Essential tactics of tissue preparation and matrix nano-spotting for successful compound imaging mass spectrometry

The ultimate goal of MALDI-Imaging Mass Spectrometry (MALDI-IMS) is to achieve spatial localization of analytes in tissue sections down to individual tissue compartments or even at the level of a few cells. With compound tissue imaging, it is possible to track the transportation of an unlabelled, inhaled reference compound within lung tissue, through the application of MALDI-IMS. The procedure for isolation and preparation of lung tissues is found to be crucial in order to preserve the anatomy and structure of the pulmonary compartments.To avoid delocalization of analytes within lung tissue compartments we have applied an in-house designed nano-spotter, based on a microdispenser mounted on an XY table, of which movement and spotting functionality were fully computer controlled. We demonstrate the usefulness of this platform in lung tissue sections isolated from rodent in vivo model, applied to compound tissue imaging as exemplified with the determination of the spatial distribution of (1α,2β,4β,7β)-7-[(hydroxidi-2-thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.0^2,4]nonane, also known as tiotropium. We provide details on tissue preparation protocols and sample spotting technology for successful identification of drug in mouse lung tissue by using MALDI-Orbitrap instrumentation.     

Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay

The Escherichia coli (E. coli) reference collection, ECOR, consists of 72 strains that are representative of the genotypic diversity, as indexed by multilocus enzyme electrophoresis (MLEE), in the species as a whole. MLEE revealed 4 main phylogenetic groups designated A, B1, B2 and D. We present a study of the relationship between the ECOR strains as determined by polymorphisms in seven variable-number of tandem repeats (VNTR) loci. Seven tandem repeats that were present in more than one of the fully sequenced E. coli strains were selected, and primers were constructed in order to amplify the targets in all species where the loci were present. The combined result for all VNTR loci was adapted as a multiple-locus variable-number tandem repeats analysis (MLVA) and showed that the ECOR collection was divided into 63 distinct genotypes. The ECOR phylogenetic groups defined by MLEE were not well conserved by MLVA. A set of 61 pathogenic isolates of both E. coli and Shigella spp. was then tested with the same set of VNTR loci, and revealed 54 distinct genotypes. In addition, the MLVA method was used to genotype isolates from patients and suspected sources in a recent outbreak of E. coli O103 in Norway. The pathogenic E. coli isolates contained the diarrhea causing categories EIEC, EAEC, STEC, ETEC and EPEC. Shigella isolates were of species S. flexneri, S. boydii, S. sonnei and S. dysenteriae. The MLVA method rapidly genotyped all isolates in the study at a Simpson's index of diversity of D=0.98.     

Variable responses of natural enemies to Salix triandra phenotypes with different secondary chemistry

Plant phenotypes often differ in their resistance to natural enemies, but the mechanism for this has seldom been identified. The aim of this study was to determine if the spatial patterns of phenotype use of a highly specialized insect herbivore (the galling sawfly Pontania triandrae ) in a natural willow population can be related to phenotypic variation in plant secondary chemistry. Furthermore, we tested if traits that confer resistance to one type of natural enemy, i.e. the galling sawfly, also confer resistance to others, in our case a leaf beetle Gonioctena linnaeana and the rust fungus Melampsora amygdalinae . We identified 18 phenotypes with high and 18 phenotypes with low gall density in our field population and determined gall densities, the degree of leaf damage and rust infection on each phenotype and collected leaves for chemical analyses. The concentration of phenolics was higher in phenotypes with high density of galls suggesting that this galling sawfly may use phenolics as oviposition cues. Rust infection showed the opposite pattern, with lower levels on clones with high concentration of phenolics, while leaf damage by G. linnaeana did not differ between clone types. This indicates that these important natural enemies may assert divergent selection on willow phenotypes and that this might provide a mechanism for maintaining phenotypic variation within willow populations.     

Structures of Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase provide new insights into catalysis

Isopentenyl diphosphate is the precursor of various isoprenoids that are essential to all living organisms. It is produced by the mevalonate pathway in humans but by an alternate route in plants, protozoa, and many bacteria. 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step of this non-mevalonate pathway, which involves an NADPH-dependent rearrangement and reduction of 1-deoxy-D-xylulose 5-phosphate to form 2-C-methyl-D-erythritol 4-phosphate. The use of different pathways, combined with the reported essentiality of the enzyme makes the reductoisomerase a highly promising target for drug design. Here we present several high resolution structures of the Mycobacterium tuberculosis 1-deoxy-D-xylulose-5-phosphate reductoisomerase, representing both wild type and mutant enzyme in various complexes with Mn(2+), NADPH, and the known inhibitor fosmidomycin. The asymmetric unit corresponds to the biological homodimer. Although crystal contacts stabilize an open active site in the B molecule, the A molecule displays a closed conformation, with some differences depending on the ligands bound. An inhibition study with fosmidomycin resulted in an estimated IC(50) value of 80 nm. The double mutant enzyme (D151N/E222Q) has lost its ability to bind the metal and, thereby, also its activity. Our structural information complemented with molecular dynamics simulations and free energy calculations provides the framework for the design of new inhibitors and gives new insights into the reaction mechanism. The conformation of fosmidomycin bound to the metal ion is different from that reported in a previously published structure and indicates that a rearrangement of the intermediate is not required during catalysis.     

