Mass spectrometry imaging with high resolution in mass and space

Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of ‘mass spectrometry imaging with high resolution in mass and space.’ Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5–10 μm. Proteins were analyzed after on-tissue tryptic digestion at 50-μm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 μm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level

Inzucht in Rinderpopulationen - Ergebnisse einer Expertenbefragung

Kinship breeding is recommended as an adequate breeding approach for organic farms, because breeders work with their own well adapted cow families and bulls. Therefore kinship breeding is promoted on organic farms in a project conducted in Switzerland and Germany. An often arising question is about a possible negative impact of inbreeding on animal health, performance and welfare, but there is not much and quite varying information available. Therefore, a literature study and 10 interviews with dairy cattle breeding experts were conducted to get a better knowledge on inbreeding impacts in small populations. Results: Inbreeding depressions were described for production, sperm quality and quantity, reproduction, calf vitality, longevity and calving traits and on hereditary diseases. Experts argued that cattle is quite resistant against inbreeding depressions. Their occurrence depends on the degree of inbreeding and on the effective population size, but even more on breeders’ knowledge about their animals’ features in past generations. Natural mating bears a smaller risk of inbreeding on population level than artificial insemination

Perfusion of the interventricular septum during ventilation with positive end-expiratory pressure (PEEP)

Objective: To determine whether regional hypoperfusion of the interventricular septum occurs during ventilation with positive end-expiratory pressure. Design: Animal study. Animals: Anesthetized, closed chest dogs (n = 8). Interventions: Induction of experimental adult respiratory distress syndrome (ARDS) and then ventilation with 10,15, and 20 cm H2O of positive end-expiratory pressure. Measurements and Main Results: Cardiac output and regional interventricular septum blood flow 'were assessed at control, at induction of experimental ARDS, and at each level of positive end-expiratory pressure. Ventilation with 20 cm H2O of positive end-expiratory pressure decreased cardiac output (-32% vs. control, p <.05), and did not change absolute, but increased relative (to cardiac output) interventricular septum blood flow. During experimental ARDS and ventilation at 20 cm H2O end-expiratory pressure, there was a redistribution of flow toward the right ventricular free wall (+93%, p < .001) and the right ventricular part of the interventricular septum (+68%, p < .01), while flow to the left ventricular interventricular septum and to the left ventricular free wall remained unchanged. Locally hypoperfused interventricular septum areas or findings indicative of interventricular septum ischemia were not observed during positive end-expiratory pressure. Conclusions: The decrease in cardiac output during positive end-expiratory pressure is not caused by impaired interventricular septum blood supply. The preferential perfusion of the right ventricular interventricular septum indicates increased local right ventricular interventricular septum oxygen-demand and suggests that during positive end-expiratory pressure, this part of the interventricular septum functionally dissociates from the left ventricular interventricular septum and the left ventricular free wall to support the stressed right ventricle

IR-MALDI Mass Spectrometry Imaging with Plasma Post-Ionization of Nonpolar Metabolites

Ambient mass spectrometry imaging (MSI) methods come with the advantage of visualizing biomolecules from tissues with no or minimal sample preparation and operation under atmospheric-pressure conditions. Similar to all other MSI methodologies, however, ambient MSI modalities suffer from a pronounced bias toward either polar or nonpolar analytes due to the underlying desorption and ionization mechanisms of the ion source. In this study, we present the design, construction, testing, and application of an in-capillary dielectric barrier discharge (DBD) module for post-ionization of neutrals desorbed by an ambient infrared matrix-assisted laser desorption/ionization (IR-MALDI) MSI source. We demonstrate that the DBD device enhances signal intensities of nonpolar compounds by up to 104 compared to IR-MALDI without affecting transmission of IR-MALDI ions. This allows performing MSI experiments of mouse tissue and Danaus plexippus caterpillar tissue sections, visualizing the distribution of sterols, fatty acids, monoglycerides, and diglycerides that are not detected in IR-MALDI MSI experiments. The pronounced signal enhancement due to IR-MALDI-DBD compared to IR-MALDI MSI enables mapping of nonpolar analytes with pixel resolutions down to 20 μm in mouse brain tissue and to discern the spatial distribution of sterol lipids characteristic for histological regions of D. plexippus

Half-Sandwich Rhodium Complexes with Releasable N-Donor Monodentate Ligands: Solution Chemical Properties and the Possibility for Acidosis Activation

Cancer chemotherapeutics usually have serious side effects. Targeting the special properties of cancer and activation of the anticancer drug in the tumor microenvironment in situ may decrease the intensity of the side effects and improve the efficacy of therapy. In this study, half-sandwich Rh complexes are introduced, which may be activated at the acidic, extracellular pH of the tumor tissue. The synthesis and aqueous stability of mixed-ligand complexes with a general formula of [Rh(η5-Cp*)(N,N/O)(N)]2+/+ are reported, where (N,N/O) indicates bidentate 8-quinolate, ethylenediamine and 1,10-phenanthroline and (N) represents the releasable monodentate ligand with a nitrogen donor atom. UV-visible spectrophotometry, 1H NMR, and pH-potentiometry were used to determine the protonation constants of the monodentate ligands, the proton dissociation constants of the coordinated water molecules in the aqua complexes, and the formation constants of the mixed-ligand complexes. The obtained data were compared to those of the analogous Ru(η6-p-cymene) complexes. The developed mixed-ligand complexes were tested in drug-sensitive and resistant colon cancer cell lines (Colo205 and Colo320, respectively) and in four bacterial strains (Gram-positive and Gram-negative, drug-sensitive, and resistant) at different pH values (5–8). The mixed-ligand complexes with 1-methylimidazole displayed sufficient stability at pH 7.4, and their activation was found in cancer cells with decreasing pH; moreover, the mixed-ligand complexes demonstrated antimicrobial activity in Gram-positive and Gram-negative bacteria, including the resistant MRSA strain. This study proved the viability of incorporating releasable monodentate ligands into mixed-ligand half-sandwich complexes, which is supported by the biological assays

