DNA-histone Complexes Research Articles

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Elevated preoperative von Willebrand factor is associated with perioperative thrombosis in infants and neonates with congenital heart disease

Essentials Perioperative thrombosis is a major cause of morbidity and mortality in congenital heart disease. Neonates and infants undergoing repair of congenital heart lesions were prospectively followed. Elevated von Willebrand factor (VWF) to ADAMTS-13 activity ratios typified the postoperative period. Thrombosis was associated with preoperative VWF activity and cryoprecipitate transfusion SUMMARY: Background The surgical repair of congenital heart malformations is frequently complicated by perioperative thrombosis of unclear etiology. An imbalance between von Willebrand factor (VWF) and ADAMTS-13 is an emerging variable in thrombosis. Objectives To describe perioperative changes to VWF, ADAMTS-13 and NETosis, and evaluate clinical and biochemical associations with postoperative thrombosis. Methods Neonates and infants undergoing palliation or definitive surgical repair of congenital heart malformations were recruited (n=133). Preoperative and postoperative plasma levels of VWF, ADAMTS-13 and markers of NETosis were determined. Patients were followed for up to 30days for the occurrence of thrombosis. Univariate and multivariate logistic regression analyses were conducted to identify variables associated with thrombosis. Results We identified significant postoperative increases in VWF activity, VWF level, DNA-histone complexes and cell-free DNA with an overall decrease in ADAMTS-13 activity. Patients experiencing postoperative thrombotic events (9%) were characterized by surgery performed at a lower intraoperative temperature, higher preoperative lactic acid levels, and higher preoperative VWF activity and level. A multivariate logistic regression model identified preoperative VWF activity (odds ratio (OR)8.39 per IUmL-1 , 95% confidence interval [CI]1.73-40.55) and transfusion of cryoprecipitate (OR1.10 per mLkg-1 , 95%CI1.03-1.17) as being associated with thrombosis. Conclusions Pediatric patients undergoing surgical repair of congenital heart malformations are exposed to high levels of VWF with diminished or minimal change to ADAMTS-13 in the immediate postoperative period. Elevated preoperative VWF activity is associated with postoperative thrombosis in pediatric congenital heart disease.

Histone isoform H2A1H promotes attainment of distinct physiological states by altering chromatin dynamics

BackgroundThe distinct functional effects of the replication-dependent histone H2A isoforms have been demonstrated; however, the mechanistic basis of the non-redundancy remains unclear. Here, we have investigated the specific functional contribution of the histone H2A isoform H2A1H, which differs from another isoform H2A2A3 in the identity of only three amino acids.ResultsH2A1H exhibits varied expression levels in different normal tissues and human cancer cell lines (H2A1C in humans). It also promotes cell proliferation in a context-dependent manner when exogenously overexpressed. To uncover the molecular basis of the non-redundancy, equilibrium unfolding of recombinant H2A1H-H2B dimer was performed. We found that the M51L alteration at the H2A–H2B dimer interface decreases the temperature of melting of H2A1H-H2B by ~ 3 °C as compared to the H2A2A3-H2B dimer. This difference in the dimer stability is also reflected in the chromatin dynamics as H2A1H-containing nucleosomes are more stable owing to M51L and K99R substitutions. Molecular dynamic simulations suggest that these substitutions increase the number of hydrogen bonds and hydrophobic interactions of H2A1H, enabling it to form more stable nucleosomes.ConclusionWe show that the M51L and K99R substitutions, besides altering the stability of histone–histone and histone–DNA complexes, have the most prominent effect on cell proliferation, suggesting that the nucleosome stability is intimately linked with the physiological effects observed. Our work provides insights into the molecular basis of the non-redundancy of the histone H2A isoforms that are being increasingly reported to be functionally important in varied physiological contexts.

Origin of DNA compaction

Chromosome Structure As a repeating unit in eukaryotic chromatin, a nucleosome wraps DNA in superhelical turns around a histone octamer. Mattiroli et al. present the crystal structure of an archaeal histone-DNA complex in which the histone-mediated DNA geometry is exactly the same as that in the nucleosome. Comparing features of archaeal and eukaryotic chromatin structures offers important insights into the evolution of eukaryotic nucleosomes. Science , this issue p. [609][1] [1]: /lookup/doi/10.1126/science.aaj1849

Structure of histone-based chromatin in Archaea.

Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.

Evaluation of assays for quantification of DNA in canine plasma as an indirect marker of NETosis.

