Publications by authors named "Claus Jorgensen"

88 Publications

Topological features of integrin adhesion complexes revealed by multiplexed proximity biotinylation.

J Cell Biol 2020 Aug;219(8)

Wellcome Centre for Cell-Matrix Research, Faculty of Biology, Medicine & Health, Manchester Academic Health Science Centre, University of Manchester, Manchester, UK.

Integrin adhesion complexes (IACs) bridge the extracellular matrix to the actin cytoskeleton and transduce signals in response to both chemical and mechanical cues. The composition, interactions, stoichiometry, and topological organization of proteins within IACs are not fully understood. To address this gap, we used multiplexed proximity biotinylation (BioID) to generate an in situ, proximity-dependent adhesome in mouse pancreatic fibroblasts. Integration of the interactomes of 16 IAC-associated baits revealed a network of 147 proteins with 361 proximity interactions. Candidates with underappreciated roles in adhesion were identified, in addition to established IAC components. Bioinformatic analysis revealed five clusters of IAC baits that link to common groups of prey, and which therefore may represent functional modules. The five clusters, and their spatial associations, are consistent with current models of IAC interaction networks and stratification. This study provides a resource to examine proximal relationships within IACs at a global level.
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http://dx.doi.org/10.1083/jcb.202003038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401799PMC
August 2020

Glucocorticoids rapidly inhibit cell migration through a novel, non-transcriptional HDAC6 pathway.

J Cell Sci 2020 Jun 11;133(11). Epub 2020 Jun 11.

Division of Diabetes, Endocrinology, and Gastroenterology, University of Manchester, Manchester, M13 9PT, UK

Glucocorticoids (GCs) act through the glucocorticoid receptor (GR, also known as NR3C1) to regulate immunity, energy metabolism and tissue repair. Upon ligand binding, activated GR mediates cellular effects by regulating gene expression, but some GR effects can occur rapidly without new transcription. Here, we show that GCs rapidly inhibit cell migration, in response to both GR agonist and antagonist ligand binding. The inhibitory effect on migration is prevented by GR knockdown with siRNA, confirming GR specificity, but not by actinomycin D treatment, suggesting a non-transcriptional mechanism. We identified a rapid onset increase in microtubule polymerisation following GC treatment, identifying cytoskeletal stabilisation as the likely mechanism of action. HDAC6 overexpression, but not knockdown of αTAT1, rescued the GC effect, implicating HDAC6 as the GR effector. Consistent with this hypothesis, ligand-dependent cytoplasmic interaction between GR and HDAC6 was demonstrated by quantitative imaging. Taken together, we propose that activated GR inhibits HDAC6 function, and thereby increases the stability of the microtubule network to reduce cell motility. We therefore report a novel, non-transcriptional mechanism whereby GCs impair cell motility through inhibition of HDAC6 and rapid reorganization of the cell architecture.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.242842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7295589PMC
June 2020

The Shb scaffold binds the Nck adaptor protein, p120 RasGAP, and Chimaerins and thereby facilitates heterotypic cell segregation by the receptor EphB2.

J Biol Chem 2020 03 14;295(12):3932-3944. Epub 2020 Feb 14.

Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario M5S 1A8, Canada

Eph receptors are a family of receptor tyrosine kinases that control directional cell movement during various biological processes, including embryogenesis, neuronal pathfinding, and tumor formation. The biochemical pathways of Eph receptors are context-dependent in part because of the varied composition of a heterotypic, oligomeric, active Eph receptor complex. Downstream of the Eph receptors, little is known about the essential phosphorylation events that define the context and instruct cell movement. Here, we define a pathway that is required for Eph receptor B2 (EphB2)-mediated cell sorting and is conserved among multiple Eph receptors. Utilizing a HEK293 model of EphB2/ephrinB1 cell segregation, we found that the scaffold adaptor protein SH2 domain-containing adaptor protein B (Shb) is essential for EphB2 functionality. Further characterization revealed that Shb interacts with known modulators of cytoskeletal rearrangement and cell mobility, including Nck adaptor protein (Nck), p120-Ras GTPase-activating protein (RasGAP), and the α- and β-Chimaerin Rac GAPs. We noted that phosphorylation of Tyr, Tyr, and Tyr of Shb is required for EphB2-ephrinB1 boundary formation, as well as binding of Nck, RasGAP, and the chimaerins, respectively. Similar complexes were formed in the context of EphA4, EphA8, EphB2, and EphB4 receptor activation. These results indicate that phosphotyrosine-mediated signaling through Shb is essential in EphB2-mediated heterotypic cell segregation and suggest a conserved function for Shb downstream of multiple Eph receptors.
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http://dx.doi.org/10.1074/jbc.RA119.009276DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7086039PMC
March 2020

A framework for advancing our understanding of cancer-associated fibroblasts.

Nat Rev Cancer 2020 03 24;20(3):174-186. Epub 2020 Jan 24.

Department of Anatomy, University of California, San Francisco, San Francisco, CA, USA.

Cancer-associated fibroblasts (CAFs) are a key component of the tumour microenvironment with diverse functions, including matrix deposition and remodelling, extensive reciprocal signalling interactions with cancer cells and crosstalk with infiltrating leukocytes. As such, they are a potential target for optimizing therapeutic strategies against cancer. However, many challenges are present in ongoing attempts to modulate CAFs for therapeutic benefit. These include limitations in our understanding of the origin of CAFs and heterogeneity in CAF function, with it being desirable to retain some antitumorigenic functions. On the basis of a meeting of experts in the field of CAF biology, we summarize in this Consensus Statement our current knowledge and present a framework for advancing our understanding of this critical cell type within the tumour microenvironment.
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http://dx.doi.org/10.1038/s41568-019-0238-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046529PMC
March 2020

Epigenetic and Transcriptomic Characterization of Pure Adipocyte Fractions From Obese Pigs Identifies Candidate Pathways Controlling Metabolism.

