| 1. |
- Ding, J., et al.
(författare)
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Inhibition of HMGCoA Reductase Reveals An Unexpected Role for Cholesterol During PGC Migration in the Mouse.
- 2008
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Ingår i: BMC developmental biology. - 1471-213X. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- ABSTRACT: BACKGROUND: Primordial germ cells (PGCs) are the embryonic precursors of the sperm and eggs. Environmental or genetic defects that alter PGC development can impair fertility or cause formation of germ cell tumors. RESULTS: We demonstrate a novel role for cholesterol during germ cell migration in mice. Cholesterol was measured in living tissue dissected from mouse embryos and was found to accumulate within the developing gonads as germ cells migrate to colonize these structures. Cholesterol synthesis was blocked in culture by inhibiting the activity of HMG CoA reductase (HMGCR) resulting in germ cell survival and migration defects. These defects were rescued by co-addition of isoprenoids and cholesterol, but neither compound alone was sufficient. In contrast, loss of the last or penultimate enzyme in cholesterol biosynthesis did not alter PGC numbers or position in vivo. However embryos that lack these enzymes do not exhibit cholesterol defects at the stage at which PGCs are migrating. This demonstrates that during gestation, the cholesterol required for PGC migration can be supplied maternally. CONCLUSIONS: In the mouse, cholesterol is required for PGC survival and motility. It may act cell-autonomously by regulating clustering of growth factor receptors within PGCs or non cell-autonomously by controlling release of growth factors required for PGC guidance and survival.
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| 2. |
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| 3. |
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| 4. |
- Kurczy, Michael, 1980, et al.
(författare)
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Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation
- 2010
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 107:7, s. 2751-2756
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry imaging has been used here to suggest that changes in membrane structure drive lipid domain formation in mating single-cell organisms. Chemical studies of lipid bilayers in both living and model systems have revealed that chemical composition is coupled to localized membrane structure. However, it is not clear if the lipids that compose the membrane actively modify membrane structure or if structural changes cause heterogeneity in the surface chemistry of the lipid bilayer. We report that time-of-flight secondary ion mass spectrometry images of mating Tetrahymena thermophila acquired at various stages during mating demonstrate that lipid domain formation, identified as a decrease in the lamellar lipid phosphatidylcholine, follows rather than precedes structural changes in the membrane. Domains are formed in response to structural changes that occur during cell-to-cell conjugation. This observation has wide implications in all membrane processes.
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| 5. |
- Kurczy, Michael, 1980, et al.
(författare)
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Nanotome Cluster Bombardment to Recover Spatial Chemistry After Preparation of Biological Samples for SIMS Imaging
- 2010
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Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 21:5, s. 833-836
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Tidskriftsartikel (refereegranskat)abstract
- A C-60(+) cluster ion projectile is employed for sputter cleaning biological surfaces to reveal spatio-chemical information obscured by contamination overlayers. This protocol is used as a supplemental sample preparation method for time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of frozen and freeze-dried biological materials. Following the removal of nanometers of material from the surface using sputter cleaning, a frozen-patterned cholesterol film and a freeze-dried tissue sample were analyzed using ToF-SIMS imaging. In both experiments, the chemical information was maintained after the sputter dose, due to the minimal chemical damage caused by C-60(+) bombardment. The damage to the surface produced by freeze-drying the tissue sample was found to have a greater effect on the loss of cholesterol signal than the sputter-induced damage. In addition to maintaining the chemical information, sputtering is not found to alter the spatial distribution of molecules on the surface. This approach removes artifacts that might obscure the surface chemistry of the sample and are common to many biological sample preparation schemes for ToF-SIMS imaging.
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| 6. |
- Lanekoff, Ingela, 1975, et al.
(författare)
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Mass spectrometry imaging of freeze-dried membrane phospholipids of dividing Tetrahymena pyriformis
- 2013
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Ingår i: Surface and Interface Analysis. - : Wiley. - 0142-2421 .- 1096-9918. ; 45:1, s. 211-214
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Tidskriftsartikel (refereegranskat)abstract
- Time of Flight secondary ion mass spectrometry (TOF-SIMS) has been used to explore the distribution of phospholipids in the plasma membrane of Tetrahymena pyriformis during cell division. The dividing cells were freeze-dried prior to analysis followed by line scan and region of interest analysis at various stages of cell division. The results showed no signs of phospholipid domain formation at the junction between the dividing cells. Instead the results showed that the sample preparation technique had a great impact on one of the examined phospholipids, namely phosphatidylcholine (PC). Phosphatidylcholine and 2-aminoethylphosphonolipid (2-AEP) have therefore been evaluated in Tetrahymena cells that have been subjected to different sample preparation techniques: freeze drying ex situ, freeze fracture, and freeze fracture with partial or total freeze drying in situ. The result suggests that freeze drying ex situ causes the celia to collapse and cover the plasma membrane.
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| 7. |
- Passarelli, Melissa, 1983, et al.