Neutrophil extracellular traps (NET), consisting of a filamentous DNA/chromatin-histone scaffold originating from neutrophils are part of the innate immune response, may be released under a variety of inflammatory conditions and are associated with an increased risk for thrombosis. The purpose of this study was to evaluate a SYTOX green fluorescence assay and a histone-DNA complex (hisDNA) ELISA for quantification of NET-related DNA in canine plasma. The influence of variations in blood sample handling on assay results was tested. Accuracy of the SYTOX green fluorescence and the hisDNA ELISA was evaluated with dilutional linearity using serial dilutions. Interference was assessed by addition of purified bilirubin or hemoglobin. Precision was determined by calculating the intra- and inter-assay CV. Preanalytic sample handling did not influence DNA measurements by either assay. Citrate and EDTA plasma samples were equivalent. For the DNA fluorescence assay, dilutional linearity was poor due to autofluorescence, which was corrected by addition of canine plasma to the diluent. The presence of bilirubin and hemoglobin also increased autofluorescence, and resulted in falsely low concentrations of DNA. On the hisDNA ELISA, pigmentemia had no effect. Both assays as modified in this study are suitable for measuring DNA in canine EDTA or citrate plasma. However, performance of the fluorescence assay was impacted by pigmentemia, and it was less sensitive than the ELISA in detecting the presence of nucleosome material in the plasma.

Post-cardiac arrest level of free-plasma DNA and DNA-histone complexes.

Plasma DNA-histone complexes and total free-plasma DNA have the potential to quantify the ischaemia-reperfusion damages occurring after cardiac arrest. Furthermore, DNA-histone complexes may have the potential of being a target for future treatment. The aim was to examine if plasma DNA-histone complexes and the levels of total free-plasma DNA were elevated in post-cardiac arrest patients compared with healthy individuals, and to examine if these biomarkers were capable of predicting mortality. We included 42 comatose out-of-hospital cardiac arrest patients and collected blood samples after 22, 46 and 70 h. Samples for DNA-histone complexes were quantified by Cell Death Detection ELISAplus . The total free-plasma DNA analyses were quantified with qPCR by analysing the Beta-2 microglobulin gene. The control group comprised 40 healthy individuals. We found no difference in the level of DNA-histone complexes between the 22-h sample and healthy individuals (P = 0.10). In the 46-h sample, there was an increased level of DNA-histone complexes in non-survivors compared with survivors 30 days after the cardiac arrest (P < 0.01) and the area under the ROC curve was 0.78 (95% confidence interval: 0.59;0.96). The level of total free-plasma DNA was increased in the 22-h sample compared with healthy individuals (P < 0.001) but no significant difference was found between non-survivors and survivors 30 days after the cardiac arrest (all P ≥ 0.06). An increased level of DNA-histone complexes was associated with increased mortality and that the level of total free-plasma DNA was elevated post-cardiac arrest.

Circulating Histone Is Elevated in Acute Leukemia and Protects Leukemic Cell Death through Enhancement of Leukemic Cell Adhesion to Endothelium

Background: Leukocytes release nuclear contents into extracellular milieu called by extracellular traps (ET) when they are stimulated by reactive oxygen species (ROS). The nuclear contents mainly compose of histone-DNA complex and neutrophil elastase. This study investigated whether the leukemic cells could release ET and the released histone could induce endothelial activation, finally resulting in leukemic progression. Methods: The circulating ET biomarkers (histone-DNA complex, cell free DNA, neutrophil elastase) were measured in 80 patients with hematologic diseases and 40 healthy controls by ELISA. ET formation and ROS level were investigated during leukemic cell proliferation in vitro. Histone-induced endothelial adhesion molecule expression and survival were measured by flow cytometry. Results: Acute leukemia showed high ET biomarkers, which correlated with peripheral blast count. Leukemic cells produced high ROS and release extracellular histone, which was significantly blocked by antioxidants. Histone significantly induced 3 endothelial adhesion molecules expression, promoting leukemic cell adhesion to endothelial cells, which was inhibited by histone inhibitors (heparin, polysialic acid, activated protein C), 3 neutralizing antibodies against adhesion molecules, and Toll like receptor(TLR)9 antagonist. When the leukemic cell co-cultured with endothelial cells, the adherent leukemic cells showed better survival than the non-adherent, demonstrating that histone-treated endothelial cells protected leukemic cells from both spontaneous and chemotherapy-induced death. Conclusion: Our data for the first time supports that extracellular histone could be released from leukemic cells through ROS dependent mechanism. The released histone could promote leukemic cell adhesion through endothelial adhesion molecule induction and finally protect from leukemic cell death. Disclosures No relevant conflicts of interest to declare.