Inter-joint coupling effects on muscle contributions to endpoint force and acceleration in a musculoskeletal model of the cat hindlimb

The biomechanical principles underlying the organization of muscle activation patterns during standing balance are poorly understood. The goal of this study was to understand the influence of biomechanical inter-joint coupling on endpoint forces and accelerations induced by the activation of individual muscles during postural tasks. We calculated induced endpoint forces and accelerations of 31 muscles in a 7 degree-of-freedom, three-dimensional model of the cat hindlimb. To test the effects of inter-joint coupling, we systematically immobilized the joints (excluded kinematic degrees of freedom) and evaluated how the endpoint force and acceleration directions changed for each muscle in 7 different conditions. We hypothesized that altered inter-joint coupling due to joint immobilization of remote joints would substantially change the induced directions of endpoint force and acceleration of individual muscles. Our results show that for most muscles crossing the knee or the hip, joint immobilization altered the endpoint force or acceleration direction by more than 90° in the dorsal and sagittal planes. Induced endpoint forces were typically consistent with behaviorally observed forces only when the ankle was immobilized. We then activated a proximal muscle simultaneous with an ankle torque of varying magnitude, which demonstrated that the resulting endpoint force or acceleration direction is modulated by the magnitude of the ankle torque. We argue that this simple manipulation can lend insight into the functional effects of co-activating muscles. We conclude that inter-joint coupling may be an essential biomechanical principle underlying the coordination of proximal and distal muscles to produce functional endpoint actions during motor tasks.     

Building blood vessels--stem cell models in vascular biology

Spheroids of differentiating embryonic stem cells, denoted embryoid bodies, constitute a high-quality model for vascular development, particularly well suited for loss-of-function analysis of genes required for early embryogenesis. This review examines vasculogenesis and angiogenesis in murine embryoid bodies and discusses the promise of stem cell-based models for the study of human vascular development.     

Organic dust-induced activation, adhesion to substrate and expression of intercellular adhesion molecules in THP-1 monocytes

Inhalation of organic dust in a swine confinement building induces systemic reactions, increased bronchial responsiveness and intense airway inflammation in previously unexposed, healthy subjects. These effects are self-limiting, but chronic respiratory symptoms are frequently observed in swine confinement workers. The present study was aimed at investigating organic dust-induced activation of the monocytic leukemia cell line, THP-1. Unstimulated THP-1 cells proliferate in suspension but cultivation for several days in medium with complete dust or 0.22-mu-filtered suspension, caused a subset of the THP-1 cells to adhere to the substratum. As assessed by transmission light- and indirect immunofluorescence microscopy, dust-stimulated adherent THP-1 cells adopted macrophage-like morphology and expressed vimentin. Intercellular adhesion molecule (ICAM)-1 was expressed in all dust-activated adherent cells, but only in 1% of the unstimulated cells in suspension. Sialoadhesin, a macrophage marker, was detected in dust-stimulated adherent THP-1 cells but not in the parental monocytes. Serum factors were required for the dust-induced expression of sialoadhesin, but not for adhesion to substrate or expression of ICAM-1. In addition, morphology and expression of vascular endothelial growth factor (VEGF) of dust-stimulated adherent cells equalled that of PMA-differentiated THP-1 cells, but the PMA-differentiated cells exhibited weak sialoadhesin labelling. In conclusion, exposure to organic dust from a swine confinement building activated a subset of THP-1 monocytes inducing expression of intercellular adhesion molecules, which are important in inflammation. The sustained adhesion to substrate indicates that organic dust from a swine confinement building may contain agents that prevent deactivation and detachment of the cells.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Investigation of electric current perception thresholds of different EHS groups

An increasing number of persons with health symptoms of unclear origin take refuge in the hypothesis that they suffer from electromagnetic hypersensitivity (EHS). So far EHS is not an accepted diagnosis and there is no validated test to verify the proposed relationship between electromagnetic fields and symptoms. Groups reporting EHS are very heterogeneous but share a belief that they have an increased sensitivity to electromagnetic fields. It was studied to which extent a quantitative indicator for electrosensitivity, the electric current perception threshold, and its variability coefficient, depend on the recruitment strategy for self-declared hypersensitive persons. Individual electrosensitivity was investigated by provocation of the lower arms to directly coupled 50 Hz electric currents. Self-declared EHS persons were selected from members of a self aid group, from responders to a newspaper call, and from persons actively asking for investigations in their search for help. It turned out that quantitative electrosensitivity was quite different among the three groups. It is interesting that the members of the EHS self aid group exhibit a considerable overlap with general population sample. Pooled together it could be shown that hypersensitive persons as a group differ significantly from the general population sample, however with a pronounced overlap with the normal range. It can be concluded that EHS groups are very inhomogeneous and contain numerous persons with no increased ability to perceive low frequency electric or magnetic fields. This investigation shows the importance of the study design, in particular of the recruitment strategies of EHS persons for the final outcome.     

Wood formation in urban Norway maple trees studied by the micro-coring method

Intra-annual wood formation in healthy and affected Norway maple (Acer platanoides L.) street trees in the City of Ljubljana, Slovenia, was studied by the micro-coring method. Samples were taken with a Trephor instrument at weekly intervals during the 2005 growing season. The beginning of wood formation corresponded to the initiation of cambial activity in the middle of April in all investigated Norway maple trees. In healthy trees, the period of wood formation was finished in mid-September, persisting 7 weeks longer than in affected ones. Small destructivity and sufficient quality of micro-cores confirmed that sampling with a Trephor is a convenient method for the study of wood formation in diffuse-porous hardwoods, growing in the harsh conditions of an urban environment.