Front Genet 2019 17;10:1268. Epub 2019 Dec 17.

Novo Nordisk Foundation Centre for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

Reprogramming of adipocyte function in obesity is implicated in metabolic disorders like type 2 diabetes. Here, we used the pig, an animal model sharing many physiological and pathophysiological similarities with humans, to perform in-depth epigenomic and transcriptomic characterization of pure adipocyte fractions. Using a combined DNA methylation capture sequencing and Reduced Representation bisulfite sequencing (RRBS) strategy in 11 lean and 12 obese pigs, we identified in 3529 differentially methylated regions (DMRs) located at close proximity to-, or within genes in the adipocytes. By sequencing of the transcriptome from the same fraction of isolated adipocytes, we identified 276 differentially expressed transcripts with at least one or more DMR. These transcripts were over-represented in gene pathways related to MAPK, metabolic and insulin signaling. Using a candidate gene approach, we further characterized 13 genes potentially regulated by DNA methylation and identified putative transcription factor binding sites that could be affected by the differential methylation in obesity. Our data constitute a valuable resource for further investigations aiming to delineate the epigenetic etiology of metabolic disorders.
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http://dx.doi.org/10.3389/fgene.2019.01268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927937PMC
December 2019

Adverse events of exercise therapy in randomised controlled trials: a systematic review and meta-analysis.

Br J Sports Med 2020 Sep 28;54(18):1073-1080. Epub 2019 Sep 28.

Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Odense, Denmark.

Objective: To evaluate the relative risk (RR) of serious and non-serious adverse events in patients treated with exercise therapy compared with those in a non-exercising control group.

Design: Systematic review and meta-analysis.

Data Sources: Primary studies were identified based on The Cochrane Database of Systematic Reviews investigating the effect of exercise therapy.

Eligibility Criteria: At least two of the authors independently evaluated all identified reviews and primary studies. Randomised controlled trials were included if they compared any exercise therapy intervention with a non-exercising control. Two authors independently extracted data. The RR of serious and non-serious adverse events was estimated separately.

Results: 180 Cochrane reviews were included and from these, 773 primary studies were identified. Of these, 378 studies (n=38 368 participants) reported serious adverse events and 375 studies (n=38 517 participants) reported non-serious adverse events. We found no increase in risk of serious adverse events (RR=0.96 (95%CI 0.90 to 1.02, I: 0.0%) due to exercise therapy. There was, however, an increase in non-serious adverse events (RR=1.19 (95%CI 1.09 to 1.30, I: 0.0%). The number needed to treat for an additional harmful outcome for non-serious adverse events was 6 [95%CI 4 to 11).

Conclusion: Participating in an exercise intervention increased the relative risk of non-serious adverse events, but not of serious adverse events. Exercise therapy may therefore be recommended as a relatively safe intervention.PROSPERO registration numberCRD42014014819.
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http://dx.doi.org/10.1136/bjsports-2018-100461DOI Listing
September 2020

Ascaris Suum Infection Downregulates Inflammatory Pathways in the Pig Intestine In Vivo and in Human Dendritic Cells In Vitro.

J Infect Dis 2018 01;217(2):310-319

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Frederiksberg, Denmark.

Ascaris suum is a helminth parasite of pigs closely related to its human counterpart, A. lumbricoides, which infects almost 1 billion people. Ascaris is thought to modulate host immune and inflammatory responses, which may drive immune hyporesponsiveness during chronic infections. Using transcriptomic analysis, we show here that pigs with a chronic A. suum infection have a substantial suppression of inflammatory pathways in the intestinal mucosa, with a broad downregulation of genes encoding cytokines and antigen-processing and costimulatory molecules. A. suum body fluid (ABF) suppressed similar transcriptional pathways in human dendritic cells (DCs) in vitro. DCs exposed to ABF secreted minimal amounts of cytokines and had impaired production of cyclooxygengase-2, altered glucose metabolism, and reduced capacity to induce interferon-gamma production in T cells. Our in vivo and in vitro data provide an insight into mucosal immune modulation during Ascaris infection, and show that A. suum profoundly suppresses immune and inflammatory pathways.
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http://dx.doi.org/10.1093/infdis/jix585DOI Listing
January 2018

Deregulation of obesity-relevant genes is associated with progression in BMI and the amount of adipose tissue in pigs.

Mol Genet Genomics 2018 Feb 14;293(1):129-136. Epub 2017 Sep 14.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Grønnegårdsvej 3, 1870, Frederiksberg C, Denmark.

