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Search Results: 1 - 10 of 301106 matches for " J. Timothy Westwood "
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A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods
Justin C Yarrow, Zachary E Perlman, Nicholas J Westwood, Timothy J Mitchison
BMC Biotechnology , 2004, DOI: 10.1186/1472-6750-4-21
Abstract: We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate.The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.When wounded or scratched, cell monolayers respond to the disruption of cell-cell contacts and an increased concentration of growth factors at the wound margin by healing the wound through a combination of proliferation and migration [1-3]; these processes reflect the behavior of individual cells as well as the properties of the cell sheet as a surrogate tissue. To perform a wound healing assay, a wound is typically introduced in a cell monolayer using an object such as a pipette tip or syringe needle and the assay is performed on an individual coverslip or in a multiwell plate. The monolayers recover and heal the wound in a process that can be observed over a timecourse of 3–24 hrs. The wound heals in a stereotyped fashion – cells polarize toward the wound, initiate protrusion, migrate, and close the wound. Progression of these events can be monitored by manuall
Whole-Genome Analysis Reveals That Active Heat Shock Factor Binding Sites Are Mostly Associated with Non-Heat Shock Genes in Drosophila melanogaster
Sarah E. Gonsalves,Alan M. Moses,Zak Razak,Francois Robert,J. Timothy Westwood
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0015934
Abstract: During heat shock (HS) and other stresses, HS gene transcription in eukaryotes is up-regulated by the transcription factor heat shock factor (HSF). While the identities of the major HS genes have been known for more than 30 years, it has been suspected that HSF binds to numerous other genes and potentially regulates their transcription. In this study, we have used a chromatin immunoprecipitation and microarray (ChIP-chip) approach to identify 434 regions in the Drosophila genome that are bound by HSF. We have also performed a transcript analysis of heat shocked Kc167 cells and third instar larvae and compared them to HSF binding sites. The heat-induced transcription profiles were quite different between cells and larvae and surprisingly only about 10% of the genes associated with HSF binding sites show changed transcription. There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites. Analysis of the locations of the HSF binding sites revealed that 57% were contained within genes with approximately 2/3rds of these sites being in introns. We also found that the insulator protein, BEAF, has enriched binding prior to HS to promoters of genes that are bound by HSF upon HS but that are not transcriptionally induced during HS. When the genes associated with HSF binding sites in promoters were analyzed for gene ontology terms, categories such as stress response and transferase activity were enriched whereas analysis of genes having HSF binding sites in introns identified those categories plus ones related to developmental processes and reproduction. These results suggest that Drosophila HSF may be regulating many genes besides the known HS genes and that some of these genes may be regulated during non-stress conditions.
Microarray Analysis of Gene Expression by Skeletal Muscle of Three Mouse Models of Kennedy Disease/Spinal Bulbar Muscular Atrophy
Kaiguo Mo,Zak Razak,Pengcheng Rao,Zhigang Yu,Hiroaki Adachi,Masahisa Katsuno,Gen Sobue,Andrew P. Lieberman,J. Timothy Westwood,D. Ashley Monks
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0012922
Abstract: Emerging evidence implicates altered gene expression within skeletal muscle in the pathogenesis of Kennedy disease/spinal bulbar muscular atrophy (KD/SBMA). We therefore broadly characterized gene expression in skeletal muscle of three independently generated mouse models of this disease. The mouse models included a polyglutamine expanded (polyQ) AR knock-in model (AR113Q), a polyQ AR transgenic model (AR97Q), and a transgenic mouse that overexpresses wild type AR solely in skeletal muscle (HSA-AR). HSA-AR mice were included because they substantially reproduce the KD/SBMA phenotype despite the absence of polyQ AR.
