Atherosclerotic Plaque Destabilization in Mice: A Comparative Study

Atherosclerosis-associated diseases are the main cause of mortality and morbidity in western societies. The progression of atherosclerosis is a dynamic process evolving from early to advanced lesions that may become rupture-prone vulnerable plaques. Acute coronary syndromes are the clinical manifestation of life-threatening thrombotic events associated with high-risk vulnerable plaques. Hyperlipidemic mouse models have been extensively used in studying the mechanisms controlling initiation and progression of atherosclerosis. However, the understanding of mechanisms leading to atherosclerotic plaque destabilization has been hampered by the lack of proper animal models mimicking this process. Although various mouse models generate atherosclerotic plaques with histological features of human advanced lesions, a consensus model to study atherosclerotic plaque destabilization is still lacking. Hence, we studied the degree and features of plaque vulnerability in different mouse models of atherosclerotic plaque destabilization and find that the model based on the placement of a shear stress modifier in combination with hypercholesterolemia represent with high incidence the most human like lesions compared to the other models.     

Silver Staining of Ultrathin Sections of Tissues Embedded in Polyester Resin

Specimens 1 mm³ from rat liver and kidney were fixed for 50 min in cold (0-2° C) 1% OsO₄ in veronal-acetate buffer, pH 7.7, and containing 0.1% MgCl₂; then dehydrated and embedded in Vestopal-W. Sections were cut in two ranges, 0.1-2 µ and 60-90 mµ thick, and attached to slides by floating on water and drying at 60° C. The thicker ones, for light microscopy, were soaked in acetone 1.5-3 hr; the thinner, for electron microscopy, 20-30 min. Both kinds were stained by Wilder's (1935) method for reticulum. Those for light microscopy were finished by dehydrating, clearing and covering in the customary manner; those for electron microscopy, by coating with 1% parlodion, drying, cutting the film about 2 mm² around the section, and freeing the section by soaking in water. The section was then mounted on a grid. The structures stained are: nuclei, basement membrane of capillaries, reticulum fibers of the liver and kidney, and in addition, the basement membrane of the kidney tubules. The mitochondria, vesicles, endoplasmic reticulum and cell membranes were not defined.     

Correlation Between Degradation of Bacteriophage T2 Deoxyribonucleic Acid and the Resistance of Escherichia coli to Infection

The ability of certain strains of Escherichia coli to degrade T2 deoxyribonucleic acid to acid-soluble fragments is correlated with their high capacity to survive T2 infection.     

Discovery of exposure markers in urine for Brassica-containing meals served with different protein sources by UPLC-qTOF-MS untargeted metabolomics

An untargeted metabolomics approach has been applied to discover and identify exposure markers in urine for nine Nordic meals. A cross-over meal study was carried out in 17 subjects. The meals included a Pie, a Soup and a Barleyotto (pearl barley based risotto), each prepared with three protein sources; meat, fish or vegetarian. Urine samples were collected in different time intervals before and after intake of the test meals, covering a total of 24 h. The samples were analyzed by UPLC-qTOF-MS. Discriminating features for meals and protein sources were selected by use of double cross-validated partial least squares discriminant analysis and two additional validation steps: (1) time-course of excretion and (2) analysis of sensitivity and specificity. In addition, eight meal studies with single foods were carried out to investigate the food sources of the markers. In total 31 potential exposure markers (PEMs) of foods were found for the meals and protein sources. Fifteen of the 31 PEMs were also found in studies with single foods. Ten PEMs were identified or putatively annotated. Among the PEMs were a range of conjugated isothiocyanates from the Brassica oleracea species. Trimethylamine N-oxide was found as a fish marker. Additional unknown PEMs were found for chicory salad, parsley and fava beans, while other PEMs were dependent on the meal matrix rather than individual foods. The study demonstrates that it is possible to find PEMs in 24 h urine samples even when foods are given as part of a complex meal.     