The aim of this study was to elucidate the relative impact of three phenotypes often used to characterize obesity on perturbation of molecular pathways involved in obesity. The three obesity-related phenotypes are (1) body mass index (BMI), (2) amount of subcutaneous adipose tissue (SATa), and (3) amount of retroperitoneal adipose tissue (RPATa). Although it is generally accepted that increasing amount of RPATa is 'unhealthy', a direct comparison of the relative impact of the three obesity-related phenotypes on gene expression has, to our knowledge, not been performed previously. We have used multiple linear models to analyze altered gene expression of selected obesity-related genes in tissues collected from 19 female pigs phenotypically characterized with respect to the obesity-related phenotypes. Gene expression was assessed by high-throughput qPCR in RNA from liver, skeletal muscle and abdominal adipose tissue. The stringent statistical approach used in the study has increased the power of the analysis compared to the classical approach of analysis in divergent groups of individuals. Our approach led to the identification of key components of cellular pathways that are modulated in the three tissues in association with changes in the three obesity-relevant phenotypes (BMI, SATa and RPATa). The deregulated pathways are involved in biosynthesis and transcript regulation in adipocytes, in lipid transport, lipolysis and metabolism, and in inflammatory responses. Deregulation seemed more comprehensive in liver (23 genes) compared to abdominal adipose tissue (10 genes) and muscle (3 genes). Notably, the study supports the notion that excess amount of intra-abdominal adipose tissue is associated with a greater metabolic disease risk. Our results provide molecular support for this notion by demonstrating that increasing amount of RPATa has a higher impact on perturbation of cellular pathways influencing obesity and obesity-related metabolic traits compared to increase in BMI and amount of SATa.
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http://dx.doi.org/10.1007/s00438-017-1369-2DOI Listing
February 2018

Cell-Specific Labeling for Analyzing Bidirectional Signaling by Mass Spectrometry.

Methods Mol Biol 2017 ;1636:219-234

Systems Oncology, CRUK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester, M20 4QL, UK.

Cell-specific proteome labeling enables global proteome-wide analysis of cell signaling in heterotypic co-cultures. Such approaches have provided unique insight in contact-initiated receptor tyrosine kinase signaling, transfer of proteomic material between heterotypic cells, and interactions between normal and oncogenic cells. Here we describe current methods for cell-specific labeling of heterotypic cells with isotopic labeled amino acids (e.g., SILAC and CTAP). We outline the advantages and disadvantages of individual approaches, describe typical experimental scenarios, and discuss where each experimental approach is optimally applied.
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http://dx.doi.org/10.1007/978-1-4939-7154-1_14DOI Listing
April 2018

Haplotypes on pig chromosome 3 distinguish metabolically healthy from unhealthy obese individuals.

PLoS One 2017 1;12(6):e0178828. Epub 2017 Jun 1.

Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

We have established a pig resource population specifically designed to elucidate the genetics involved in development of obesity and obesity related co-morbidities by crossing the obesity prone Göttingen Minipig breed with two lean production pig breeds. In this study we have performed genome wide association (GWA) to identify loci with effect on blood lipid levels. The most significantly associated single nucleotide polymorphisms (SNPs) were used for linkage disequilibrium (LD) and haplotype analyses. Three separate haploblocks which influence the ratio between high density lipoprotein cholesterol and total cholesterol (HDL-C/CT), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) levels respectively were identified on Sus Scrofa chromosome 3 (SSC3). Large additive genetic effects were found for the HDL-C/CT and LDL-C haplotypes. Haplotypes segregating from Göttingen Minipigs were shown to impose a positive effect on blood lipid levels. Thus, the genetic profile of the Göttingen Minipig breed seems to support a phenotype comparable to the metabolic healthy obese (MHO) phenotype in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453593PMC
September 2017

Trajectory of self-reported pain and function and knee extensor muscle strength in young patients undergoing arthroscopic surgery for meniscal tears: A systematic review and meta-analysis.

J Sci Med Sport 2017 Aug 21;20(8):712-717. Epub 2017 Feb 21.

Research Unit for Musculoskeletal Function and Physiotherapy, Department of Sports Science and Clinical Biomechanics, University of Southern Denmark, Denmark; Department of Rehabilitation, Copenhagen University Hospital, Denmark.

Objectives: To investigate the trajectory of patient reported pain and function and knee extensor muscle strength over time in young individuals undergoing arthroscopic meniscal surgery.

Design: Systematic review and meta-analysis METHODS: Six databases were searched up to October 13th, 2016.

Patients And Intervention: People aged 30 years or younger undergoing surgery for a meniscal tear.

Outcomes: and comparator: (1) Self-reported pain and function in patients undergoing meniscal surgery compared to a non-operative control group (2). Knee extensor strength in the leg undergoing surgery compared to a healthy control group or the contra-lateral leg. Methodological quality was assessed using the SIGN 50 guidelines.

Results: No studies were found on patient reported pain and function. Six studies, including 137 patients were included in the analysis on knee extensor muscle strength. Knee extensor muscle strength was impaired in the injured leg prior to surgery and was still reduced compared with control data up to 12 months after surgery (SMD: -1.16) (95% CI: -1.83; -0.49). All included studies were assessed to have a high risk of bias.

Conclusions: No studies were found comparing the trajectory of self-reported pain and function in patients undergoing arthroscopic surgery compared with non-operative treatments for young patients with meniscal tears. Knee extensor strength seemed to be impaired up to 12 months after surgery in young patients undergoing surgery for meniscal tears. The results of the present study should be interpreted with caution due to a limited number of available studies with high risk of bias including relatively few patients.
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http://dx.doi.org/10.1016/j.jsams.2017.02.004DOI Listing
August 2017

Transcriptional immune response in mesenteric lymph nodes in pigs with different levels of resistance to Ascaris suum.