Genome-wide analysis of the maternal-to-zygotic transition in Drosophila primordial germ cells
Najeeb U Siddiqui, Xiao Li, Hua Luo, Angelo Karaiskakis, Huayun Hou, Thomas Kislinger, J Timothy Westwood, Quaid Morris, Howard D Lipshitz
Genome Biology , 2012, DOI: 10.1186/gb-2012-13-2-r11
Abstract: We purified PGCs from Drosophila embryos, defined their proteome and transcriptome, and assessed the content, scale and dynamics of their MZT. Transcripts encoding proteins that implement particular types of biological functions group into nine distinct expression profiles, reflecting coordinate control at the transcriptional and posttranscriptional levels. mRNAs encoding germ-plasm components and cell-cell signaling molecules are rapidly degraded while new transcription produces mRNAs encoding the core transcriptional and protein synthetic machineries. The RNA-binding protein Smaug is essential for the PGC MZT, clearing transcripts encoding proteins that regulate stem cell behavior, transcriptional and posttranscriptional processes. Computational analyses suggest that Smaug and AU-rich element binding proteins function independently to control transcript elimination.The scale of the MZT is similar in the soma and PGCs. However, the timing and content of their MZTs differ, reflecting the distinct developmental imperatives of these cell types. The PGC MZT is delayed relative to that in the soma, likely because relief of PGC-specific transcriptional silencing is required for zygotic genome activation as well as for efficient maternal transcript clearance.During early animal embryogenesis the maternal-to-zygotic transition (MZT) accomplishes two things: elimination of a subset of maternally encoded gene products and transcriptional activation of the zygote's genome. The scale of these two processes is very similar in all animals, with about a third of the maternal mRNAs being cleared from embryos and about a fifth of the zygote's genome being transcribed at high levels (reviewed in [1,2]). While the scale of these events is highly conserved, the choice of specific transcripts for either elimination or production differs extensively, probably because the vast differences in cellular and developmental processes in early embryos of different phyla impose distinct requirem
Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics
Maria DLA Jaime, Luis V Lopez-Llorca, Ana Conesa, Anna Y Lee, Michael Proctor, Lawrence E Heisler, Marinella Gebbia, Guri Giaever, J Timothy Westwood, Corey Nislow
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-267
Abstract: Three different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS.Overexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS’s mechanism of action is different from other commonly studied fungicides that target membranes, sugges
Epigenetic Regulation of Learning and Memory by Drosophila EHMT/G9a
Jamie M. Kramer,Korinna Kochinke,Merel A. W. Oortveld,Hendrik Marks,Daniela Kramer,Eiko K. de Jong,Zoltan Asztalos,J. Timothy Westwood,Hendrik G. Stunnenberg,Marla B. Sokolowski,Krystyna Keleman,Huiqing Zhou,Hans van Bokhoven,Annette Schenck
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1000569
Abstract: The epigenetic modification of chromatin structure and its effect on complex neuronal processes like learning and memory is an emerging field in neuroscience. However, little is known about the “writers” of the neuronal epigenome and how they lay down the basis for proper cognition. Here, we have dissected the neuronal function of the Drosophila euchromatin histone methyltransferase (EHMT), a member of a conserved protein family that methylates histone 3 at lysine 9 (H3K9). EHMT is widely expressed in the nervous system and other tissues, yet EHMT mutant flies are viable. Neurodevelopmental and behavioral analyses identified EHMT as a regulator of peripheral dendrite development, larval locomotor behavior, non-associative learning, and courtship memory. The requirement for EHMT in memory was mapped to 7B-Gal4 positive cells, which are, in adult brains, predominantly mushroom body neurons. Moreover, memory was restored by EHMT re-expression during adulthood, indicating that cognitive defects are reversible in EHMT mutants. To uncover the underlying molecular mechanisms, we generated genome-wide H3K9 dimethylation profiles by ChIP-seq. Loss of H3K9 dimethylation in EHMT mutants occurs at 5% of the euchromatic genome and is enriched at the 5′ and 3′ ends of distinct classes of genes that control neuronal and behavioral processes that are corrupted in EHMT mutants. Our study identifies Drosophila EHMT as a key regulator of cognition that orchestrates an epigenetic program featuring classic learning and memory genes. Our findings are relevant to the pathophysiological mechanisms underlying Kleefstra Syndrome, a severe form of intellectual disability caused by mutations in human EHMT1, and have potential therapeutic implications. Our work thus provides novel insights into the epigenetic control of cognition in health and disease.
Epigenetic Regulation of Learning and Memory by Drosophila EHMT/G9a
Jamie M. Kramer,Korinna Kochinke,Merel A. W. Oortveld,Hendrik Marks,Daniela Kramer,Eiko K. de Jong,Zoltan Asztalos,J. Timothy Westwood,Hendrik G. Stunnenberg,Marla B. Sokolowski,Krystyna Keleman,Huiqing Zhou,Hans van Bokhoven ,Annette Schenck
PLOS Biology , 2011, DOI: 10.1371/journal.pbio.1000569
Abstract: The epigenetic modification of chromatin structure and its effect on complex neuronal processes like learning and memory is an emerging field in neuroscience. However, little is known about the “writers” of the neuronal epigenome and how they lay down the basis for proper cognition. Here, we have dissected the neuronal function of the Drosophila euchromatin histone methyltransferase (EHMT), a member of a conserved protein family that methylates histone 3 at lysine 9 (H3K9). EHMT is widely expressed in the nervous system and other tissues, yet EHMT mutant flies are viable. Neurodevelopmental and behavioral analyses identified EHMT as a regulator of peripheral dendrite development, larval locomotor behavior, non-associative learning, and courtship memory. The requirement for EHMT in memory was mapped to 7B-Gal4 positive cells, which are, in adult brains, predominantly mushroom body neurons. Moreover, memory was restored by EHMT re-expression during adulthood, indicating that cognitive defects are reversible in EHMT mutants. To uncover the underlying molecular mechanisms, we generated genome-wide H3K9 dimethylation profiles by ChIP-seq. Loss of H3K9 dimethylation in EHMT mutants occurs at 5% of the euchromatic genome and is enriched at the 5′ and 3′ ends of distinct classes of genes that control neuronal and behavioral processes that are corrupted in EHMT mutants. Our study identifies Drosophila EHMT as a key regulator of cognition that orchestrates an epigenetic program featuring classic learning and memory genes. Our findings are relevant to the pathophysiological mechanisms underlying Kleefstra Syndrome, a severe form of intellectual disability caused by mutations in human EHMT1, and have potential therapeutic implications. Our work thus provides novel insights into the epigenetic control of cognition in health and disease.
