Publications by authors named "David J Baker"

45 Publications

Behavioural and neurochemical mechanisms underpinning the feeding-suppressive effect of GLP-1/CCK combinatorial therapy.

Mol Metab 2021 01 19;43:101118. Epub 2020 Nov 19.

MRC Metabolic Diseases Unit, University of Cambridge Metabolic Research Laboratories, WT-MRC Institute of Metabolic Science, University of Cambridge, Cambridge CB2 OQQ, UK. Electronic address:

Objectives: Combinatorial therapies are under intense investigation to develop more efficient anti-obesity drugs; however, little is known about how they act in the brain to produce enhanced anorexia and weight loss. The goal of this study was to identify the brain sites and neuronal populations engaged during the co-administration of GLP-1R and CCK1R agonists, an efficient combination therapy in obese rodents.

Methods: We measured acute and long-term feeding and body weight responses and neuronal activation patterns throughout the neuraxis and in specific neuronal subsets in response to GLP-1R and CCK1R agonists administered alone or in combination in lean and high-fat diet fed mice. We used PhosphoTRAP to obtain unbiased molecular markers for neuronal populations selectively activated by the combination of the two agonists.

Results: The initial anorectic response to GLP-1R and CCK1R co-agonism was mediated by a reduction in meal size, but over a few hours, a reduction in meal number accounted for the sustained feeding suppressive effects. The nucleus of the solitary tract (NTS) is one of the few brain sites where GLP-1R and CCK1R signalling interact to produce enhanced neuronal activation. None of the previously categorised NTS neuronal subpopulations relevant to feeding behaviour were implicated in this increased activation. However, we identified NTS/AP Calcrl neurons as treatment targets.

Conclusions: Collectively, these studies indicated that circuit-level integration of GLP-1R and CCK1R co-agonism in discrete brain nuclei including the NTS produces enhanced rapid and sustained appetite suppression and weight loss.
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http://dx.doi.org/10.1016/j.molmet.2020.101118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7720077PMC
January 2021

Soil Bacterial Communities Exhibit Strong Biogeographic Patterns at Fine Taxonomic Resolution.

mSystems 2020 Jul 21;5(4). Epub 2020 Jul 21.

School of Biological Sciences, Monash University, Clayton, VIC, Australia

Bacteria have been inferred to exhibit relatively weak biogeographic patterns. To what extent such findings reflect true biological phenomena or methodological artifacts remains unclear. Here, we addressed this question by analyzing the turnover of soil bacterial communities from three data sets. We applied three methodological innovations: (i) design of a hierarchical sampling scheme to disentangle environmental from spatial factors driving turnover; (ii) resolution of 16S rRNA gene amplicon sequence variants to enable higher-resolution community profiling; and (iii) application of the new metric zeta diversity to analyze multisite turnover and drivers. At fine taxonomic resolution, rapid compositional turnover was observed across multiple spatial scales. Turnover was overwhelmingly driven by deterministic processes and influenced by the rare biosphere. The communities also exhibited strong distance decay patterns and taxon-area relationships, with values within the interquartile range reported for macroorganisms. These biogeographical patterns were weakened upon applying two standard approaches to process community sequencing data: clustering sequences at 97% identity threshold and/or filtering the rare biosphere (sequences lower than 0.05% relative abundance). Comparable findings were made across local, regional, and global data sets and when using shotgun metagenomic markers. Altogether, these findings suggest that bacteria exhibit strong biogeographic patterns, but these signals can be obscured by methodological limitations. We advocate various innovations, including using zeta diversity, to advance the study of microbial biogeography. It is commonly thought that bacterial distributions show lower spatial variation than for multicellular organisms. In this article, we present evidence that these inferences are artifacts caused by methodological limitations. Through leveraging innovations in sampling design, sequence processing, and diversity analysis, we provide multifaceted evidence that bacterial communities in fact exhibit strong distribution patterns. This is driven by selection due to factors such as local soil characteristics. Altogether, these findings suggest that the processes underpinning diversity patterns are more unified across all domains of life than previously thought, which has broad implications for the understanding and management of soil biodiversity.
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http://dx.doi.org/10.1128/mSystems.00540-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7566276PMC
July 2020

Chronic glucokinase activator treatment activates liver Carbohydrate response element binding protein and improves hepatocyte ATP homeostasis during substrate challenge.

Diabetes Obes Metab 2020 11 7;22(11):1985-1994. Epub 2020 Jul 7.

Biosciences Institute, Newcastle University, Medical School, Newcastle upon Tyne, UK.

Aim: To test the hypothesis that glucokinase activators (GKAs) induce hepatic adaptations that alter intra-hepatocyte metabolite homeostasis.

Methods: C57BL/6 mice on a standard rodent diet were treated with a GKA (AZD1656) acutely or chronically. Hepatocytes were isolated from the mice after 4 or 8 weeks of treatment for analysis of cellular metabolites and gene expression in response to substrate challenge.

Results: Acute exposure of mice to AZD1656 or a liver-selective GKA (PF-04991532), before a glucose tolerance test, or challenge of mouse hepatocytes with GKAs ex vivo induced various Carbohydrate response element binding protein (ChREBP) target genes, including Carbohydrate response element binding protein beta isoform (ChREBP-β), Gckr and G6pc. Both glucokinase activation and ChREBP target gene induction by PF-04991532 were dependent on the chirality of the molecule, confirming a mechanism linked to glucokinase activation. Hepatocytes from mice treated with AZD1656 for 4 or 8 weeks had lower basal glucose 6-phosphate levels and improved ATP homeostasis during high substrate challenge. They also had raised basal ChREBP-β mRNA and AMPK-α mRNA (Prkaa1, Prkaa2) and progressively attenuated substrate induction of some ChREBP target genes and Prkaa1 and Prkaa2.