Extracellular Histone Released from Leukemic Cells Increases Their Adhesion to Endothelium and Protects them from Spontaneous and Chemotherapy-Induced Leukemic Cell Death.

IntroductionWhen leukocytes are stimulated by reactive oxygen species (ROS), they release nuclear contents into the extracellular milieu, called by extracellular traps (ET). The nuclear contents are mainly composed of the histone–DNA complex and neutrophil elastase. This study investigated whether leukemic cells could release ET and the released histone could induce endothelial activation, eventually resulting in leukemic progression.MethodsThe circulating ET were measured in 80 patients with hematologic diseases and 40 healthy controls. ET formation and ROS levels were investigated during leukemic cell proliferation in vitro. Histone-induced endothelial adhesion molecules expression and cell survival were measured by flow cytometry.ResultsAcute leukemia patients had high levels of ET, which correlated with peripheral blast count. Leukemic cells produced high ROS levels and released extracellular histone, which was significantly blocked by antioxidants. Histone significantly induced 3 endothelial adhesion molecules expression, and promoted leukemic cell adhesion to endothelial cells, which was inhibited by histone inhibitors (heparin, polysialic acid, and activated protein C), neutralizing antibodies against these adhesion molecules, and a Toll like receptor(TLR)9 antagonist. When leukemic cells were co-cultured with endothelial cells, adherent leukemic cells showed better survival than the non-adherent ones, demonstrating that histone-treated endothelial cells protected leukemic cells from both spontaneous and chemotherapy-induced death.ConclusionOur data demonstrate for the first time that extracellular histone can be released from leukemic cells through a ROS-dependent mechanism. The released histone promotes leukemic cell adhesion by inducting the surface expression of endothelial adhesion molecules and eventually protects leukemic cells from cell death.

Porcine endothelium induces DNA-histone complex formation in human whole blood: a harmful effect of histone on coagulation and endothelial activation.

Neutrophils play a role in xenograft rejection. When neutrophils are stimulated, they eject the DNA-histone complex into the extracellular space, called neutrophil extracellular traps (NET). We investigated whether NET formation actively occurs in the xenograft and contributes to coagulation and endothelial activation. Human whole blood was incubated with porcine aortic endothelial cells (pEC) from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. In the supernatant plasma from human blood, the level of the DNA-histone complex was measured by ELISA, and thrombin generation was measured using a calibrated automated thrombogram. Histone-induced tissue factor and adhesion molecule expression were measured by flow cytometry. pEC from both wild-type and GTKO pigs significantly induced DNA-histone complex formation in human whole blood. The DNA-histone complex produced shortened the thrombin generation time and clotting time. Histone alone dose-dependently induced tissue factor and adhesion molecule expression in pEC. Aurintricarboxylic acid pretreatment partially inhibited pEC-induced DNA-histone complex formation. DNA-histone complex actively generated upon xenotransplantation is a novel target to inhibit coagulation and endothelial activation. To prevent tissue factor and adhesion molecule expression, a strategy to block soluble histone may be required in xenotransplantation.

Current Pathological and Laboratory Considerations in the Diagnosis of Disseminated Intravascular Coagulation.

Systemically sustained thrombin generation in vivo is the hallmark of disseminated intravascular coagulation (DIC). Typically, this is in response to a progressing disease state that is associated with significant cellular injury. The etiology could be infectious or noninfectious, with the main pathophysiological mechanisms involving cross-activation among coagulation, innate immunity, and inflammatory responses. This leads to consumption of both pro- and anticoagulant factors as well as endothelial dysfunction and disrupted homeostasis at the blood vessel wall interface. In addition to the release of tissue plasminogen activator (tPA) and soluble thrombomodulin (sTM) following cellular activation and damage, respectively, there is the release of damage-associated molecular patterns (DAMPs) such as extracellular histones and cell-free DNA. Extracellular histones are increasingly recognized as significantly pathogenic in critical illnesses through direct cell toxicity, the promotion of thrombin generation, and the induction of neutrophil extracellular trap (NET) formation. Clinically, high circulating levels of histones and histone–DNA complexes are associated with multiorgan failure, DIC, and adverse patient outcomes. Their measurements as well as that of other DAMPs and molecular markers of thrombin generation are not yet applicable in the routine diagnostic laboratory. To provide a practical diagnostic tool for acute DIC, a composite scoring system using rapidly available coagulation tests is recommended by the International Society on Thrombosis and Haemostasis. Its usefulness and limitations are discussed alongside the advances and unanswered questions in DIC pathogenesis.

