Histone increases leukemic cell adhesion to endothelial cells (EA.hy926; hEC).

(A) Calf thymus histone (50 μg/mL) was pre-mixed with 62.5 μg/mL polysialic acid, 100 U/mL heparin, or 100 nM activated protein C (APC) for 1 h, 10 min, and 30 min, respectively, and then added to hEC for 5 h. Then, U937 cells were added onto histone-treated or untreated hEC layers for 30 min. After washing, the adherent small round U937 cells on hEC layers were counted under a microscope (per ×100 field). (B) The numbers of adherent U937 cells are shown as mean ± SEM of 4 experiments. *P #P < 0.05 versus histone-treated.</p

The baseline characteristics and laboratory results of the study populations.

<p>The baseline characteristics and laboratory results of the study populations.</p

dCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9’s specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant

dCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9’s specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant

PFS and OS according to pomalidomide response.

(DOCX)</p

Additional file 1: Table S1. of Identification of somatic mutations using whole-exome sequencing in Korean patients with acute myeloid leukemia

Details of the 36 AML patients. Table S2. Functional information for 15 significantly mutated genes in 36 Korean AML patients. Table S3. Results of gene ontology and KEGG pathway analyses. (DOCX 38 kb

Extracellular traps are released from leukemic cells.

<p>Three leukemic cell lines (U937, THP-1, HL-60) were cultured in DMEM with 10% fetal calf serum without medium exchange for 5 days. (A) The numbers of autonomously proliferated cells are expressed as mean ± SEM of 4 experiments. (B, C) The levels of the histone–DNA complex and cell-free dsDNA were measured in the corresponding culture supernatants. (D) U937 cells (fresh or cultured for 5 days) were stained with SYTOX green and DAPI and observed using a confocal laser microscope. Images shown are representative of 3 independent experiments. (E) Total ROS activity was measured in lysates of U937 cells prepared before and after culturing for 5 days (mean±SEM of 3 experiments). (F) U937 cells were pretreated with an inhibitor of extracellular trap formation (200 μM Cl-amidine) or with antioxidants (ATA, 100 μM; quercetin, 100 μM; NAC, 50 μM; or APDC, 100 μM) and the histone–DNA complex levels were measured in the supernatants after 5 day-culture. *<i>P</i> < 0.05 versus each leukemic cell line at 0 h. ^#<i>P</i> < 0.05 versus no inhibitor.</p

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

<div><p>Introduction</p><p>When 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.</p><p>Methods</p><p>The 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 <i>in vitro</i>. Histone-induced endothelial adhesion molecules expression and cell survival were measured by flow cytometry.</p><p>Results</p><p>Acute 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.</p><p>Conclusion</p><p>Our 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.</p></div

dCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9’s specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant

Table_1_Clonal hematopoiesis as a novel risk factor for type 2 diabetes mellitus in patients with hypercholesterolemia.docx

IntroductionClonal hematopoiesis of indeterminate potential (CHIP) is associated with atherosclerosis and cardiovascular disease. It has been suggested that CHIP may be related to diabetes, so we investigated the association between CHIP and new-onset type 2 diabetes.MethodsThis study included 4,047 subjects aged >=40 years without diabetes. To detect CHIP, targeted gene sequencing of genomic DNA from peripheral blood cells was performed. The incidence of new-onset type 2 diabetes during the follow-up period was evaluated.ResultsOf the total subjects, 635 (15.7%) had CHIP. During the median follow-up of 5.1 years, the incidence of new-onset diabetes was significantly higher in CHIP carriers than in subjects without CHIP (11.8% vs. 9.1%, p = 0.039). In a univariate analysis, CHIP significantly increased the risk of new-onset diabetes (HR 1.32, 95% CI 1.02–1.70, p = 0.034), but in a multivariate analysis, it was not significant. The CHIP-related risk of new onset diabetes differed according to LDL cholesterol level. In the hyper-LDL cholesterolemia group, CHIP significantly increased the risk of diabetes (HR 1.64, 95% CI 1.09–2.47, p = 0.018), but it did not increase the risk in the non-hyper-LDL cholesterolemia group. The subjects with CHIP and hyper-LDL-cholesterolemia had approximately twice the risk of diabetes than subjects without CHIP and with low LDL cholesterol (HR 2.05, 95% CI 1.40–3.00, p ConclusionThe presence of CHIP was a significant risk factor for new-onset type 2 diabetes, especially in subjects with high LDL cholesterol. These results show the synergism between CHIP and high LDL cholesterol as a high-risk factor for diabetes.</p