Paediatric admissions to hospitals in the Cape Town Metro district: A survey
A Westwood, M Levin, J Hageman
South African Journal of Child Health , 2012,
Abstract: A point prevalence survey of 381 paediatric medical inpatients in the 11 public hospitals in Cape Town in November 2008 showed that 70% of them were in central hospitals, with 39.4% requiring level 3 (sub-specialist) care. Numbers of children in hospital and their levels of health care requirement did not vary by sub-district of residence. Seventy-seven per cent of patients were under 5 years of age; 5% were teenagers. Few patients changed level of care during admission, but 10% did not need to be in hospital at the time of review. Median length of stay was 4 days, with children with level 3 needs having the longest lengths of stay. An under-provision of level 1 beds was demonstrated. HIV infection had been identified in 12% of admissions. While children with level 3 problems were well catered for in terms of bed provision, level 1 and step-down/home care provision were deficient or inefficiently utilised.
Powder River Basin Coal: Powering America  [PDF]
Timothy J. Considine
Natural Resources (NR) , 2013, DOI: 10.4236/nr.2013.48063
Abstract:

Powder River Basin (PRB) coal in Wyoming and Montana is used to produce 18 percent of the electricity consumed in the United States. Coal production from the PRB more than doubled between 1994 and 2009. PRB coal companies produced greater amounts of coal at declining real prices over much of this period through investment in equipment and production systems that achieved massive economies of scale. The bulk of PRB coal is shipped to the middle part of America from Texas in the south to Michigan in the north and New York in the east. States that consume significant amounts of PRB coal have electricity rates well below the national average. The largest industrial users of electricity are in these regions. Replacing PRB coal would require almost 5.5 trillion cubic feet of natural gas per year, representing a 26 percent increase in demand. Such an increase in gas consumption would increase prices for natural gas by roughly 76 percent. In such a world, U.S. energy users would pay $107 billion more each year for electricity and natural gas. Hence, by using PRB coal, the U.S. economy avoids $107 billion per year in higher energy costs. Estimates reported in the literature indicate that the gross environmental damages from PRB coal production are $27 billion. Hence, the net social benefits of PRB coal are $80 billion per year. Given the large size and low cost of these reserves, PRB coal will likely supply societal energy needs well into the future as long as the public and their elected officials are willing to accept the environmental impacts in return for the substantial economic benefits from using PRB coal.

An Integrative Socio-Technical Enterprise Approach to Urban Design/Planning for Sustainable Development  [PDF]
Timothy J. Downs
Open Journal of Civil Engineering (OJCE) , 2018, DOI: 10.4236/ojce.2018.82015
Abstract: Human society locally and globally needs to better understand and respond to ever-more complex, interwoven problems: environmental degradation; climate instability; persistent poverty; disparities in human health; growing income/wealth inequality; economies and infrastructures vulnerable to climate shock; and mounting socio-political unrest. Cities are where most people live, urbanization is a strong upward global trend, and cities bring all these problems into sharp, compelling focus. Since outcomes stem from processes and systems, we argue transformative changes depend on re-imagining the Urban Design, Urban Planning and Urban Development Practice (UD/UP/UDP) process. While there has been insufficient attention to process innovation— with technological aspects tending to dominate UD/UP/UDP work—emerging systems views of cities, and disenchantment with existing modes are enabling. We propose an empirically based integrative frame to tackle recognized conundrums, and inform an adaptive UD/UP/UDP process—from concept through design, assessment, planning, implementation, project functioning and monitoring. The frame contemplates six domains (6-D): 1) Project ethos, concept, and framing; 2) sectors, topics, and issues; 3) Varying spatial and temporal scales; 4) Stakeholder interests, relationships and capacities; 5) Knowledge types, modes and methods; and 6) Socio-technical capacities and networks. The frame, process and outcomes constitute a socio-technical enterprise (STE) approach to UD/UP/UDP work, with implications for education, training, and professional practice. We highlight the pivotal role Integrators and Universities play, and the scalability of STE knowledge/capacity networks. The case of Greater Mexico City/Central Mexico Urban Region illustrates the utility of the approach in a hyper-complex, climate-change vulnerable regional context.
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