Transient Receptor Potential Channel M4 and M5 in Magnocellular Cells in Rat Supraoptic and Paraventricular Nuclei

The neurohypophysial hormones, vasopressin (VP) and oxytocin (OT), are synthesised by magnocellular cells in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) of the hypothalamus. The release of VP into the general circulation from the neurohypophysis increases during hyperosmolality, hypotension and hypovolaemia. VP neurones increase hormone release by increasing their firing rate as a result of adopting a phasic bursting. Depolarising after potentials (DAPs) following a series of action potentials are considered to be involved in the generation of the phasic bursts by summating to plateau potentials. We recently discovered a fast DAP (fDAP) in addition to the slower DAP characterised previously. Almost all VP neurones expressed the fDAP, whereas only 16% of OT neurones had this property, which implicates the involvement of fDAP in the generation of the firing patterns in VP neurones. Our findings obtained from electrophysiological experiments suggested that the ionic current underlying the fDAP is mediated by those of two closely-related Ca 2+-activated cation channels: the melastatin-related subfamily of transient receptor potential channels, TRPM4 and TRPM5. In the present study, double/triple immunofluorescence microscopy and reverse transcriptase-polymerase chain reaction techniques were employed to evaluate whether TRPM4 and TRPM5 are specifically located in VP neurones. Using specific antibodies against these channels, TRPM5 immunoreactivity was found almost exclusively in VP neurones, but not in OT neurones in both the SON and PVN. The most prominent TRPM5 immunoreactivity was in the dendrites of VP neurones. By contrast, most TRPM4 immunoreactivity occurred in cell bodies of both VP and OT neurones. TRPM4 and TRPM5 mRNA were both found in a cDNA library derived from SON punches. These results indictate the possible involvement of TRPM5 in the generation of the fDAP, and these channels may play an important role in determining the distinct firing properties of VP neurones in the SON. © 2011 The Authors. Journal of Neuroendocrinology © 2011 Blackwell Publishing Ltd

Simulating the Mammalian Blastocyst - Molecular and Mechanical Interactions Pattern the Embryo

Mammalian embryogenesis is a dynamic process involving gene expression and mechanical forces between proliferating cells. The exact nature of these interactions, which determine the lineage patterning of the trophectoderm and endoderm tissues occurring in a highly regulated manner at precise periods during the embryonic development, is an area of debate. We have developed a computational modeling framework for studying this process, by which the combined effects of mechanical and genetic interactions are analyzed within the context of proliferating cells. At a purely mechanical level, we demonstrate that the perpendicular alignment of the animal-vegetal (a-v) and embryonic-abembryonic (eb-ab) axes is a result of minimizing the total elastic conformational energy of the entire collection of cells, which are constrained by the zona pellucida. The coupling of gene expression with the mechanics of cell movement is important for formation of both the trophectoderm and the endoderm. In studying the formation of the trophectoderm, we contrast and compare quantitatively two hypotheses: (1) The position determines gene expression, and (2) the gene expression determines the position. Our model, which couples gene expression with mechanics, suggests that differential adhesion between different cell types is a critical determinant in the robust endoderm formation. In addition to differential adhesion, two different testable hypotheses emerge when considering endoderm formation: (1) A directional force acts on certain cells and moves them into forming the endoderm layer, which separates the blastocoel and the cells of the inner cell mass (ICM). In this case the blastocoel simply acts as a static boundary. (2) The blastocoel dynamically applies pressure upon the cells in contact with it, such that cell segregation in the presence of differential adhesion leads to the endoderm formation. To our knowledge, this is the first attempt to combine cell-based spatial mechanical simulations with genetic networks to explain mammalian embryogenesis. Such a framework provides the means to test hypotheses in a controlled in silico environment

Characteristics of equine summer eczema with emphasis on differences between Finnhorses and Icelandic horses in a 11-year study

