Interphase chromosome positioning in in vitro porcine cells and ex vivo porcine tissues

Copyright @ 2012 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and 85 reproduction in any medium, provided the original author and source are credited. The article was made available through the Brunel University Open Access Publishing Fund.BACKGROUND: In interphase nuclei of a wide range of species chromosomes are organised into their own specific locations termed territories. These chromosome territories are non-randomly positioned in nuclei which is believed to be related to a spatial aspect of regulatory control over gene expression. In this study we have adopted the pig as a model in which to study interphase chromosome positioning and follows on from other studies from our group of using pig cells and tissues to study interphase genome re-positioning during differentiation. The pig is an important model organism both economically and as a closely related species to study human disease models. This is why great efforts have been made to accomplish the full genome sequence in the last decade. RESULTS: This study has positioned most of the porcine chromosomes in in vitro cultured adult and embryonic fibroblasts, early passage stromal derived mesenchymal stem cells and lymphocytes. The study is further expanded to position four chromosomes in ex vivo tissue derived from pig kidney, lung and brain. CONCLUSIONS: It was concluded that porcine chromosomes are also non-randomly positioned within interphase nuclei with few major differences in chromosome position in interphase nuclei between different cell and tissue types. There were also no differences between preferred nuclear location of chromosomes in in vitro cultured cells as compared to cells in tissue sections. Using a number of analyses to ascertain by what criteria porcine chromosomes were positioned in interphase nuclei; we found a correlation with DNA content.This study is partly supported by Sygen International PLC

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in $\textit{Caenorhabditis elegans}$. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense.A.C.B. was supported by an HFSP grant to E.A.M. (RPG0014/2015). This work was supported by Cancer Research UK (C13474/A18583, C6946/A14492), the Wellcome Trust (104640/Z/14/Z, 092096/Z/10/Z), and The European Research Council (ERC, grant 260688). The work of P.M. and X.Z. is supported by NIH grant R01GM113242 and NIH grant R01GM122080. R.M. was a Commonwealth Scholar, funded by the UK Government. J.M.C., A.N., and C.J.W. were supported by the CIHR (MOP-274660) and the Canada Research Chairs Program. A.I.L. was supported by a Wellcome Trust Programme Grant (108058/Z/15/Z) and M.L was supported by 2013/RSE/SCOTGOV/ MARIECURIE

Acidithiobacillus thiooxidans secretome containing a newly described lipoprotein Licanantase enhances chalcopyrite bioleaching rate

The nature of the mineral–bacteria interphase where electron and mass transfer processes occur is a key element of the bioleaching processes of sulfide minerals. This interphase is composed of proteins, metabolites, and other compounds embedded in extracellular polymeric substances mainly consisting of sugars and lipids (Gehrke et al., Appl Environ Microbiol 64(7):2743–2747, 1998). On this respect, despite Acidithiobacilli—a ubiquitous bacterial genera in bioleaching processes (Rawlings, Microb Cell Fact 4(1):13, 2005)—has long been recognized as secreting bacteria (Jones and Starkey, J Bacteriol 82:788–789, 1961; Schaeffer and Umbreit, J Bacteriol 85:492–493, 1963), few studies have been carried out in order to clarify the nature and the role of the secreted protein component: the secretome. This work characterizes for the first time the sulfur (meta)secretome of Acidithiobacillus thiooxidans strain DSM 17318 in pure and mixed cultures with Acidithiobacillus ferrooxidans DSM 16786, identifying the major component of these secreted fractions as a single lipoprotein named here as Licanantase. Bioleaching assays with the addition of Licanantase-enriched concentrated secretome fractions show that this newly found lipoprotein as an active protein additive exerts an increasing effect on chalcopyrite bioleaching rate

NCAM180 Regulates Ric8A Membrane Localization and Potentiates β-Adrenergic Response

Cooperation between receptors allows integrated intracellular signaling leading to appropriate physiological responses. The Neural Cell Adhesion Molecule (NCAM) has three main isoforms of 120, 140 and 180 kDa, with adhesive and signaling properties, but their respective functions remains to be fully identified. Here we show that the human NCAM180 intracellular domain is a novel interactor of the human guanosine exchange factor (GEF) Ric8A using the yeast two hybrid system and immunoprecipitation. Furthermore, NCAM, Ric8A and Gαs form a tripartite complex. Colocalization experiments by confocal microscopy revealed that human NCAM180 specifically induces the recruitment of Ric8A to the membrane. In addition, using an in vitro recombinant system, and in vivo by comparing NCAM knock-out mouse brain to NCAM heterozygous and wild type brains, we show that NCAM expression dose dependently regulates Ric8A redistribution in detergent resistent membrane microdomains (DRM). Previous studies have demonstrated essential roles for Ric8 in Gα protein activity at G protein coupled receptors (GPCR), during neurotransmitter release and for asymmetric cell division. We observed that inhibition of Ric8A by siRNA or its overexpression, decreases or increases respectively, cAMP production following β-adrenergic receptor stimulation. Furthermore, in human HEK293T recombinant cells, NCAM180 potentiates the Gαs coupled β-adrenergic receptor response, in a Ric8A dependent manner, whereas NCAM120 or NCAM140 do not. Finally, in mouse hippocampal neurons expressing endogenously NCAM, NCAM is required for the agonist isoproterenol to induce cAMP production, and this requirement depends on Ric8A. These data illustrate a functional crosstalk between a GPCR and an IgCAM in the nervous system