A conceptual framework for the spatial analysis of landscape genetic data

Understanding how landscape heterogeneity constrains gene flow and the spread of adaptive genetic variation is important for biological conservation given current global change. However, the integration of population genetics, landscape ecology and spatial statistics remains an interdisciplinary challenge at the levels of concepts and methods. We present a conceptual framework to relate the spatial distribution of genetic variation to the processes of gene flow and adaptation as regulated by spatial heterogeneity of the environment, while explicitly considering the spatial and temporal dynamics of landscapes, organisms and their genes. When selecting the appropriate analytical methods, it is necessary to consider the effects of multiple processes and the nature of population genetic data. Our framework relates key landscape genetics questions to four levels of analysis: (i) node-based methods, which model the spatial distribution of alleles at sampling locations (nodes) from local site characteristics; these methods are suitable for modeling adaptive genetic variation while accounting for the presence of spatial autocorrelation. (ii) Link-based methods, which model the probability of gene flow between two patches (link) and relate neutral molecular marker data to landscape heterogeneity; these methods are suitable for modeling neutral genetic variation but are subject to inferential problems, which may be alleviated by reducing links based on a network model of the population. (iii) Neighborhood-based methods, which model the connectivity of a focal patch with all other patches in its local neighborhood; these methods provide a link to metapopulation theory and landscape connectivity modeling and may allow the integration of node- and link-based information, but applications in landscape genetics are still limited. (iv) Boundary-based methods, which delineate genetically homogeneous populations and infer the location of genetic boundaries; these methods are suitable for testing for barrier effects of landscape features in a hypothesis-testing framework. We conclude that the power to detect the effect of landscape heterogeneity on the spatial distribution of genetic variation can be increased by explicit consideration of underlying assumptions and choice of an appropriate analytical approach depending on the research question.     

Biomanipulation as an Application of Food-Chain Theory: Constraints, Synthesis, and Recommendations for Temperate Lakes

The aim of this review is to identify problems, find general patterns, and extract recommendations for successful biomanipulation. An important conclusion is that the pelagic food chain from fish to algae may not be the only process affected by a biomanipulation. Instead, this process should be viewed as the “trigger” for secondary processes, such as establishment of submerged macrophytes, reduced internal loading of nutrients, and reduced resuspension of particles from the sediment. However, fish reduction also leads to a high recruitment of young-of-the-year (YOY) fish, which feed extensively on zooplankton. This expansion of YOY the first years after fish reduction is probably a major reason for less successful biomanipulations. Recent, large-scale biomanipulations have made it possible to update earlier recommendations regarding when, where, and how biomanipulation should be performed. More applicable recommendations include (1) the reduction in the biomass of planktivorous fish should be 75% or more; (2) the fish reduction should be performed efficiently and rapidly (within 1–3 years); (3) efforts should be made to reduce the number of benthic feeding fish; (4) the recruitment of YOY fish should be reduced; (5) the conditions for establishment of submerged macrophytes should be improved; and (6) the external input of nutrients (phosphorus and nitrogen) should be reduced as much as possible before the biomanipulation. Recent biomanipulations have shown that, correctly performed, the method also achieves results in large, relatively deep and eutrophic lakes, at least in a 5-year perspective. Although repeated measures may be necessary, the general conclusion is that biomanipulation is not only possible, but also a relatively inexpensive and attractive method for management of eutrophic lakes, and in particular as a follow-up measure to reduced nutrient load. Received 14 April 1998; accepted 31 August 1998     

Fecundity selection does not vary along a large geographical cline of trait means in a passerine bird

Local environmental and ecological conditions are commonly expected to result in local adaptation, although there are few examples of variation in phenotypic selection across continent‐wide spatial scales. We collected standardized data on selection with respect to the highly variable plumage coloration of pied flycatcher (Ficedula hypoleuca Pall.) males from 17 populations across the species' breeding range. The observed selection on multiple male coloration traits via the annual number of fledged young was generally relatively weak. The main aim of the present study, however, was to examine whether the current directional selection estimates are associated with distance to the sympatric area with the collared flycatcher (Ficedula albicollis Temminck), a sister species with which the pied flycatcher is showing character displacement. This pattern was expected because plumage traits in male pied flycatchers are changing with the distance to these areas of sympatry. However, we did not find such a pattern in current selection on coloration. There were no associations between current directional selection on ornamentation and latitude or longitude either. Interestingly, current selection on coloration traits was not associated with the observed mean plumage traits of the populations. Thus, there do not appear to be geographical gradients in current directional fecundity selection on male plumage ornamentation. The results of the present study do not support the idea that constant patterns in directional fecundity selection would play a major role in the maintenance of coloration among populations in this species. By contrast, the tendency for relatively weak mosaic‐like variation in selection among populations could reflect just a snapshot of temporally variable, potentially environment‐dependent, selection, as suggested by other studies in this system. Such fine‐grained variable selection coupled with gene flow could maintain extensive phenotypic variation across populations. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 808–827.     

