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PloS One 2022It is currently unknown why obesity leads in some patients to prediabetes and metabolic syndrome. Microinflammation potentially caused by extracellular DNA is supposed...
It is currently unknown why obesity leads in some patients to prediabetes and metabolic syndrome. Microinflammation potentially caused by extracellular DNA is supposed to be involved. The aim of this cross-sectional study in healthy mice was to analyze the association between plasma extracellular DNA and glucose metabolism. Fasting glycemia and insulin were measured in healthy adult female mice that subsequently underwent an oral glucose tolerance test. Indices of glucose metabolism and insulin sensitivity were calculated. DNA was isolated from plasma and quantified fluorometrically. Deoxyribonuclease (DNase) activity of plasma was measured using the single radial enzyme diffusion method. Fasting glycemia correlated negatively with both, extracellular DNA and DNase (r = -0.44 and r = -0.32, respectively). DNase was associated positively with the incremental area under curve (r = 0.35), while extracellular DNA correlated negatively with total area under curve of glycemia during oral glucose tolerance test (r = -0.34). Measures of insulin sensitivity were found to be associated with neither extracellular DNA, nor DNase. The hypothesis of an association of low DNase with increased fasting glucose was partially proved. Surprisingly, low extracellular DNA is associated with higher fasting glucose and lower glucose tolerance in mice. As novel therapeutic targets for prediabetes and metabolic syndrome are highly needed, this study provides novel unexpected associations within the limitations of the focus on physiological variability as it was conducted on healthy mice. The causality of these associations should be proved in further interventional experiments.
Topics: Animals; Blood Glucose; Cross-Sectional Studies; DNA; Deoxyribonucleases; Female; Insulin; Insulin Resistance; Metabolic Syndrome; Mice; Prediabetic State
PubMed: 35385497
DOI: 10.1371/journal.pone.0265099 -
Nature Oct 2022RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop...
RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.
Topics: CRISPR-Cas Systems; Deoxyribonuclease I; DNA; RNA, Guide, CRISPR-Cas Systems; Cryoelectron Microscopy; CRISPR-Associated Proteins
PubMed: 36224386
DOI: 10.1038/s41586-022-05324-6 -
Journal of Veterinary Internal Medicine Sep 2017Immune-mediated hemolytic anemia (IMHA) in dogs has a high risk of thrombosis and is associated with marked neutrophilia and necrosis. Cell death and release of... (Observational Study)
Observational Study
BACKGROUND
Immune-mediated hemolytic anemia (IMHA) in dogs has a high risk of thrombosis and is associated with marked neutrophilia and necrosis. Cell death and release of neutrophil extracellular traps contribute to increased serum concentrations of cell-free DNA, and in human autoimmune disease reduced DNase activity further increases cell-free DNA. Free DNA in blood has prothrombotic properties and could contribute to hypercoagulability in IMHA.
HYPOTHESIS
Cell-free DNA is elevated and DNase activity reduced in dogs with IMHA compared to healthy dogs.
ANIMALS
Dogs presenting to two referral hospitals with IMHA (n = 28) and healthy controls (n = 20).
METHODS
Prospective observational study. Blood was collected and death and thrombotic events occurring in the first 14 days after hospitalization recorded. DNA was extracted from plasma with a commercial kit and quantified by PicoGreen fluorescence. DNase activity of serum was measured by radial diffusion assay.
RESULTS
Cell-free DNA was significantly higher in cases (median: 45 ng/mL, range: 10-2334 ng/mL) than controls (26 ng/mL, range 1-151 ng/mL, P = 0.0084). DNase activity was not different between cases and controls (P = 0.36). Four cases died and there were five suspected or confirmed thrombotic events. Cell-free DNA concentration was associated with death (odds ratio for upper quartile versus lower 3 quartiles: 15; 95% confidence interval 1.62-201; P = 0.03) but not thrombosis (P = 0.57).
CONCLUSIONS AND CLINICAL IMPORTANCE
Cell-free DNA is elevated in dogs with IMHA and likely reflects increased release rather than impaired degradation of DNA. Cell-free DNA concentration is potentially associated with death and might be a prognostic indicator, but this requires confirmation in a larger population.
Topics: Anemia, Hemolytic, Autoimmune; Animals; Case-Control Studies; Cell-Free Nucleic Acids; Deoxyribonucleases; Dog Diseases; Dogs; Female; Male; Prospective Studies
PubMed: 28833583
DOI: 10.1111/jvim.14808 -
Drug Delivery Dec 2023Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function....
