Extracellular Vesicles From Epicardial Fat Facilitate Atrial Fibrillation.

BACKGROUND: The role of epicardial fat (eFat)-derived extracellular vesicles (EVs) in the pathogenesis of atrial fibrillation (AF) has never been studied. We tested the hypothesis that eFat-EVs transmit proinflammatory, profibrotic, and proarrhythmic molecules that induce atrial myopathy and fibrillation. METHODS: We collected eFat specimens from patients with (n=32) and without AF (n=30) during elective heart surgery. eFat samples were grown as organ cultures, and the culture medium was collected every 2 days. We then isolated and purified eFat-EVs from the culture medium, and analyzed the EV number, size, morphology, specific markers, encapsulated cytokines, proteome, and microRNAs. Next, we evaluated the biological effects of unpurified and purified EVs on atrial mesenchymal stromal cells and endothelial cells in vitro. To establish a causal association between eFat-EVs and vulnerability to AF, we modeled AF in vitro using induced pluripotent stem cell-derived cardiomyocytes. RESULTS: Microscopic examination revealed excessive inflammation, fibrosis, and apoptosis in fresh and cultured eFat tissues. Cultured explants from patients with AF secreted more EVs and harbored greater amounts of proinflammatory and profibrotic cytokines, and profibrotic microRNA, as well, than those without AF. The proteomic analysis confirmed the distinctive profile of purified eFat-EVs from patients with AF. In vitro, purified and unpurified eFat-EVs from patients with AF had a greater effect on proliferation and migration of human mesenchymal stromal cells and endothelial cells, compared with eFat-EVs from patients without AF. Last, whereas eFat-EVs from patients with and without AF shortened the action potential duration of induced pluripotent stem cell-derived cardiomyocytes, only eFat-EVs from patients with AF induced sustained reentry (rotor) in induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: We show, for the first time, a distinctive proinflammatory, profibrotic, and proarrhythmic signature of eFat-EVs from patients with AF. Our findings uncover another pathway by which eFat promotes the development of atrial myopathy and fibrillation.

Nucleosome Core Particle Disassembly and Assembly Kinetics Studied Using Single-Molecule Fluorescence.

The stability of the nucleosome core particle (NCP) is believed to play a major role in regulation of gene expression. To understand the mechanisms that influence NCP stability, we studied stability and dissociation and association kinetics under different histone protein (NCP) and NaCl concentrations using single-pair Förster resonance energy transfer and alternating laser excitation techniques. The method enables distinction between folded, unfolded, and intermediate NCP states and enables measurements at picomolar to nanomolar NCP concentrations where dissociation and association reactions can be directly observed. We reproduced the previously observed nonmonotonic dependence of NCP stability on NaCl concentration, and we suggest that this rather unexpected behavior is a result of interplay between repulsive and attractive forces within positively charged histones and between the histones and the negatively charged DNA. Higher NaCl concentrations decrease the attractive force between the histone proteins and the DNA but also stabilize H2A/H2B histone dimers, and possibly (H3/H4)2 tetramers. An intermediate state in which one DNA arm is unwrapped, previously observed at high NaCl concentrations, is also explained by this salt-induced stabilization. The strong dependence of NCP stability on ion and histone concentrations, and possibly on other charged macromolecules, may play a role in chromosomal morphology.

Rational design of DNA motors: fuel optimization through single-molecule fluorescence.

While numerous DNA-based molecular machines have been developed in recent years, high operational yield and speed remain a major challenge. To understand the reasons for the limited performance, and to find rational solutions, we applied single-molecule fluorescence techniques and conducted a detailed study of the reactions involved in the operation of a model system comprised of a bipedal DNA walker that strides on a DNA origami track powered by interactions with fuel and antifuel strands. Analysis of the kinetic profiles of the leg-lifting reactions indicates a pseudo-first-order antifuel binding mechanism leading to a rapid and complete leg-lifting, indicating that the fuel-removal reaction is not responsible for the 1% operational yield observed after six steps. Analysis of the leg-placing reactions showed that although increased concentrations of fuel increase the reaction rate, they decrease the yield by consecutively binding the motor and leading to an undesirable trapped state. Recognizing this, we designed asymmetrical hairpin-fuels that by regulating the reaction hierarchy avoid consecutive binding. Motors operating with the improved fuels show 74% yield after 12 consecutive reactions, a dramatic increase over the 1% observed for motors operating with nonhairpin fuels. This work demonstrates that studying the mechanisms of the reactions involved in the operation of DNA-based molecular machines using single-molecule fluorescence can facilitate rationally designed improvements that increase yield and speed and promote the applicability of DNA-based machines.