Acta Parasitol 2017 Mar;62(1):141-153

A single nucleotide polymorphism on chromosome 4 (SNP TXNIP) has been reported to be associated with roundworm (Ascaris suum) burden in pigs. The objective of the present study was to analyse the immune response to A. suum mounted by pigs with genotype AA (n = 24) and AB (n = 23) at the TXNIP locus. The pigs were repeatedly infected with A. suum from eight weeks of age until necropsy eight weeks later. An uninfected control group (AA; n = 5 and AB; n = 5) was also included. At post mortem, we collected mesenteric lymph nodes and measured the expression of 28 selected immune-related genes. Recordings of worm burdens confirmed our previous results that pigs of the AA genotype were more resistant to infection than AB pigs. We estimated the genotype difference in relative expression levels in infected and uninfected animals. No significant change in expression levels between the two genotypes due to infection was observed for any of the genes, although IL-13 approached significance (P = 0.08; Punadjusted = 0.003). Furthermore, statistical analysis testing for the effect of infection separately in each genotype showed significant up-regulation of IL-13 (P<0.05) and CCL17 (P<0.05) following A. suum infection in the 'resistant' AA genotype and not in the 'susceptible' AB genotype. Pigs of genotype AB had higher expression of the high-affinity IgG receptor (FCGR1A) than AA pigs in both infected and non-infected animals (P = 1.85*10-11).
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http://dx.doi.org/10.1515/ap-2017-0017DOI Listing
March 2017

Proteomics profiling of interactome dynamics by colocalisation analysis (COLA).

Mol Biosyst 2016 Dec;13(1):92-105

Institute of Cancer Research, Division of Cancer Biology, 237 Fulham Road, London SW3 6JB, UK.

Localisation and protein function are intimately linked in eukaryotes, as proteins are localised to specific compartments where they come into proximity of other functionally relevant proteins. Significant co-localisation of two proteins can therefore be indicative of their functional association. We here present COLA, a proteomics based strategy coupled with a bioinformatics framework to detect protein-protein co-localisations on a global scale. COLA reveals functional interactions by matching proteins with significant similarity in their subcellular localisation signatures. The rapid nature of COLA allows mapping of interactome dynamics across different conditions or treatments with high precision.
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http://dx.doi.org/10.1039/c6mb00701eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315029PMC
December 2016

α1,2-Fucosyllactose Does Not Improve Intestinal Function or Prevent Escherichia coli F18 Diarrhea in Newborn Pigs.

J Pediatr Gastroenterol Nutr 2017 02;64(2):310-318

*Comparative Pediatrics and Nutrition, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen †National Veterinary Institute, Technical University of Denmark, Lyngby ‡Department of Food Science, Faculty of Science, University of Copenhagen, Frederiksberg §Arla Foods Amba, Brabrand, Viby J, Denmark.

Objectives: Infectious diarrhea, a leading cause of morbidity and deaths, is less prevalent in breastfed infants compared with infants fed infant formula. The dominant human milk oligosaccharide (HMO), α-1,2-fucosyllactose (2'-FL), has structural homology to bacterial adhesion sites in the intestine and may in part explain the protective effects of human milk. We hypothesized that 2'-FL prevents diarrhea via competitive inhibition of pathogen adhesion in a pig model for sensitive newborn infants.

Methods: Intestinal cell studies were coupled with studies on cesarean-delivered newborn pigs (n = 24) without (control) or with inoculation of enterotoxigenic Escherichia coli F18 (7.5 × 10/day for 8 days) fed either no (F18) or 10 g/L 2'-FL (2FL-F18).

Results: In vitro studies revealed decreased pathogen adhesion to intestinal epithelial cells with 2'-FL (5 g/L; P < 0.001). F18 pigs showed more diarrhea than control pigs (P < 0.01). Administration of 2'-FL to F18 pigs failed to prevent diarrhea, although the relative weight loss tended to be reduced (-19 vs -124 g/kg, P = 0.12), higher villi were observed in the distal small intestine (P < 0.05), and a trend toward increased proportion of mucosa and activities of some brush border enzymes in the proximal small intestine. In situ abundance of α-1,2-fucose and E coli was similar between groups, whereas sequencing showed higher abundance of Enterobacteriaceae in F18, Enterococcus in control and Lachnospiraceae in 2FL-F18 pigs.

Conclusions: 2'-FL inhibited in vitro adhesion of E coli F18 to epithelial cells, but had limited effects on diarrhea and mucosal health in newborn pigs challenged with E coli F18.
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http://dx.doi.org/10.1097/MPG.0000000000001276DOI Listing
February 2017

Oncogenic KRAS Regulates Tumor Cell Signaling via Stromal Reciprocation.

Cell 2016 May 14;165(4):910-20. Epub 2016 Apr 14.

The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK; Cancer Research UK Manchester Institute, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. Electronic address:

Oncogenic mutations regulate signaling within both tumor cells and adjacent stromal cells. Here, we show that oncogenic KRAS (KRAS(G12D)) also regulates tumor cell signaling via stromal cells. By combining cell-specific proteome labeling with multivariate phosphoproteomics, we analyzed heterocellular KRAS(G12D) signaling in pancreatic ductal adenocarcinoma (PDA) cells. Tumor cell KRAS(G12D) engages heterotypic fibroblasts, which subsequently instigate reciprocal signaling in the tumor cells. Reciprocal signaling employs additional kinases and doubles the number of regulated signaling nodes from cell-autonomous KRAS(G12D). Consequently, reciprocal KRAS(G12D) produces a tumor cell phosphoproteome and total proteome that is distinct from cell-autonomous KRAS(G12D) alone. Reciprocal signaling regulates tumor cell proliferation and apoptosis and increases mitochondrial capacity via an IGF1R/AXL-AKT axis. These results demonstrate that oncogene signaling should be viewed as a heterocellular process and that our existing cell-autonomous perspective underrepresents the extent of oncogene signaling in cancer. VIDEO ABSTRACT.
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http://dx.doi.org/10.1016/j.cell.2016.03.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868820PMC
May 2016

Phosphoproteomic analysis of interacting tumor and endothelial cells identifies regulatory mechanisms of transendothelial migration.

