Publications by authors named "Edwin C A Stigter"

10 Publications

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Pancreatic cancer organoids recapitulate disease and allow personalized drug screening.

Proc Natl Acad Sci U S A 2019 Dec 9. Epub 2019 Dec 9.

Oncode Institute, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands;

We report the derivation of 30 patient-derived organoid lines (PDOs) from tumors arising in the pancreas and distal bile duct. PDOs recapitulate tumor histology and contain genetic alterations typical of pancreatic cancer. In vitro testing of a panel of 76 therapeutic agents revealed sensitivities currently not exploited in the clinic, and underscores the importance of personalized approaches for effective cancer treatment. The PRMT5 inhibitor EZP015556, shown to target (a gene commonly lost in pancreatic cancer)-negative tumors, was validated as such, but also appeared to constitute an effective therapy for a subset of MTAP-positive tumors. Taken together, the work presented here provides a platform to identify novel therapeutics to target pancreatic tumor cells using PDOs.
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http://dx.doi.org/10.1073/pnas.1911273116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6936689PMC
December 2019

A Single Complex Allele in a Patient With Partial Lipodystrophy.

Front Physiol 2018 26;9:1363. Epub 2018 Sep 26.

Center for Molecular Medicine, University Medical Centre Utrecht, Utrecht University, Utrecht, Netherlands.

Genetic lipodystrophies are a group of rare syndromes associated with major metabolic complications - including severe insulin resistance, type 2 diabetes mellitus, and hypertriglyceridemia - which are classified according to the distribution of adipose tissue. Lipodystrophies can be present at birth or develop during life and can range from local to partial and general. With at least 18 different genes implicated so far, definite diagnosis can be challenging due to clinical and genetic heterogeneity. In an adult female patient with clinical and metabolic features of partial lipodystrophy we identified via whole genome sequencing (WGS) a single complex allele [V67M;V167A], functionally equivalent to heterozygosity. encodes for an acyltransferase implicated in the biosynthesis of triacylglycerol and glycerophospholipids. So far homozygous and compound heterozygous mutations in have only been associated with generalized lipodystrophy. A SNP risk score analysis indicated that the index patient is not predisposed to lipodystrophy based on her genetic background. The partial phenotype in our patient is therefore more likely associated to the genetic variants in To test whether the resulting double-mutant AGPAT2 protein is functional we analyzed its enzymatic activity via mass spectrometry. The resulting AGPAT2 double mutant is enzymatically inactive. Our data support the view that the current classification of lipodystrophies as strictly local, partial or generalized may have to be re-evaluated and viewed more as a continuum, both in terms of clinical presentation and underlying genetic causes. Better molecular understanding of lipodystrophies may lead to new therapies to treat adipose tissue dysfunction in common and rare diseases.
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http://dx.doi.org/10.3389/fphys.2018.01363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6168662PMC
September 2018

Aqueous Humor Analysis Identifies Higher Branched Chain Amino Acid Metabolism as a Marker for Human Leukocyte Antigen-B27 Acute Anterior Uveitis and Disease Activity.

Am J Ophthalmol 2019 02 9;198:97-110. Epub 2018 Oct 9.

Ophthalmo-Immunology Unit, University Medical Center Utrecht, Utrecht, the Netherlands; Department of Ophthalmology, University Medical Center Utrecht, Utrecht, the Netherlands; Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, the Netherlands.

Purpose: Human leukocyte antigen-B27 (HLA-B27)-positive acute anterior uveitis (AAU) has a higher recurrence rate and shows more anterior chamber cell infiltration compared with HLA-B27-negative patients, suggesting distinct etiologies of these clinically overlapping conditions. To advance our understanding of the biology of AAU, we characterized the metabolic profile of aqueous humor (AqH) of patients with HLA-B27-associated AAU (B27-AAU) and noninfectious idiopathic AAU (idiopathic AAU).

Design: Experimental laboratory study.

Methods: AqH samples from 2 independent cohorts totaling 30 patients with B27-AAU, 16 patients with idiopathic AAU, and 20 patients with cataracts underwent 2 individual rounds of direct infusion mass spectrometry. Features predicted by direct infusion mass spectrometry that facilitated maximum separation between the disease groups in regression models were validated by liquid chromatography/tandem mass spectrometry-based quantification with appropriate standards.