Rheumatoid arthritis (RA), an autoimmune disease, is characterized by inflammatory cell infiltration that damages cartilage, disrupts bone, and impairs joint function. The therapeutic efficacy of RA treatments with the severely affected side remains unsatisfactory despite current treatment methods that primarily focus on anti-inflammatory activity, largely because of the complicatedly pathological mechanisms. A recently identified mechanism for RA development involves the interaction of RA autoantibodies with various proinflammatory cytokines to facilitate the formation of neutrophil extracellular traps (NETs), which increased inflammatory responses to express inflammatory cytokines and chemokines. Therefore, NETs architecture digestion may inhibit the positive-feedback inflammatory signal pathway and lessen joint damage in RA. In this work, deoxyribonuclease I (DNase) is connected to oxidized hyaluronic acid (OHA) via Schiff base reaction to extend the half-life of DNase. The modification does not influence the DNase activity for plasmid deoxyribonucleic acid hydrolysis and NETs' architecture disruption. Carboxymethyl chitosan is crosslinked with DNase-functionalised OHA (DHA) to form an injectable, degradable, and biocompatible hydrogel (DHY) to further strengthen the adhesive capability of DHA. Importantly, the collagen-induced arthritis model demonstrates that intra-articular injection of DHY can significantly reduce inflammatory cytokine expression and alleviate RA symptoms, which can be significantly improved by combining methotrexate. Here, a DNase-functionalised hydrogel has been developed for RA treatment by constantly degrading the novel drug target of NETs to decrease inflammatory response in RA.
Topics: Humans; Extracellular Traps; Hydrogels; Arthritis, Rheumatoid; Cytokines; Deoxyribonucleases; Neutrophils
PubMed: 36724178
DOI: 10.1080/10717544.2023.2173332 -
Nature Communications Sep 2023CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic...
CRISPR/Cas9-mediated gene editing has great potential utility for treating genetic diseases. However, its therapeutic applications are limited by unintended genomic alterations arising from DNA double-strand breaks and random integration of exogenous DNA. In this study, we propose NICER, a method for correcting heterozygous mutations that employs multiple nicks (MNs) induced by Cas9 nickase and a homologous chromosome as an endogenous repair template. Although a single nick near the mutation site rarely leads to successful gene correction, additional nicks on homologous chromosomes strongly enhance gene correction efficiency via interhomolog homologous recombination (IH-HR). This process partially depends on BRCA1 and BRCA2, suggesting the existence of several distinct pathways for MN-induced IH-HR. According to a genomic analysis, NICER rarely induces unintended genomic alterations. Furthermore, NICER restores the expression of disease-causing genes in cells derived from genetic diseases with compound heterozygous mutations. Overall, NICER provides a precise strategy for gene correction.
Topics: Homologous Recombination; Mutation; Anti-Bacterial Agents; DNA Breaks, Double-Stranded; Deoxyribonuclease I
PubMed: 37714828
DOI: 10.1038/s41467-023-41048-5 -
FEBS Letters Aug 2014Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and... (Review)
Review
Genomic DNA is constantly challenged from endogenous as well as exogenous sources. The DNA damage response (DDR) mechanism has evolved to combat these challenges and ensure genomic integrity. In this review, we will focus on repair of DNA double-strand breaks (DSB) by homologous recombination and the role of several nucleases and other recombination factors as suitable targets for cancer therapy. Their inactivation as well as overexpression have been shown to sensitize cancer cells by increasing toxicity to DNA-damaging agents and radiation or to be responsible for resistance of cancer cells. These factors can also be used in targeted cancer therapy by taking advantage of specific genetic abnormalities of cancer cells that are not present in normal cells and that result in cancer cell lethality.
Topics: Animals; DNA Repair Enzymes; Deoxyribonucleases; Enzyme Inhibitors; Homologous Recombination; Humans; Molecular Targeted Therapy; Neoplasms; Recombinational DNA Repair
PubMed: 24928444
DOI: 10.1016/j.febslet.2014.06.010 -
PLoS Genetics Jul 2022Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We...