Evaluation of Circulating Markers of Neutrophil Extracellular Trap (NET) Formation as Risk Factors for Diabetic Retinopathy in a Case-Control Association Study.

Background: Inflammatory stimuli can induce neutrophils to release nuclear DNA combined with histones into the extracellular space, forming neutrophil extracellular traps. Because inflammation contributes to diabetic retinopathy, it is plausible that neutrophil extracellular trap formation actively occurs in diabetic retinopathy. This case-control study investigated the clinical relevance of circulating levels of neutrophil extracellular trap components as risk factors of diabetic retinopathy, and further evaluated whether glucose induced neutrophil extracellular trap formation in vitro using whole blood from healthy volunteers. Methods: Circulating levels of DNA-histone complexes, cell free double-stranded DNA, and polymorphonuclear neutrophil elastase, considered to be markers of neutrophil extracellular trap formation, were measured in patients with diabetic retinopathy (n=28) and without (n=62) and in 28 healthy controls. Results: Circulating DNA-histone complex and polymorphonuclear neutrophil elastase levels were significantly increased in patients with diabetic retinopathy compared with those without retinopathy. Multivariable logistic regression analysis, adjusted for glycated hemoglobin levels and fasting blood glucose, revealed that DNA-histone complex and polymorphonuclear neutrophil elastase levels were significant independent risk factors of retinopathy. In vitro experiments also showed that glucose significantly increased markers of neutrophil extracellular trap formation in a dose-dependent manner. Conclusions: Markers of neutrophil extracellular trap formation were independent risk factors of diabetic retinopathy. This finding provides a new insight into the potential therapeutic and preventive approaches to dampen neutrophil extracellular trap formation.

Iron-chelating agent desferrioxamine stimulates formation of neutrophil extracellular traps (NETs) in human blood-derived neutrophils.

Neutrophil extracellular trap (NET) formation is a significant innate immune defense mechanism against microbial infection that complements other neutrophil functions including phagocytosis and degranulation of antimicrobial peptides. NETs are decondensed chromatin structures in which antimicrobial components (histones, antimicrobial peptides and proteases) are deployed and mediate immobilization of microbes. Here we describe an effect of iron chelation on the phenotype of NET formation. Iron-chelating agent desferrioxamine (DFO) showed a modest but significant induction of NETs by freshly isolated human neutrophils as visualized and quantified by immunocytochemistry against histone-DNA complexes. Further analyses revealed that NET induction by iron chelation required NADPH-dependent production of reactive oxygen species (ROS) as well as protease and peptidyl-arginine-deiminase 4 (PAD4) activities, three key mechanistic pathways previously linked to NET formation. Our results demonstrate that iron chelation by DFO contributes to the formation of NETs and suggest a target for pharmacological manipulation of NET activity.

Classification of Promoters Based on the Combination of Core Promoter Elements Exhibits Different Histone Modification Patterns.

Four different histones (H2A, H2B, H3, and H4; two subunits each) constitute a histone octamer, around which DNA wraps to form histone-DNA complexes called nucleosomes. Amino acid residues in each histone are occasionally modified, resulting in several biological effects, including differential regulation of transcription. Core promoters that encompass the transcription start site have well-conserved DNA motifs, including the initiator (Inr), TATA box, and DPE, which are collectively called the core promoter elements (CPEs). In this study, we systematically studied the associations between the CPEs and histone modifications by integrating the Drosophila Core Promoter Database and time-series ChIP-seq data for histone modifications (H3K4me3, H3K27ac, and H3K27me3) during development in Drosophila melanogaster via the modENCODE project. We classified 96 core promoters into four groups based on the presence or absence of the TATA box or DPE, calculated the histone modification ratio at the core promoter region, and transcribed region for each core promoter. We found that the histone modifications in TATA-less groups were static during development and that the core promoters could be clearly divided into three types: i) core promoters with continuous active marks (H3K4me3 and H3K27ac), ii) core promoters with a continuous inactive mark (H3K27me3) and occasional active marks, and iii) core promoters with occasional histone modifications. Linear regression analysis and non-linear regression by random forest showed that the TATA-containing groups included core promoters without histone modifications, for which the measured RNA expression values were not predictable accurately from the histone modification status. DPE-containing groups had a higher relative frequency of H3K27me3 in both the core promoter region and transcribed region. In summary, our analysis showed that there was a systematic link between the existence of the CPEs and the dynamics, frequency and influence on transcriptional activity of histone modifications.