Results: Partial least square-discriminant analysis revealed metabolite profiles that were able to separate patients with B27-AAU from those with iodiopathic AAU. Pathway enrichment analysis, based on metabolites on which separation of the groups in the partial least square-discriminant analysis model was based, demonstrated the involvement of branched-chain amino acid biosynthesis, ascorbate and aldarate metabolism, the tricarboxylic acid cycle, and glycolysis-diverting pathways (eg, serine biosynthesis) across all investigated cohorts. Notably, the metabolite ketoleucine was elevated in B27-AAU across all 3 runs and moderately-but robustly-correlated with anterior chamber cell count (correlation coefficient range 0.41-0.81).

Conclusions: These results illustrate metabolic heterogeneity between HLA-B27-positive and HLA-B27-negative AAU, including an increase of branched-chain amino acid biosynthesis, that reflects disease activity in AAU.
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http://dx.doi.org/10.1016/j.ajo.2018.10.004DOI Listing
February 2019

Intestinal Failure and Aberrant Lipid Metabolism in Patients With DGAT1 Deficiency.

Gastroenterology 2018 07 29;155(1):130-143.e15. Epub 2018 Mar 29.

Ludwig Boltzmann Institute for Rare and Undiagnosed Diseases, Vienna, Austria; CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria; Department of Pediatrics and Adolescent Medicine, Medical University of Vienna, Vienna, Austria; St. Anna Kinderspital and Children's Cancer Research Institute, Department of Pediatrics, Medical University of Vienna, Vienna, Austria. Electronic address:

Background & Aims: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids.

Methods: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7.

Results: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids.

Conclusions: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.
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http://dx.doi.org/10.1053/j.gastro.2018.03.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058035PMC
July 2018

Bacterial surface layer proteins as a novel capillary coating material for capillary electrophoretic separations.

Anal Chim Acta 2016 Jun 8;923:89-100. Epub 2016 Apr 8.

Division of Analytical Biosciences, Leiden Academic Centre for Drug Research, Universiteit Leiden, Einsteinweg 55, 2300, RA, Leiden, The Netherlands.

A novel concept for stable coating in capillary electrophoresis, based on recrystallization of surface layer proteins on hydrophobized fused silica capillaries, was demonstrated. Surface layer protein A (SlpA) from Lactobacillus acidophilus bacteria was extracted, purified and used for coating pre-silanized glass substrates presenting different surface wettabilities (either hydrophobic or hydrophilic). Contact angle determination on SlpA-coated hydrophobic silica slides showed that the surfaces turned to hydrophilic after coating (53 ± 5°), due to a protein monolayer formation by protein-surface hydrophobic interactions. Visualization by atomic force microscopy demonstrated the presence of a SlpA layer on methylated silica slides displaying a surface roughness of 0.44 ± 0.02 nm. Additionally, a protein layer was visualized by fluorescence microscopy in methylated silica capillaries coated with SlpA and fluorescein isothiocyanate-labeled. The SlpA-coating showed an outstanding stability, even after treatment with 20 mM NaOH (pH 12.3). The electroosmotic flow in coated capillaries showed a partial suppression at pH 7.50 (3.8 ± 0.5 10(-9) m(2) V(-1) s(-1)) when compared with unmodified fused silica (5.9 ± 0.1 10(-8) m(2) V(-1) s(-1)). To demonstrate the potential of this novel coating, the SlpA-coated capillaries were applied for the first time for electrophoretic separation, and proved to be very suitable for the isotachophoretic separation of lipoproteins in human serum. The separations showed a high degree of repeatability (absolute migration times with 1.1-1.8% coefficient-of-variation (CV) within a day) and 2-3% CV inter-capillary reproducibility. The capillaries were stable for more than 100 runs at pH 9.40, and showed to be an exceptional alternative for challenging electrophoretic separations at long-term use.
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http://dx.doi.org/10.1016/j.aca.2016.04.005DOI Listing
June 2016

Insulin/IGF-1-mediated longevity is marked by reduced protein metabolism.