Disentangling subpopulations in single-molecule FRET and ALEX experiments with photon distribution analysis.

Among the advantages of the single-molecule approach when used to study biomolecular structural dynamics and interaction is its ability to distinguish between and independently observe minor subpopulations. In a single-molecule Förster resonance energy transfer (FRET) and alternating laser excitation diffusion experiment, the various populations are apparent in the resultant histograms. However, because histograms are calculated based on the per-burst mean FRET and stoichiometry ratio and not on the internal photon distribution, much of the acquired information is lost, thereby reducing the capabilities of the method. Here we suggest what to our knowledge is a novel statistical analysis tool that significantly enhances these capabilities, and we use it to identify and isolate static and dynamic subpopulations. Based on a kernel density estimator and a proper photon distribution analysis, for each individual burst, we calculate scores that reflect properties of interest. Specifically, we determine the FRET efficiency and brightness ratio distributions and use them to reveal 1), the underlying structure of a two-state DNA-hairpin and a DNA hairpin that is bound to DNA origami; 2), a minor doubly labeled dsDNA subpopulation concealed in a larger singly labeled dsDNA; and 3), functioning DNA origami motors concealed within a larger subpopulation of defective motors. Altogether, these findings demonstrate the usefulness of the proposed approach. The method was developed and tested using simulations, its rationality is described, and a computer algorithm is provided.

Extracellular Vesicular Transmission of miR-423-5p from HepG2 Cells Inhibits the Differentiation of Hepatic Stellate Cells.

Liver fibrosis (LF) is a major cause of morbidity and mortality worldwide. Hepatic stellate cells (HSCs) are the primary source of extracellular matrix in the liver and their activation is a central event in LF development. Extracellular vesicles (EVs) are intercellular communication agents, which play important roles in physiological processes in chronic liver diseases. The aim of this study was to examine the crosstalk between hepatocytes and HSCs mediated by hepatocyte-secreted EVs. EVs were purified from primary mouse hepatocytes, HepG2 cell lines, under normal or stressed conditions. The effect of EVs on primary HSCs (pHSCs) differentiation was evaluated by measuring of differentiation markers. In addition, their impact on the carbon tetrachloride (CCl4)-induced fibrosis mouse model was evaluated. The results demonstrated that HepG2-EVs regulate HSC differentiation and that under stress conditions, promoted pHSCs differentiation into the myofibroblast phenotype. The evaluation of miRNA sequences in the HepG2 secreted EVs demonstrated high levels of miR-423-5p. The examination of EV cargo following stress conditions identified a significant reduction of miR-423-5p in HepG2-EVs relative to HepG2-EVs under normal conditions. In addition, pHSCs transfected with miR-423-5p mimic and exhibit lower mRNA levels of alpha smooth muscle actin and Collagen type 1 alpha, and the mRNA expression level of genes targeted the family with sequence-similarity-3 (FAM3) and Monoacylglycerol lipase (Mgll). This study strengthened the hypothesis that EVs are involved in LF and that their cargo changes in stress conditions. In addition, miR-423-5p was shown to be involved in HSCs differentiation and hence, fibrosis development.

Interleukin-1α and Interleukin-1ß play a central role in the pathogenesis of fulminant hepatic failure in mice.

