Nucleosome Levels Research Articles

Association of circulating nucleosomes levels with FIT performance for advanced adenomas in a symptomatic population.

170 Background: The effectiveness of screening using either fecal immunochemical testing (FIT) or colonoscopy in reducing colorectal cancer (CRC) mortality has been demonstrated in previous studies. Nevertheless, the single test sensitivity of FIT for relevant precursor lesions, especially high-risk neoplasms like invasive cancer or advanced adenomas (AA), is a concern. Improving the sensitivity for high-risk neoplasm is critical to improving the effectiveness of screening in reducing CRC incidence and mortality. Dysregulation of histone post-translational modifications (PTMs) has been linked with several cancers including CRC. We investigated the clinical performance of circulating nucleosome levels containing specific histone PTMs in blood, in combination with FIT from symptomatic subjects referred to colonoscopy to evaluate their discriminant power towards CRC and AA. Methods: 10mls whole blood was obtained prior to colonoscopy from 476 patients referred to colonoscopy in National Taiwan University Hospital for surveillance or secondary to bowel symptoms. Patients were classified into 5 groups based on their colonoscopy reports: (i) patient with CRC (n = 67), (ii) patients with AA (n = 60, including 22 with AA≥2cm); (iii) patients with non-AA (n = 123); (iv) patients with non-neoplastic polyps (n = 29); (vi) healthy controls with no endoscopic lesion (n = 197). Plasma samples were analyzed for nucleosome levels using 7 different quantitative immunoassays (Nu.Q assays; Belgian Volition SRL, Belgium) targeting H3.1-nucleosomes or different histone modifications (H3K27Me3-, H3K36Me3-, H3K9Me3-, H3K14Ac-, H3K27Ac- or H3R8Cit-nucleosomes). All study subjects were asked to collect a stool sample at home within 48-hours before the colonoscopy. The fecal samples were assayed with the OC-SENSOR FIT kit (Eiken Chemical Co., Ltd., Tokyo, Japan) for all subjects. Results: At a cut-off of 20µg/g feces, the FIT test showed a sensitivity of 83.5% at 82.1% specificity for CRC +AA vs. Controls. All the CRC patients were detected but 35% of AA were missed, and 7 of which were high-risk adenomas (AA≥2cm). A combination of 2 Nu.Q biomarkers: H3K36Me3 and H3K9Me3 with FIT in a decision tree model showed an improved sensitivity of 98.4% allowing the detection of all CRC patients and 97% of the patients with AA including all high-risk adenomas. The 2 missed AA were one AA below 1cm and one between 1 and 2cm. Unnecessary colonoscopies could be reduced by 28.9% of the control and 20.7% of the non-neoplastic polyps subgroups as both are found negative with this panel of assays. Conclusions: At present, all the symptomatic patients are sent to colonoscopy in clinical practice. Our results indicate that H3K36Me3- and H3K9Me3-nucleosome levels, in combination with FIT in a decision tree model, could detect all CRC patients and all high-risk adenomas and help reduce unnecessary colonoscopies.

Circulating nucleosomes for detection of colorectal cancer and high-risk advanced adenomas.