Urinary cell-free DNA (ucfDNA) is a potential biomarker for bladder cancer detection. However, the biological characteristics of ucfDNA are not well understood. We explored the roles of deoxyribonuclease 1 (DNASE1) and deoxyribonuclease 1-like 3 (DNASE1L3) in the fragmentation of ucfDNA using mouse models. The deletion of Dnase1 in mice (Dnase1-/-) caused aberrations in ucfDNA fragmentation, including a 24-fold increase in DNA concentration, and a 3-fold enrichment of long DNA molecules, with a relative decrease of fragments with thymine ends and reduction of jaggedness (i.e., the presence of single-stranded protruding ends). In contrast, such changes were not observed in mice with Dnase1l3 deletion (Dnase1l3-/-). These results suggested that DNASE1 was an important nuclease contributing to the ucfDNA fragmentation. Western blot analysis revealed that the concentration of DNASE1 protein was higher in urine than DNASE1L3. The native-polyacrylamide gel electrophoresis zymogram showed that DNASE1 activity in urine was higher than that in plasma. Furthermore, the proportion of ucfDNA fragment ends within DNase I hypersensitive sites (DHSs) was significantly increased in Dnase1-deficient mice. In humans, patients with bladder cancer had lower proportions of ucfDNA fragment ends within the DHSs when compared with participants without bladder cancer. The area under the curve (AUC) for differentiating patients with and without bladder cancer was 0.83, suggesting the analysis of ucfDNA fragmentation in the DHSs may have potential for bladder cancer detection. This work revealed the intrinsic links between the nucleases in urine and ucfDNA fragmentomics.
Topics: Animals; Cell-Free Nucleic Acids; DNA; Deoxyribonuclease I; Endodeoxyribonucleases; Endonucleases; Humans; Mice; Mice, Knockout; Urinary Bladder Neoplasms
PubMed: 35793278
DOI: 10.1371/journal.pgen.1010262 -
Sensors (Basel, Switzerland) Jul 2014Nucleolytic enzymes are associated with various diseases, and several methods have been developed for their detection. DNase expression is modulated in such diseases as... (Review)
Review
Nucleolytic enzymes are associated with various diseases, and several methods have been developed for their detection. DNase expression is modulated in such diseases as acute myocardial infarction, transient myocardial ischemia, oral cancer, stomach cancer, and malignant lymphoma, and DNase I is used in cystic fibroma therapy. RNase is used to treat mesothelial cancer because of its antiproliferative, cytotoxic, and antineoplastic activities. Angiogenin, an angiogenic factor, is a member of the RNase A family. Angiogenin inhibitors are being developed as anticancer drugs. In this review, we describe fluorometric and electrochemical techniques for detecting DNase and RNase in disease. Oligonucleotides having fluorescence resonance energy transfer (FRET)-causing chromophores are non-fluorescent by themselves, yet become fluorescent upon cleavage by DNase or RNase. These oligonucleotides serve as a powerful tool to detect activities of these enzymes and provide a basis for drug discovery. In electrochemical techniques, ferrocenyl oligonucleotides with or without a ribonucleoside unit are used for the detection of RNase or DNase. This technique has been used to monitor blood or serum samples in several diseases associated with DNase and RNase and is unaffected by interferents in these sample types.
Topics: Biological Assay; DNA; Deoxyribonucleases; Electrochemistry; Fluorescence Resonance Energy Transfer; RNA; Ribonucleases
PubMed: 25019631
DOI: 10.3390/s140712437 -
Trends in Biotechnology May 2015Genome editing (GE) entails the modification of specific genomic sequences in living cells for the purpose of determining, changing, or expanding their function(s).... (Review)
Review
Genome editing (GE) entails the modification of specific genomic sequences in living cells for the purpose of determining, changing, or expanding their function(s). Typically, GE occurs after delivering sequence-specific designer nucleases (e.g., ZFNs, TALENs, and CRISPR/Cas9) and donor DNA constructs into target cells. These designer nucleases can generate gene knockouts or gene knock-ins when applied alone or in combination with donor DNA templates, respectively. We review progress in this field, with an emphasis on designer nuclease and donor template delivery into mammalian target cell populations. We also discuss the impact that incremental improvements to these tools are having on the specificity and fidelity attainable with state-of-the-art DNA-editing procedures. Finally, we identify areas that warrant further investigation.
Topics: Animals; DNA; Deoxyribonucleases; Gene Targeting; Genetic Engineering; Genomics; Humans; Models, Genetic; Protein Engineering
PubMed: 25819765
DOI: 10.1016/j.tibtech.2015.02.011 -
Respiratory Research Mar 2023COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs)....
BACKGROUND
COVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment.
METHODS
Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers.
RESULTS
DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I.
CONCLUSIONS
Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.
Topics: Animals; Humans; Mice; COVID-19; SARS-CoV-2; Extracellular Traps; COVID-19 Drug Treatment; Disease Models, Animal; Neutrophils; Acute Lung Injury; Deoxyribonuclease I
PubMed: 36864506
DOI: 10.1186/s12931-023-02336-2