Sci Signal 2016 Feb 9;9(414):ra15. Epub 2016 Feb 9.

Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. Cancer Research UK Manchester Institute, The University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.

The exit of metastasizing tumor cells from the vasculature, extravasation, is regulated by their dynamic interactions with the endothelial cells that line the internal surface of vessels. To elucidate signals controlling tumor cell adhesion to the endothelium and subsequent transendothelial migration, we performed phosphoproteomic analysis to map cell-specific changes in protein phosphorylation that were triggered by contact between metastatic MDA-MB-231 breast cancer cells and endothelial cells. From the 2669 unique phosphorylation sites identified, 77 and 43 were differentially phosphorylated in the tumor cells and endothelial cells, respectively. The receptor tyrosine kinase ephrin type A receptor 2 (EPHA2) exhibited decreased Tyr(772) phosphorylation in the cancer cells upon endothelial contact. Knockdown of EPHA2 increased adhesion of the breast cancer cells to human umbilical vein endothelial cells (HUVECs) and their transendothelial migration in coculture cell assays, as well as early-stage lung colonization in vivo. EPHA2-mediated inhibition of transendothelial migration of breast cancer cells depended on interaction with the ligand ephrinA1 on HUVECs and phosphorylation of EPHA2-Tyr(772). When EPHA2 phosphorylation dynamics were compared between cell lines of different metastatic ability, EPHA2-Tyr(772) was rapidly dephosphorylated after ephrinA1 stimulation specifically in cells targeting the lung. Knockdown of the phosphatase LMW-PTP reduced adhesion and transendothelial migration of the breast cancer cells. Overall, cell-specific phosphoproteomic analysis provides a bidirectional map of contact-initiated signaling between tumor and endothelial cells that can be further investigated to identify mechanisms controlling the transendothelial cell migration of cancer cells.
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http://dx.doi.org/10.1126/scisignal.aac5820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485367PMC
February 2016

Altered Methylation Profile of Lymphocytes Is Concordant with Perturbation of Lipids Metabolism and Inflammatory Response in Obesity.

J Diabetes Res 2016 21;2016:8539057. Epub 2015 Dec 21.

Inflammation and Infection Research, School of Medical Sciences, UNSW Australia, Sydney, NSW 2052, Australia.

Obesity is associated with immunological perturbations that contribute to insulin resistance. Epigenetic mechanisms can control immune functions and have been linked to metabolic complications, although their contribution to insulin resistance still remains unclear. In this study, we investigated the link between metabolic dysfunction and immune alterations with the epigenetic signature in leukocytes in a porcine model of obesity. Global DNA methylation of circulating leukocytes, adipose tissue leukocyte trafficking, and macrophage polarisation were established by flow cytometry. Adipose tissue inflammation and metabolic function were further characterised by quantification of metabolites and expression levels of genes associated with obesity and inflammation. Here we show that obese pigs showed bigger visceral fat pads, higher levels of circulating LDL cholesterol, and impaired glucose tolerance. These changes coincided with impaired metabolism, sustained macrophages infiltration, and increased inflammation in the adipose tissue. Those immune alterations were linked to global DNA hypermethylation in both B-cells and T-cells. Our results provide novel insight into the possible contribution of immune cell epigenetics into the immunological disturbances observed in obesity. The dramatic changes in the transcriptomic and epigenetic signature of circulating lymphocytes reinforce the concept that epigenetic processes participate in the increased immune cell activation and impaired metabolic functions in obesity.
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http://dx.doi.org/10.1155/2016/8539057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4698937PMC
October 2016

Rho-associated kinase (ROCK) function is essential for cell cycle progression, senescence and tumorigenesis.

Elife 2016 Jan 14;5:e12994. Epub 2016 Jan 14.

Division of Cancer Biology, Institute of Cancer Research, London, United Kingdom.

Rho-associated kinases 1 and 2 (ROCK1/2) are Rho-GTPase effectors that control key aspects of the actin cytoskeleton, but their role in proliferation and cancer initiation or progression is not known. Here, we provide evidence that ROCK1 and ROCK2 act redundantly to maintain actomyosin contractility and cell proliferation and that their loss leads to cell-cycle arrest and cellular senescence. This phenotype arises from down-regulation of the essential cell-cycle proteins CyclinA, CKS1 and CDK1. Accordingly, while the loss of either Rock1 or Rock2 had no negative impact on tumorigenesis in mouse models of non-small cell lung cancer and melanoma, loss of both blocked tumor formation, as no tumors arise in which both Rock1 and Rock2 have been genetically deleted. Our results reveal an indispensable role for ROCK, yet redundant role for isoforms 1 and 2, in cell cycle progression and tumorigenesis, possibly through the maintenance of cellular contractility.
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http://dx.doi.org/10.7554/eLife.12203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4798951PMC
January 2016

Genome-wide association study reveals a locus for nasal carriage of Staphylococcus aureus in Danish crossbred pigs.

BMC Vet Res 2015 Nov 26;11:290. Epub 2015 Nov 26.

Department of Veterinary Clinical and Animal Sciences, University of Copenhagen, Frederiksberg, Denmark.

Background: Staphylococcus aureus is an important human opportunistic pathogen residing on skin and mucosae of healthy people. Pigs have been identified as a source of human colonization and infection with methicillin-resistant Staphylococcus aureus (MRSA) and novel measures are needed to control zoonotic transmission. A recent longitudinal study indicated that a minority of pigs characterized by high nasal load and stable carriage may be responsible for the maintenance of S. aureus within farms. The primary objective of the present study was to detect genetic loci associated with nasal carriage of S. aureus in Danish crossbred pigs (Danish Landrace/Yorkshire/Duroc).

