Research Note: Jejunum phosphatases and systemic myo-inositol in broiler chickens fed without or with supplemented phytase.

As a constituent of animal cells, myo-inositol (MI) has been hypothesized to be crucial in several metabolic and regulatory pathways. Recently, it was shown that dietary phytase contributes to release of MI from phytate in the poultry digestive tract, increasing its systemic concentrations. This study investigated the activities of phosphatases in the jejunum and systemic plasma MI concentration in broilers not supplemented or supplemented with phytase through analyses based on modifications from commercial enzyme activity kits. Three hundred sixty male Ross 308 broilers were randomly allocated to 24 pens (15 birds per pen) in 4 dietary groups. The positive control group was fed with an adequate basal diet. The negative control group (NC) was fed with a reduced level of P and Ca. Groups Phy1500 and Phy3000 were fed with the NC diet plus 1,500 or 3,000 FTU of phytase per kilogram of feed, respectively. One bird per pen was selected for the measurement of jejunal phosphatase activity; MI concentration in plasma, the liver, and the kidney; and key MI enzyme concentrations (liver inositol monophosphatase 1 [IMPase 1] and kidney myo-inositol oxygenase [MIOX]). Endogenous phytase and alkaline phosphatase activity as well as IMPase 1 and MIOX expression were not statistically different among the dietary groups. The supplementation of 1500 FTU of phytase per kilogram of feed resulted in increase of plasma (P < 0.001) and kidney (P < 0.05) but not liver MI concentrations. The results indicated that systemic MI might reflect MI released from dietary sources; however, it did not appear to change expression of enzymes related to endogenous MI synthesis in the liver and catabolism in the kidney. New and larger studies are necessary to reach stronger evidence on the effects of dietary phytase on intestinal and systemic MI concentrations in broilers.

Arabidopsis phosphatidylinositol 4-phosphate 5-kinase 2 contains a functional nuclear localization sequence and interacts with alpha-importins.

The Arabidopsis phosphoinositide kinase PIP5K2 has been implicated in the control of membrane trafficking and is important for development and growth. In addition to cytosolic functions of phosphoinositides, a nuclear phosphoinositide system has been proposed, but evidence for nuclear phosphoinositides in plants is limited. Fluorescence-tagged variants of PIP5K2 reside in the nucleus of Arabidopsis root meristem cells, in addition to reported plasma membrane localization. Here we report on the interaction of PIP5K2 with alpha-importins and characterize its nuclear localization sequences (NLSs). The PIP5K2 sequence contains four putative NLSs (NLSa-NLSd) and only a PIP5K2 fragment containing NLSs is imported into nuclei of onion epidermis cells upon transient expression. PIP5K2 interacts physically with alpha-importin isoforms in cytosolic split-ubiquitin-based yeast two-hybrid tests, in dot-blot experiments and in immuno-pull-downs. A 27-amino-acid fragment of PIP5K2 containing NLSc is necessary and sufficient to mediate the nuclear import of a large cargo fusion consisting of two mCherry markers fused to RubisCO large subunit. Substitution of basic residues in NLSc results in reduced import of PIP5K2 or other cargoes into plant nuclei. The data suggest that PIP5K2 is subject to active, alpha-importin-mediated nuclear import, consistent with a nuclear role for PIP5K2 in addition to its reported cytosolic functions. The detection of both substrate and product of PIP5K2 in plant nuclei according to reporter fluorescence and immunofluorescence further supports the notion of a nuclear phosphoinositide system in plants. Variants of PIP5K2 with reduced nuclear residence might serve as tools for the future functional study of plant nuclear phosphoinositides.

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro.

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research.

Immunophenotyping of immune cell populations in the raccoon (Procyon lotor).

The raccoon (Procyon lotor) is a highly adaptable carnivore that has rapidly conquered Europe over the last decades and represents a potential candidate as pathogen reservoir, bearing the risk for transmission of infectious agents, as zoonosis or spill-over, to other wild life and domestic animals and man. Comprehensive investigations of infectious diseases in raccoons require a detailed knowledge of the participating immune cell populations. To close this gap of knowledge, various antibodies were tested for cross-reactivity with leukocytes in lymphoid organs and peripheral blood of raccoons using immunohistochemistry and flow cytometry, respectively. Eight out of 16 antibodies, directed against CD3, CD79α, Pax-5, IgG, CD44, MHC class II, myeloid/histiocyte antigen (MAC387), and Iba-1 exhibited a specific immunoreaction with cells in distinct anatomical compartments in formalin-fixed paraffin-embedded lymphoid tissues. Flow cytometric analysis revealed that 7 out of 18 antibodies directed against CD11c, CD14, CD21, CD44, CD79α, MHC class I and II cross-reacted with peripheral blood-derived raccoon leukocytes. Summarized, the usefulness of several cross-reacting antibodies was determined for the characterization of raccoon immune cells in immunohistochemistry and flow cytometry, offering the opportunity to study the raccoon immune system under normal and diseased conditions.

Passage-dependent morphological and phenotypical changes of a canine histiocytic sarcoma cell line (DH82 cells).

DH82 cells represent a permanent macrophage cell line isolated from a dog with histiocytic sarcoma (HS) and are commonly used in various fields of research upon infection and cancer, respectively. Despite its frequent use, data on cell surface antigen expression of this cell line are fragmentary and in part inconsistent. We therefore aimed at a detailed morphological and antigenic characterization of DH82 cells with respect to passage-dependent differences. Cellular morphology of early (≤ 13) and late (≥ 66) passages of DH82 cells was evaluated via scanning electron microscopy. Moreover, cells were labelled with 10 monoclonal antibodies directed against CD11c, CD14, CD18, CD44, CD45, CD80, CD86, MHC-I, MHC-II, and ICAM-1 for flow cytometric analysis. Early passage cells were characterized by round cell bodies with abundant small cytoplasmic projections whereas later passages exhibited a spindle-shaped morphology with large processes. The percentage of CD11c-, CD14-, CD18-, CD45-, and CD80 positive cells significantly decreased in late passages whereas the expression of CD44, CD86, MHC-I, MHC-II and ICAM-1 remained unchanged. DH82 cells represent a remarkably heterogeneous cell line with divergent antigenic and morphologic properties. The present findings have important implications for future studies, which should consider distinct characteristics with regard to the used passage.

Disentangling the effect of genes, the environment and chance on sex ratio variation in a wild bird population.

Sex ratio theory proposes that the equal sex ratio typically observed in birds and mammals is the result of natural selection. However, in species with chromosomal sex determination, the same 1 : 1 sex ratio is expected under random Mendelian segregation. Here, we present an analysis of 14 years of sex ratio data for a population of song sparrows (Melospiza melodia) on Mandarte Island, at the nestling stage and at independence from parental care. We test for the presence of variance in sex ratio over and above the binomial variance expected under Mendelian segregation, and thereby quantify the potential for selection to shape sex ratio. Furthermore, if sex ratio variation is to be shaped by selection, we expect some of this extra-binomial variation to have a genetic basis. Despite ample statistical power, we find no evidence for the existence of either genetic or environmentally induced variation in sex ratio, in the nest or at independence. Instead, the sex ratio variation observed matches that expected under random Mendelian segregation. Using one of the best datasets of its kind, we conclude that female song sparrows do not, and perhaps cannot, adjust the sex of their offspring. We discuss the implications of this finding and make suggestions for future research.