Factor XII-mediated contact activation related to poor prognosis in disseminated intravascular coagulation

BackgroundThe contact system that initiates the intrinsic coagulation pathway plays a role in thrombus formation. Since neutrophil extracellular traps (NET), which are mainly composed of histone and DNA, are actively formed in disseminated intravascular coagulation (DIC) and the NET can activate factor XII, it is plausible that a NET component strongly activates the contact system in patients with DIC. MethodsIn 146 patients suspected of having DIC, the plasma levels of contact system factors including factor XII, activated factor XII (XIIa), prekallikrein, high-molecular-weight kininogen (HMWK), bradykinin, extrinsic factor VII and histone–DNA complex were measured. In an in vitro plasma clotting assay, factor XII–deficient plasma was stimulated with silica or histone. ResultsThe levels of not only extrinsic coagulation factor VII but also intrinsic coagulation factors including factors XI and XII were significantly decreased in patients with overt DIC in comparison with those with no overt DIC. Factor XIIa and histone-DNA complex were also significantly increased in patients with overt DIC. However, HMWK, prekallikrein and bradykinin were not significantly different between patients with and without overt DIC. Interestingly, factors XII and XIIa were revealed as significantly independent potential prognostic markers for DIC. The histone-DNA complex level significantly contributed to the factor XIIa level (20.6%). In an in vitro clotting assay, histone, a major component of NET, activated coagulation that was dependent, in part, on the presence of factor XII. ConclusionThese findings suggest that active NET formation can induce factor XII-mediated coagulation activation in patients with DIC with poor prognosis. The resulting factor XIIa release can be used as an independent potential prognostic marker for DIC. Activation of factor XII-mediated coagulation may be a potential therapeutic target in DIC,

DNA-histone complexes as ligands amplify cell penetration and nuclear targeting of anti-DNA antibodies via energy-independent mechanisms.

We have generated three monoclonal cell-penetrating antibodies (CPAbs) from a non-immunized lupus-prone (NZB × NZW)F1 mouse that exhibited high anti-DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA-histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell-entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis-specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy-independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant.

Histones Differentially Modulate the Anticoagulant and Profibrinolytic Activities of Heparin, Heparin Derivatives, and Dabigatran.

The antithrombin activity of unfractionated heparin (UFH) is offset by extracellular histones, which, along with DNA, represent a novel mediator of thrombosis and a structural component of thrombi. Here, we systematically evaluated the effect of histones, DNA, and histone-DNA complexes on the anticoagulant and profibrinolytic activities of UFH, its derivatives enoxaparin and fondaparinux, and the direct thrombin inhibitor dabigatran. Thrombin generation was assessed by calibrated automated thrombinography, inhibition of factor Xa and thrombin by synthetic substrates, tissue plasminogen activator-mediated clot lysis by turbidimetry, and thrombin-activatable fibrinolysis inhibitor (TAFI) activation by a functional assay. Histones alone delayed coagulation and slightly stimulated fibrinolysis. The anticoagulant activity of UFH and enoxaparin was markedly inhibited by histones, whereas that of fondaparinux was enhanced. Histones neutralized both the anti-Xa and anti-IIa activities of UFH and preferentially blocked the anti-IIa activity of enoxaparin. The anti-Xa activity of fondaparinux was not influenced by histones when analyzed by chromogenic substrates, but was potentiated in a plasma prothrombinase assay. Histones inhibited the profibrinolytic activity of UFH and enoxaparin and enhanced that of fondaparinux by acting on the modulation of TAFI activation by anticoagulants. Histone H1 was mainly responsible for these effects. Histone-DNA complexes, as well as intact neutrophil extracellular traps, impaired the activities of UFH, enoxaparin, and fondaparinux. Dabigatran was not noticeably affected by histones and/or DNA, whatever the assay performed. In conclusion, histones and DNA present in the forming clot may variably influence the antithrombotic activities of anticoagulants, suggesting a potential therapeutic advantage of dabigatran and fondaparinux over heparins.