Results: Fifty-six persistent carriers and 65 non-carriers selected from 15 farms surveyed in the previous longitudinal study were genotyped using Illumina's Porcine SNP60 beadchip. In addition, spa typing was performed on 126 S. aureus isolates from 37 pigs to investigate possible relationships between host and S. aureus genotypes. A single SNP (MARC0099960) on chromosome 12 was found to be associated with nasal carriage of S. aureus at a genome-wide level after permutation testing (p = 0.0497) whereas the association of a neighboring SNP was found to be borderline (p = 0.114). Typing of S. aureus isolates led to detection of 11 spa types belonging to the three main S. aureus clonal complexes (CC) previously described in pigs (CC9, CC30 and CC398). Individual carriers often harbored multiple S. aureus genotypes and the host-pathogen interaction seems to be independent of S. aureus genotype.

Conclusion: Our results suggest it may be possible to select pigs genetically resistant to S. aureus nasal colonization as a tool to control transmission of livestock-associated MRSA to humans.
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http://dx.doi.org/10.1186/s12917-015-0599-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4662016PMC
November 2015

Comparative Analyses of QTLs Influencing Obesity and Metabolic Phenotypes in Pigs and Humans.

PLoS One 2015 8;10(9):e0137356. Epub 2015 Sep 8.

Department of Veterinary Clinical and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

The pig is a well-known animal model used to investigate genetic and mechanistic aspects of human disease biology. They are particularly useful in the context of obesity and metabolic diseases because other widely used models (e.g. mice) do not completely recapitulate key pathophysiological features associated with these diseases in humans. Therefore, we established a F2 pig resource population (n = 564) designed to elucidate the genetics underlying obesity and metabolic phenotypes. Segregation of obesity traits was ensured by using breeds highly divergent with respect to obesity traits in the parental generation. Several obesity and metabolic phenotypes were recorded (n = 35) from birth to slaughter (242 ± 48 days), including body composition determined at about two months of age (63 ± 10 days) via dual-energy x-ray absorptiometry (DXA) scanning. All pigs were genotyped using Illumina Porcine 60k SNP Beadchip and a combined linkage disequilibrium-linkage analysis was used to identify genome-wide significant associations for collected phenotypes. We identified 229 QTLs which associated with adiposity- and metabolic phenotypes at genome-wide significant levels. Subsequently comparative analyses were performed to identify the extent of overlap between previously identified QTLs in both humans and pigs. The combined analysis of a large number of obesity phenotypes has provided insight in the genetic architecture of the molecular mechanisms underlying these traits indicating that QTLs underlying similar phenotypes are clustered in the genome. Our analyses have further confirmed that genetic heterogeneity is an inherent characteristic of obesity traits most likely caused by segregation or fixation of different variants of the individual components belonging to cellular pathways in different populations. Several important genes previously associated to obesity in human studies, along with novel genes were identified. Altogether, this study provides novel insight that may further the current understanding of the molecular mechanisms underlying human obesity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137356PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4562524PMC
May 2016

Gender and Obesity Specific MicroRNA Expression in Adipose Tissue from Lean and Obese Pigs.

PLoS One 2015 29;10(7):e0131650. Epub 2015 Jul 29.

Animal Genetics, Department of Veterinary Clinical and Animal Science, Faculty of Health Sciences, University of Copenhagen, Frederiksberg, Denmark; Center for non-coding RNA in Technology and Health, Computational Biology and Bioinformatics, Department of Veterinary Clinical and Animal Science, Faculty of Health Sciences, University of Copenhagen, Frederiksberg, Denmark.

Obesity is a complex condition that increases the risk of life threatening diseases such as cardiovascular disease and diabetes. Studying the gene regulation of obesity is important for understanding the molecular mechanisms behind the obesity derived diseases and may lead to better intervention and treatment plans. MicroRNAs (miRNAs) are short non-coding RNAs regulating target mRNA by binding to their 3'UTR. They are involved in numerous biological processes and diseases, including obesity. In this study we use a mixed breed pig model designed for obesity studies to investigate differentially expressed miRNAs in subcutaneous adipose tissue by RNA sequencing (RNAseq). Both male and female pigs are included to explore gender differences. The RNAseq study shows that the most highly expressed miRNAs are in accordance with comparable studies in pigs and humans. A total of six miRNAs are differentially expressed in subcutaneous adipose tissue between the lean and obese group of pigs, and in addition gender specific significant differential expression is observed for a number of miRNAs. The differentially expressed miRNAs have been verified using qPCR. The results of these studies in general confirm the trends found by RNAseq. Mir-9 and mir-124a are significantly differentially expressed with large fold changes in subcutaneous adipose tissue between lean and obese pigs. Mir-9 is more highly expressed in the obese pigs with a fold change of 10 and a p-value < 0.001. Mir-124a is more highly expressed in the obese pigs with a fold change of 114 and a p-value < 0.001. In addition, mir-124a is significantly higher expressed in abdominal adipose tissue in male pigs with a fold change of 119 and a p-value < 0.05. Both miRNAs are also significantly higher expressed in the liver of obese male pigs where mir-124a has a fold change of 12 and mir-9 has a fold change of 1.6, both with p-values < 0.05.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131650PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4519260PMC
April 2016

Integrative analysis of kinase networks in TRAIL-induced apoptosis provides a source of potential targets for combination therapy.

