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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.


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.


Sul J.H.,University of California at Los Angeles | Han B.,Harvard University | Han B.,The Broad Institute of MIT and Harvard | Ye C.,The Broad Institute of MIT and Harvard | And 2 more authors.
PLoS Genetics | Year: 2013

Gene expression data, in conjunction with information on genetic variants, have enabled studies to identify expression quantitative trait loci (eQTLs) or polymorphic locations in the genome that are associated with expression levels. Moreover, recent technological developments and cost decreases have further enabled studies to collect expression data in multiple tissues. One advantage of multiple tissue datasets is that studies can combine results from different tissues to identify eQTLs more accurately than examining each tissue separately. The idea of aggregating results of multiple tissues is closely related to the idea of meta-analysis which aggregates results of multiple genome-wide association studies to improve the power to detect associations. In principle, meta-analysis methods can be used to combine results from multiple tissues. However, eQTLs may have effects in only a single tissue, in all tissues, or in a subset of tissues with possibly different effect sizes. This heterogeneity in terms of effects across multiple tissues presents a key challenge to detect eQTLs. In this paper, we develop a framework that leverages two popular meta-analysis methods that address effect size heterogeneity to detect eQTLs across multiple tissues. We show by using simulations and multiple tissue data from mouse that our approach detects many eQTLs undetected by traditional eQTL methods. Additionally, our method provides an interpretation framework that accurately predicts whether an eQTL has an effect in a particular tissue. © 2013 Sul et al.


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.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 474.29K | Year: 2016

DESCRIPTION provided by applicant Astrocytes are increasingly understood to be active players in the etiology and treatment of Major Depressive Disorders MDD or depression and offer a new biological area for development of novel antidepressant drugs The goal of this project is to understand the molecular mechanisms of action of antidepressant drugs on astrocytes The knowledge gained will create opportunities to identify novel targets for discovery of new antidepressant drugs and biomarkers that predict or stratify drug response We have developed an innovative high throughput in vitro platform for genomewide association studies IV GWAS of drug response In this project we will use a genetically diverse population of astrocytes differentiated from mouse embryonic stem cells IV GWAS vastly increases the number of genetic screens over conventional live animal or human GWAS Further the in vitro environment can be tightly controlled which increases statistical power and enables more precise experimental designs We will identify genes that modify phenotypes relevant to depression in astrocytes treated with antidepressant drugs We will test almost all of the antidepressant drugs in current use a set of compounds In Phase I we will pre screen the compounds and phenotypes to identify those exhibiting the largest genetic variability andquot Heritabilityandquot In Phase II we will conduct IV GWAS using cell lines to identify genes that regulate or predict variable response to drug treatment We will validate these findings in both mouse and human astrocytes PUBLIC HEALTH RELEVANCE A more complete understanding of the molecular pathways underlying Major Depressive Disorders MDD is a critical bottleneck in the development of new therapies for depression We want to understand the molecular mechanisms underlying how antidepressant drugs act on astrocytes the most abundant cell type in the brain This will drive discovery of new antidepressant drugs


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.92K | Year: 2016

Abstract Brain swelling is a serious complication of multiple disease conditions including liver failure metastatic tumors traumatic brain injury and ischemic stroke TBI and stroke afflict M and K persons per year in the US alone and is a costly health care burden and a devastating social burden Current treatments for brain swelling are limited and generally ineffective highlighting the dramatic unmet need for better therapeutics A better understanding of the molecular pathways and cellular mechanisms is sorely needed to identify new drug targets as well as more predictive biomarkers that can stratify patients for clinical treatment decisions The goal of this project is to identify new drug targets and biomarkers of response for cytotoxic edema of astrocytes We propose to use a novel and innovative technology that we have developed that will take an unbiased approach to functionally identifying the causal mediators of astrocyte swelling Our approach uses a large panel of genetically diverse astrocyte lines to identify the genes and pathways that mechanistically underlie cytotoxic edema In Phase I we will develop two high throughput kinetic assays for astrocyte swelling that are robust scalable and automatable for screening compounds that induce or block swelling An impedance based morphological assay will measure swelling and recovery and a rapid calcium flux assay will measure cationic influx In Phase II we will use these assays to screen genetically diverse astrocyte lines and then map and identify the genes that mediate response to inducers and blockers of cell swelling Validation of candidate target genes will be conducted in both human and mouse astrocytes Human genes that modify human astrocyte response to compound are the ultimate aims and end products of this project Those genes and sequence variants in the human population will also be evaluated as potential prognostic biomarkers using existing clinical data in retrospective analyses Brain swelling cerebral edema is a serious consequence of many disorders such as acute liver failure metastatic cancer stroke and traumatic brain injury A more complete understanding of the molecular pathways underlying cerebral edema is a critical bottleneck in the development of new therapies for brain swelling We want to understand the molecular mechanisms underlying swelling in astrocytes the most abundant cell type in the brain toward the discovery of more effective treatments


Grant
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


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 224.91K | Year: 2013

We propose to develop a high throughput in vitro assay to identify genetic factors affecting toxicant impact on neuronal differentiation. This project uses a novel genetic mapping resource developed at Predictive Biology, the Diversity Outbred (DO) Mouse ES Cell Panel. Using robust ES cell engineering methods, we will incorporate neuronal differentiation reporters into DO ES lines. Neuronal Stem Cell (NSC) lines will be derived from the panel of modified DO ES lines, and the NSC panel will be used to quantify neuronal differentiation after toxicant exposure. These data will be used to map loci and identify genes that mediate variable response to neurotoxicants. The project design prioritizes rapid, sensitive and highly scalable assay technology, and featuresa cassette approach to vector design to facilitate construction of multiple assay panels to track toxicant effects on specific types or classes of neurons and neuronal cells. This technology will enable primary screening of large numbers of compounds at HTS scale, and will be useful for prioritizing compounds for follow up studies in vitro and in vivo using both mouse and human cells as well as live animal models. The genes and toxicological pathways identified using this technology will ultimately improvehuman health by providing a more detailed mechanistic understanding of human developmental neurotoxicity, and will contribute to a genetically informed approach to estimating risk associated with exposure to neurotoxicants. PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: This project will ultimately improve human health by providing a more detailed understanding of developmental neurotoxicity, and will contribute to a genetically informed approach to estimating risk associated with exposure to neurotoxicants.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 199.13K | Year: 2014

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


Grant
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.

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