Intracellular localisation and innate immune responses following Francisella noatunensis infection of Atlantic cod (Gadus morhua) macrophages

The facultative intracellular bacterium Francisella noatunensis causes francisellosis in Atlantic cod (Gadus morhua), but little is known about its survival strategies or how these bacteria evade the host immune response. In this study we show intracellular localisation of F. noatunensis in cod macrophages using indirect immunofluorescence techniques and green fluorescent labelled bacteria. Transmission electron microscopy revealed that F. noatunensis was enclosed by a phagosomal membrane during the initial phase of infection. Bacteria were at a later stage of the infection found in large electron-lucent zones, apparently surrounded by a partially intact or disintegrated membrane. Immune electron microscopy demonstrated the release of bacterial derived vesicles from intracellular F. noatunensis, an event suspected of promoting phagosomal membrane degradation and allowing escape of the bacteria to cytoplasm. Studies of macrophages infected with F. noatunensis demonstrated a weak activation of the inflammatory response genes as measured by increased expression of the Interleukin (IL)-1β and IL-8. In comparison, a stronger induction of gene expression was found for the anti-inflammatory IL-10 indicating that the bacterium exhibits a role in down-regulating the inflammatory response. Expression of the p40 subunit of IL-12/IL-17 genes was highly induced during infection suggesting that F. noatunensis promotes T cell polarisation. The host macrophage responses studied here showed low ability to distinguish between live and inactivated bacteria, although other types of responses could be of importance for such discriminations. The immunoreactivity of F. noatunensis lipopolysaccharide (LPS) was very modest, in contrast to the strong capacity of Escherichia coli LPS to induce inflammatory responsive genes. These results suggest that F. noatunensis virulence mechanisms cover many strategies for intracellular survival in cod macrophages.     

Differential sensitivity of Ctenocephalides felis and Drosophila melanogaster nicotinic acetylcholine receptor α1 and α2 subunits in recombinant hybrid receptors to nicotinoids and neonicotinoid insecticides

Nicotinic acetylcholine receptors (nAChRs) are the binding sites for nicotinoid drugs, such as nicotine and epibatidine, and are the molecular targets of the selectively insecticidal neonicotinoids. In this study we report the full length cDNA cloning of the three Ctenocephalides (C.) felis (cat flea) nAChR α subunits Cfα1, Cfα2, and Cfα3. When expressed in Xenopus oocytes as hybrid receptors with the Gallus gallus (chicken) β2 (Ggβ2) subunit, these cat flea α subunits formed acetylcholine-responsive ion channels. Acetylcholine-evoked currents of Cfα2/Ggβ2 were resistant to α-bungarotoxin, while those of Cfα1/Ggβ2 were sensitive to this snake toxin. The pharmacological profiles of Cfα1/Ggβ2, Cfα2/Ggβ2 and the chicken neuronal receptor Ggα4/Ggβ2 for acetylcholine, two nicotinoids and 6 insecticidal neonicotinoids were determined and compared. Particularly remarkable was the finding that Cfα1/Ggβ2 was far more sensitive to acetylcholine, nicotine and neonicotinoid agonists than either Cfα2/Ggβ2 or Ggα4/Ggβ2: for the anti flea neonicotinoid market compound imidacloprid the respective EC??s were 0.02 μM, 1.31 μM and 10 μM. These results were confirmed for another insect species, Drosophila melanogaster, where the pharmacological profile of the Dmα1 and Dmα2 subunits as hybrid receptors with Ggβ2 in Xenopus oocyte expressions resulted in a similar sensitivity pattern as those identified for the C. felis orthologs. Our results show that at least in a Ggβ2 hybrid receptor setting, insect α1 subunits confer higher sensitivity to neonicotinoids than α2 subunits, which may contribute in vivo to the insect-selective action of this pesticide class.     

Peptidomic profiling of human cerebrospinal fluid identifies YPRPIHPA as a novel substrate for prolylcarboxypeptidase

Prolylcarboxypeptidase (PRCP) is a serine protease that catalyzes the cleavage of C‐terminal amino acids linked to proline in peptides. It is ubiquitously expressed and is involved in regulating blood pressure, proliferation, inflammation, angiogenesis, and weight maintenance. To identify the candidate proximal target engagement markers for PRCP inhibition in the central nervous system, we profiled the peptidome of human cerebrospinal fluid to look for PRCP substrates using a MS‐based in vitro substrate profiling assay. These experiments identified a single peptide, with the sequence YPRPIHPA, as a novel substrate for PRCP in human cerebrospinal fluid. The peptide YPRPIHPA is from the extracellular portion of human endothelin B receptor‐like protein 2.