Sci Signal 2015 Apr 7;8(371):rs3. Epub 2015 Apr 7.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario M5G 1X5, Canada.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous secreted peptide and, in preclinical studies, preferentially induces apoptosis in tumor cells rather than in normal cells. The acquisition of resistance in cells exposed to TRAIL or its mimics limits their clinical efficacy. Because kinases are intimately involved in the regulation of apoptosis, we systematically characterized kinases involved in TRAIL signaling. Using RNA interference (RNAi) loss-of-function and cDNA overexpression screens, we identified 169 protein kinases that influenced the dynamics of TRAIL-induced apoptosis in the colon adenocarcinoma cell line DLD-1. We classified the kinases as sensitizers or resistors or modulators, depending on the effect that knockdown and overexpression had on TRAIL-induced apoptosis. Two of these kinases that were classified as resistors were PX domain-containing serine/threonine kinase (PXK) and AP2-associated kinase 1 (AAK1), which promote receptor endocytosis and may enable cells to resist TRAIL-induced apoptosis by enhancing endocytosis of the TRAIL receptors. We assembled protein interaction maps using mass spectrometry-based protein interaction analysis and quantitative phosphoproteomics. With these protein interaction maps, we modeled information flow through the networks and identified apoptosis-modifying kinases that are highly connected to regulated substrates downstream of TRAIL. The results of this analysis provide a resource of potential targets for the development of TRAIL combination therapies to selectively kill cancer cells.
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http://dx.doi.org/10.1126/scisignal.2005700DOI Listing
April 2015

Micro-flow-injection analysis (μFIA) immunoassay of herbicide residue 2,6-dichlorobenzamide – towards automated at-line monitoring using modular microfluidics.

Analyst 2015 Mar;140(5):1616-23

Department of Micro- and Nanotechnology, Technical University of Denmark, Ørsteds Plads, Bygning 345Ø, 2800 Kgs. Lyngby, Denmark.

As a part of developing new systems for continuously monitoring the presence of pesticides in groundwater, a microfluidic amperometric immunosensor was developed for detecting the herbicide residue 2,6-dichlorobenzamide (BAM) in water. A competitive immunosorbent assay served as the sensing mechanism and amperometry was applied for detection. Both the immunoreaction chip (IRC) and detection (D) unit are integrated on a modular microfluidic platform with in-built micro-flow-injection analysis (μFIA) function. The immunosorbent, immobilized in the channel of the IRC, was found to have high long-term stability and withstand many regeneration cycles, both of which are key requirements for systems utilized in continuous monitoring. The IRC was regenerated during 51 cycles in a heterogeneous competitive assay out of which 27 were without the analyte (the highest possible signal level) in order to assess the regeneration capability of the immunosorbent. Detection of BAM standard solutions was performed in the concentration range from 62.5 μg L(-1) to 0.0008 μg L(-1). Non-linear regression of the data using the four-parameter logistic equation generated a sigmoidal standard curve showing an IC50 value (concentration that reduces the signal by 50%) of 0.25 μg L(-1). The strongest signal variation is observed in the concentration range between 0.02 and 2.5 μg L(-1), which includes the 0.1 μg L(-1) threshold limit set by the European Commission for BAM in drinking water. The presented results demonstrate the potential of the constructed μFIA immunosensor as an at-line monitoring system for controlling the quality of ground water supply.
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http://dx.doi.org/10.1039/c4an01576bDOI Listing
March 2015

Reproducible automated phosphopeptide enrichment using magnetic TiO2 and Ti-IMAC.

Anal Chem 2014 Oct 2;86(20):10296-302. Epub 2014 Oct 2.

The Institute of Cancer Research , 237 Fulham Road, London SW3 6JB, United Kingdom.

Reproducible, comprehensive phosphopeptide enrichment is essential for studying phosphorylation-regulated processes. Here, we describe the application of hyper-porous magnetic TiO2 and Ti-IMAC microspheres for uniform automated phosphopeptide enrichment. Combining magnetic microspheres with a magnetic particle-handling robot enables rapid (45 min), reproducible (r2 ≥ 0.80) and high-fidelity (>90% purity) phosphopeptide purification in a 96-well format. Automated phosphopeptide enrichment demonstrates reproducible synthetic phosphopeptide recovery across 2 orders of magnitude, "well-to-well" quantitative reproducibility indistinguishable to internal SILAC standards, and robust "plate-to-plate" reproducibility across 5 days of independent enrichments. As a result, automated phosphopeptide enrichment enables statistical analysis of label-free phosphoproteomic samples in a high-throughput manner. This technique uses commercially available, off-the-shelf components and can be easily adopted by any laboratory interested in phosphoproteomic analysis. We provide a free downloadable automated phosphopeptide enrichment program to facilitate uniform interlaboratory collaboration and exchange of phosphoproteomic data sets.
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http://dx.doi.org/10.1021/ac5025842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4206527PMC
October 2014

Systematic evaluation of quantotypic peptides for targeted analysis of the human kinome.

Nat Methods 2014 Oct 24;11(10):1041-4. Epub 2014 Aug 24.

1] Division of Cancer Biology, The Institute of Cancer Research, London, UK. [2].

In targeted proteomics it is critical that peptides are not only proteotypic but also accurately represent the level of the protein (quantotypic). Numerous approaches are used to identify proteotypic peptides, but quantotypic properties are rarely assessed. We show that measuring ratios of proteotypic peptides across biological samples can be used to empirically identify peptides with good quantotypic properties. We applied this technique to identify quantotypic peptides for 21% of the human kinome.
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http://dx.doi.org/10.1038/nmeth.3072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180722PMC
October 2014

PKA-regulated VASP phosphorylation promotes extrusion of transformed cells from the epithelium.