Summer eczema, allergic dermatitis of the horse, was studied on 275 affected horses in Finland in 1997–2007. Features of the horses, clinical signs of the disease and owners' opinions of aggravating factors were recorded. Differences, especially, between two of the native Scandinavian horse breeds, the Finnhorse and the Icelandic horse, were evaluated. The study was based on clinical examination and information from the owners. Of the horses, 50% were Finnhorses, 26% Icelandic horses and 24% consisted of different breeds of ponies and other horses. Of the Finnhorses, 76% had summer eczema by the age of 5 years, but in the Icelandic horses born in Finland the average age at onset was 7 years. The vast majority of the horses, 75%, had moderate clinical signs, while 16% showed severe and 9% mild. The severity of clinical signs did not depend on the duration of the disease nor was it related to the age at onset. The only linkage to severity was the breed of the horse or import from Iceland; New Forest ponies and imported Icelandic horses showed severe clinical signs significantly more often than Finnhorses. Of the owners, 38% regarded insects as the only aggravating factor, 24% mentioned several simultaneous factors, including grass fodder and sunlight, while 22% could not specify any. In Finland, a typical horse breed suffering from summer eczema is the Finnhorse and the characteristics of the disease are mainly uniform with the other breeds affected. Equine summer eczema seems to be aggravated by various combinations of environmental factors

Plasma-wall interaction on the divertor tiles of JET ITER-like wall from the viewpoint of micro/nanoscopic observations

Micro/nanoscopic observations on the surface of the divertor tiles used in the first campaign (2011-2012) of the JET tokamak with ITER-like Wall (JET ILW) have been carried out by means of several material analysis techniques. Previous results from the inner divertor were reported for a single poloidal section of the tile numbers 1, 3 and 4, i.e., upper, vertical and horizontal targets, respectively. The formation of the thick stratified mixed-material deposition layer on tiles 1 and 4, and erosion on tile 3 were identified. This study is mostly focused on the outer divertor: tiles 6, 7 and 8. In contrast to the inner tile, remarkable surface modifications have not been observed on the vertical target (tiles 7 and 8) where sputtering erosion and impurity deposition would have been almost balanced. Only a specific part of tile 6 (horizontal target) located near the exhaust channel was covered with a stratified ("geological-like") mixed-material deposition layer which mainly included Be and Ni with the thickness of similar to 2 mu m. Special feature of this mixed layer was that a certain amount of nitrogen (N) was clearly detected in the layer. Since the concentration of N varied with the depth position, it could be depended on the amount of that gas puffed for plasma edge cooling during the JET experimental campaign. In addition to the outer divertor tiles, a very interesting feature of the local erosion and deposition effects is reported in this paper.Peer reviewe

Generation of the Sotos syndrome deletion in mice

Haploinsufficiency of the human 5q35 region spanning the NSD1 gene results in a rare genomic disorder known as Sotos syndrome (Sotos), with patients displaying a variety of clinical features, including pre- and postnatal overgrowth, intellectual disability, and urinary/renal abnormalities. We used chromosome engineering to generate a segmental monosomy, i.e., mice carrying a heterozygous 1.5-Mb deletion of 36 genes on mouse chromosome 13 (4732471D19Rik-B4galt7), syntenic with 5q35.2–q35.3 in humans (Df(13)Ms2Dja(+/−) mice). Surprisingly Df(13)Ms2Dja(+/−) mice were significantly smaller for their gestational age and also showed decreased postnatal growth, in contrast to Sotos patients. Df(13)Ms2Dja(+/−) mice did, however, display deficits in long-term memory retention and dilation of the pelvicalyceal system, which in part may model the learning difficulties and renal abnormalities observed in Sotos patients. Thus, haploinsufficiency of genes within the mouse 4732471D19Rik–B4galt7 deletion interval play important roles in growth, memory retention, and the development of the renal pelvicalyceal system. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00335-012-9416-0) contains supplementary material, which is available to authorized users

Automatic Extraction of Nuclei Centroids of Mouse Embryonic Cells from Fluorescence Microscopy Images

