| 1. |
- Aref, Mohaddeseh A., et al.
(author)
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Intracellular injection of phospholipids directly alters exocytosis and the fraction of chemical release in chromaffin cells as measured by nano-electrochemistry
- 2020
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In: Chemical Science. - : Royal Society of Chemistry (RSC). - 2041-6520 .- 2041-6539. ; 11:43, s. 11869-11876
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Journal article (peer-reviewed)abstract
- Using a nano-injection method, we introduced phospholipids having different intrinsic geometries into single secretory cells and used single cell amperometry (SCA) and intracellular vesicle impact electrochemical cytometry (IVIEC) with nanotip electrodes to monitor the effects of intracellular incubation on the exocytosis process and vesicular storage. Combining tools, this work provides new information to understand the impact of intracellular membrane lipid engineering on exocytotic release, vesicular content and fraction of chemical release. We also assessed the effect of membrane lipid alteration on catecholamine storage of isolated vesicles by implementing another amperometric technique, vesicle impact electrochemical cytometry (VIEC), outside the cell. Exocytosis analysis reveals that the intracellular nano-injection of phosphatidylcholine and lysophosphatidylcholine decreases the number of released catecholamines, whereas phosphatidylethanolamine shows the opposite effect. These observations support the emerging hypothesis that lipid curvature results in membrane remodeling through secretory pathways, and also provide new evidence for a critical role of the lipid localization in modulating the release process. Interestingly, the IVIEC data imply that total vesicular content is also affected by in situ supplementation of the cells with some lipids, while, the corresponding VIEC results show that the neurotransmitter content in isolated vesicles is not affected by altering the vesicle membrane lipids. This suggests that the intervention of phospholipids inside the cell has its effect on the cellular machinery for vesicle release rather than vesicle structure, and leads to the somewhat surprising conclusion that modulating release has a direct effect on vesicle structure, which is likely due to the vesicles opening and closing again during exocytosis. These findings could lead to a novel regulatory mechanism for the exocytotic or synaptic strength based on lipid heterogeneity across the cell membrane.
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| 2. |
- Aref, Mohaddeseh, et al.
(author)
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Potentiometric pH Nanosensor for Intracellular Measurements: Real-Time and Continuous Assessment of Local Gradients
- 2021
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 93:47, s. 15744-15751
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Journal article (peer-reviewed)abstract
- We present a pH nanosensor conceived for single intracellular measurements. The sensing architecture consisted of a two-electrode system evaluated in the potentiometric mode. We used solid-contact carbon nanopipette electrodes tailored to produce both the indicator (pH nanosensor) and reference electrodes. The indicator electrode was a membrane-based ion-selective electrode containing a receptor for hydrogen ions that provided a favorable selectivity for intracellular measurements. The analytical features of the pH nanosensor revealed a Nernstian response (slope of -59.5 mV/pH unit) with appropriate repeatability and reproducibility (variation coefficients of <2% for the calibration parameters), a fast response time (<5 s), adequate medium-term drift (0.7 mV h(-)(1)), and a linear range of response including physiological and abnormal cell pH levels (6.0-8.5). In addition, the position and configuration of the reference electrode were investigated in cell-based experiments to provide unbiased pH measurements, in which both the indicator and reference electrodes were located inside the same cell, each of them inside two neighboring cells, or the indicator electrode inside the cell and the reference electrode outside of (but nearby) the studied cell. Finally, the pH nanosensor was applied to two cases: (i) the tracing of the pH gradient from extra-to intracellular media over insertion into a single PC12 cell and (ii) the monitoring of variations in intracellular pH in response to exogenous administration of pharmaceuticals. It is anticipated that the developed pH nanosensor, which is a label-free analytical tool, has high potential to aid in the investigation of pathological states that manifest in cell pH misregulation, with no restriction in the type of targeted cells.
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| 3. |
- Philipsen, Mai H., 1988, et al.