Conclusions: Chronic GKA treatment of C57BL/6 mice for 8 weeks activates liver ChREBP and improves the resilience of hepatocytes to compromised ATP homeostasis during high-substrate challenge. These changes are associated with raised mRNA levels of ChREBP-β and both catalytic subunits of AMP-activated protein kinase.
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http://dx.doi.org/10.1111/dom.14111DOI Listing
November 2020

Developing an Arrayed CRISPR-Cas9 Co-Culture Screen for Immuno-Oncology Target ID.

SLAS Discov 2020 07 6;25(6):581-590. Epub 2020 May 6.

Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, UK.

Immunotherapies including PD-L1 blockade have shown remarkable increases in the T cell-directed antitumor response; however, efficacy is seen only in a minority of patients. Recently, pooled CRISPR-Cas9 knockout (CRISPRn) screens in tumor/immune co-culture systems have identified a number of genes that confer resistance to T cell killing in pathways including antigen presentation and cytokine signaling, providing insight into tumor mechanisms that cause resistance to immunotherapies. The development of an arrayed CRISPRn screen in a tumor/immune co-culture system would allow the identification of novel targets for immuno-oncology, characterization of hits from pooled screens, and multiple assay endpoints to be measured per gene. Here, a small-scale arrayed CRISPRn screen was successfully developed to investigate the effects on a co-culture of T cells and Cas9-expressing PC9 lung adenocarcinoma cells modified to express anti-CD3 antibody on the cell surface (PC9-OKT3 T cell system). A focused CRISPRn library was designed to target genes involved in known resistance mechanisms (including antigen presentation, cytokine signaling, and apoptosis) as well as genes involved in immune synapse interactions. The viability of PC9 cells was assessed in two-dimensional adherent co-cultures via longitudinal imaging analysis. Knockout of epidermal growth factor receptor (EGFR) and PLK1 in tumor cells cultured alone or with T cells resulted in increased tumor cell death, as expected, whereas knockout of the test gene ICAM1 showed subtle donor-specific resistance to T cell killing. Taken together, these data provide proof of concept for arrayed CRISPRn screens in tumor/immune co-culture systems and warrant further investigation of in vitro co-culture models.
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http://dx.doi.org/10.1177/2472555220916457DOI Listing
July 2020

A lack of genetically compatible mates constrains the spread of an invasive weed.

New Phytol 2020 06 23;226(6):1864-1872. Epub 2020 Mar 23.

School of Biological Sciences, Monash University, Building 18, Clayton, Vic., 3800, Australia.

Introduced populations often experience lag times before invasion, but the mechanisms constraining rapid expansions of introduced populations are unclear. Solidago altissima is a North American native plant with highly invasive Japanese populations and introduced Australian populations that are not invasive despite the climatic and ecological suitability of the region. By contrasting Australian with Japanese populations, we tested the hypothesis that Australian population growth is limited by a lack of long-distance dispersal via seeds owing to a limited number of compatible mates. In the field, Australian populations rarely produced viable seeds. A cross-pollination experiment found that Australian plants are fertile, yet lack compatible mates within Australia. Genetic analysis revealed that Australian individuals descend from a small set of self-incompatible genetic clones, which explains the negligible seed set within Australia. Our results show that low genetic diversity, leading to mate incompatibility, inhibits invasiveness of Australian S.  altissima, and provides compelling evidence for genetic, rather than ecological, factors constraining invasion in Australia.
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http://dx.doi.org/10.1111/nph.16496DOI Listing
June 2020

Efficient development of stable and highly functionalised peptides targeting the CK2α/CK2β protein-protein interaction.

Chem Sci 2019 May 12;10(19):5056-5063. Epub 2019 Apr 12.

Department of Chemistry , University of Cambridge , Lensfield Road , CB2 1EW , Cambridge , UK . Email:

The discovery of new Protein-Protein Interaction (PPI) modulators is currently limited by the difficulties associated with the design and synthesis of selective small molecule inhibitors. Peptides are a potential solution for disrupting PPIs; however, they typically suffer from poor stability and limited tissue penetration hampering their wide spread use as new chemical biology tools and potential therapeutics. In this work, a combination of CuAAC chemistry, molecular modelling, X-ray crystallography, and biological validation allowed us to develop highly functionalised peptide PPI inhibitors of the protein CK2. The lead peptide, , prevents the formation of the holoenzyme assembly , slows down proliferation, induces apoptosis in cancer cells and is stable in human serum. could aid the development of novel CK2 inhibitors acting at the interface and help to fully understand the intracellular pathways involving CK2. Importantly, the approach adopted herein could be applied to many PPI targets and has the potential to ease the study of PPIs by efficiently providing access to functionalised peptides.
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http://dx.doi.org/10.1039/c9sc00798aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530537PMC
May 2019

Vaspin promotes insulin sensitivity of elderly muscle and is upregulated in obesity.

J Endocrinol 2019 Feb 1. Epub 2019 Feb 1.

S Jones, Institute of Inflammation and Ageing, University of Birmingham, Birmingham, B15 2WB, United Kingdom of Great Britain and Northern Ireland.

