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Lopez-Contreras A.J.,Genomic Instability Group | Ruppen I.,Proteomics Unit | Nieto-Soler M.,Genomic Instability Group | Murga M.,Genomic Instability Group | And 8 more authors.
Cell Reports | Year: 2013

DNA replication is facilitated by multiple factors that concentrate in the vicinity of replication forks. Here, we developed an approach that combines the isolation of proteins on nascent DNA chains with mass spectrometry (iPOND-MS), allowing a comprehensive proteomic characterization of the human replisome and replisome-associated factors. In addition to known replisome components, we provide a broad list of proteins that reside in the vicinity of the replisome, some of which were not previously associated with replication. For instance, our data support a link between DNA replication and the Williams-Beuren syndrome and identify ZNF24 as a replication factor. In addition, we reveal that SUMOylation is widespread for factors that concentrate near replisomes, which contrasts with lower UQylation levels at these sites. This resource provides a panoramic view of the proteins that concentrate in the surroundings of the replisome, which should facilitate future investigations on DNA replication and genome maintenance. © 2013 The Authors. Source

Nel A.E.,University of California at Los Angeles | Nel A.E.,Predictive Biology
Journal of Internal Medicine | Year: 2013

Nanotechnology introduces a new field that requires novel approaches and methods for hazard and risk assessment. For an appropriate scientific platform for safety assessment, nanoscale properties and functions of engineered nanomaterials (ENMs), including how the physicochemical properties of the materials relate to mechanisms of injury at the nano-bio interface, must be considered. Moreover, this rapidly advancing new field requires novel test strategies that allow multiple toxicants to be screened in robust, mechanism-based assays in which the bulk of the investigation can be carried out at the cellular and biomolecular level whilst maintaining limited animal use and is based on the contribution of toxicological pathways to the pathophysiology of disease. First, a predictive toxicological approach for the safety assessment of ENMs will be discussed against the background of a '21st-century vision' for using alternative test strategies (ATSs) to perform toxicological assessment of large numbers of untested chemicals, thereby reducing a backlog that could otherwise become a problem for nanotechnology. An ATS is defined here as an alternative to animal experiments or refinement/reduction alternative to traditional animal testing. Secondly, the approach of selecting pathways of toxicity to screen for the pulmonary hazard potential of carbon nanotubes and metal oxides will be discussed, as well as how to use these pathways to perform high-content or high-throughput testing and how the data can be used for hazard ranking, risk assessment, regulatory decision-making and 'safer-by-design' strategies. Finally, the utility and disadvantages of this predictive toxicological approach to ENM safety assessment, and how it can assist the 21st-century vision, will be addressed. © 2013 The Association for the Publication of the Journal of Internal Medicine. Source

Manes S.,CSIC - National Center for Biotechnology | Fuentes G.,Bioinformatics Institute A STAR | Peregil R.M.,CSIC - National Center for Biotechnology | Rojas A.M.,Predictive Biology | Lacalle R.A.,CSIC - National Center for Biotechnology
FASEB Journal | Year: 2010

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)-β participates in establishing polarity during leukocyte chemotaxis. Its final 83 amino acids localize PIP5KIβ to the uropod of chemotaxing neutrophils and T cells, and interact with ezrin-radixin-moesin (ERM) proteins and EBP50 (4.1-ERM-binding phosphoprotein 50), a scaffold protein with 2 PDZ (PSD-95, disc large, ZO-1) domains. The structural motifs at the PIP5KIβ C terminus that confer signaling specificity are, nonetheless, unknown. We show that the last 4 residues of PIP5KIβ constitute an atypical PDZ-binding motif, which steers PIP5KIβ to the uropod by binding to both EBP50 PDZ domains. Molecular modeling and mutagenesis indicated that PDZ-binding motif is necessary for PIP5KIβ localization and for chemoattractant-induced neutrophil polarization. Polarity in cells that express PIP5KIβ mutants lacking the PDZ-binding motif was restored by overexpression of PIP5KIβ, but not of PIP5KIγ-i2, another isoform that localizes to the neutrophil uropod. Our results identify an isoform-specific PDZ-binding motif in PIP5KIβ, which confers specificity for PIP5KIβ signaling at the uropod during leukocyte chemotaxis. © FASEB. Source

Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 438.91K | Year: 2012

DESCRIPTION (provided by applicant): Strain background can strongly influence the outcome of toxicity tests in animals. Time and cost constraints preclude in vivo approaches to longstanding concerns about the lack of genetic diversity in animal models. Anin vitro platform is vastly more efficient for understanding the role of genetic background in toxicology testing, and sacrifices no animals. In this project we propose to build a large panel of genetically diverse ES cells from the Diversity Outcross line of mice. The DO mice are an advanced intercross of the 8 founder strains used for the Collaborative Cross. The DO strains capture almost all the sequence variants in laboratory strains, and harbor hundreds to thousands more recombinational breakpoints than F2 mice. They ideally suited for facilitate complex trait mapping. ES cells offer tremendous flexibility and is also ideal for an in vitro genetics platform. DO ES cell lines immortalize DO genomes, creating a renewable resource. In principle, ES cellscan provide access to almost any cell type by directed differentiation. In this way, the DO ES panel is extremely versatile as a permanent, renewable resource. In Phase I, we will establish feasibility by deriving 90 independent, validated DO ES lines andestimate the genetic component of cytotoxicity observed for 15 model toxicant compounds in the Phase I panel. In Phase II we will complete the 600 ES line panel, identify compounds with a significant genetic component to cytotoxic response using a 1400 reference compound set, and map the loci mediating that genetic component in the identified compounds. PUBLIC HEALTH RELEVANCE: There is a longstanding interest in how genetics impacts toxicology, but cost effective, highly scalable tools to investigate this relationship were not available. This project will improve human health and reduce the risk of environmental toxicants by developing a high throughput in vitro platform for investigating the genetic basis of variable response to toxicant exposure.

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 990.71K | Year: 2015

DESCRIPTION provided by applicant We are interested in the genetic basis of drug induced cardiotoxicity and how these genetic factors may underlie serious adverse drug reactions that affect a small but significant fraction of individuals therapeutic circumstances o both We will conduct genomewide screens for factors affecting cardiomyocyte contractile deficit or arrhythmia caused by anthracycline and tyrosine kinase inhibitor induced contractile deficit or arrhythmia The ultimate research goal of this project is to discover novel mechanisms targets and chemical structures underlying variability in drug induced cardiotoxicity The proposed two stage approach is to first conduct high throughput genomewide association studies in vitro using genetically diverse mouse ES derived cardiomyocytes The genes found in those studies will be functionally validated in human IPS derived cardiomyocytes by reducing their expression via gene knockdown This approach takes advantage of the mapping power of mouse genetics with the biological relevance of human cell responses PUBLIC HEALTH RELEVANCE We are developing a high throughput platform for understanding how genes and drugs can interact to damage the heart This research will ultimately improve treatment of many diseases by reducing the likelihood of unexpected drug induced heart damage

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