Accurate identification of cell nuclei and their tracking using three dimensional (3D) microscopic images is a demanding task in many biological studies. Manual identification of nuclei centroids from images is an error-prone task, sometimes impossible to accomplish due to low contrast and the presence of noise. Nonetheless, only a few methods are available for 3D bioimaging applications, which sharply contrast with 2D analysis, where many methods already exist. In addition, most methods essentially adopt segmentation for which a reliable solution is still unknown, especially for 3D bio-images having juxtaposed cells. In this work, we propose a new method that can directly extract nuclei centroids from fluorescence microscopy images. This method involves three steps: (i) Pre-processing, (ii) Local enhancement, and (iii) Centroid extraction. The first step includes two variations: first variation (Variant-1) uses the whole 3D pre-processed image, whereas the second one (Variant-2) modifies the preprocessed image to the candidate regions or the candidate hybrid image for further processing. At the second step, a multiscale cube filtering is employed in order to locally enhance the pre-processed image. Centroid extraction in the third step consists of three stages. In Stage-1, we compute a local characteristic ratio at every voxel and extract local maxima regions as candidate centroids using a ratio threshold. Stage-2 processing removes spurious centroids from Stage-1 results by analyzing shapes of intensity profiles from the enhanced image. An iterative procedure based on the nearest neighborhood principle is then proposed to combine if there are fragmented nuclei. Both qualitative and quantitative analyses on a set of 100 images of 3D mouse embryo are performed. Investigations reveal a promising achievement of the technique presented in terms of average sensitivity and precision (i.e., 88.04% and 91.30% for Variant-1; 86.19% and 95.00% for Variant-2), when compared with an existing method (86.06% and 90.11%), originally developed for analyzing C. elegans images

Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos

<p>Abstract</p> <p>Background</p> <p>The use of genetically-encoded fluorescent proteins has revolutionized the fields of cell and developmental biology and in doing so redefined our understanding of the dynamic morphogenetic processes that shape the embryo. With the advent of more accessible and sophisticated imaging technologies as well as an abundance of fluorescent proteins with different spectral characteristics, the dynamic processes taking place <it>in situ </it>in living cells and tissues can now be probed. Photomodulatable fluorescent proteins are one of the emerging classes of genetically-encoded fluorescent proteins.</p> <p>Results</p> <p>We have compared PA-GFP, PS-CFP2, Kaede and KikGR four readily available and commonly used photomodulatable fluorescent proteins for use in ES cells and mice. Our results suggest that the green-to-red photoconvertible fluorescent protein, Kikume Green-Red (KikGR), is most suitable for cell labeling and lineage studies in ES cells and mice because it is developmentally neutral, bright and undergoes rapid and complete photoconversion. We have generated transgenic ES cell lines and strains of mice exhibiting robust widespread expression of KikGR. By efficient photoconversion of KikGR we labeled subpopulations of ES cells in culture, and groups of cells within <it>ex utero </it>cultured mouse embryos. Red fluorescent photoconverted cells and their progeny could be followed for extended periods of time.</p> <p>Conclusion</p> <p>Transgenic ES cells and mice exhibiting widespread readily detectable expression of KikGR are indistinguishable from their wild type counterparts and are amenable to efficient photoconversion. They represent novel tools for non-invasive selective labeling specific cell populations and live imaging cell dynamics and cell fate. Genetically-encoded photomodulatable proteins such as KikGR represent emergent attractive alternatives to commonly used vital dyes, tissue grafts and genetic methods for investigating dynamic behaviors of individual cells, collective cell dynamics and fate mapping applications.</p

Shared and Distinct Functions of the Transcription Factors IRF4 and IRF8 in Myeloid Cell Development