Adipokines have emerged as central mediators of insulin sensitivity and metabolism, in part due to the known association of obesity with metabolic syndrome disorders such as type 2 diabetes. Recent studies in rodents have identified the novel adipokine vaspin, as playing a protective role in inflammatory metabolic diseases by functioning to promote insulin sensitivity during metabolic stress. However, at present the skeletal muscle and adipose tissue expression of vaspin in humans is poorly characterised. Furthermore, the functional role of vaspin in skeletal muscle insulin sensitivity has not been studied. Since skeletal muscle is the major tissue for insulin-stimulated glucose uptake understanding the functional role of vaspin in human muscle insulin signalling is critical in determining its role in glucose homeostasis. The objective of this study was to profile the skeletal muscle and subcutaneous adipose tissue expression of vaspin in humans of varying adiposity and to determine the functional role of vaspin in mediating insulin signalling and glucose uptake in human skeletal muscle. Our data shows that vaspin is secreted from both human subcutaneous adipose tissue and skeletal muscle, and is more highly expressed in obese older individuals compared to lean older individuals. Furthermore, we demonstrate that vaspin induces activation of the PI3K/AKT axis, independent of insulin receptor activation, promotes GLUT4 expression and translocation and sensitises older obese human skeletal muscle to insulin-mediated glucose uptake.
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http://dx.doi.org/10.1530/JOE-18-0528DOI Listing
February 2019

Conserving the abundance of nonthreatened species.

Conserv Biol 2019 04 24;33(2):319-328. Epub 2018 Oct 24.

School of Biological Sciences, Monash University, Clayton, VIC, 3800, Australia.

Human modification of the environment is driving declines in population size and distributional extent of much of the world's biota. These declines extend to many of the most abundant and widespread species, for which proportionally small declines can result in the loss of vast numbers of individuals, biomass, and interactions. These losses could have major localized effects on ecological and cultural processes and services without elevating a species' global extinction risk. Although most conservation effort is directed at species threatened with extinction in the very near term, the value of retaining abundance regardless of global extinction risk is justifiable based on many biodiversity or ecosystem service metrics, including cultural services, at scales from local to global. The challenges of identifying conservation priorities for widespread and abundant species include quantifying the effects of species' abundance on services and understanding how these effects are realized as populations decline. Negative effects of population declines may be disconnected from the threat processes driving declines because of species movements and environment flows (e.g., hydrology). Conservation prioritization for these species shares greater similarity with invasive species risk assessments than extinction risk assessments because of the importance of local context and per capita effects of abundance on other species. Because conservation priorities usually focus on preventing the extinction of threatened species, the rationale and objectives for incorporating declines of nonthreatened species must be clearly articulated, going beyond extinction risk to encompass the range of likely harmful effects (e.g., secondary extinctions, loss of ecosystem services) if declines persist or are not reversed. Research should focus on characterizing the effects of local declines in species that are not threatened globally across a range of ecosystem services and quantifying the spatial distribution of these effects through the distribution of abundance. The case for conserving abundance in nonthreatened species can be made most powerfully when the costs of losing this abundance are better understood.
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http://dx.doi.org/10.1111/cobi.13197DOI Listing
April 2019

The role of adipokines in skeletal muscle inflammation and insulin sensitivity.

J Inflamm (Lond) 2018 9;15. Epub 2018 May 9.

1MRC-ARUK Centre for Musculoskeletal Ageing Research, Medical School, Queen Elizabeth Hospital, University of Birmingham, Birmingham, B15 2WB UK.

Background: There is currently an unmet clinical need to develop better pharmacological treatments to improve glucose handling in Type II Diabetes patients with obesity. To this end, determining the effect of obesity-associated adipokines on skeletal muscle insulin sensitivity has emerged as an important area of drug discovery research. This review draws together the data on the functional role of adipokines on skeletal muscle insulin signalling, highlights several understudied novel adipokines and provides a perspective on the direction of future research.

Main Body: The adipokines leptin, resistin, visfatin and adiponectin have all been shown to affect skeletal muscle insulin sensitivity by impacting on the activity of components within insulin signalling pathways, affecting GLUT4 translocation and modulating insulin-mediated skeletal muscle glucose uptake. Furthermore, proteomic analysis of the adipose tissue secretome has recently identified several novel adipokines including vaspin, chemerin and pref-1 that are associated with obesity and insulin resistance in humans and functionally impact on insulin signalling pathways. However, predominantly, these functional findings are the result of studies in rodents, with in vitro studies utilising either rat L6 or murine C2C12 myoblasts and/or myotubes. Despite the methodology to isolate and culture human myoblasts and to differentiate them into myotubes being established, the use of human muscle in vitro models for the functional validation of adipokines on skeletal muscle insulin sensitivity is limited.

Conclusion: Understanding the mechanism of action and function of adipokines in mediating insulin sensitivity in skeletal muscle may lead to the development of novel therapeutics for patients with type 2 diabetes. However, to date, studies conducted in human skeletal muscle cells and tissues are limited. Such human in vitro studies should be prioritised in order to reduce the risk of candidate drugs failing in the clinic due to the assumption that rodent skeletal muscle target validation studies will to translate to human.
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http://dx.doi.org/10.1186/s12950-018-0185-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944154PMC
May 2018

Molecular architecture of potassium chloride co-transporter KCC2.

Sci Rep 2017 11 28;7(1):16452. Epub 2017 Nov 28.

CALIXAR, 60 avenue Rockefeller, 69008, Lyon, France.

