Relative quantification of cellular sections with molecular depth profiling ToF-SIMS imaging

We report the use of secondary ion mass spectrometry (SIMS) imaging to quantify the relative difference in the amount of lipid between two sections, the plasma membrane and the cytoplasm, of single cells from two different populations. Cells were each labeled with lipophillic dyes, frozen, fractured and analyzed in a ToF-SIMS mass spectrometer equipped with a 40 keV C60+ ion source. In addition to identifying cells from separate populations, the lipophilic dyes can be used as a marker for the outer leaflet of the cell membrane and therefore as a depth finder. Here, we show that it is possible to compare the amount of lipids with particular headgroups in the cell membrane of a treated cell to the membrane of a control cell. Following erosion of the cell membranes, the amount of the two specific lipid head groups in the cytoplasm of the treated cell can be compared to those lipids in a control cell. Here we take the first step in this experimental design and display the ability to analyze multiple sections of frozen cells following a single fracture

Interactive XCMS Online: simplifying advanced metabolomic data processing and subsequent statistical analyses.

XCMS Online (xcmsonline.scripps.edu) is a cloud-based informatic platform designed to process and visualize mass-spectrometry-based, untargeted metabolomic data. Initially, the platform was developed for two-group comparisons to match the independent, "control" versus "disease" experimental design. Here, we introduce an enhanced XCMS Online interface that enables users to perform dependent (paired) two-group comparisons, meta-analysis, and multigroup comparisons, with comprehensive statistical output and interactive visualization tools. Newly incorporated statistical tests cover a wide array of univariate analyses. Multigroup comparison allows for the identification of differentially expressed metabolite features across multiple classes of data while higher order meta-analysis facilitates the identification of shared metabolic patterns across multiple two-group comparisons. Given the complexity of these data sets, we have developed an interactive platform where users can monitor the statistical output of univariate (cloud plots) and multivariate (PCA plots) data analysis in real time by adjusting the threshold and range of various parameters. On the interactive cloud plot, metabolite features can be filtered out by their significance level (p-value), fold change, mass-to-charge ratio, retention time, and intensity. The variation pattern of each feature can be visualized on both extracted-ion chromatograms and box plots. The interactive principal component analysis includes scores, loadings, and scree plots that can be adjusted depending on scaling criteria. The utility of XCMS functionalities is demonstrated through the metabolomic analysis of bacterial stress response and the comparison of lymphoblastic leukemia cell lines

Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation

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

Global Isotope Metabolomics Reveals Adaptive Strategies for Nitrogen Assimilation

Nitrogen cycling is a microbial metabolic process essential for global ecological/agricultural balance. To investigate the link between the well-established ammonium and the alternative nitrate assimilation metabolic pathways, global isotope metabolomics was employed to examine three nitrate reducing bacteria using ¹⁵NO₃ as a nitrogen source. In contrast to a control (<i>Pseudomonas stutzeri</i> RCH2), the results show that two of the isolates from Oak Ridge, Tennessee (<i>Pseudomonas</i> N2A2 and N2E2) utilize nitrate and ammonia for assimilation concurrently with differential labeling observed across multiple classes of metabolites including amino acids and nucleotides. The data reveal that the N2A2 and N2E2 strains conserve nitrogen-containing metabolites, indicating that the nitrate assimilation pathway is a conservation mechanism for the assimilation of nitrogen. Co-utilization of nitrate and ammonia is likely an adaption to manage higher levels of nitrite since the denitrification pathways utilized by the N2A2 and N2E2 strains from the Oak Ridge site are predisposed to the accumulation of the toxic nitrite. The use of global isotope metabolomics allowed for this adaptive strategy to be investigated, which would otherwise not have been possible to decipher

Amperometric Detection of Single Vesicle Acetylcholine Release Events from an Artificial Cell

Acetylcholine is a highly abundant nonelectroactive neurotransmitter in the mammalian central nervous system. Neurochemical release occurs on the millisecond time scale, requiring a fast, sensitive sensor such as an enzymatic amperometric electrode. Typically, the enzyme used for enzymatic electrochemical sensors is applied in excess to maximize signal. Here, in addition to sensitivity, we have also sought to maximize temporal resolution, by designing a sensor that is sensitive enough to work at near monolayer enzyme coverage. Reducing the enzyme layer thickness increases sensor temporal resolution by decreasing the distance and reducing the diffusion time for the enzyme product to travel to the sensor surface for detection. In this instance, the sensor consists of electrodeposited gold nanoparticle modified carbon fiber microelectrodes (CFMEs). Enzymes often are sensitive to curvature upon surface adsorption; thus, it was important to deposit discrete nanoparticles to maintain enzyme activity while depositing as much gold as possible to maximize enzyme coverage. To further enhance sensitivity, the enzymes acetylcholinesterase (AChE) and choline oxidase (ChO) were immobilized onto the gold nanoparticles at the previously determined optimal ratio (1:10 AChE/ChO) for most efficient sequential enzymatic activity. This optimization approach has enabled the rapid detection to temporally resolve single vesicle acetylcholine release from an artificial cell. The sensor described is a significant advancement in that it allows for the recording of acetylcholine release on the order of the time scale for neurochemical release in secretory cells

Great Britain Ministry of Information – Daily Press Notices and Bulletins (1943-08-10)

From the UK National Archives: http://www.nationalarchives.gov.uk/theartofwar/inf3.htm “Formed on September 4th 1939, the day after Britain’s declaration of war, the Ministry of Information (MOI) was the central government department responsible for publicity and propaganda in the Second World War. The initial functions of the MOI were threefold: news and press censorship; home publicity; and oversees publicity in Allied and neutral countries”. This collection contains Press Notices and Bulletins published by the MOI between 1939-1945. The Press Notices and Bulletins are among many publications and films issued by the agency during the war.UT Librarie