Dysfunction in atox-1 and ceruloplasmin alters labile Cu levels and consequently Cu homeostasis in C. elegans.

Copper (Cu) is an essential trace element, however an excess is toxic due to its redox properties. Cu homeostasis therefore needs to be tightly regulated via cellular transporters, storage proteins and exporters. An imbalance in Cu homeostasis has been associated with neurodegenerative disorders such as Wilson's disease, but also Alzheimer's or Parkinson's disease. In our current study, we explored the utility of using Caenorhabditis elegans (C. elegans) as a model of Cu dyshomeostasis. The application of excess Cu dosing and the use of mutants lacking the intracellular Cu chaperone atox-1 and major Cu storage protein ceruloplasmin facilitated the assessment of Cu status, functional markers including total Cu levels, labile Cu levels, Cu distribution and the gene expression of homeostasis-related genes. Our data revealed a decrease in total Cu uptake but an increase in labile Cu levels due to genetic dysfunction, as well as altered gene expression levels of Cu homeostasis-associated genes. In addition, the data uncovered the role ceruloplasmin and atox-1 play in the worm's Cu homeostasis. This study provides insights into suitable functional Cu markers and Cu homeostasis in C. elegans, with a focus on labile Cu levels, a promising marker of Cu dysregulation during disease progression.

Subcellular detection of PEBCA particles in macrophages: combining darkfield microscopy, confocal Raman microscopy, and ToF-SIMS analysis.

The detection of biomedical organic nanocarriers in cells and tissues is still an experimental challenge. Here we developed an imaging strategy for the label-free detection of poly (ethylbutyl cyanoacrylate) (PEBCA) particles. Experiments were carried out with phagocytic NR8383 macrophages exposed to non-toxic and non-activating concentrations of fluorescent (PEBCA NR668 and PEBCA NR668/IR), non-fluorescent (PEBCA), and cabazitaxel-loaded PEBCA particles (PEBCA CBZ). Exposure to PEBCA NR668 revealed an inhomogeneous particle uptake similar to what was obtained with the free modified Nile Red dye (NR668). In order to successfully identify the PEBCA-loaded cells under label-free conditions, we developed an imaging strategy based on enhanced darkfield microscopy (DFM), followed by confocal Raman microscopy (CRM) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Nitrile groups of the PEBCA matrix and PEBCA ions were used as suitable analytes for CRM and ToF-SIMS, respectively. Masses found with ToF-SIMS were further confirmed by Orbitrap-SIMS. The combined approach allowed to image small (< 1 µm) PEBCA-containing phagolysosomes, which were identified as PEBCA-containing compartments in NR8383 cells by electron microscopy. The combination of DFM, CRM, and ToF-SIMS is a promising strategy for the label-free detection of PEBCA particles.

Evaluation of Time-of-Flight Secondary Ion Mass Spectrometry Spectra of Peptides by Random Forest with Amino Acid Labels: Results from a Versailles Project on Advanced Materials and Standards Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the identification of peptide sample TOF-SIMS spectra by machine learning. More than 1000 time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra of six peptide model samples (one of them was a test sample) were collected using 27 TOF-SIMS instruments from 25 institutes of six countries, the U. S., the U. K., Germany, China, South Korea, and Japan. Because peptides have systematic and simple chemical structures, they were selected as model samples. The intensity of peaks in every TOF-SIMS spectrum was extracted using the same peak list and normalized to the total ion count. The spectra of the test peptide sample were predicted by Random Forest with 20 amino acid labels. The accuracy of the prediction for the test spectra was 0.88. Although the prediction of an unknown peptide was not perfect, it was shown that all of the amino acids in an unknown peptide can be determined by Random Forest prediction and the TOF-SIMS spectra. Moreover, the prediction of peptides, which are included in the training spectra, was almost perfect. Random Forest also suggests specific fragment ions from an amino acid residue Q, whose fragment ions detected by TOF-SIMS have not been reported, in the important features. This study indicated that the analysis using Random Forest, which enables translation of the mathematical relationships to chemical relationships, and the multi labels representing monomer chemical structures, is useful to predict the TOF-SIMS spectra of an unknown peptide.

Distribution of cholesterol and galactosylceramide in rat cerebellar white matter.

White matter and the inner granular layer of rat cerebellum was analysed by imaging time-of-flight secondary-ion mass spectrometry (TOF-SIMS) equipped with a Bi+ ion cluster gun. Samples were prepared by high pressure freezing, freeze-fracturing and freeze drying or by plunge freezing and cryostat sectioning. The identified and localized chemical species were: sodium, potassium, phosphocholine, cholesterol and galactosylceramide (GalC) with carbon chain lengths C18:0 (N-stearoyl-galactosylceramide) and C24:0 (N-lignoceroylgalactosylceramide) with CH24:0 (hydroxy-lignoceroylgalactosylceramide). We report new findings regarding the organization of myelin in white matter. One is cholesterol-rich, ribbon-shaped 10-20 microm areas excluding Na+ and K+. The second finding is the different distribution of GalC C18 and GalC C24 in relation to these areas, where GalC C18 was localized in cholesterol-rich areas and GalC C24 was localized in Na/K-enriched areas. The distribution of GalC was in small spots, homogeneous in size, of 0.8-1.5 microm. Sample preparation with high pressure freezing allowed separate localization of sodium and potassium in tissue samples.

Imaging TOF-SIMS of rat kidney prepared by high-pressure freezing.

Phosphocholine, potassium ions, and sodium ions were localized in rat kidney with imaging TOF-SIMS. Tissue preparation was performed with high-pressure freezing, freeze-fracturing and freeze-drying. The distribution of sodium ions was visualized by imaging the signal at m/z 23 of positively charged secondary ions, and the distribution of potassium ions was visualized by imaging the signal at m/z 39. Potassium was found localized within cells of the proximal tubulus epithelium and within cells of the glomeruli. High signals of sodium ions were seen in the interstitial tissue and also in epithelial cells of the collecting ducts and in glomeruli. The overlay image showed that the distribution of sodium ions and potassium ions were largely complementary with color mixing in glomeruli and in the interstitium surrounding proximal tubules. The ion distribution was further analyzed by correlation analysis. Phosphocholine-containing phospholipids were visualized by imaging the phosphocholine head group at m/z 184 of positively charged ions. The m/z 184 signal shows a ubiquitous distribution with a high intensity of phosphocholine in epithelial cells. Overlay image of m/z 184, m/z 39, and m/z 23 and multivariate analysis showed that the localization of high levels of phosphocholine colocalizes with high levels of potassium ions, as expected for an ion with intracellular localization.

Tissue molecular ion imaging by gold cluster ion bombardment.

The use of gold cluster focused ion beams produced by a liquid metal ion gun in a TOF-SIMS mass spectrometer is shown to dramatically enhance secondary ion emission of phospholipids and peptides. The method has been successfully tested with cells grown onto plastic slips and with mouse brain slices, without any treatment of the samples. Very reliable time-of-flight mass spectra are acquired with a low primary ion dose of a few 10(7) ions, and high lateral resolution molecular ion images are obtained for heavy ions of great biological interest. This approach offers new opportunities in pharmacological and biological research fields by localizing compounds of interest such as drugs or metabolites in tissues.