KCC2 is a neuron specific K-Cl co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into "head" and "core" domains connected by a flexible "linker". Dimers are asymmetrical and display a bent "S-shape" architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.
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http://dx.doi.org/10.1038/s41598-017-15739-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705597PMC
November 2017

Development and characterisation of a novel glucagon like peptide-1 receptor antibody.

Diabetologia 2018 03 9;61(3):711-721. Epub 2017 Nov 9.

University of Cambridge Metabolic Research Laboratories, WT-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Hills Road, Cambridge, CB2 0QQ, UK.

Aims/hypothesis: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo.

Methods: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice.

Results: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs.

Conclusions/interpretation: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.
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http://dx.doi.org/10.1007/s00125-017-4491-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890879PMC
March 2018

-Ethylmaleimide increases KCC2 cotransporter activity by modulating transporter phosphorylation.

J Biol Chem 2017 12 1;292(52):21253-21263. Epub 2017 Nov 1.

From the AstraZeneca Tufts Laboratory for Basic and Translational Neuroscience, Boston, Massachusetts 02111.

K/Cl cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABA) receptors, which are Cl-permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which -ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.
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http://dx.doi.org/10.1074/jbc.M117.817841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5766942PMC
December 2017

Gut check on diabesity: leveraging gut mechanisms for the treatment of type 2 diabetes and obesity.

Curr Opin Pharmacol 2017 12 10;37:10-15. Epub 2017 Aug 10.

Cardiovascular and Metabolic Disease, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, UK. Electronic address:

Gut hormones have long been understood to regulate food intake and metabolism. Bariatric surgery significantly elevates circulating gut hormone levels and is proven to affect acute remission of type 2 diabetes before any weight loss is observed. Subsequent weight loss is accrued over weeks to months but is sustained into the long term. Hence, there exists great enthusiasm to recapitulate these changes in gut hormones in the form of novel combination drugs for type 2 diabetes and obesity.
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http://dx.doi.org/10.1016/j.coph.2017.07.010DOI Listing
December 2017

Perception of Leitmotives in Richard Wagner's Der Ring des Nibelungen.

Front Psychol 2017 4;8:662. Epub 2017 May 4.

Music, Mind and Brain Lab, Department of Psychology, Goldsmiths, University of LondonLondon, UK.

The music of Richard Wagner tends to generate very diverse judgments indicative of the complex relationship between listeners and the sophisticated musical structures in Wagner's music. This paper presents findings from two listening experiments using the music from Wagner's that explores musical as well as individual listener parameters to better understand how listeners are able to hear leitmotives, a compositional device closely associated with Wagner's music. Results confirm findings from a previous experiment showing that specific expertise with Wagner's music can account for a greater portion of the variance in an individual's ability to recognize and remember musical material compared to measures of generic musical training. Results also explore how acoustical distance of the leitmotives affects memory recognition using a chroma similarity measure. In addition, we show how characteristics of the compositional structure of the leitmotives contributes to their salience and memorability. A final model is then presented that accounts for the aforementioned individual differences factors, as well as parameters of musical surface and structure. Our results suggest that that future work in music perception may consider both individual differences variables beyond musical training, as well as symbolic features and audio commonly used in music information retrieval in order to build robust models of musical perception and cognition.
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http://dx.doi.org/10.3389/fpsyg.2017.00662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5415611PMC
May 2017

Use of CRISPR/Cas9-engineered INS-1 pancreatic β cells to define the pharmacology of dual GIPR/GLP-1R agonists.

Biochem J 2016 09 15;473(18):2881-91. Epub 2016 Jul 15.

Cardiovascular and Metabolic Disease, MedImmune, Granta Park, Cambridge CB21 6GH, U.K.

Dual-agonist molecules combining glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) activity represent an exciting therapeutic strategy for diabetes treatment. Although challenging due to shared downstream signalling pathways, determining the relative activity of dual agonists at each receptor is essential when developing potential novel therapeutics. The challenge is exacerbated in physiologically relevant cell systems expressing both receptors. To this end, either GIP receptors (GIPR) or GLP-1 receptors (GLP-1R) were ablated via RNA-guided clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonucleases in the INS-1 pancreatic β-cell line. Multiple clonal cell lines harbouring gene disruptions for each receptor were isolated and assayed for receptor activity to identify functional knockouts (KOs). cAMP production in response to GIPR or GLP-1R activation was abolished and GIP- or GLP-1-induced potentiation of glucose-stimulated insulin secretion (GSIS) was attenuated in the cognate KO cell lines. The contributions of individual receptors derived from cAMP and GSIS assays were confirmed in vivo using GLP-1R KO mice in combination with a monoclonal antibody antagonist of GIPR. We have successfully applied CRISPR/Cas9-engineered cell lines to determining selectivity and relative potency contributions of dual-agonist molecules targeting receptors with overlapping native expression profiles and downstream signalling pathways. Specifically, we have characterised molecules as biased towards GIPR or GLP-1R, or with relatively balanced potency in a physiologically relevant β-cell system. This demonstrates the broad utility of CRISPR/Cas9 when applied to native expression systems for the development of drugs that target multiple receptors, particularly where the balance of receptor activity is critical.
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http://dx.doi.org/10.1042/BCJ20160476DOI Listing
September 2016

Choice of baseline climate data impacts projected species' responses to climate change.

Glob Chang Biol 2016 07 19;22(7):2392-404. Epub 2016 Apr 19.

