Publications by authors named "Jef Hooyberghs"

40 Publications

Constrained Standardization of Count Data from Massive Parallel Sequencing.

J Mol Biol 2021 May 29;433(11):166966. Epub 2021 Mar 29.

Universiteit Hasselt, Data Science Institute (DSI), Interuniversity Institute for Biostatistics and Statistical Bioinformatics (I-BioStat), Agoralaan, Diepenbeek BE 3590, Belgium; Universiteit Antwerpen, Centre for Proteomics, Groenenborgerlaan 171, Antwerpen BE 2020, Belgium. Electronic address:

In high-throughput omics disciplines like transcriptomics, researchers face a need to assess the quality of an experiment prior to an in-depth statistical analysis. To efficiently analyze such voluminous collections of data, researchers need triage methods that are both quick and easy to use. Such a normalization method for relative quantitation, CONSTANd, was recently introduced for isobarically-labeled mass spectra in proteomics. It transforms the data matrix of abundances through an iterative, convergent process enforcing three constraints: (I) identical column sums; (II) each row sum is fixed (across matrices) and (III) identical to all other row sums. In this study, we investigate whether CONSTANd is suitable for count data from massively parallel sequencing, by qualitatively comparing its results to those of DESeq2. Further, we propose an adjustment of the method so that it may be applied to identically balanced but differently sized experiments for joint analysis. We find that CONSTANd can process large data sets at well over 1 million count records per second whilst mitigating unwanted systematic bias and thus quickly uncovering the underlying biological structure when combined with a PCA plot or hierarchical clustering. Moreover, it allows joint analysis of data sets obtained from different batches, with different protocols and from different labs but without exploiting information from the experimental setup other than the delineation of samples into identically processed sets (IPSs). CONSTANd's simplicity and applicability to proteomics as well as transcriptomics data make it an interesting candidate for integration in multi-omics workflows.
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http://dx.doi.org/10.1016/j.jmb.2021.166966DOI Listing
May 2021

CONSTANd: An Efficient Normalization Method for Relative Quantification in Small- and Large-Scale Omics Experiments in R BioConductor and Python.

J Proteome Res 2021 04 11;20(4):2151-2156. Epub 2021 Mar 11.

Data Science Institute (DSI), Interuniversity Institute for Biostatistics and Statistical Bioinformatics (I-BioStat), Universiteit Hasselt, Agoralaan, Diepenbeek 3590, Belgium.

For differential expression studies in all omics disciplines, data normalization is a crucial step that is often subject to a balance between speed and effectiveness. To keep up with the data produced by high-throughput instruments, researchers require fast and easy-to-use yet effective methods that fit into automated analysis pipelines. The CONSTANd normalization method meets these criteria, so we have made its source code available for R/BioConductor and Python. We briefly review the method and demonstrate how it can be used in different omics contexts for experiments of any scale. Widespread adoption across omics disciplines would ease data integration in multiomics experiments.
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http://dx.doi.org/10.1021/acs.jproteome.0c00977DOI Listing
April 2021

PRiSM: A prototype for exhaustive, restriction-free database searching for mass spectrometry-based identification.

Rapid Commun Mass Spectrom 2020 Oct 2:e8962. Epub 2020 Oct 2.

Universiteit Hasselt, Data Science Institute (DSI), Theoretical Physics, Diepenbeek, Belgium.

Rationale: The current methods for identifying peptides in mass spectral product ion data still struggle to do so for the majority of spectra. Based on the experimental setup and other assumptions, such methods restrict the search space to speed up computations, but at the cost of creating blind spots. The proteomics community would greatly benefit from a method that is capable of covering the entire search space without using any restrictions, thus establishing a baseline for identification.

Methods: We conceived the "mass pattern paradigm" (MPP) that enables the creation of such an identification method, and we implemented it into a prototype database search engine "PRiSM" (PRotein-Spectrum Matching). We then assessed its operational characteristics by applying it to publicly available high-precision mass spectra of low and high identification difficulty. We used those characteristics to gain theoretical insights into trade-offs between sensitivity and speed when trying to establish a baseline for identification.

Results: Of 100 low difficulty spectra, PRiSM and SEQUEST agree on 84 identifications (of which 75 are statistically significant). Of 15 of 100 spectra not identified in a previous study (using SEQUEST), 13 are considered reliable after visual inspection and represent 3 proteins (out of 9 in total) not detected previously.

Conclusions: Despite leaving noise intact, the simple PRiSM prototype can make statistically reliable identifications, while controlling the false discovery rate by fitting a null distribution. It also identifies some spectra previously unidentifiable in an "extremely open" SEQUEST search, paving the way to establishing a baseline for identification in proteomics.
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http://dx.doi.org/10.1002/rcm.8962DOI Listing
October 2020

Leveraging European infrastructures to access 1 million human genomes by 2022.

Nat Rev Genet 2019 11 27;20(11):693-701. Epub 2019 Aug 27.

Global Alliance for Genomics and Health, Toronto, Ontario, Canada.

Human genomics is undergoing a step change from being a predominantly research-driven activity to one driven through health care as many countries in Europe now have nascent precision medicine programmes. To maximize the value of the genomic data generated, these data will need to be shared between institutions and across countries. In recognition of this challenge, 21 European countries recently signed a declaration to transnationally share data on at least 1 million human genomes by 2022. In this Roadmap, we identify the challenges of data sharing across borders and demonstrate that European research infrastructures are well-positioned to support the rapid implementation of widespread genomic data access.
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http://dx.doi.org/10.1038/s41576-019-0156-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7115898PMC
November 2019

Enhancing the Performance of DNA Surface-Hybridization Biosensors through Target Depletion.