Interferon regulatory factor (IRF) 8 and IRF4 are structurally-related, hematopoietic cell-specific transcription factors that cooperatively regulate the differentiation of dendritic cells and B cells. Whilst in myeloid cells IRF8 is known to modulate growth and differentiation, the role of IRF4 is poorly understood. In this study, we show that IRF4 has activities similar to IRF8 in regulating myeloid cell development. The ectopic expression of IRF4 in myeloid progenitor cells in vitro inhibits cell growth, promotes macrophages, but hinders granulocytic cell differentiation. We also show that IRF4 binds to and activates transcription through the IRF-Ets composite sequence (IECS). Furthermore, we demonstrate that Irf8-/-Irf4-/- mice exhibit a more severe chronic myeloid leukemia (CML)-like disease than Irf8-/- mice, involving a disproportionate expansion of granulocytes at the expense of monocytes/macrophages. Irf4-/- mice, however, display no obvious abnormality in myeloid cell development, presumably because IRF4 is expressed at a much lower level than IRF8 in granulocyte-macrophage progenitors. Our results also suggest that IRF8 and IRF4 have not only common but also specific activities in myeloid cells. Since the expression of both the IRF8 and IRF4 genes is downregulated in CML patients, these results may add to our understanding of CML pathogenesis

Evolution and Survival on Eutherian Sex Chromosomes

Since the two eutherian sex chromosomes diverged from an ancestral autosomal pair, the X has remained relatively gene-rich, while the Y has lost most of its genes through the accumulation of deleterious mutations in nonrecombining regions. Presently, it is unclear what is distinctive about genes that remain on the Y chromosome, when the sex chromosomes acquired their unique evolutionary rates, and whether X-Y gene divergence paralleled that of paralogs located on autosomes. To tackle these questions, here we juxtaposed the evolution of X and Y homologous genes (gametologs) in eutherian mammals with their autosomal orthologs in marsupial and monotreme mammals. We discovered that genes on the X and Y acquired distinct evolutionary rates immediately following the suppression of recombination between the two sex chromosomes. The Y-linked genes evolved at higher rates, while the X-linked genes maintained the lower evolutionary rates of the ancestral autosomal genes. These distinct rates have been maintained throughout the evolution of X and Y. Specifically, in humans, most X gametologs and, curiously, also most Y gametologs evolved under stronger purifying selection than similarly aged autosomal paralogs. Finally, after evaluating the current experimental data from the literature, we concluded that unique mRNA/protein expression patterns and functions acquired by Y (versus X) gametologs likely contributed to their retention. Our results also suggest that either the boundary between sex chromosome strata 3 and 4 should be shifted or that stratum 3 should be divided into two strata

Effects of small interfering RNA targeting thymidylate synthase on survival of ACC3 cells from salivary adenoid cystic carcinoma

<p>Abstract</p> <p>Background</p> <p>Thymidylate synthase (TS) is an important target for chemotherapeutic treatment of cancer and high expression of TS has been associated with poor prognosis or refractory disease in several cancers including colorectal and head and neck cancer. Although TS is known to regulate cell cycles and transcription factors, its potency as a therapeutic target has not been fully explored in adenoid cystic carcinoma (ACC).</p> <p>Methods</p> <p>An ACC cell line (ACC3) was transfected with siRNA targeting the TS gene and inhibition of cell growth and induction of apoptosis-associated molecules were evaluated <it>in vitro</it>. In addition, the <it>in vivo </it>effect of TS siRNA on tumor progression was assessed using a xenograft model.</p> <p>Results</p> <p>Our results demonstrated that ACC3 cells showed significantly higher TS expression than non-cancer cell lines and the induction of TS siRNA led to inhibition of cell proliferation. The effect was associated with an increase in p53, p21, and active caspase-3 and S-phase accumulation. We also found up-regulation of spermidine/spermine N1-acetyltransferase (SSAT), a polyamine metabolic enzyme. Furthermore, treatment with TS siRNA delivered by atelocollagen showed a significant cytostatic effect through the induction of apoptosis in a xenograft model.</p> <p>Conclusion</p> <p>TS may be an important therapeutic target and siRNA targeting TS may be of potential therapeutic value in ACC.</p