School of Biological & Biomedical Sciences, Durham University, Mountjoy Site, Durham, DH1 3LE, UK.

Climate data created from historic climate observations are integral to most assessments of potential climate change impacts, and frequently comprise the baseline period used to infer species-climate relationships. They are often also central to downscaling coarse resolution climate simulations from General Circulation Models (GCMs) to project future climate scenarios at ecologically relevant spatial scales. Uncertainty in these baseline data can be large, particularly where weather observations are sparse and climate dynamics are complex (e.g. over mountainous or coastal regions). Yet, importantly, this uncertainty is almost universally overlooked when assessing potential responses of species to climate change. Here, we assessed the importance of historic baseline climate uncertainty for projections of species' responses to future climate change. We built species distribution models (SDMs) for 895 African bird species of conservation concern, using six different climate baselines. We projected these models to two future periods (2040-2069, 2070-2099), using downscaled climate projections, and calculated species turnover and changes in species-specific climate suitability. We found that the choice of baseline climate data constituted an important source of uncertainty in projections of both species turnover and species-specific climate suitability, often comparable with, or more important than, uncertainty arising from the choice of GCM. Importantly, the relative contribution of these factors to projection uncertainty varied spatially. Moreover, when projecting SDMs to sites of biodiversity importance (Important Bird and Biodiversity Areas), these uncertainties altered site-level impacts, which could affect conservation prioritization. Our results highlight that projections of species' responses to climate change are sensitive to uncertainty in the baseline climatology. We recommend that this should be considered routinely in such analyses.
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http://dx.doi.org/10.1111/gcb.13273DOI Listing
July 2016

Lysosomal and phagocytic activity is increased in astrocytes during disease progression in the SOD1 (G93A) mouse model of amyotrophic lateral sclerosis.

Front Cell Neurosci 2015 15;9:410. Epub 2015 Oct 15.

Department of Neuroscience, Sheffield Institute for Translational Neuroscience, University of Sheffield Sheffield, UK.

Astrocytes are key players in the progression of amyotrophic lateral sclerosis (ALS). Previously, gene expression profiling of astrocytes from the pre-symptomatic stage of the SOD1(G93A) model of ALS has revealed reduced lactate metabolism and altered trophic support. Here, we have performed microarray analysis of symptomatic and late-stage disease astrocytes isolated by laser capture microdissection (LCM) from the lumbar spinal cord of the SOD1(G93A) mouse to complete the picture of astrocyte behavior throughout the disease course. Astrocytes at symptomatic and late-stage disease show a distinct up-regulation of transcripts defining a reactive phenotype, such as those involved in the lysosome and phagocytic pathways. Functional analysis of hexosaminidase B enzyme activity in the spinal cord and of astrocyte phagocytic ability has demonstrated a significant increase in lysosomal enzyme activity and phagocytic activity in SOD1(G93A) vs. littermate controls, validating the findings of the microarray study. In addition to the increased reactivity seen at both stages, astrocytes from late-stage disease showed decreased expression of many transcripts involved in cholesterol homeostasis. Staining for the master regulator of cholesterol synthesis, SREBP2, has revealed an increased localization to the cytoplasm of astrocytes and motor neurons in late-stage SOD1(G93A) spinal cord, indicating that down-regulation of transcripts may be due to an excess of cholesterol in the CNS during late-stage disease possibly due to phagocytosis of neuronal debris. Our data reveal that SOD1(G93A) astrocytes are characterized more by a loss of supportive function than a toxic phenotype during ALS disease progression and future studies should focus upon restorative therapies.
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http://dx.doi.org/10.3389/fncel.2015.00410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4606544PMC
November 2015

Geographical variation in species' population responses to changes in temperature and precipitation.

Proc Biol Sci 2015 11;282(1818):20151561

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK.

Despite increasing concerns about the vulnerability of species' populations to climate change, there has been little overall synthesis of how individual population responses to variation in climate differ between taxa, with trophic level or geographically. To address this, we extracted data from 132 long-term (greater than or equal to 20 years) studies of population responses to temperature and precipitation covering 236 animal and plant species across terrestrial and freshwater habitats. Our results identify likely geographical differences in the effects of climate change on populations and communities in line with macroecological theory. Temperature tended to have a greater overall impact on populations than precipitation, although the effects of increased precipitation varied strongly with latitude, being most positive at low latitudes. Population responses to increased temperature were generally positive, but did not vary significantly with latitude. Studies reporting significant climatic trends through time tended to show more negative effects of temperature and more positive effects of precipitation upon populations than other studies, indicating climate change has already impacted many populations. Most studies of climate change impacts on biodiversity have focused on temperature and are from middle to high northern latitudes. Our results suggest their findings may be less applicable to low latitudes.
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http://dx.doi.org/10.1098/rspb.2015.1561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4650153PMC
November 2015

Meteorological conditions, climate change, new emerging factors, and asthma and related allergic disorders. A statement of the World Allergy Organization.

World Allergy Organ J 2015 14;8(1):25. Epub 2015 Jul 14.

Department of Pediatrics, Hanyang University College of Medicine, Seoul, Korea.

