Defect-Induced Supercollision Cooling of Photoexcited Carriers in Graphene

Defects play a fundamental role in the energy relaxation of hot photoexcited carriers in graphene, thus a complete understanding of these processes are vital for improving the development of graphene devices. Recently, it has been theoretically predicted and experimentally demonstrated that defect-assisted acoustic phonon supercollision, the collision between a carrier and both an acoustic phonon and a defect, is an important energy relaxation process for carriers with excess energy below the optical phonon emission. Here, we studied samples with defects optically generated in a controlled manner to experimentally probe the supercollision model as a function of the defect density. We present pump and probe transient absorption measurements showing that the decay time decreases as the density of defect increases as predicted by the supercollision model

Treatment with MβCD leads to changes in membrane raft organization of cardiomyocytes.

<p>Confocal images of control (A) and cardiomyocytes pre-treated with 10 mM MβCD (B) or HγCD (C). Cells were washed, fixed and then labeled with CTXb-Alexa 488, which recognizes GM1, a raft marker. In comparison to control cells, which show a homogenous strong labeling for GM1, cholesterol-depleted cardiomyocytes reveals a more discrete labeling. Cells treated with HγCD show GM1 labeling similar to control cells whereas cholesterol-replenished cells (D) exhibit both patterns of cholesterol-depleted (arrows) as well as control (asterisks) GM1 labeling. Scale bar: 0.9 µm.</p

<i>T. cruzi</i> invasion of cells and association with LAMP-1 in cardiomyocytes decreases after cholesterol depletion.

<p>Cardiomyocytes pre-treated or not with different cyclodextrins were washed and challenged with <i>T. cruzi</i> trypomastigotes at a M.O.I of 50, for 40 minutes at 37°C, then fixed and processed for immunofluorescence detection of total intracellular parasites, as well as intracellular parasites associated with LAMP-1 (a lysosomal marker). Both <i>T. cruzi</i> internalization (A) and association with host LAMP-1 (B) diminishes after incubation with 10 and 15 mM of MβCD but not after treatment with 10 and 15 mM of HγCD. Cholesterol replenishment after treatment with 15 mM MβCD reverts the effect of the drug on parasite cell invasion (A), and at least partially on LAMP-1 association (B). The average number of cardiomyocytes ±SD per 10 counted fields in each coverslip is shown above the bars (A). Data are shown as mean of triplicates ±SD. Asteriks indicate statistically significant differences (p < 0.05, Student's t test) between control and treated cells. (C) Representative panels of <i>T. cruzi</i> invasion and association with host cell lysosomes, revealed by immunocytochemistry. Total cell and parasite nuclei, as well as parasite kinteoplast DNA were labeled with DAPI; lysosomes were labeled with anti-LAMP 1 antibody followed by secondary IgG labeled with Alexa Fluor 488; extracellular parasites in the field were labeled with anti-<i>T.cruzi</i> antibody followed by secondary IgG labeled with Alexa Fluor 546. From top to bottom: control cells, 15 mM MβCD treated cells, 15 mM HγCD treated cells and 15 mM MβCD treated cells followed by incubation with 0.05 mM of WSC. Blue arrows show total <i>T. cruzi</i> trypomastigotes in the field, yellow ellipsoids show lysosomal associated trypomastigotes, red triangles points out extracellular trypomastigotes and the last column shows the merge of the three previous. Scale bar: 10 µm.</p

MβCD but not HγCD cell incubation leads to lysosomal exocytosis in cardiomyocytes.

<p>Cardiomyocytes were exposed to either 10 mM MβCD or HγCD for 10, 20 or 40 minutes at 37°C, in the absence (white bars) or presence (black bars) of calcium. Both extracellular media and lysates were collected and exposed to 4-methylumbelliferyl-N-acetyl-B-D-glucosaminide, the fluorescent substrate of beta-hexosaminidase, an enzyme resident within lysosomes. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001583#s3" target="_blank">Results</a> are shown as ratio between β-hexosaminidase activity in extracellular media and total β-hexosaminidase activity (extracellular media over extracellular media plus β- hexosaminidase cell lysate hexosaminidase activity). Cells treated with 10 µM Ionomycin (Calbiochem) for 10 minutes were used as lysosomal exocytosis positive control. Data are shown as mean of triplicates ±SD. Asteriks indicate statistically significant differences (p < 0.05,Student's t test) between control and treated cells.</p

Cholesterol depletion leads to changes in lysosomal distribution within cells.

<p>Representative panels of lysosomal distribution in control cardiomyocyte (A) and cardiomyocytes pre-treated either with 10 mM (B) or 15 mM MβCD (C), 10 mM or (D) 15 mM HγCD (E), or 15 mM MβCD followed by 0.05 mM WSC (F). MβCD treated cardiomyocytes show significant changes in lysosomal dispersion in cell cytoplasm. After drug treatment, cells were washed and incubated with <i>T. cruzi</i> TCTs, M.O.I. of 50 parasites per cell for 40 minutes at 37°C. After cell invasion, cells were washed, fixed and immunostained for LAMP-1 (green) DAPI (blue) and analyzed under fluorescence microscope. In comparison to untreated cardiomyocytes (A), which exhibit homogenous and well distributed LAMP-1 labeling, MβCD treated cardiomyocytes, (B) and (C), show a heterogeneous LAMP-1 labeling with lysosomes localized predominantly near cell nuclei. On the other hand, HγCD treated cardiomyocytes and cholesterol-replenished cells, (E), (F) and (D), present lysosomal distribution similar to control cells. Scale bar: 10 µm.</p

Lysosomal exocytosis events after cholesterol depletion are not due to cell death.

<p>After treatment with MβCD or HγCD, in the absence (white bars) or presence (black bars) of calcium, cardiomyocytes were trypsinized, collected and incubated with HFS solution, containing propidium iodide (PI). Cells that became inviable after drug treatment acquired PI labeling in their nuclei due to membrane permeability, and were counted as dead cells by flow cytometer. Data are shown as mean of triplicates ±SD.</p

MβCD treatment is effective in sequestering cholesterol from the plasma membrane.

<p>(A) Control, (B) 15 mM MβCD treated and (C) 15 mM MβCD followed by 0.05 mM WSC treated cardiomyocytes show significant changes in Filipin labeling. Cholesterol-depleted cells (B) reveal very little Filipin labeling, whereas cholesterol-replenished cells show strong labeling for cholesterol, similar to control cells. (D) Cardiomyocytes treated with 5, 10 or 15 mM of MβCD reveal a substantial decrease in Filipin labeling in a dose-dependent manner whereas cholesterol replenishment with 0.05 mM of WSC returns Filipin fluorescence to control values. Normalized data are shown as mean of triplicates ±SD. Asteriks indicate statistically significant differences (* p < 0.05 and ** p < 0.01, One way ANOVA followed by Newman-Keuls) between control and treated cells. Scale bar: 10 µm.</p