One of the most sensitive, time-consuming, and variable steps of chromatin immunoprecipitation (ChIP) is chromatin sonication. Traditionally, this process can take hours to properly sonicate enough chromatin for multiple ChIP assays. Further, the length of sheared DNA is often inconsistent. In order to faithfully measure chemical and structural changes at the chromatin level, sonication needs to be reliable. Thus, chromatin fragmentation by sonication represents a significant bottleneck to downstream quantitative analysis. To improve the consistency and efficiency of chromatin sonication, we developed and tested a cavitation enhancing reagent based on sonically active nanodroplets. Here, we show that nanodroplets increase sonication efficiency by 16-fold and provide more consistent levels of chromatin fragmentation. Using the previously characterized chromatin in vivo assay (CiA) platform, we generated two distinct chromatin states in order to test nanodroplet-assisted sonication sensitivity in measuring post-translational chromatin marks. By comparing euchromatin to chemically induced heterochromatin at the same CiA:Oct4 locus, we quantitatively measure the capability of our new sonication technique to resolve differences in chromatin structure. We confirm that nanodroplet-assisted sonication results are indistinguishable from those of samples processed with traditional sonication in downstream applications. While the processing time for each sample was reduced from 38.4 to 2.3 min, DNA fragment distribution sizes were significantly more consistent with a coefficient of variation 2.7 times lower for samples sonicated in the presence of nanodroplets. In conclusion, sonication utilizing the nanodroplet cavitation enhancement reagent drastically reduces the amount of processing time and provides consistently fragmented chromatin of high quality for downstream applications.
Chromatin accessibility states that influence gene expression and other nuclear processes can be altered in disease. The constellation of transcription factors and chromatin regulatory complexes in cells results in characteristic patterns of chromatin accessibility. The study of these patterns in tissues has been limited because existing chromatin accessibility assays are ineffective for archival formalin-fixed, paraffin-embedded (FFPE) tissues. We have developed a method to efficiently extract intact chromatin from archival tissue via enhanced cavitation with a nanodroplet reagent consisting of a lipid shell with a liquid perfluorocarbon core. Inclusion of nanodroplets during the extraction of chromatin from FFPE tissues enhances the recovery of intact accessible and nucleosome-bound chromatin. We show that the addition of nanodroplets to the chromatin accessibility assay formaldehyde-assisted isolation of regulatory elements (FAIRE), does not affect the accessible chromatin signal. Applying the technique to FFPE human tumor xenografts, we identified tumor-relevant regions of accessible chromatin shared with those identified in primary tumors. Further, we deconvoluted non-tumor signal to identify cellular components of the tumor microenvironment. Incorporation of this method of enhanced cavitation into FAIRE offers the potential for extending chromatin accessibility to clinical diagnosis and personalized medicine, while also enabling the exploration of gene regulatory mechanisms in archival samples.
Acute pulmonary embolism (PE) is the third most common cause of cardiovascular death. 1 Although there is little debate about how to manage low-or high-risk acute PE, therapeutic strategies for patients who have intermediaterisk acute PE are continuously evolving. This condition is associated with a substantial risk of death at 90 days and with possible progression to acute hemodynamic decompensation. In the Fibrinolysis for Patients with Intermediate Risk Pulmonary Embolism (PEITHO) trial, 2 normotensive patients were divided into 2 groups: one group was given tenecteplase plus heparin, and the other, placebo plus heparin. In the anticoagulation-only group, 28 of 499 patients (5.6%) died or progressed to hemodynamic decompensation, compared with 13 of 506 (2.6%) in the tenecteplase group; however, the rates of major bleeding and stroke were significantly higher in the tenecteplase group. The investigators recommended great caution in considering fibrinolytic therapy for hemodynamically stable patients with intermediate-risk PE, right ventricular (RV) dysfunction, and elevated cardiac troponin levels. 2 Systemic fibrinolytic therapy reduced the risk of hemodynamic decompensation but significantly increased the risk of stroke and intracranial hemorrhage (ICH). 2 Patients with acute PE and RV strain have a low cardiac reserve and are subject to sudden decompensation. The therapeutic goal in patients with intermediate-risk PE, particularly in those at high intermediate risk, is to improve symptoms while lowering the risk classification or preventing near-term hemodynamic decompensation. Minimizing the risk of severe hemorrhagic complications while accelerating thrombus resolution is guiding new therapies for intermediate-risk acute PE.
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