The prevalence of allergic airway diseases such as asthma and rhinitis has increased dramatically to epidemic proportions worldwide. Besides air pollution from industry derived emissions and motor vehicles, the rising trend can only be explained by gross changes in the environments where we live. The world economy has been transformed over the last 25 years with developing countries being at the core of these changes. Around the planet, in both developed and developing countries, environments are undergoing profound changes. Many of these changes are considered to have negative effects on respiratory health and to enhance the frequency and severity of respiratory diseases such as asthma in the general population. Increased concentrations of greenhouse gases, and especially carbon dioxide (CO2), in the atmosphere have already warmed the planet substantially, causing more severe and prolonged heat waves, variability in temperature, increased air pollution, forest fires, droughts, and floods - all of which can put the respiratory health of the public at risk. These changes in climate and air quality have a measurable impact not only on the morbidity but also the mortality of patients with asthma and other respiratory diseases. The massive increase in emissions of air pollutants due to economic and industrial growth in the last century has made air quality an environmental problem of the first order in a large number of regions of the world. A body of evidence suggests that major changes to our world are occurring and involve the atmosphere and its associated climate. These changes, including global warming induced by human activity, have an impact on the biosphere, biodiversity, and the human environment. Mitigating this huge health impact and reversing the effects of these changes are major challenges. This statement of the World Allergy Organization (WAO) raises the importance of this health hazard and highlights the facts on climate-related health impacts, including: deaths and acute morbidity due to heat waves and extreme meteorological events; increased frequency of acute cardio-respiratory events due to higher concentrations of ground level ozone; changes in the frequency of respiratory diseases due to trans-boundary particle pollution; altered spatial and temporal distribution of allergens (pollens, molds, and mites); and some infectious disease vectors. According to this report, these impacts will not only affect those with current asthma but also increase the incidence and prevalence of allergic respiratory conditions and of asthma. The effects of climate change on respiratory allergy are still not well defined, and more studies addressing this topic are needed. Global warming is expected to affect the start, duration, and intensity of the pollen season on the one hand, and the rate of asthma exacerbations due to air pollution, respiratory infections, and/or cold air inhalation, and other conditions on the other hand.
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http://dx.doi.org/10.1186/s40413-015-0073-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4499913PMC
July 2015

Mechanisms underpinning climatic impacts on natural populations: altered species interactions are more important than direct effects.

Glob Chang Biol 2014 Jul 28;20(7):2221-9. Epub 2014 Mar 28.

British Trust for Ornithology, The Nunnery, Thetford IP24 2PU, UK.

Shifts in species' distribution and abundance in response to climate change have been well documented, but the underpinning processes are still poorly understood. We present the results of a systematic literature review and meta-analysis investigating the frequency and importance of different mechanisms by which climate has impacted natural populations. Most studies were from temperate latitudes of North America and Europe; almost half investigated bird populations. We found significantly greater support for indirect, biotic mechanisms than direct, abiotic mechanisms as mediators of the impact of climate on populations. In addition, biotic effects tended to have greater support than abiotic factors in studies of species from higher trophic levels. For primary consumers, the impact of climate was equally mediated by biotic and abiotic mechanisms, whereas for higher level consumers the mechanisms were most frequently biotic, such as predation or food availability. Biotic mechanisms were more frequently supported in studies that reported a directional trend in climate than in studies with no such climatic change, although sample sizes for this comparison were small. We call for more mechanistic studies of climate change impacts on populations, particularly in tropical systems.
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http://dx.doi.org/10.1111/gcb.12559DOI Listing
July 2014

A pan-European study of capabilities to manage mass casualties from the release of chemical agents: the MASH project.

Am J Disaster Med 2013 ;8(1):13-23

SAMU de Paris, Hopital Necker - Enfants Malades, Paris, France.

The European Union (EU) Mass Casualties and Health (MASH) project that ran between 2008 and 2010 was designed to study the management of mass casualties from chemical and radiological releases and associated health implications. One area of study for this project concerned arrangements within EU Member States for the management of mass casualties following a chemical release. This was undertaken via a confidential online questionnaire that was sent to selected points of contact throughout the EU. Responses were obtained from 18 states from respondents holding senior positions in chemical planning and incident response. Information gathered shows a lack of uniformity within the EU about the organization of responses to chemical releases and the provision of medical care. This article presents the overall findings of the study demonstrating differences between countries on planning and organization, decontamination, prehospital emergency medical responses, clinical diagnoses, and therapy and aftercare. Although there may be an understandable reluctance from national respondents to share information on security and other grounds, the findings, nevertheless, revealed substantial differences between current planning and operational responses within the EU states for the management of mass chemical casualties. The existing international networks for response to radiation incidents are not yet matched by equivalent networks for chemical responses yet sufficient information was available from the study to identify potential deficiencies, identify common casualty management pathways, and to make recommendations for future operations within the EU. Improvements in awareness and training and the application of modern information and communications will help to remedy this situation. Specialized advanced life support and other medical care for chemical casualties appear lacking in some countries. A program of specialized training and action are required to apply the findings revealed by the MASH study into a unified cross-border emergency medical response.
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http://dx.doi.org/10.5055/ajdm.2013.0107DOI Listing
August 2013

Explosions and human health: the long-term effects of blast injury.

Prehosp Disaster Med 2012 Aug 17;27(4):385-91. Epub 2012 Jul 17.

Extreme Events and Health Protection Section, Centre for Radiological, Chemical and Environmental Hazards, United Kingdom Health Protection Agency, London, United Kingdom.