Mol Syst Biol 2013 Jul 2;9:679. Epub 2013 Jul 2.

University Medical Center Utrecht, Wilhelmina Children's Hospital, Department of Molecular Cancer Research, Section Metabolic Diseases, Utrecht, The Netherlands.

Mutations in the daf-2 gene of the conserved Insulin/Insulin-like Growth Factor (IGF-1) pathway double the lifespan of the nematode Caenorhabditis elegans. This phenotype is completely suppressed by deletion of Forkhead transcription factor daf-16. To uncover regulatory mechanisms coordinating this extension of life, we employed a quantitative proteomics strategy with daf-2 mutants in comparison with N2 and daf-16; daf-2 double mutants. This revealed a remarkable longevity-specific decrease in proteins involved in mRNA processing and transport, the translational machinery, and protein metabolism. Correspondingly, the daf-2 mutants display lower amounts of mRNA and 20S proteasome activity, despite maintaining total protein levels equal to that observed in wild types. Polyribosome profiling in the daf-2 and daf-16;daf-2 double mutants confirmed a daf-16-dependent reduction in overall translation, a phenotype reminiscent of Dietary Restriction-mediated longevity, which was independent of germline activity. RNA interference (RNAi)-mediated knockdown of proteins identified by our approach resulted in modified C. elegans lifespan confirming the importance of these processes in Insulin/IGF-1-mediated longevity. Together, the results demonstrate a role for the metabolism of proteins in the Insulin/IGF-1-mediated extension of life.
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http://dx.doi.org/10.1038/msb.2013.35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3734508PMC
July 2013

Development and validation of a quantitative LC-tandem MS assay for hexadeca-4,7,10,13-tetraenoic acid in human and mouse plasma.

J Chromatogr B Analyt Technol Biomed Life Sci 2013 Apr 24;925:16-9. Epub 2013 Jan 24.

University Medical Centre Utrecht, Department of Metabolic Diseases and Netherlands Metabolomics Centre, Lundlaan 6, 3584 EA Utrecht, The Netherlands.

Upon exposure to platinum analogs, mesenchymal stem cells were recently found to excrete minute amounts of specific lipid mediators that induce chemotherapy resistance. One of these lipids is hexadeca-4,7,10,13-tetraenoic acid (FA(16:4)n-3). Importantly, FA(16:4)n-3 is present in high concentrations in certain fish oils and algae and oral intake of these products also potently induced chemotherapy resistance. These findings suggested that certain foods could negatively affect clinical chemotherapy treatment. In order to allow further study of the relation between FA(16:4)n-3 and clinical chemotherapy resistance, a method for the detection and quantification of FA(16:4)n-3 in plasma is required. Therefore, a quantification method for FA(16:4)n-3 in human and mouse plasma was developed consisting of a liquid-liquid extraction, solid phase clean-up and LC-MS/MS (MRM) analysis. The method was fully validated over a period of three weeks according to the standard protocols and requirements. The linearity range of the method is 1-100 nmol/L (r(2)>0.99) using deuterated FA(16:3)n-3 as an internal standard. The limits of quantification and detection are 1.0 nmol/L and 0.8 nmol/L, respectively. Recoveries for spiked concentrations range from 103 to 108%. The intra-day and inter-day mean precision amounts to 98-106% and 100-108%, respectively. No significant loss of FA(16:4)n-3 is observed upon storage at -80 °C. The developed assay for the detection and quantification of FA(16:4)n-3 in human plasma is robust and reproducible. The validation parameters are within limits of acceptance.
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http://dx.doi.org/10.1016/j.jchromb.2013.01.012DOI Listing
April 2013

Quantification of in vivo oxidative damage in Caenorhabditis elegans during aging by endogenous F3-isoprostane measurement.

Aging Cell 2013 Apr 30;12(2):214-23. Epub 2013 Jan 30.

University Medical Center Utrecht, Department of Metabolic Diseases and Netherlands Metabolomics Center, Utrecht, 3508 AB, The Netherlands.

