, IL-8) and NETosis biomarkers (MPO-DNA, MPO, cfDNA) were up to 4-fold higher in PGD3 recipients compared to non-PGD3 (p = 0.041 to 0.001). In summary, perioperative elevation of NETosis biomarkers is associated with PGD3 following human lung transplantation and these biomarkers might serve to identify recipients at risk of PGD3 and initiate preventive therapies. Full article

Atherosclerosis (AS) is the leading cause of cardiovascular diseases (CVDs) with a high rate of mortality worldwide. Plasma cell-free DNA (cfDNA), mainly originating from apoptosis, necrosis, and active secretion, has been recognized as a promising biomarker for the diagnosis and prognosis of multiple cancers, whereas there are no reports about cfDNA in CVDs. Here, we found an increased quantity and decreased integrity of cfDNA (cfDI) in the serum from AS patients compared with normal controls. Moreover, the reduced cfDI is inversely correlated with serum LDL levels, carotid plaque size, and carotid plaque thickness in the progression of AS. Consistently, in vivo experiments confirmed that the release and cleavage of cfDNA were increased concomitantly with the development and progression of AS in ApoE⁻/⁻ mice. Our study sheds light on the potential of cfDNA and cfDI as molecular biomarkers for detecting and monitoring AS. Full article

Sclerotium rot causes damping-off and stem rot in seedlings and mature mungbeans, which negatively impacts cultivation. The use of a rhizobacterium to control soil-borne diseases is an alternative method to the excess use of synthetic fungicides; therefore, this study aims to screen rhizosphere actinobacteria with fungicidal activities against Sclerotium rolfsii, the pathogen that causes sclerotium rot in mungbeans. Primary screening showed that the Streptomyces sp. isolate Z1-04-02 displayed the highest effectiveness against S. rolfsii in dual culture plates, with a percentage inhibition of 74.28%. An assay containing enzymes that degrade cell walls, of the cell-free culture filtrate (CF) of Z1-04-02, showed that the activities of chitinase and β-1,3-glucanase were 0.0209 and 1.0210 U/mL, respectively, which was significantly higher than that of the control (media alone). The cell-free CF of Z1-04-02, incubated at 37 °C and 100 °C, using agar well diffusion, effectively inhibited the growth of S. rolfsii with inhibition percentages of 37.78% and 27.78%, respectively. Solid-phase microextraction (SPME) was applied to trap volatiles released from Z1-04-02 and gas chromatography–mass spectrometry (GC/MS); volatile antifungal compounds were tentatively identified as bicyclic monoterpene (1R)-(-)-myrtenal. The application of the cell-free CF, and the spore suspension of Z1-04-02, showed disease severity indexes (DSIs) of 12.5% and 8.25%, respectively, which were significantly lower than those showing inoculation by S. rolfsii alone. The identification of this strain by morphology, biochemistry tests, and 16s rDNA sequences revealed that Z1-04-02 was Streptomyces albulus. This finding revealed that S. albulus Z1-04-02 displayed diverse fungicidal activities against S. rolfsii, and it has the potential to act as a biological control agent in terms of inhibiting sclerotium rot in mungbeans. Full article

Rationale: Infection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics or single functional assays. Cell functions are interwoven pathways, and understanding the effect across the spectrum of neutrophil function may identify therapeutic targets. Objectives: Examine neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia patients (CAP). Methods: Isolated neutrophils underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NETosis and receptor expression. Circulating DNAse 1 activity, levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI were measured and correlated to clinical outcome. Serial sampling on day three to five post hospitalization were also measured. The effect of ex vivo PI3K inhibition was measured in a further cohort of 18 COVID-19 patients. Results: Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p = 0.0397) and NETosis (p = 0.0332), and impaired phagocytosis (p = 0.0036) associated with impaired ROS generation (p < 0.0001). The percentage of CD54+ neutrophils (p < 0.001) was significantly increased, while surface expression of CD11b (p = 0.0014) and PD-L1 (p = 0.006) were significantly decreased in COVID-19. COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p = 0.0396) and impaired DNAse activity (p < 0.0001). The ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils (p = 0.0129). Conclusions: COVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration and NETosis, and impaired antimicrobial responses. These changes highlight that targeting neutrophil function may help modulate COVID-19 severity. Full article

Unique bits of genetic, biological and pathological information occur in differently sized cell-free DNA (cfDNA) populations. This is a significant discovery, but much of the phenomenon remains to be explored. We investigated cfDNA fragmentation patterns in cultured human bone cancer (143B) cells using increasingly sensitive electrophoresis assays, including four automated microfluidic capillary electrophoresis assays from Agilent, i.e., DNA 1000, High Sensitivity DNA, dsDNA 915 and dsDNA 930, and an optimized manual agarose gel electrophoresis protocol. This comparison showed that (i) as the sensitivity and resolution of the sizing methods increase incrementally, additional nucleosomal multiples are revealed (hepta-nucleosomes were detectable with manual agarose gel electrophoresis), while the estimated size range of high molecular weight (HMW) cfDNA fragments narrow correspondingly; (ii) the cfDNA laddering pattern extends well beyond the 1–3 nucleosomal multiples detected by commonly used methods; and (iii) the modal size of HMW cfDNA populations is exaggerated due to the limited resolving power of electrophoresis, and instead consists of several poly-nucleosomal subpopulations that continue the series of DNA laddering. Furthermore, the most sensitive automated assay used in this study (Agilent dsDNA 930) revealed an exponential decay in the relative contribution of increasingly longer cfDNA populations. This power-law distribution suggests the involvement of a stochastic inter-nucleosomal DNA cleavage process, wherein shorter populations accumulate rapidly as they are fed by the degradation of all larger populations. This may explain why similar size profiles have historically been reported for cfDNA populations originating from different processes, such as apoptosis, necrosis, accidental cell lysis and purported active release. These results not only demonstrate the diversity of size profiles generated by different methods, but also highlight the importance of caution when drawing conclusions on the mechanisms that generate different cfDNA size populations, especially when only a single method is used for sizing. Full article

All cell and tissue types constantly release DNA fragments into human body fluids by various mechanisms including programmed cell death, accidental cell degradation and active extrusion. Particularly, cell-free DNA (cfDNA) in plasma or serum has been utilized for minimally invasive molecular diagnostics. Disease onset or pathological conditions that lead to increased cell death alter the contribution of different tissues to the total pool of cfDNA. Because cfDNA molecules retain cell-type specific epigenetic features, it is possible to infer tissue-of-origin from epigenetic characteristics. Recent research efforts demonstrated that analysis of, e.g., methylation patterns, nucleosome occupancy, and fragmentomics determined the cell- or tissue-of-origin of individual cfDNA molecules. This novel tissue-of origin-analysis enables to estimate the contributions of different tissues to the total cfDNA pool in body fluids and find tissues with increased cell death (pathologic condition), expanding the portfolio of liquid biopsies towards a wide range of pathologies and early diagnosis. In this review, we summarize the currently available tissue-of-origin approaches and point out the next steps towards clinical implementation. Full article