Unveiling the spatial distribution of aflatoxin B1 and plant defense metabolites in maize using AP‐SMALDI mass spectrometry imaging

SUMMARYIn order to cope with the presence of unfavorable compounds, plants can biotransform xenobiotics, translocate both parent compounds and metabolites, and perform compartmentation and segregation at the cellular or tissue level. Such a scenario also applies to mycotoxins, fungal secondary metabolites with a pre‐eminent role in plant infection. In this work, we aimed to describe the effect of the interplay between Zea mays (maize) and aflatoxin B1 (AFB1) at the tissue and organ level. To address this challenge, we used atmospheric pressure scanning microprobe matrix‐assisted laser desorption/ionization mass spectrometry imaging (AP‐SMALDI MSI) to investigate the biotransformation, localization and subsequent effects of AFB1 on primary and secondary metabolism of healthy maize plants, both in situ and from a metabolomics standpoint. High spatial resolution (5 µm) provided fine localization of AFB1, which was located within the root intercellular spaces, and co‐localized with its phase‐I metabolite aflatoxin M2. We provided a parallel visualization of maize metabolic changes, induced in different organs and tissues by an accumulation of AFB1. According to our untargeted metabolomics investigation, anthocyanin biosynthesis and chlorophyll metabolism in roots are most affected. The biosynthesis of these metabolites appears to be inhibited by AFB1 accumulation. On the other hand, metabolites found in above‐ground organs suggest that the presence of AFB1 may also activate the biochemical response in the absence of an actual fungal infection; indeed, several plant secondary metabolites known for their antimicrobial or antioxidant activities were localized in the outer tissues, such as phenylpropanoids, benzoxazinoids, phytohormones and lipids

Candida albicans adhesion to central venous catheters: Impact of blood plasma-driven germ tube formation and pathogen-derived adhesins

Candida albicans-related bloodstream infections are often associated with infected central venous catheters (CVC) triggered by microbial adhesion and biofilm formation. We utilized single-cell force spectroscopy (SCFS) and flow chamber models to investigate the adhesion behavior of C. albicans yeast cells and germinated cells to naïve and human blood plasma (HBP)-coated CVC tubing. Germinated cells demonstrated up to 56.8-fold increased adhesion forces to CVC surfaces when compared to yeast cells. Coating of CVCs with HBP significantly increased the adhesion of 60-min germinated cells but not of yeast cells and 30-min germinated cells. Under flow conditions comparable to those in major human veins, germinated cells displayed a flow directional-orientated adhesion pattern to HBP-coated CVC material, suggesting the germ tip to serve as the major adhesive region. None of the above-reported phenotypes were observed with germinated cells of an als3Δ deletion mutant, which displayed similar adhesion forces to CVC surfaces as the isogenic yeast cells. Germinated cells of the als3Δ mutant also lacked a clear flow directional-orientated adhesion pattern on HBP-coated CVC material, indicating a central role for Als3 in the adhesion of germinated C. albicans cells to blood exposed CVC surfaces. In the common model of C. albicans, biofilm formation is thought to be mediated primarily by yeast cells, followed by surface-triggered the formation of hyphae. We suggest an extension of this model in which C. albicans germ tubes promote the initial adhesion to blood-exposed implanted medical devices via the germ tube-associated adhesion protein Als3

Identifying an interaction site between MutH and the C-terminal domain of MutL by crosslinking, affinity purification, chemical coding and mass spectrometry

To investigate protein–protein interaction sites in the DNA mismatch repair system we developed a crosslinking/mass spectrometry technique employing a commercially available trifunctional crosslinker with a thiol-specific methanethiosulfonate group, a photoactivatable benzophenone moiety and a biotin affinity tag. The XACM approach combines photocrosslinking (X), in-solution digestion of the crosslinked mixtures, affinity purification via the biotin handle (A), chemical coding of the crosslinked products (C) followed by MALDI-TOF mass spectrometry (M). We illustrate the feasibility of the method using a single-cysteine variant of the homodimeric DNA mismatch repair protein MutL. Moreover, we successfully applied this method to identify the photocrosslink formed between the single-cysteine MutH variant A223C, labeled with the trifunctional crosslinker in the C-terminal helix and its activator protein MutL. The identified crosslinked MutL-peptide maps to a conserved surface patch of the MutL C-terminal dimerization domain. These observations are substantiated by additional mutational and chemical crosslinking studies. Our results shed light on the potential structures of the MutL holoenzyme and the MutH–MutL–DNA complex