The immediate patterns of injury from explosions are well documented, from both military and civil experience. However, few studies have focused on less immediately apparent health consequences and latent effects of explosions in survivors, emergency responders and the surrounding community. This review aimed to analyze the risks to health following an explosion in a civil setting. A comprehensive review of the open literature was conducted, and data on 10 relevant military, civilian and industrial events were collected. Events were selected according to availability of published studies and involvement of large numbers of people injured. In addition, structured interviews with experts in the field were conducted, and existing national guidelines reviewed. The review revealed significant and potentially long-term health implications affecting various body systems and psychological well-being following exposure to an explosion. An awareness of the short- and long-term health effects of explosions is essential in screening for blast injuries, and identifying latent pathologies that could otherwise be overlooked in stressful situations with other visually distracting injuries and, often, mass casualties. Such knowledge would guide responsible medical staff in implementing early appropriate interventions to reduce the burden of long-term sequelae. Effective planning and response strategies would ensure accessibility of appropriate health care resources and evidence-based information in the aftermath of an explosion.
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http://dx.doi.org/10.1017/S1049023X12000891DOI Listing
August 2012

D-dimer assays for the identification of menstrual blood.

Forensic Sci Int 2011 Oct 7;212(1-3):210-4. Epub 2011 Jul 7.

Forensic Science Service Ltd, Trident Court 2960 Solihull Parkway, Birmingham Business Park, Birmingham B37 7YN, United Kingdom.

A method to reliably distinguish menstrual blood from blood in the normal circulation (peripheral blood) would be of considerable use in the forensic analysis and interpretation of evidence in sexual offence investigations. Previous attempts to address this issue have explored microscopy, lactate dehydrogenase isozyme identification, mRNA and miRNA profiling, and identification of the products of fibrinolysis. Here, four assays for D-dimer, a terminal degradation product of fibrinolysis, are evaluated for their specificity and sensitivity in detection of menstrual blood. In addition the effect of exercise, and sample storage upon D-dimer detection was investigated. Comparison of different assays revealed significant differences in results given. Nevertheless, no positive results for D-dimer were obtained using peripheral blood, mixtures of peripheral blood with semen, or peripheral blood taken from donors after moderate exercise. D-dimer was found to be detectable in 100% of menstrual blood samples after 1 week at room temperature and also in samples stored long-term (>3 years) at -20 °C. D-dimer may be an effective, simple to use tool for the presumptive identification of menstrual blood identification.
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http://dx.doi.org/10.1016/j.forsciint.2011.06.013DOI Listing
October 2011

Safe management of mass fatalities following chemical, biological, and radiological incidents.

Prehosp Disaster Med 2009 May-Jun;24(3):180-8

Health Protection Agency, London, UK.

Contaminated mass fatalities following the release of chemical, biological, or radiological agents pose a potential major health hazard. A United Kingdom government investigation has identified a number of areas of risk. This paper presents an outline of the findings of the study and describes specific pathways for the management of contaminated and non-contaminated fatalities. Factors determining the choice between cremation and burial are discussed. Effective decontamination remains a neglected area of study for both fatalities and casualties.
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http://dx.doi.org/10.1017/s1049023x00006786DOI Listing
September 2010

PPARdelta agonism inhibits skeletal muscle PDC activity, mitochondrial ATP production and force generation during prolonged contraction.

J Physiol 2009 Jan 10;587(1):231-9. Epub 2008 Nov 10.

Centre for Integrated Systems Biology and Medicine, Queens Medical Centre, University of Nottingham Medical School, Nottingham NG7 2UH, UK.

We have recently shown that PPARdelta agonism, used clinically to treat insulin resistance, increases fat oxidation and up-regulates mitochondrial PDK4 mRNA and protein expression in resting skeletal muscle. We hypothesized that PDK4 up-regulation, which inhibits pyruvate dehydrogenase complex (PDC)-dependent carbohydrate (CHO) oxidation, would negatively affect muscle function during sustained contraction where the demand on CHO is markedly increased. Three groups of eight male Wistar rats each received either vehicle or a PPARdelta agonist (GW610742X) at two doses (5 and 100 mg (kg body mass (bm))(-1) orally for 6 days. On the seventh day, the gastrocnemius-soleus-plantaris muscle group was isolated and snap frozen, or underwent 30 min of electrically evoked submaximal intensity isometric contraction using a perfused hindlimb model. During contraction, the rate of muscle PDC activation was significantly lower at 100 mg (kg bm)(-1) compared with control (P < 0.01). Furthermore, the rates of muscle PCr hydrolysis and lactate accumulation were significantly increased at 100 mg (kg bm)(-1) compared with control, reflecting lower mitochondrial ATP generation. Muscle tension development during contraction was significantly lower at 100 mg (kg bm)(-1) compared with control (25%; P < 0.05). The present data demonstrate that PPARdelta agonism inhibits muscle CHO oxidation at the level of PDC during prolonged contraction, and is paralleled by the activation of anaerobic metabolism, which collectively impair contractile function.
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http://dx.doi.org/10.1113/jphysiol.2008.164210DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2670036PMC
January 2009

Decontamination of multiple casualties who are chemically contaminated: a challenge for acute hospitals.

Prehosp Disaster Med 2008 Mar-Apr;23(2):175-81

Emergency Department, Frimley Park NHS Foundation Trust, Portsmouth Road, Frimley, Camberley, Surrey GU16 7UJ, UK.