J Cell Sci 2014 Aug 24;127(Pt 16):3425-33. Epub 2014 Jun 24.

MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, University College London, London WC1E 6BT, UK Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University, Sapporo 060-0815, Japan

At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in Ras(V12)-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA-VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis.
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http://dx.doi.org/10.1242/jcs.149674DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132389PMC
August 2014

Cell-specific labeling enzymes for analysis of cell-cell communication in continuous co-culture.

Mol Cell Proteomics 2014 Jul 12;13(7):1866-76. Epub 2014 May 12.

From the ‡Division of Cancer Biology, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK;

We report the orthologous screening, engineering, and optimization of amino acid conversion enzymes for cell-specific proteomic labeling. Intracellular endoplasmic-reticulum-anchored Mycobacterium tuberculosis diaminopimelate decarboxylase (DDC(M.tub-KDEL)) confers cell-specific meso-2,6-diaminopimelate-dependent proliferation to multiple eukaryotic cell types. Optimized lysine racemase (Lyr(M37-KDEL)) supports D-lysine specific proliferation and efficient cell-specific isotopic labeling. When ectopically expressed in discrete cell types, these enzymes confer 90% cell-specific isotopic labeling efficiency after 10 days of co-culture. Moreover, DDC(M.tub-KDEL) and Lyr(M37-KDEL) facilitate equally high cell-specific labeling fidelity without daily media exchange. Consequently, the reported novel enzyme pairing can be used to study cell-specific signaling in uninterrupted, continuous co-cultures. Demonstrating the importance of increased labeling stability for addressing novel biological questions, we compare the cell-specific phosphoproteome of fibroblasts in direct co-culture with epithelial tumor cells in both interrupted (daily media exchange) and continuous (no media exchange) co-cultures. This analysis identified multiple cell-specific phosphorylation sites specifically regulated in the continuous co-culture. Given their applicability to multiple cell types, continuous co-culture labeling fidelity, and suitability for long-term cell-cell phospho-signaling experiments, we propose DDC(M.tub-KDEL) and Lyr(M37-KDEL) as excellent enzymes for cell-specific labeling with amino acid precursors.
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http://dx.doi.org/10.1074/mcp.O113.037119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4083121PMC
July 2014

Functional study of a genetic marker allele associated with resistance to Ascaris suum in pigs.

Parasitology 2014 May;141(6):777-87

Parasitology and Aquatic Diseases, Department of Veterinary Disease Biology, University of Copenhagen, Dyrlægevej 100, DK-1870 Frederiksberg C, Denmark.

Two single nucleotide polymorphisms (SNP TXNIP and SNP ARNT), both on chromosome 4, have been reported to be associated with roundworm (Ascaris suum) burden in pigs. In the present study, we selected pigs with two SNP TXNIP genotypes (AA; n = 24 and AB; n = 24), trickle-infected them with A. suum from 8 weeks of age until necropsy 8 weeks later, and tested the hypothesis that pigs with the AA genotype would have higher levels of resistance than pigs of AB genotype. We used different indicators of resistance (worm burden, fecal egg counts (FEC), number of liver white spots and A. suum-specific serum IgG antibody levels). Pigs of the AA genotype had lower mean macroscopic worm burden (2.4 vs 19.3; P = 0.06), lower mean total worm burden (26.5 vs 70.1; P = 0.09) and excreted fewer A. suum eggs at week 8 PI (mean number of eggs/g feces: 238 vs 1259; P = 0.14) than pigs of the AB genotype, as expected based on prior associations. The pigs were also genotyped at another locus (SNP ARNT) which showed a similar trend. This study provides suggestive evidence that resistant pigs may be selected using a genetic marker, TXNIP, and provides further support to the quantitative trait locus on chromosome 4.
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http://dx.doi.org/10.1017/S0031182013002175DOI Listing
May 2014

Identification of hypoxia-regulated proteins using MALDI-mass spectrometry imaging combined with quantitative proteomics.

J Proteome Res 2014 May 24;13(5):2297-313. Epub 2014 Apr 24.

Hypoxia and Metastasis Team and ‡Cell Communications Team, Cancer Research U.K. Tumour Cell Signalling Unit, Division of Cancer Biology, The Institute of Cancer Research , London, United Kingdom.

Hypoxia is present in most solid tumors and is clinically correlated with increased metastasis and poor patient survival. While studies have demonstrated the role of hypoxia and hypoxia-regulated proteins in cancer progression, no attempts have been made to identify hypoxia-regulated proteins using quantitative proteomics combined with MALDI-mass spectrometry imaging (MALDI-MSI). Here we present a comprehensive hypoxic proteome study and are the first to investigate changes in situ using tumor samples. In vitro quantitative mass spectrometry analysis of the hypoxic proteome was performed on breast cancer cells using stable isotope labeling with amino acids in cell culture (SILAC). MS analyses were performed on laser-capture microdissected samples isolated from normoxic and hypoxic regions from tumors derived from the same cells used in vitro. MALDI-MSI was used in combination to investigate hypoxia-regulated protein localization within tumor sections. Here we identified more than 100 proteins, both novel and previously reported, that were associated with hypoxia. Several proteins were localized in hypoxic regions, as identified by MALDI-MSI. Visualization and data extrapolation methods for the in vitro SILAC data were also developed, and computational mapping of MALDI-MSI data to IHC results was applied for data validation. The results and limitations of the methodologies described are discussed.
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http://dx.doi.org/10.1021/pr401056cDOI Listing
May 2014