BACKGROUND AND AIMS: Fulminant hepatitis failure (FHF) is marked by the sudden loss of hepatic function, with a severe life-threatening course in persons with no prior history of liver disease. Interleukin (IL)-1α and IL-1ß are key inflammatory cytokines but little is known about their role in the development of FHF. The aim of this study was to assess the involvement of IL-1α and IL-1ß in the progression of LPS/GalN-induced FHF. METHODS: WT, IL-1α or IL-1ß deficient mice were injected with LPS/GalN. Blood and liver tissue were collected at different time points, FHF related pathways were examined. RESULTS: After FHF induction the survival of both IL-1α and IL-1ß KO mice was longer than that of WT mice. Lower serum liver enzyme levels, demonstrated reduced hepatic injury in the IL-1α and IL-1ßKO mice. Histologically detected liver injury and apoptotic hepatocytes were significantly reduced in the IL-1αand IL-1ßKO mice compared to WT mice. Reduced hepatic IkB levels and upregulated NFκB activity in WT mice remained inhibited in IL-1α and IL-1ß KO mice. Hepatic expression levels of TNFα and IL-6 were significantly increased in WT mice but not in IL-1α and IL-1ß KO mice. CONCLUSIONS: IL-1α and IL-1ß play a central role in the pathogenesis of LPS/GalN-induced FHF. These interleukins are associated with the activation of NFκB signaling, upregulation of the pro-inflammatory cytokines and liver damage and apoptosis. Since neither IL-1α nor IL-1ß depletions completely rescued the phenotype, we believe that IL-1α and IL-1ß have a similar and probably complementary role in FHF progression.

Detailed study of DNA hairpin dynamics using single-molecule fluorescence assisted by DNA origami.

The dynamics of two DNA hairpins (5'-TCGCCT-A31-AGGCGA-3' and 5'-TCGCCG-A31-CGGCGA-3') were studied using immobilization-based and diffusion-based single-molecule fluorescence techniques. The techniques enabled separated and detailed investigation of the states and of the transition reactions. Only two states, open and closed, were identified from analysis of the FRET histograms; metastable states with lifetimes longer than the technique resolution (0.3 ms) were not observed. The opening and closing reaction rates were determined directly from the FRET time trajectories, and the Gibbs free energies of these states and of the transition state were calculated using the Kramer theory. The rates, which are undoubtedly of transitions between the fully closed and the fully open states and ranged from 2 to 90 s(-1), were lower (∼10-fold) than the rates previously determined from fluorescence correlation spectroscopy. The heights of the barriers for closing were almost identical for the two hairpins. The barrier for opening the hairpin with the stronger stem was higher (4.3 kJ/mol) than that for the hairpin with the weaker stem, in very good agreement with the difference in stability calculated by the nearest-neighbor method. The barrier for closing the hairpin decreased (∼8 kJ/mol) and the barrier for opening increased (∼4 kJ/mol) with increasing NaCl concentration (10-100 mM), indicating that higher ionic strength stabilizes the folded state with respect to the transition state and stabilizes the transition state relative to the unfolded state. The very good agreements in the dynamics measured for free hairpins, for hairpins anchored to origami, and for hairpins anchored to the coverslip and the very good agreement between the two single-molecule techniques demonstrate that neither the origami nor the coverslip influence the hairpin dynamics, supporting a previous demonstration that origami can serve as a platform for biophysical investigations.

Studying the structural dynamics of bipedal DNA motors with single-molecule fluorescence spectroscopy.

We present a test case example of a detailed single-molecule fluorescence study of one of the most sophisticated and complex DNA devices introduced to date, a recently published autonomous bipedal DNA motor. We used the diffusion-based single-molecule Förster resonance energy transfer technique, coupled to alternating laser excitation (sm-FRET-ALEX), to monitor the motor assembly and operation. The study included verification of the formation of the correct structures, and of the correct motor operation, determination of the formation and stepping reaction yields, and identification of side products. Finally, the mechanisms of the motor assembly and operation were elucidated by measuring the reaction kinetics profile of track-walker binding and of lifting of the walker's leg upon fuel addition. The profiles revealed a fast phase, in which about half of the reaction was completed, followed by a slow phase which adds somewhat to the yield, reflecting the incomplete motor assembly and operation identified in the equilibrium experiments. Although further study is needed to fully understand the reasons for the incomplete assembly and operation, this work demonstrates that single-molecule fluorescence, based on its ability to provide detailed in situ structural dynamics information, inaccessible for traditional methods, constitutes an excellent tool for chaperoning the development of DNA-based technology.