Transgenic quails reveal dynamic TCF/ß-catenin signaling during avian embryonic development.

The Wnt/ß-catenin signaling pathway is highly conserved throughout evolution, playing crucial roles in several developmental and pathological processes. Wnt ligands can act at a considerable distance from their sources and it is therefore necessary to examine not only the Wnt-producing but also the Wnt-receiving cells and tissues to fully appreciate the many functions of this pathway. To monitor Wnt activity, multiple tools have been designed which consist of multimerized Wnt signaling response elements (TCF/LEF binding sites) driving the expression of fluorescent reporter proteins (e.g. GFP, RFP) or of LacZ. The high stability of those reporters leads to a considerable accumulation in cells activating the pathway, thereby making them easily detectable. However, this makes them unsuitable to follow temporal changes of the pathway's activity during dynamic biological events. Even though fluorescent transcriptional reporters can be destabilized to shorten their half-lives, this dramatically reduces signal intensities, particularly when applied in vivo. To alleviate these issues, we developed two transgenic quail lines in which high copy number (12× or 16×) of the TCF/LEF binding sites drive the expression of destabilized GFP variants. Translational enhancer sequences derived from viral mRNAs were used to increase signal intensity and specificity. This resulted in transgenic lines efficient for the characterization of TCF/ß-catenin transcriptional dynamic activities during embryogenesis, including using in vivo imaging. Our analyses demonstrate the use of this transcriptional reporter to unveil novel aspects of Wnt signaling, thus opening new routes of investigation into the role of this pathway during amniote embryonic development.

Local Immune Changes in Early Stages of Inflammation and Carcinogenesis Correlate with the Collagen Scaffold Changes of the Colon Mucosa.

Continuous activation of the immune system inside a tissue can lead to remodelling of the tissue structure and creation of a specific microenvironment, such as during the tumour development. Chronic inflammation is a central player in stimulating changes that alter the tissue stroma and can lead to fibrotic evolution. In the colon mucosa, regulatory mechanisms, including TGF-ß1, avoid damaging inflammation in front of the continuous challenge by the intestinal microbiome. Inducing either DSS colitis or AOM colorectal carcinogenesis in AVN-Wistar rats, we evaluated at one month after the end of each treatment whether immunological changes and remodelling of the collagen scaffold were already in development. At this time point, we found in both models a general downregulation of pro-inflammatory cytokines and even of TGF-ß1, but not of IL-6. Moreover, we demonstrated by multi-photon microscopy the simultaneously presence of pro-fibrotic remodelling of the collagen scaffold, with measurable changes in comparison to the control mucosa. The scaffold was significantly modified depending on the type of induced stimulation. These results suggest that at one month after the end of the DSS or AOM inductions, a smouldering inflammation is present in both induced conditions, since the pro-inflammatory cytokines still exceed, in proportion, the local homeostatic regulation of which TGF-ß1 is a part (inflammatory threshold). Such an inflammation appears sufficient to sustain remodelling of the collagen scaffold that may be taken as a possible pathological marker for revealing pre-neoplastic inflammation.

Bacteriophage uptake by mammalian cell layers represents a potential sink that may impact phage therapy.

It is increasingly apparent that bacteriophages, viruses that infect bacteria and more commonly referred to as simply phages, have tropisms outside their bacterial hosts. Using live tissue culture cell imaging, we demonstrate that cell type, phage size, and morphology play a major role in phage internalization. Uptake was validated under physiological conditions using a microfluidic device. Phages adhered to mammalian tissues, with adherent phages being subsequently internalized by macropinocytosis, with functional phages accumulating intracellularly. We incorporated these results into a pharmacokinetic model demonstrating the potential impact of phage accumulation by cell layers, which represents a potential sink for circulating phages in the body. During phage therapy, high doses of phages are directly administered to a patient in order to treat a bacterial infection, thereby facilitating broad interactions between phages and mammalian cells. Understanding these interactions will have important implications on innate immune responses, phage pharmacokinetics, and the efficacy of phage therapy.

Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

BACKGROUND: The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na+/K+-ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. METHODS: Na+/K+-ATPase level was determined by [3H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. RESULTS: Na+/K+-ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. CONCLUSIONS: Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na+/K+-ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. GENERAL SIGNIFICANCE: Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na+/K+-ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane.

Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by ß-cyclodextrin (ß-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in ß-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and ß-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

Flo11p, drug efflux pumps, and the extracellular matrix cooperate to form biofilm yeast colonies.