169 Background: Detection and treatment of early colorectal cancer (CRC) or advanced adenomas (AA) is the key for superior outcomes and fecal immunochemical testing (FIT)-based screening has proven effective in reducing CRC death and the risk of advanced-stage CRC but the discrepancy in the effectiveness was observed between proximal and distal cancers. On the other hand, false-positive results of FIT with 60 % of negative colonoscopy is another concern, considering the burden to the screening participants and the healthcare providers. A blood-based test as an alternative or supplement to FIT has the potential to concurrently enhance the detection of high-risk neoplasms and identify individuals at risk who should then be referred to colonoscopy. We investigated the levels of circulating free nucleosomes containing different epigenetic modifications in patients referred for colonoscopy. Methods: 10mls whole blood was obtained from 520 asymptomatic patients prior to colonoscopy at National Taiwan University Hospital. Patients were classified into 5 groups based on their colonoscopy reports: (i) patient with CRC (n = 33), (ii) patients with AA (n = 123, including 18 with AA > 2cm); (iii) patients with non-AA (n = 168); (iv) patients with non-neoplastic polyps (n = 30); (vi) healthy controls with no endoscopic lesion (n = 166). Plasma samples were analyzed for nucleosome levels using 7 different quantitative immunoassays (Nu.Q assays; Belgian Volition SRL, Belgium) targeting H3.1-nucleosomes or different histone modifications (H3K27Me3-, H3K36Me3-, H3K9Me3-, H3K14Ac-, H3K27Ac- or H3R8Cit-nucleosomes). All study subjects were asked to collect a stool sample at home within 48-hours before the colonoscopy. The fecal samples were assayed with the Eiken OC-SENSOR FIT kit (Eiken Chemical Co., Ltd., Tokyo, Japan) for all study subjects. Results: At a cut-off of 20µg/g feces, the FIT test showed a sensitivity of 92,9% at 17.5% specificity for CRC +AA vs. Controls. All the CRC patients and 83.3% of the high-risk AA (> 2cm) patients were FIT positive. A combination of 2 Nu.Q biomarkers: H3K27Me3 and H3R8Cit with FIT in a logistic regression model showed improved sensitivity of 95% at 20.6% specificity allowing the detection of all CRC patients and 94.3% of AA patients including all the high-risk adenomas. The FIT test detected 46 out of 55 proximal adenomas whereas the combined model could detect 50 proximal adenomas including 3 proximal AA > 2cm not being detected by FIT (p < 0.05). The same combination would reduce unnecessary colonoscopies by 21.8% of the control and 23.3% of the non-neoplastic polyps subgroups compared to 17.5% and 6.7%, respectively with the FIT test alone. Conclusions: Our results indicate that H3K27Me3 and H3R8Cit-nucleosome levels in combination with FIT could improve the detection of proximal high-risk AA and could also provide a non-invasive method to reduce unnecessary colonoscopy.

Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging

Nucleosomes are the basic units of chromatin and critical for storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks, in which post-translational modifications of histones play a key role. However, the mode of action and the structural implications at the single-molecule level of nucleosomes is still poorly understood. Here we apply a high-throughput atomic force microscopy imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants three additional methyl groups at lysine 36 of histone H3 (H3K36me3), phosphorylation of H3 histones at serine 10 (H3S10phos), and acetylation of H4 histones at lysines 5, 8, 12, and 16 (H4K5/8/12/16ac). Our data set of more than 25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5-bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild-type nucleosomes and additionally unwrap stochastically from both sides, similar to centromere protein A (CENP-A) nucleosomes and in contrast to the highly anticooperative unwrapping of wild-type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild-type nucleosomes and also retain the same level of anticooperativity. Our findings help to put the mode of action of these modifications into context. Although H3K36me3 likely acts partially by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.

Mapping Mammalian 3D Genomes by Micro-C.

3D genome mapping aims at connecting the physics of chromatin folding to the underlying biological events, and applications of various chromosomal conformation capture (3C) assays continue to discover critical roles of genome folding in regulating nuclear functions. To interrogate the full spectrum of chromatin folding ranging from the level of nucleosomes to full chromosomes in mammals, we developed an enhanced 3C-based method called Micro-C. The protocol employs Micrococcal nuclease (MNase) to fragment the genome, which overcomes the resolution limit of restriction enzyme-based methods, enabling the estimation of contact frequencies between proximal nucleosomes. Such improvements successfully resolve the fine-scale level of chromatin folding, including enhancer-promoter or promoter-promoter interactions, genic and nucleosomal folding, and boost the signal-to-noise ratio in detecting loops and substructures underlying TADs. In this chapter, we will thoroughly discuss the details of the Micro-C protocol and critical parameters to consider for generating high-quality Micro-C maps.

