A high-throughput in vivo micronucleus assay for genome instability screening in mice.

We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labor-intensive, microscopy-based techniques. Here we describe a 2-d, 96-well plate-based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 μl of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability.Acknowledgments We thank M. Hitcham and N. Harman for assistance with blood collections, W. Cheng for assistance with flow cytometry during high-throughput screening and K. Dry for comments on the manuscript. R.E.M. is supported by Cancer Research UK (CRUK; project grant C20510/A12401). D.J.A. is supported by CRUK. D.J.A. and B.L.N. are supported by the Wellcome Trust. Research in the Jackson Laboratory is funded by CRUK program grant no. C6/A11224, the European Research Council and the European Community Seventh Framework Programme grant agreement no. HEALTH-F2-2010-259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. G.B. is funded by CRUK program grant no. C6/A11224.This is the accepted manuscript for a paper published in Nature Protocols 10, 205–215 (2015) doi:10.1038/nprot.2015.010, Published online 31 December 201

Bone marrow cell derived arginase I is the major source of allergen-induced lung arginase but is not required for airway hyperresponsiveness, remodeling and lung inflammatory responses in mice

<p>Abstract</p> <p>Background</p> <p>Arginase is significantly upregulated in the lungs in murine models of asthma, as well as in human asthma, but its role in allergic airway inflammation has not been fully elucidated in mice.</p> <p>Results</p> <p>In order to test the hypothesis that arginase has a role in allergic airway inflammation we generated arginase I-deficient bone marrow (BM) chimeric mice. Following transfer of arginase I-deficient BM into irradiated recipient mice, arginase I expression was not required for hematopoietic reconstitution and baseline immunity. Arginase I deficiency in bone marrow-derived cells decreased allergen-induced lung arginase by 85.8 ± 5.6%. In contrast, arginase II-deficient mice had increased lung arginase activity following allergen challenge to a similar level to wild type mice. BM-derived arginase I was not required for allergen-elicited sensitization, recruitment of inflammatory cells in the lung, and proliferation of cells. Furthermore, allergen-induced airway hyperresponsiveness and collagen deposition were similar in arginase-deficient and wild type mice. Additionally, arginase II-deficient mice respond similarly to their control wild type mice with allergen-induced inflammation, airway hyperresponsiveness, proliferation and collagen deposition.</p> <p>Conclusion</p> <p>Bone marrow cell derived arginase I is the predominant source of allergen-induced lung arginase but is not required for allergen-induced inflammation, airway hyperresponsiveness or collagen deposition.</p

Multi-membership gene regulation in pathway based microarray analysis

This article is available through the Brunel Open Access Publishing Fund. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Background: Gene expression analysis has been intensively researched for more than a decade. Recently, there has been elevated interest in the integration of microarray data analysis with other types of biological knowledge in a holistic analytical approach. We propose a methodology that can be facilitated for pathway based microarray data analysis, based on the observation that a substantial proportion of genes present in biochemical pathway databases are members of a number of distinct pathways. Our methodology aims towards establishing the state of individual pathways, by identifying those truly affected by the experimental conditions based on the behaviour of such genes. For that purpose it considers all the pathways in which a gene participates and the general census of gene expression per pathway. Results: We utilise hill climbing, simulated annealing and a genetic algorithm to analyse the consistency of the produced results, through the application of fuzzy adjusted rand indexes and hamming distance. All algorithms produce highly consistent genes to pathways allocations, revealing the contribution of genes to pathway functionality, in agreement with current pathway state visualisation techniques, with the simulated annealing search proving slightly superior in terms of efficiency. Conclusions: We show that the expression values of genes, which are members of a number of biochemical pathways or modules, are the net effect of the contribution of each gene to these biochemical processes. We show that by manipulating the pathway and module contribution of such genes to follow underlying trends we can interpret microarray results centred on the behaviour of these genes.The work was sponsored by the studentship scheme of the School of Information Systems, Computing and Mathematics, Brunel Universit

Targeting of NAT10 enhances healthspan in a mouse model of human accelerated aging syndrome.

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare, but devastating genetic disease characterized by segmental premature aging, with cardiovascular disease being the main cause of death. Cells from HGPS patients accumulate progerin, a permanently farnesylated, toxic form of Lamin A, disrupting the nuclear shape and chromatin organization, leading to DNA-damage accumulation and senescence. Therapeutic approaches targeting farnesylation or aiming to reduce progerin levels have provided only partial health improvements. Recently, we identified Remodelin, a small-molecule agent that leads to amelioration of HGPS cellular defects through inhibition of the enzyme N-acetyltransferase 10 (NAT10). Here, we show the preclinical data demonstrating that targeting NAT10 in vivo, either via chemical inhibition or genetic depletion, significantly enhances the healthspan in a Lmna G609G HGPS mouse model. Collectively, the data provided here highlights NAT10 as a potential therapeutic target for HGPS

Erratum: Author Correction: Identification of genes required for eye development by high-throughput screening of mouse knockouts.

[This corrects the article DOI: 10.1038/s42003-018-0226-0.]

Использование барий-стронциевого карбонатита при изготовлении сварочных флюсов на основе техногенных отходов металлургического производства

В данной работе рассмотрена возможность использования барий-стронциевого карбонатита при изготовлении сварочных флюсов на основе шлака производства силикомарганца, а так же на основе ковшевых электросталеплавильных шлаков, образованных при производстве рельсовых марок стали. В серии опытов в лабораторных условиях изготавливали и исследовали различные составы сварочных флюсов, были определены химические составы наплавленного металла, проведен металлографический анализ.In this paper the possibility of using barium-strontium carbonatite in the manufacture of welding fluxes on the basis of slag from the production of silicomanganese, and based on ladle steelmaking slags formed in the production of rail steel grades. In a series of experiments in the laboratory have produced and investigated different compositions of welding fluxes, were determined the chemical compositions of the weld metal metallographic analysis

Evidence synthesis as the basis for decision analysis: a method of selecting the best agricultural practices for multiple ecosystem services

Agricultural management practices have impacts not only on crops and livestock, but also on soil, water, wildlife, and ecosystem services. Agricultural research provides evidence about these impacts, but it is unclear how this evidence should be used to make decisions. Two methods are widely used in decision making: evidence synthesis and decision analysis. However, a system of evidence-based decision making that integrates these two methods has not yet been established. Moreover, the standard methods of evidence synthesis have a narrow focus (e.g., the effects of one management practice), but the standard methods of decision analysis have a wide focus (e.g., the comparative effectiveness of multiple management practices). Thus, there is a mismatch between the outputs from evidence synthesis and the inputs that are needed for decision analysis. We show how evidence for a wide range of agricultural practices can be reviewed and summarized simultaneously (“subject-wide evidence synthesis”), and how this evidence can be assessed by experts and used for decision making (“multiple-criteria decision analysis”). We show how these methods could be used by The Nature Conservancy (TNC) in California to select the best management practices for multiple ecosystem services in Mediterranean-type farmland and rangeland, based on a subject-wide evidence synthesis that was published by Conservation Evidence (www.conservationevidence.com). This method of “evidence-based decision analysis” could be used at different scales, from the local scale (farmers deciding which practices to adopt) to the national or international scale (policy makers deciding which practices to support through agricultural subsidies or other payments for ecosystem services). We discuss the strengths and weaknesses of this method, and we suggest some general principles for improving evidence synthesis as the basis for multi-criteria decision analysis