(författare)
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C-60-SIMS studies of glycerophospholipid in a LIPID MAPS model system: KDO2-Lipid A stimulated RAW 264.7 cells
- 2013
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Ingår i: Surface and Interface Analysis. - : Wiley. - 0142-2421 .- 1096-9918. ; 45:1, s. 298-301
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Tidskriftsartikel (refereegranskat)abstract
- Although secondary ion mass spectrometry (SIMS) has been successfully employed for mapping lipid distributions at the cellular level, the identification of intact lipid species in situ is often complicated by isobaric interference. The high mass resolution and tandem MS capabilities of a C60-QSTAR hybrid instrument has been utilized to identify over 50 lipid species from mouse macrophages (RAW 264.7). In this investigation, lipid assignments made based on mass accuracy were confirmed with tandem MS analyses. Data obtained from C60-SIMS was compared to liquid chromatography (LC)-MS data obtained by the LIPID MAPS consortium. A majority of the lipids detected with LC-MS, but not detected with C60-SIMS, were present at concentrations below 2.0 pmol/µg of DNA. Matrix-related effects prevented the detection of lipids with the glycerophosphoethanolamine (PE) headgroup, glycerophosphoserine (PS) headgroup and lipids with polyunsaturated fatty acyl chains in the C60-SIMS analyses. Lipid distributions obtained from a lawn of RAW 264.7 cells stimulated with the endotoxin KDO2-Lipid A were also studied. The results obtained with C60-SIMS agreed with the established LC-MS data for the glycerophosphoinositol lipid class (PI) with adequate molecular sensitivity achieved with as few as 500 cells.
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| 8. |
- Passarelli, Melissa, 1983, et al.
(författare)
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Single-Cell Lipidomics: Characterizing and Imaging Lipids on the Surface of Individual Aplysia californica Neurons with Cluster Secondary Ion Mass Spectrometry
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 85:4, s. 2231-2238
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Tidskriftsartikel (refereegranskat)abstract
- Neurons isolated from Aplysia californica, an organism with a well-defined neural network, were imaged with secondary ion mass spectrometry, C-60-SIMS. A major lipid component of the neuronal membrane was identified as 1-hexadecyl-2-octadecenoyl-sn-glycero-3-phosphocholine [PC(16:0e/18:1)] using tandem mass spectrometry (MS/MS). The assignment was made directly off the sample surface using a C-60-QSTAR instrument, a prototype instrument that combines an ion source with a commercial electrospray ionization/matrix-assisted laser desorption ionization (ESI/MALDI) mass spectrometer, Normal phase liquid chromatography mass spectrometry (NP-LC-MS) was used to confirm the assignment. Cholesterol and vitamin E were also identified with in situ tandem MS analyses that were compared to reference spectra obtained from purified compounds. In order to improve sensitivity on the single-cell level, the tandem MS spectrum of vitamin E reference material was used to extract and compile all the vitamin E related peaks from the cell image. The mass spectrometry images heterogeneous distributions cif intact lipid species, PC(16:0e/18:1), vitamin E, and cholesterol on the surface of a single neuron. The ability to detect these molecules and determine their relative distribution on the single-cell level shows that the C-60-QSTAR is a potential platform for studying important biochemical processes, such as neuron degeneration.
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| 9. |
- Piwowar, A. M., et al.
(författare)
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C60-ToF SIMS imaging of frozen hydrated HeLa cells
- 2013
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Ingår i: Surface and Interface Analysis. - : Wiley. - 0142-2421 .- 1096-9918. ; 45:1, s. 302-304
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Tidskriftsartikel (refereegranskat)abstract
- Sample preparation continues to be a major challenge for SIMS studies of biological materials. Maintaining the native hydrated state of the material is important for preserving both chemical and spatial information. Here, we discuss a method that combines a sample wash and dry protocol followed by plunge-freezing in liquid ethane for a frozen-hydrated analysis of mammalian cells (HeLa). This method allows for the removal of the growth medium and maintains the hydrated state of the cells so that they can be prepared frozen-hydrated without the need for a freeze-fracture device. The cells, which were grown on silicon, were successfully regrown after the cleaning procedure, confirming that a significant portion of the cells remain undamaged during the wash and dry procedure. Results from preliminary SIMS measurements show that is it possible to detect a large variety of biomolecular signals, including intact lipids from the plasma membrane in the mass range of 700–900Da from single cells, with little external water interference at the surface.
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| 10. |
- Shard, A. G., et al.
(författare)
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Measuring Compositions in Organic Depth Profiling: Results from a VAMAS Interlaboratory Study
- 2015
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Ingår i: Journal of Physical Chemistry B. - : American Chemical Society (ACS). - 1520-6106 .- 1520-5207. ; 119:33, s. 10784-10797
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Tidskriftsartikel (refereegranskat)abstract
- We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the measurement of composition in organic depth profiling. Layered samples with known binary compositions of Irganox 1010 and either Irganox 1098 or Fmoc-pentafluoro-L-phenylalanine in each layer were manufactured in a single batch and distributed to more than 20 participating laboratories. The samples were analyzed using argon cluster ion sputtering and either X-ray photoelectron spectroscopy (XPS) or time-of-flight secondary ion mass spectrometry (ToF-SIMS) to generate depth profiles. Participants were asked to estimate the volume fractions in two of the layers and were provided with the compositions of all other layers. Participants using XPS provided volume fractions within 0.03 of the nominal values. Participants using ToF-SIMS either made no attempt, or used various methods that gave results ranging in error from 0.02 to over 0.10 in volume fraction, the latter representing a 50% relative error for a nominal volume fraction of 0.2. Error was predominantly caused by inadequacy in the ability to compensate for primary ion intensity variations and the matrix effect in SIMS. Matrix effects in these materials appear to be more pronounced as the number of atoms in both the primary analytical ion and the secondary ion increase. Using the participants' data we show that organic SIMS matrix effects can be measured and are remarkably consistent between instruments. We provide recommendations for identifying and compensating for matrix effects. Finally, we demonstrate, using a simple normalization method, that virtually all ToF-SIMS participants could have obtained estimates of volume fraction that were at least as accurate and consistent as XPS.
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