DeNOPA: decoding nucleosome positions sensitively with sparse ATAC-seq data.

As the basal bricks, the dynamics and arrangement of nucleosomes orchestrate the higher architecture of chromatin in a fundamental way, thereby affecting almost all nuclear biology processes. Thanks to its rather simple protocol, assay for transposase-accessible chromatin using sequencing (ATAC)-seq has been rapidly adopted as a major tool for chromatin-accessible profiling at both bulk and single-cell levels; however, to picture the arrangement of nucleosomes per se remains a challenge with ATAC-seq. In the present work, we introduce a novel ATAC-seq analysis toolkit, named decoding nucleosome organization profile based on ATAC-seq data (deNOPA), to predict nucleosome positions. Assessments showed that deNOPA outperformed state-of-the-art tools with ultra-sparse ATAC-seq data, e.g. no more than 0.5 fragment per base pair. The remarkable performance of deNOPA was fueled by the short fragment reads, which compose nearly half of sequenced reads in the ATAC-seq libraries and are commonly discarded by state-of-the-art nucleosome positioning tools. However, we found that the short fragment reads enrich information on nucleosome positions and that the linker regions were predicted by reads from both short and long fragments using Gaussian smoothing. Last, using deNOPA, we showed that the dynamics of nucleosome organization may not directly couple with chromatin accessibility in the cis-regulatory regions when human cells respond to heat shock stimulation. Our deNOPA provides a powerful tool with which to analyze the dynamics of chromatin at nucleosome position level with ultra-sparse ATAC-seq data.

The Significance of Circulating Cell-Free DNA Markers in Chronic Hepatitis B Patients with Hepatocellular Carcinoma.

(1) Background: Hepatocellular carcinoma (HCC) is the most serious complication of chronic hepatitis B (CHB). Recently, the detection of circulating cell-free (cf) DNA and nucleosomes has found numerous applications in oncology. This study aimed to examine the levels of serum cfDNA markers and nucleosomes in CHB patients with and without HCC and assess their potential association with HCC patients’ survival. (2) Methods: Nineteen patients with CHB and HCC and 38 matched patients with CHB without cancer development during 5 years of antiviral therapy were included. Stored serum samples were analyzed for cfDNA species, including the cfDNA concentration and levels of Alu115, Alu247, and nucleosomes. DNA integrity was expressed as the Alu247/Alu115 ratio. (3) Results: Compared to controls, HCC patients had higher median Alu247 levels (64.2 vs. 23.2 genomic equivalent, p = 0.004) and DNA integrity (1.0 vs. 0.7, p < 0.001) and a trend for a higher median cfDNA concentration (36.0 vs. 19.5 ng/mL, p = 0.064). Increased DNA integrity (Alu247/Alu115 > 1) was associated with an increased risk of death during the first year after HCC diagnosis (p = 0.016). (4) Conclusions: Levels of Alu247 and DNA integrity in serum cfDNA are elevated in CHB patients with HCC, whereas increased DNA integrity seems to be associated with a worse short-term prognosis in this setting.

Uremic serum damages endothelium by provoking excessive neutrophil extracellular trap formation

Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality, or vascular access failure. In vitro, HD-derived uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.

Oxidative Stress and Inflammatory Mediators in Exhaled Breath Condensate of Patients with Pulmonary Tuberculosis. A Pilot Study with a Biomarker Perspective.