Granulomatous hepatitis due to Bartonella henselae infection in an immunocompetent patient

<p>Abstract</p> <p>Background</p> <p><it>Bartonella henselae </it>(<it>B. henselae</it>) is considered a rare cause of granulomatous hepatitis. Due to the fastidious growth characteristics of the bacteria, the limited sensitivity of histopathological stains, and the non-specific histological findings on liver biopsy, the diagnosis of hepatic bartonellosis can be difficult to establish. Furthermore, the optimal treatment of established hepatic bartonellosis remains controversial.</p> <p>Case presentation</p> <p>We present a case of hepatic bartonellosis in an immunocompetent woman who presented with right upper quadrant pain and a five cm right hepatic lobe mass on CT scan. The patient underwent a right hepatic lobectomy. Surgical pathology revealed florid necrotizing granulomatous hepatitis, favoring an infectious etiology. Despite extensive histological and serological evaluation a definitive diagnosis was not established initially. Thirteen months after initial presentation, hepatic bartonellosis was diagnosed by PCR studies from surgically excised liver tissue. Interestingly, the hepatic granulomas persisted and <it>Bartonella henselae </it>was isolated from the patient's enriched blood culture after several courses of antibiotic therapy.</p> <p>Conclusion</p> <p>The diagnosis of hepatic bartonellosis is exceedingly difficult to establish and requires a high degree of clinical suspicion. Recently developed, PCR-based approaches may be required in select patients to make the diagnosis. The optimal antimicrobial therapy for hepatic bartonellosis has not been established, and close follow-up is needed to ensure successful eradication of the infection.</p

Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections

Early In Vitro Differentiation of Mouse Definitive Endoderm Is Not Correlated with Progressive Maturation of Nuclear DNA Methylation Patterns

The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6), and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells. Utilizing this in vitro cell system as a model for assessing the relationship between differentiation and nuclear DNA methylation patterns, we found that differentiating cell populations display an increasing number of cells with a gain in DNA methylation load: first within their euchromatin, then extending into heterochromatic areas of the nucleus, which also results in significant changes of methylcytosine/global DNA codistribution patterns. We were also able to co-visualize and quantify the concomitant stochastic marker expression on a per-cell basis, for which we did not measure any correlation to methylcytosine loads or distribution patterns. We observe that the progression of global DNA methylation is not correlated with the standard transcription factors associated with endodermal development. Further studies are needed to determine whether the progression of global methylation could represent a useful signature of cellular differentiation. This concept of tracking epigenetic progression may prove useful in the selection of cell phenotypes for future regenerative medicine applications

Predictors of binge drinking in adolescents: ultimate and distal factors - a representative study

<p>Abstract</p> <p>Background</p> <p>As epidemiological surveys have shown, binge drinking is a constant and wide-spread problem behavior in adolescents. It is not rare to find that more than half of all adolescents engage in this behavior when assessing only the last 4 weeks of time independent of the urbanity of the region they live in. There have been several reviews on predictors of substance consumption in adolescents in general, but there has been less high quality research on predictors of binge drinking, and most studies have not been theoretically based. The current study aimed to analyze the ultimate and distal factors predicting substance consumption according to Petraitis' theory of triadic influence. We assessed the predictive value of these factors with respect to binge drinking in German adolescents, including the identification of influence direction.</p> <p>Methods</p> <p>In the years 2007/2008, a representative written survey of N = 44,610 students in the 9^thgrade of different school types in Germany was carried out (net sample). The return rate of questionnaires was 88% regarding all students whose teachers or school directors had agreed to participate in the study. In this survey, prevalence of binge drinking was investigated as well as potential predictors from the social/interpersonal, the attitudinal/environmental, and the intrapersonal fields (3 factors of Petraitis). In a multivariate logistic regression analysis, these variables were included after testing for multicollinearity in order to assess their ability to predict binge drinking.</p> <p>Results</p> <p>Prevalence of binge drinking in the last 30 days was 52.3% for the surveyed adolescents with a higher prevalence for boys (56.9%) than for girls (47.5%). The two most influential factors found to protect against binge drinking with <it>p </it>< .001 were low economic status and importance of religion. The four most relevant risk factors for binge drinking (<it>p </it>< .001) were life-time prevalence of school absenteeism/truancy, academic failure, suicidal thoughts, and violence at school in the form of aggressive behavior of teachers. The model of Petraitis was partly confirmed for Binge Drinking in German adolescents and the direction of influence factors was clarified.</p> <p>Conclusions</p> <p>Whereas some of the risk and protective factors for binge drinking are not surprising since they are known for substance abuse in general, there are two points that could be targeted in interventions that do not focus on adolescents alone: (a) training teachers in positive, reassuring behavior and constructive criticism and (b) a focus on high risk adolescents either because they have a lack of coping strategies when in a negative mood or because of their low academic achievement in combination with absenteeism from school.</p