Oxidative damage is thought to be a major cause in development of pathologies and aging. However, quantification of oxidative damage is methodologically difficult. Here, we present a robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach for accurate, sensitive, and linear in vivo quantification of endogenous oxidative damage in the nematode Caenorhabditis elegans, based on F3-isoprostanes. F3-isoprostanes are prostaglandin-like markers of oxidative damage derived from lipid peroxidation by Reactive Oxygen Species (ROS). Oxidative damage was quantified in whole animals and in multiple cellular compartments, including mitochondria and peroxisomes. Mutants of the mitochondrial electron transport proteins mev-1 and clk-1 showed increased oxidative damage levels. Furthermore, analysis of Superoxide Dismutase (sod) and Catalase (ctl) mutants uncovered that oxidative damage levels cannot be inferred from the phenotype of resistance to pro-oxidants alone and revealed high oxidative damage in a small group of chemosensory neurons. Longitudinal analysis of aging nematodes revealed that oxidative damage increased specifically with postreproductive age. Remarkably, aging of the stress-resistant and long-lived daf-2 insulin/IGF-1 receptor mutant involved distinct daf-16-dependent phases of oxidative damage including a temporal increase at young adulthood. These observations are consistent with a hormetic response to ROS.
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http://dx.doi.org/10.1111/acel.12043DOI Listing
April 2013

Mesenchymal stem cells induce resistance to chemotherapy through the release of platinum-induced fatty acids.

Cancer Cell 2011 Sep;20(3):370-83

Department of Medical Oncology, University Medical Center Utrecht, The Netherlands.

The development of resistance to chemotherapy is a major obstacle for lasting effective treatment of cancer. Here, we demonstrate that endogenous mesenchymal stem cells (MSCs) become activated during treatment with platinum analogs and secrete factors that protect tumor cells against a range of chemotherapeutics. Through a metabolomics approach, we identified two distinct platinum-induced polyunsaturated fatty acids (PIFAs), 12-oxo-5,8,10-heptadecatrienoic acid (KHT) and hexadeca-4,7,10,13-tetraenoic acid (16:4(n-3)), that in minute quantities induce resistance to a broad spectrum of chemotherapeutic agents. Interestingly, blocking central enzymes involved in the production of these PIFAs (cyclooxygenase-1 and thromboxane synthase) prevents MSC-induced resistance. Our findings show that MSCs are potent mediators of resistance to chemotherapy and reveal targets to enhance chemotherapy efficacy in patients.
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http://dx.doi.org/10.1016/j.ccr.2011.08.010DOI Listing
September 2011

Rapid surface plasmon resonance-based inhibition assay of deoxynivalenol.

J Agric Food Chem 2003 Sep;51(20):5843-8

Processing Quality & Safety, NIZO Food Research, Kernhemseweg 2, 6718 ZB Ede, The Netherlands.

Deoxynivalenol belongs to a group of highly toxic fungal metabolites produced by Fusarium species that may contaminate food and animal feed, mostly grains. Three different monoclonal mouse anti-deoxynivalenol antibodies were compared for the development of a surface plasmon resonance (SPR)-based immunoassay for the selective and quantitative determination of deoxynivalenol in naturally contaminated matrices. A conjugate of deoxynivalenol with the protein casein was prepared and immobilized on the sensor chip surface. An excess of antibody was added to each test solution before the measurement. The assay was based on the competition for antibody binding between the immobilized deoxynivalenol conjugate on the sensor and the free deoxynivalenol molecules in the test solution. The deoxynivalenol-casein sensor could be reused more than 500 times without significant loss of activity using 6 M guanidine chloride solution for regeneration. The cross-reactivity of the three antibodies in the SPR assay was tested with other trichothecene mycotoxins (3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, HT2-toxin, and T2-toxin). The only sample preparation was extraction with max 80 vol % acetonitrile and 10-fold dilution with the running buffer. The assay had an optimal range between 2.5 and 30 ng/mL deoxynivalenol in the test solution. Most results of the SPR-based assay were in agreement with liquid chromatography/tandem mass spectrometry measurements of naturally contaminated wheat samples.
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http://dx.doi.org/10.1021/jf030244dDOI Listing
September 2003