Tuberculosis (TB) is one of the highest infectious burdens worldwide. An excess of inflammation and inadequate antioxidant defense mechanisms are believed to lead to chronic inflammation and lung damage in tuberculosis (TB). However, circulating metabolites do not always replicate lung-associated biomarkers that define the pathobiology of the disease. The objective of this study was to determine the utility of exhaled breath condensate (EBC), a non-invasive and straightforward sample, to evaluate alveolar space-derived metabolites of redox state and inflammation. We assessed the levels of exhaled oxidant/antioxidant parameters (8-isoprostane, MDA, GSH), inflammatory markers, such as nucleosomes, cytokines (IL-2, IL-4, IL-6 and IL-8, IL-10, GM-CSF, TNF-α, and IFN-γ) and lipid mediators (PGE2, LTB4, RvD1, and Mar1), in patients with recently diagnosed pulmonary TB and healthy controls’ EBC and serum. The TB patients showed 36% lower GSH levels, and 2-, 1.4-, 1.1-, and 50-fold higher levels of 8-isoprostanes, nucleosomes, IL-6, and LTB4, respectively, in EBC. There was no correlation between EBC and serum, highlighting the importance of measuring local biomarkers. Quantitation of local inflammatory molecules and redox states in EBC would help find biomarkers useful for pharmacological and follow-up studies in pulmonary tuberculosis.

NucHMM: a method for quantitative modeling of nucleosome organization identifying functional nucleosome states distinctly associated with splicing potentiality

We develop a novel computational method, NucHMM, to identify functional nucleosome states associated with cell type-specific combinatorial histone marks and nucleosome organization features such as phasing, spacing and positioning. We test it on publicly available MNase-seq and ChIP-seq data in MCF7, H1, and IMR90 cells and identify 11 distinct functional nucleosome states. We demonstrate these nucleosome states are distinctly associated with the splicing potentiality of skipping exons. This advances our understanding of the chromatin function at the nucleosome level and offers insights into the interplay between nucleosome organization and splicing processes.

Characterizing circulating nucleosomes in the plasma of dogs with lymphoma

BackgroundNucleosomes consist of DNA wrapped around a histone octamer core like beads on a string so that DNA can be condensed as chromatin into chromosomes. Diseases such as cancer or inflammation lead to cell death where chromatin is fragmentated and released as mononucleosomes into the blood. The Nu.Q™ H3.1 assay measures total nucleosome concentration in plasma of humans and has been used to detect and identify cancer even at early stages. The objectives of this study were to determine if nucleosome levels could be used to distinguish between healthy dogs and dogs with various stages of lymphoma (LSA) using the Nu.Q™ H3.1 assay.A total of 126 dogs diagnosed with LSA and 134 healthy controls were recruited for this study. Plasma was collected from each dog and stored in K2-EDTA tubes. The LSA patient samples were recruited from TAMU or purchased from various biobanks. All control cases were recruited from TAMU.ResultsDogs with LSA had an approximately 7-fold increase in their plasma nucleosome concentrations compared to controls (AUC 87.8%). Nucleosome concentrations increased with cancer stage and dogs with B cell lymphomas had significantly higher nucleosome concentrations than dogs with T cell lymphomas.ConclusionsThe Nu.Q™ H3.1 assay was able to reliably detect elevated nucleosome concentrations in the plasma of dogs with LSA. Furthermore, it appears that nucleosomes are useful for differentiating cancer from healthy individuals in canines.

Plant Metabolic Gene Clusters: Evolution, Organization, and Their Applications in Synthetic Biology.

Plants are a remarkable source of high-value specialized metabolites having significant physiological and ecological functions. Genes responsible for synthesizing specialized metabolites are often clustered together for a coordinated expression, which is commonly observed in bacteria and filamentous fungi. Similar to prokaryotic gene clustering, plants do have gene clusters encoding enzymes involved in the biosynthesis of specialized metabolites. More than 20 gene clusters involved in the biosynthesis of diverse metabolites have been identified across the plant kingdom. Recent studies demonstrate that gene clusters are evolved through gene duplications and neofunctionalization of primary metabolic pathway genes. Often, these clusters are tightly regulated at nucleosome level. The prevalence of gene clusters related to specialized metabolites offers an attractive possibility of an untapped source of highly useful biomolecules. Accordingly, the identification and functional characterization of novel biosynthetic pathways in plants need to be worked out. In this review, we summarize insights into the evolution of gene clusters and discuss the organization and importance of specific gene clusters in the biosynthesis of specialized metabolites. Regulatory mechanisms which operate in some of the important gene clusters have also been briefly described. Finally, we highlight the importance of gene clusters to develop future metabolic engineering or synthetic biology strategies for the heterologous production of novel metabolites.