Patients who have been contaminated by chemical compounds present a number of difficulties to emergency departments, in particular, the risk of secondary contamination of healthcare staff and facilities. The Department of Health in the United Kingdom has provided equipment to decontaminate chemically contaminated casualties who present at emergency departments. The capacity of this equipment is limited, and although both the ambulance and fire services have equipment to cope with mass casualties at the scene of a chemical incident, there is still the possibility that acute hospitals will be overwhelmed by large numbers of self-presenting patients. The risks and potential consequences of this gap in resilience are discussed and a number of possible practical solutions are proposed.
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http://dx.doi.org/10.1017/s1049023x00005811DOI Listing
August 2008

Exercise training in late middle-aged male Fischer 344 x Brown Norway F1-hybrid rats improves skeletal muscle aerobic function.

Exp Physiol 2008 Jul 20;93(7):863-71. Epub 2008 Mar 20.

Faculty of Kinesiology, University of Calgary, 2500 University Drive NW, Calgary, Alberta, Canada T2N 1N4.

The Fischer 344 x Brown Norway F1-hybrid (F344BN) rat has become an increasingly popular and useful strain for studying age-related declines in skeletal muscle function because this strain lives long enough to experience significant declines in muscle mass. Since exercise is often considered a mechanism to combat age-related declines in muscle function, determining the utility of this strain of rat for studying the effects of exercise on the ageing process is necessary. The purpose of this study was to evaluate the plasticity of skeletal muscle aerobic function in late middle-aged male rats following 7 weeks of treadmill exercise training. Training consisted of 60 min per day, 5 days per week with velocity gradually increasing over the training period according to the capabilities of individual rats. The final 3 weeks involved 2 min high-intensity intervals to increase the training stimulus. We used in situ skeletal muscle aerobic metabolic responses and in vitro assessment of muscle mitochondrial oxidative capacity to describe the adaptations of aerobic function from the training. Training increased running endurance from 11.3 +/- 0.6 to 15.5 +/- 0.8 min, an improvement of approximately 60%. Similarly, distal hindlimb muscles from trained rats exhibited a higher maximal oxygen consumption in situ (23.2 +/- 1.3 versus 19.7 +/- 0.8 mumol min(-1) for trained versus sedentary rats, respectively) and greater citrate synthase and complex IV enzyme activities in gastrocnemius (29 and 19%, respectively) and plantaris muscles (24 and 28%, respectively) compared with age-matched sedentary control animals. Our results demonstrate that skeletal muscles from late middle-aged rats adapt to treadmill exercise by improving skeletal muscle aerobic function and mitochondrial enzyme activities. This rat strain seems suitable for further investigations using exercise as an intervention to combat ageing-related declines of skeletal muscle aerobic function.
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http://dx.doi.org/10.1113/expphysiol.2008.042069DOI Listing
July 2008

Nucleotide excision repair eliminates unique DNA-protein cross-links from mammalian cells.

J Biol Chem 2007 Aug 16;282(31):22592-604. Epub 2007 May 16.

Biology Division, Beckman Research Institute, City of Hope National Medical Center, Duarte, California 91010, USA.

DNA-protein cross-links (DPCs) present a formidable obstacle to cellular processes because they are "superbulky" compared with the majority of chemical adducts. Elimination of DPCs is critical for cell survival because their persistence can lead to cell death or halt cell cycle progression by impeding DNA and RNA synthesis. To study DPC repair, we have used DNA methyltransferases to generate unique DPC adducts in oligodeoxyribonucleotides or plasmids to monitor both in vitro excision and in vivo repair. We show that HhaI DNA methyltransferase covalently bound to an oligodeoxyribonucleotide is not efficiently excised by using mammalian cell-free extracts, but protease digestion of the full-length HhaI DNA methyltransferase-DPC yields a substrate that is efficiently removed by a process similar to nucleotide excision repair (NER). To examine the repair of that unique DPC, we have developed two plasmid-based in vivo assays for DPC repair. One assay shows that in nontranscribed regions, DPC repair is greater than 60% in 6 h. The other assay based on host cell reactivation using a green fluorescent protein demonstrates that DPCs in transcribed genes are also repaired. Using Xpg-deficient cells (NER-defective) with the in vivo host cell reactivation assay and a unique DPC indicates that NER has a role in the repair of this adduct. We also demonstrate a role for the 26 S proteasome in DPC repair. These data are consistent with a model for repair in which the polypeptide chain of a DPC is first reduced by proteolysis prior to NER.
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http://dx.doi.org/10.1074/jbc.M702856200DOI Listing
August 2007

Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells.

Anal Biochem 2007 Jun 21;365(2):246-59. Epub 2007 Mar 21.

Department of Biology, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.

We introduce a method to follow DNA repair that is suitable for both clinical and laboratory samples. An episomal construct with a unique 8-oxoguanine (8-oxoG) base at a defined position was prepared in vitro using single-stranded phage harboring a 678-bp tract from exons 5 to 9 of the human P53 gene. Mixing curve experiments showed that the real-time PCR method has a linear response to damage, suggesting that it is useful for DNA repair studies. The episomal construct with a unique 8-oxoG base was introduced into AD293 cells or human peripheral blood mononuclear cells, and plasmids were recovered as a function of time. The quantitative real-time PCR assay demonstrated that repair of the 8-oxoG was 80% complete in less than 48 h in AD293 cells. Transfection of small interfering RNAs down-regulated OGG1 expression in AD293 cells and reduced the repair of 8-oxoG to 30%. Transfection of the episome into unstimulated white blood cells showed that 8-oxoG repair had a half-life of 2 to 5h. This method is a rapid, reproducible, and robust way to monitor repair of specific adducts in virtually any cell type.
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http://dx.doi.org/10.1016/j.ab.2007.03.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614353PMC
June 2007