Much like other microorganisms, wild yeasts preferentially form surface-associated communities, such as biofilms and colonies, that are well protected against hostile environments and, when growing as pathogens, against the host immune system. However, the molecular mechanisms underlying the spatiotemporal development and environmental resistance of biofilms and colonies remain largely unknown. In this paper, we show that a biofilm yeast colony is a finely tuned, complex multicellular organism in which specialized cells jointly execute multiple protection strategies. These include a Pdr1p-regulated mechanism whereby multidrug resistance transporters Pdr5p and Snq2p expel external compounds solely within the surface cell layers as well as developmentally regulated production by internal cells of a selectively permeable extracellular matrix. The two mechanisms act in concert during colony development, allowing growth of new cell generations in a well-protected internal cavity of the colony. Colony architecture is strengthened by intercellular fiber connections.

Visualization of Reinke's crystals in normal and cryptorchid testis.

Within the human testis, Reinke's crystals are found in Leydig cells but their nature and function are poorly understood. The aim of our study was to investigate the properties of Reinke's crystals in man with the normal morphology of the testis (control group) and infertile patients diagnosed with cryptorchidism. 20 biopsies from infertile patients and six biopsies from men with regular spermatogenesis (20-30 years.) were used. Sections of the testis tissue were stained with haematoxylin and eosin and a modified Masson's method. Specimens were observed by bright field, confocal and transmission electron microscopy (TEM). The number of Reinke's crystals in investigated groups was determined applying stereological methods. In both groups, Reinke's crystals were noted within the cytoplasm and nuclei of Leydig cells. Some "free" crystals were found within the interstitial space, outside Leydig cells. Confocal microscopy proved to be very useful in the assessment of the shape and 3D reconstruction of the crystal. TEM analysis confirmed a hexagonal form of the crystal, while crystallographic data on sections of 70-300 nm thickness provided a better insight into the organization of the crystal lattice. Stereological analysis revealed a significant increase in the number of crystals in cryptorchid testes when compared with controls. Increased number of crystals in cryptorchid specimens leads to the assumption that the prolonged exposure to higher (abdominal) temperature might stimulate enzymes involved in the synthesis of the proteins of the crystal. However, the exact molecular nature of the crystal lattice remains in both normal and cryptorchid testis obscure.

Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.

Yeasts, when growing on solid surfaces, form organized multicellular structures, colonies, in which cells differentiate and thus possess different functions and undergo dissimilar fate. Understanding the principles involved in the formation of these structures requires new approaches that allow the study of individual cells directly in situ without needing to remove them from the microbial community. Here we introduced a new approach to the analysis of whole yeast microcolonies either containing specific proteins labelled by fluorescent proteins or stained with specific dyes, by two-photon excitation confocal microscopy. It revealed that the colonies are covered with a thin protective skin-like surface cell layer which blocks penetration of harmful compounds. The cells forming the layer are tightly connected via cell walls, the presence of which is essential for keeping of protective layer function. Viewing the colonies from different angles allowed us to reconstruct a three-dimensional profile of the cells producing ammonium exporter Ato1p within developing microcolonies growing either as individuals or within a group of microcolonies. We show that neighbouring microcolonies coordinate production of Ato1p-GFP. Ato1p itself appears synchronously in cells, which do not originate from the same ancestor, but occupy specific position within the colony.

Imaging of mouse experimental melanoma in vivo and ex vivo by combination of confocal and nonlinear microscopy.

We investigated possibilities of the combination of the one- and two-photon excitation microscopy for examination of the experimental melanoma tissue in vivo, in mice under general anesthesia, and ex vivo on freshly harvested specimens. Our aim was to obtain sufficiently informative images of unstained tumor tissues and their modifications after hyperthermia treatment. The mouse experimental melanoma structure was studied and compared with normal tissue from the same animal by using confocal and nonlinear microscopy techniques based on (i) one-photon excitation (1PE) fluorescence, (ii) 1PE reflectance, (iii) second harmonic generation imaging, and (iv) two-photon excitation autofluorescence. We checked different spectral conditions and other settings of image acquisition, as well as combinations of the above imaging modalities, to fully exploit the potential of these techniques in the evaluation of treated and untreated cancer tissue morphology. Our approach enabled to reveal the collagen fiber network in relation with the other tissues, and to identify invasive tumor cells. It also proved to be useful for the examination of interrelationships between functional and morphological aspects based on optical properties of the tissues, especially in studies of changes between the tumor and control tissue, as well as changes induced by physical treatments, e.g., delivery of microwave hyperthermia treatment. These differences were also evaluated quantitatively, when we found out that the maximum Euler-Poincaré characteristic reflects well the melanoma morphological structure. The results showed that the proposed investigative approach could be suitable also for a direct evaluation of tissue modifications induced by clinical interventions.