Epigenetic differences in an identical genetic background modulate alternative splicing in A. thaliana

How stable and temperature-dependent variations in DNA methylation and nucleosome occupancy influence alternative splicing (AS) remains poorly understood in plants. To answer this, we generated transcriptome, whole-genome bisulfite, and MNase sequencing data for an epigenetic Recombinant Inbred Line (epiRIL) of A. thaliana at normal and cold temperature. For comparative analysis, the same data sets for the parental ecotype Columbia (Col-0) were also generated, whereas for DNA methylation, previously published high confidence methylation profiles of Col-0 were used. Significant epigenetic differences in an identical genetic background were observed between Col-0 and epiRIL lines under normal and cold temperatures. Our transcriptome data revealed that differential DNA methylation and nucleosome occupancy modulate expression levels of many genes and AS in response to cold. Collectively, DNA methylation and nucleosome levels exhibit characteristic patterns around intron-exon boundaries at normal and cold conditions, and any perturbation in them, in an identical genetic background is sufficient to modulate AS in Arabidopsis.

Neutrophil extracellular traps and heparin-induced antibodies contribute to vascular access thrombosis in hemodialysis patients.

BackgroundAnti-heparin/platelet factor 4 (PF4) antibodies may trigger severe thrombotic complications in hemodialysis (HD) patients. Tetrameric PF4 has a high affinity for extracellular DNA, which is a key component of neutrophil extracellular traps (NETs); therefore, the interactions between anti-heparin/PF4 antibodies and NETs can contribute to prothrombotic events.MethodsAnti-heparin/PF4 antibody levels were measured by enzyme-linked immunosorbent assay and an optical density > 1.8 was regarded as clinically significant. We additionally measured serum nucleosome levels as representative markers of NETs, and the contributions of anti-heparin/PF4 and increased serum nucleosome levels to the primary functional patency loss of vascular access was assessed.ResultsThe frequency of anti-heparin/PF4 antibodies was significantly higher in incident HD patients compared to prevalent HD patients (23.6% vs. 7.7%). Serum nucleosome levels, as well as the white blood cell counts, neutrophil counts, and high- sensitivity C-reactive protein levels, were significantly higher in anti-heparin/PF4 antibody-positive patients compared to the control. Platelet counts tended to be lower in the patients with anti-heparin/PF4 of >1.8 than in the controls. Relative risk calculations showed that the presence of anti-heparin/PF4 antibodies increased the risk of primary functional patency failure by 4.28-fold, and this risk increased further with higher nucleosome levels. Furthermore, in the anti-heparin/PF4 antibody-positive group, the time to first vascular intervention was much shorter, and the risk of repeated intervention was higher, compared to the controls.ConclusionIn incident HD patients, the presence of anti-heparin/PF4 antibodies was associated with increased NET formation; this could be a strong predictor of vascular access complications

Uremic Serum Damages Endothelium by Provoking Excessive Neutrophil Extracellular Trap Formation

Background: Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Methods: Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, we differentiated HL-60 cells into neutrophil-like cells (dHL-60) by applying retinoic acid and determined the effect of uremic serum on these dHL-60 and human umbilical vein EC (HUVEC). Findings: The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality or vascular access failure. In vitro, HD-derived serum significantly increased NET formation by dHL-60, and these NETs decreased EC viability and induced their apoptosis. In addition, the ICAM-1 level in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Interpretation: Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction. Funding: This research was supported by National Research Foundation of Korea (NRF) grant funded by the Korea government (2020R1A2C110138611). Declaration of Interests: No conflicts of interest in this study. Ethics Approval Statement: This study was approved by the Institutional Ethics Committee and informed consent was obtained from the HD patients and HVs.