(author)
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Mass Spectrometry Imaging Shows Modafinil, A Student Study Drug, Changes the Lipid Composition of the Fly Brain
- 2021
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In: Angewandte Chemie-International Edition. - : Wiley. - 1433-7851 .- 1521-3773. ; 60:32, s. 17378-17382
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Journal article (peer-reviewed)abstract
- Modafinil, a widely used psychoactive drug, has been shown to exert a positive impact on cognition and is used to treat sleep disorders and hyperactivity. Using time-of-flight secondary ion mass spectrometric imaging, we studied the changes of brain lipids of Drosophila melanogaster induced by modafinil to gain insight into the functional mechanism of modafinil in the brain. We found that upon modafinil treatment, the abundance of phosphatidylcholine and sphingomyelin species in the central brain of Drosophila is significantly decreased, whereas the levels of phosphatidylethanolamine and phosphatidylinositol in the brains show significant enhancement compared to the control flies. The alteration of brain lipids caused by modafinil is consistent with previous studies about cognition-related drugs and offers a plausible mechanism regarding the action of modafinil in the brain as well as a potential target for the treatment of certain disorders.
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| 4. |
- Ranjbari, Elias, et al.
(author)
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Analytical Techniques: Shedding Light upon Nanometer-Sized Secretory Vesicles
- 2019
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In: Trends in Chemistry. - : Elsevier BV. - 2589-5974. ; 1:4, s. 440-451
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Journal article (peer-reviewed)abstract
- Vesicles, small synaptic and large dense core, are the main organelles responsible for cell-to-cell communication. Analysis of nanometer-sized vesicles and accurate assessment of their physiology are major analytical challenges. This review briefly discusses the achievements made in these areas using some of the strongest techniques comprising transmission electron microscopy, stimulated emission depletion microscopy, nanoscale secondary-ion mass spectrometry, and electrochemical cytometry. These techniques provide high spatial and temporal resolution to precisely delve into the structure of these subcellular organelles and shed light on the processes involved in cell signaling. Correlating results of these techniques has furthered our understanding of the relationship between vesicle structure and function. Both the methods and their use in evaluating function are discussed. © 2019 Elsevier Inc.
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| 5. |
- Ranjbari, Elias, et al.
(author)
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Combined electrochemistry and mass spectrometry imaging to interrogate the mechanism of action of modafinil, a cognition-enhancing drug, at the cellular and sub-cellular level
- 2021
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In: QRB Discovery. - : Cambridge University Press (CUP). - 2633-2892. ; 2
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Journal article (peer-reviewed)abstract
- (A) Microscope view during the intracellular vesicle impact electrochemical cytometry experiment (scale bar = 20 μm). (B) Normalised frequency histogram for the number of molecules stored in vesicles in control (blue) and modafinil-Treated cells (red). The distribution fits are shown for control cells with a dashed line and modafinil-Treated cells with a solid line. (C) Average of the number of molecules stored per vesicle in PC12 cells treated without (control) or with 10 μM modafinil. Number of analysed cells: control cells (15 cells) and modafinil-Treated cells (18 cells). The results were compared by a two-Tailed Mann-Whitney rank-sum test,*p < 0.05, p < 0.01 and***p < 0.001. ©
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| 6. |
- Ranjbari, Elias, et al.
(author)
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Direct Measurement of Total Vesicular Catecholamine Content with Electrochemical Microwell Arrays
- 2020
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In: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 92:16, s. 11325-11331
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Journal article (peer-reviewed)abstract
- We have designed and fabricated a microwell array chip (MWAC) to trap and detect the entire content of individual vesicles after disruption of the vesicular membrane by an applied electrical potential. To understand the mechanism of vesicle impact electrochemical cytometry (VIEC) in microwells, we simulated the rupture of the vesicles and subsequent diffusion of entrapped analytes. Two possibilities were tested: (i) the vesicle opens toward the electrode, and (ii) the vesicle opens away from the electrode. These two possibilities were simulated in the different microwells with varied depth and width. Experimental VIEC measurements of the number of molecules for each vesicle in the MWAC were compared to VIEC on a gold microdisk electrode as a control, and the quantified catecholamines between these two techniques was the same. We observed a prespike foot in a significant number of events (similar to 20%) and argue this supports the hypothesis that the vesicles rupture toward the electrode surface with a more complex mechanism including the formation of a stable pore intermediate. This study not only confirms that in standard VIEC experiments the whole content of the vesicle is oxidized and quantified at the surface of the microdisk electrode but actively verifies that the adsorbed vesicle on the surface of the electrode forms a pore in the vicinity of the electrode rather than away from it. The fabricated MWAC promotes our ability to quantify the content of vesicles accurately, which is fundamentally important in bioanalysis of the vesicles.
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