Significance of the DNA-Histone Complex Level as a Predictor of Major Adverse Cardiovascular Events in Hemodialysis Patients: The Effect of Uremic Toxin on DNA-Histone Complex Formation

Background: Neutrophils can release the DNA-histone complex into circulation following exposure to inflammatory stimuli. This prospective study investigated whether the DNA-histone complex and other biomarkers could predict major cardiovascular adverse events (MACEs) in hemodialysis (HD) patients. Methods: The levels of circulating DNA-histone complexes, cell-free DNA, interleukin (IL)-6, and neutrophil elastase were measured in 60 HD patients and 28 healthy controls. MACE was assessed at 24 months. Uremic toxin-induced neutrophil released contents were measured in vitro. Results: Compared with controls, HD patients showed higher levels of DNA-histone complexes and IL-6. The DNA-histone complex level was inversely associated with the Kt/V. In a multivariable Cox analysis, the high level of DNA-histone complexes was a significant independent predictor of MACE. The uremic toxins induced DNA-histone complex formation in normal neutrophils in vitro. Conclusion: The DNA-histone complex is a potentially useful marker to predict MACE in HD patients. Uremic toxins induced DNA-histone complex formation in vitro.

Extracellular histones induce tissue factor expression in vascular endothelial cells via TLR and activation of NF-κB and AP-1

Extracellular histones have been recognized recently as proinflammatory mediators; they are released from dying cells in response to inflammatory challenge, contributing to endothelial cell dysfunction, thrombin formation, organ failure, and death during sepsis. Clinical studies suggest that the plasma concentration of the histone-DNA complex is correlated with the severity of DIC and is a poor independent prognostic marker in sepsis. In addition, platelet activation stimulates thrombus formation. Whether histones contribute to procoagulant activity in other ways remains elusive. In this study, we confirmed that histones induce tissue factor (TF) expression in a concentration- and time-dependent manner in vascular endothelial cells (ECs) and macrophages. However, histones did not affect TF pathway inhibitor expression. Moreover, blocking the cell surface receptors TLR4 and TLR2 with specific neutralizing antibodies significantly reduced histone-induced TF expression. Furthermore, histones enhanced the nuclear translocation of NF-κB (c-Rel/p65) and AP-1 expression in a time-dependent manner in ECs. Mutating NF-κB and AP-1 significantly reduced histone-induced TF expression. Altogether, our experiments suggest that histone induces TF expression in ECs via cell surface receptors TLR4 and TLR2, simultaneously depending on the activation of the transcription factors NF-κB and AP-1.

Stabilization of DNA Structures with Poly(ethylene sodium phosphate).

The structure and stability of biomolecules under molecular crowding conditions are of interest because such information clarifies how biomolecules behave under cell-mimicking conditions. The anionic surfaces of chromatin, which is composed of DNA strands and histone complexes, are concentrated in cell nuclei and thus generate a polyanionic crowding environment. In this study, we designed and synthesized an anionic polymer, poly(ethylene sodium phosphate) (PEP·Na), which has a nucleic acid phosphate backbone and created a cell nucleus-like environment. The effects of molecular crowding with PEP·Na on the thermodynamics of DNA duplexes, triplexes, and G-quadruplexes were systematically studied. Thermodynamic analysis demonstrated that PEP·Na significantly stabilized the DNA structures; e.g., a free energy change at 25 °C for duplex formation decreased from -6.6 to -12.8 kcal/mol with 20 wt % PEP·Na. Thermodynamic parameters further indicated that the factors for the stabilization of the DNA structures were dependent on sodium ion concentration. At lower polymer concentrations, the stabilization was attributed to a shielding of the electrostatic repulsion between DNA strands by the sodium ions of PEP·Na. In contrast, at higher polymer concentrations, the DNA structures were entropically stabilized by volume exclusion, which could be enhanced by electrostatic repulsion between phosphate groups in DNA strands and in PEP·Na. Additionally, increasing PEP·Na concentration resulted in increasing enthalpy of the DNA duplex but decreasing enthalpy of DNA G-quadruplex, indicating that the polymers also promoted dehydration of the DNA strands. Thus, polyanionic crowding affects the thermodynamics of DNA structures via the sodium ions, volume exclusion, and hydration. The stabilization of DNA by the cell nucleus-like polyanionic crowding provides new information regarding DNA structures and allows for modeling reactions in cell nuclei.