RoboCOP: jointly computing chromatin occupancy profiles for numerous factors from chromatin accessibility data.

Chromatin is a tightly packaged structure of DNA and protein within the nucleus of a cell. The arrangement of different protein complexes along the DNA modulates and is modulated by gene expression. Measuring the binding locations and occupancy levels of different transcription factors (TFs) and nucleosomes is therefore crucial to understanding gene regulation. Antibody-based methods for assaying chromatin occupancy are capable of identifying the binding sites of specific DNA binding factors, but only one factor at a time. In contrast, epigenomic accessibility data like MNase-seq, DNase-seq, and ATAC-seq provide insight into the chromatin landscape of all factors bound along the genome, but with little insight into the identities of those factors. Here, we present RoboCOP, a multivariate state space model that integrates chromatin accessibility data with nucleotide sequence to jointly compute genome-wide probabilistic scores of nucleosome and TF occupancy, for hundreds of different factors. We apply RoboCOP to MNase-seq and ATAC-seq data to elucidate the protein-binding landscape of nucleosomes and 150 TFs across the yeast genome, and show that our model makes better predictions than existing methods. We also compute a chromatin occupancy profile of the yeast genome under cadmium stress, revealing chromatin dynamics associated with transcriptional regulation.

Endothelium Activation Markers in Severe Hospitalized COVID-19 Patients: Role in Mortality Risk Prediction.

Introduction Endothelial damage and hypercoagulability are major players behind the hemostatic derangement of SARS-CoV-2 infection. Aim In this prospective study we assessed endothelial and inflammatory biomarkers in a cohort of COVID-19 patients, aiming to identify predictive factors of in-hospital mortality. Methods COVID-19 patients hospitalized in intensive care (ICU) and non-ICU units at 2 Bergamo (Italy) hospitals from March 23 to May 30, 2020, were enrolled. Markers of endothelium activation including von-Willebrand factor (vWF), soluble thrombomodulin (sTM), and fibrinolytic proteins (t-PA and PAI-1) were measured. Additionally, D-dimer, Fibrinogen, FVIII, nucleosomes, C reactive protein (CRP) and procalcitonin were assessed. Results Sixty-three (45 ICU, and 18 non-ICU) patients, with a median age of 62 years were analyzed. Increased plasma levels of D-dimer, FVIII, fibrinogen, nucleosomes, CRP, and procalcitonin were observed in the whole cohort. Extremely elevated vWF levels characterized all patients (highest values in ICU-subjects). After a median time of 30 days, death occurred in 13 (21%) patients. By multivariable analysis, vWF-activity, neutrophil-count and PaO2/FiO2 were significantly associated with death. Using these variables, a linear score with 3-risk groups was generated that provided a cumulative incidence of death of 0% in the low-, 32% in the intermediate-, and 78% in the high-risk group. Conclusions COVID-19-induced hemostatic abnormalities are exacerbated by the severity of the disease and strongly correlate with the inflammatory status, underlying the link between coagulation, endothelial activation, and inflammation. Our study provides evidence for a role of vWF, together with neutrophils and PaO2/FiO2, as a significant predictor of in-hospital mortality by SARSCoV-2 infection.

Biomarkers of Endothelial Function in Sepsis-Associated Disseminated Intravascular Coagulation

Introduction: Sepsis-associated disseminated intravascular coagulation (DIC) is a complex clinical syndrome with high mortality. The pathogenesis of DIC involves significant interactions between multiple pathophysiologic processes including coagulation, inflammation, infection response, platelets and endothelial dysfunction. The role of the endothelium in DIC is multifaceted and may contribute significantly to its severity and outcome. In sepsis, increased inflammation can lead to activation of the endothelium and the development of a procoagulant phenotype, characterized in part by elevated levels of cell surface tissue factor (TF). The endothelial response to inflammation is also characterized by the production of other factors that may be descriptive of the status of the endothelium, including endocan. The endothelium is also largely responsible for the production of endogenous anticoagulants, such as Tissue Factor Pathway Inhibitor (TFPI) and Protein C. The derangement of the endogenous anticoagulant system is critical to the pathogenesis of DIC and may also be reflective of the state of the endothelium. Investigation of factors that may prove damaging to the endothelium, including extracellular nuclear material such as nucleosomes and the protein High Mobility Group Box 1 (HMGB-1) may also provide insight into endothelial status and associated patient prognosis. The purpose of this study was to quantify measures of endothelial function, including markers of activation (TF and endocan), endogenous anticoagulants (TFPI and Protein C), and damage-associated factors (nucleosomes and HMGB-1) in the plasma of patients with sepsis and DIC and to determine the relationship of these factors with severity of illness and outcome.Materials and Methods: Citrated plasma samples were prospectively collected from 103 patients with sepsis and suspected DIC at ICU admission (day 0) and again serially on ICU days 4 and 8 under an IRB approved protocol. DIC score was determined using the ISTH scoring algorithm (e.g. platelet count, PT/INR, fibrinogen, and D-Dimer). Plasma from healthy individuals was purchased from a commercial laboratory (George King Biomedical, Overland, KS). Plasma levels of endocan, TF, TFPI, nucleosomes, and HMGB-1 were evaluated using commercially available ELISA kits. Protein C levels were measured using a functional clot-based assay.Results: In the DIC patients, endocan, TF, TFPI, nucleosomes, and HMGB-1 were significantly elevated compared to normal on ICU days 0, 4, and 8. Protein C was significantly reduced compared to normal at all time points.Patients were stratified into groups of sepsis and DIC based on the ISTH DIC score, and levels of markers were compared between groups. Markers reflective of overall endothelial activation (endocan and TF) showed no variation based on DIC score. Protein C showed significant variation based on DIC score, with a marked drop in Protein C levels with increasing severity of illness; however, TFPI showed no variation. Increasing levels of factors associated with endothelial damage (nucleosomes and HMGB-1) were observed with increasing severity of illness. Results are shown in Table 1.Differences in biomarker levels between survivors and non-survivors were also evaluated. Endocan (p = 0.0252) and HMGB-1 (p = 0.0306) showed significant increases in non-survivors compared to survivors while Protein C showed a significant reduction (p = 0.0093) in non-survivors compared to survivors. TF, TFPI, and nucleosomes showed no variation based on survival.Conclusion: Overall, levels of markers of endothelial activation, endogenous anticoagulants, and factors contributing to endothelial damage were elevated in the plasma of patients with sepsis-associated DIC compared to the healthy population. In particular, factors associated with endothelial damage, namely the toxic nuclear factors nucleosomes and HMGB-1, showed elevated levels with increasing severity of coagulopathy, suggesting that the endothelial damage caused by these factors may contribute to the development of coagulation dysfunction. Levels of endocan, Protein C, and HMGB-1 also showed significant association with mortality, underscoring the relevance of all aspects of endothelial physiology to patient outcome in DIC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Two Arabidopsis Homologs of Human Lysine-Specific Demethylase Function in Epigenetic Regulation of Plant Defense Responses.

Epigenetic marks such as covalent histone modification and DNA methylation are crucial for mitotically and meiotically inherited cellular memory-based plant immunity. However, the roles of individual players in the epigenetic regulation of plant immunity are not fully understood. Here we reveal the functions of two Arabidopsis thaliana homologs of human lysine-specific demethylase1-like1, LDL1 and LDL2, in the maintenance of methyl groups at lysine 4 of histone H3 and in plant immunity to Pseudomonas syringae infection. The growth of virulent P. syringae strains was reduced in ldl1 and ldl2 single mutants compared to wild-type plants. Local and systemic disease resistance responses, which coincided with the rapid, robust transcription of defense-related genes, were more stably expressed in ldl1 ldl2 double mutants than in the single mutants. At the nucleosome level, mono-methylated histone H3K4 accumulated in ldl1 ldl2 plants genome-wide and in the mainly promoter regions of the defense-related genes examined in this study. Furthermore, in silico comparative analysis of RNA-sequencing and chromatin immunoprecipitation data suggested that several WRKY transcription factors, e.g., WRKY22/40/70, might be partly responsible for the enhanced immunity of ldl1 ldl2. These findings suggest that LDL1 and LDL2 control the transcriptional sensitivity of a group of defense-related genes to establish a primed defense response in Arabidopsis.

Cryo-EM structure of SETD2/Set2 methyltransferase bound to a nucleosome containing oncohistone mutations

Substitution of lysine 36 with methionine in histone H3.3 (H3.3K36M) is an oncogenic mutation that inhibits SETD2-mediated histone H3K36 tri-methylation in tumors. To investigate how the oncohistone mutation affects the function of SETD2 at the nucleosome level, we determined the cryo-EM structure of human SETD2 associated with an H3.3K36M nucleosome and cofactor S-adenosylmethionine (SAM), and revealed that SETD2 is attached to the N-terminal region of histone H3 and the nucleosome DNA at superhelix location 1, accompanied with the partial unwrapping of nucleosome DNA to expose the SETD2-binding site. These structural features were also observed in the previous cryo-EM structure of the fungal Set2–nucleosome complex. By contrast with the stable association of SETD2 with the H3.3K36M nucleosome, the EM densities of SETD2 could not be observed on the wild-type nucleosome surface, suggesting that the association of SETD2 with wild-type nucleosome might be transient. The linker histone H1, which stabilizes the wrapping of nucleosome DNA at the entry/exit sites, exhibits an inhibitory effect on the activities of SETD2 and displays inversely correlated genome distributions with that of the H3K36me3 marks. Cryo-EM analysis of yeast H3K36 methyltransferase Set2 complexed with nucleosomes further revealed evolutionarily conserved structural features for nucleosome recognition in eukaryotes, and provides insights into the mechanism of activity regulation. These findings have advanced our understanding of the structural basis for the tumorigenesis mechanism of the H3.3K36M mutation and highlight the effect of nucleosome conformation on the regulation of histone modification.

Circulating Nucleosomes as Potential Markers to Monitor COVID-19 Disease Progression.

The severity of coronavirus disease 2019 (COVID-19) varies significantly with cases spanning from asymptomatic to lethal with a subset of individuals developing Severe Acute Respiratory Syndrome (SARS) and death from respiratory failure. To determine whether global nucleosome and citrullinated nucleosome levels were elevated in COVID-19 patients, we tested two independent cohorts of COVID-19 positive patients with quantitative nucleosome immunoassays and found that nucleosomes were highly elevated in plasma of COVID-19 patients with a severe course of the disease relative to healthy controls and that both histone 3.1 variant and citrullinated nucleosomes increase with disease severity. Elevated citrullination of circulating nucleosomes is indicative of neutrophil extracellular trap formation, neutrophil activation and NETosis in severely affected individuals. Importantly, using hospital setting (outpatient, inpatient or ICU) as a proxy for disease severity, nucleosome levels increased with disease severity and may serve as a guiding biomarker for treatment. Owing to the limited availability of mechanical ventilators and extracorporal membrane oxygenation (ECMO) equipment, there is an urgent need for effective tools to rapidly assess disease severity and guide treatment selection. Based on our studies of two independent cohorts of COVID-19 patients from Belgium and Germany, we suggest further investigation of circulating nucleosomes and citrullination as biomarkers for clinical triage, treatment allocation and clinical drug discovery.