Langmuir 2019 09 3;35(37):12276-12283. Epub 2019 Sep 3.

Flemish Institute for Technological Research (VITO) , Boeretang 200 , B-2400 Mol , Belgium.

DNA surface-hybridization biosensors utilize the selective hybridization of target sequences in solution to surface-immobilized probes. In this process, the target is usually assumed to be in excess, so that its concentration does not significantly vary while hybridizing to the surface-bound probes. If the target is initially at low concentrations and/or if the number of probes is very large, and they have high affinity for the target, the DNA in solution may become depleted. In this paper we analyze the equilibrium and kinetics of hybridization of DNA biosensors in the case of strong target depletion, by extending the Langmuir adsorption model. We focus, in particular, on the detection of a small amount of a single-nucleotide "mutant" sequence (concentration ) in a solution, which differs by one or more nucleotides from an abundant "wild-type" sequence (concentration ≫ ). We show that depletion can give rise to a strongly enhanced sensitivity of the biosensors. Using representative values of rate constants and hybridization free energies, we find that in the depletion regime one could detect relative concentrations / that are up to 3 orders of magnitude smaller than in the conventional approach. The kinetics is surprisingly rich and exhibits a nonmonotonic adsorption with no counterpart in the no-depletion case. Finally, we show that, alongside enhanced detection sensitivity, this approach offers the possibility of sample enrichment, by substantially increasing the relative amount of the mutant over the wild-type sequence.
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http://dx.doi.org/10.1021/acs.langmuir.9b01761DOI Listing
September 2019

MIND: A Double-Linear Model To Accurately Determine Monoisotopic Precursor Mass in High-Resolution Top-Down Proteomics.

Anal Chem 2019 08 23;91(15):10310-10319. Epub 2019 Jul 23.

UA-VITO Center for Proteomics , University of Antwerp , 2000 Antwerp , Belgium.

Top-down proteomics approaches are becoming ever more popular, due to the advantages offered by knowledge of the intact protein mass in correctly identifying the various proteoforms that potentially arise due to point mutation, alternative splicing, post-translational modifications, etc. Usually, the average mass is used in this context; however, it is known that this can fluctuate significantly due to both natural and technical causes. Ideally, one would prefer to use the monoisotopic precursor mass, but this falls below the detection limit for all but the smallest proteins. Methods that predict the monoisotopic mass based on the average mass are potentially affected by imprecisions associated with the average mass. To address this issue, we have developed a framework based on simple, linear models that allows prediction of the monoisotopic mass based on the exact mass of the most-abundant (aggregated) isotope peak, which is a robust measure of mass, insensitive to the aforementioned natural and technical causes. This linear model was tested experimentally, as well as in silico, and typically predicts monoisotopic masses with an accuracy of only a few parts per million. A confidence measure is associated with the predicted monoisotopic mass to handle the off-by-one-Da prediction error. Furthermore, we introduce a correction function to extract the "true" (i.e., theoretically) most-abundant isotope peak from a spectrum, even if the observed isotope distribution is distorted by noise or poor ion statistics. The method is available online as an R shiny app: https://valkenborg-lab.shinyapps.io/mind/.
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http://dx.doi.org/10.1021/acs.analchem.9b02682DOI Listing
August 2019

Role of nanoparticle size and sialic acids in the distinct time-evolution profiles of nanoparticle uptake in hematopoietic progenitor cells and monocytes.

J Nanobiotechnology 2019 May 13;17(1):62. Epub 2019 May 13.

Health Department, Flemish Institute For Technological Research (VITO), Boeretang 200, 2400, Mol, Belgium.

Background: Human hematopoietic progenitor cells (HPCs) are important for cell therapy in cancer and tissue regeneration. In vitro studies have shown a transient association of 40 nm polystyrene nanoparticles (PS NPs) with these cells, which is of interest for intelligent design and application of NPs in HPC-based regenerative protocols. In this study, we aimed to investigate the involvement of nanoparticles' size and membrane-attached glycan molecules in the interaction of HPCs with PS NPs, and compared it with monocytes. Human cord blood-derived HPCs and THP-1 cells were exposed to fluorescently labelled, carboxylated PS NPs of 40, 100 and 200 nm. Time-dependent nanoparticle membrane association and/or uptake was observed by measuring fluorescence intensity of exposed cells at short time intervals using flow cytometry. By pretreating the cells with neuraminidase, we studied the possible effect of membrane-associated sialic acids in the interaction with NPs. Confocal microscopy was used to visualize the cell-specific character of the NP association.

Results: Confocal images revealed that the majority of PS NPs was initially observed to be retained at the outer membrane of HPCs, while the same NPs showed immediate internalization by THP-1 monocytic cells. After prolonged exposure up to 4 h, PS NPs were also observed to enter the HPCs' intracellular compartment. Cell-specific time courses of NP association with HPCs and THP-1 cells remained persistent after cells were enzymatically treated with neuraminidase, but significantly increased levels of NP association could be observed, suggesting a role for membrane-associated sialic acids in this process.

Conclusions: We conclude that the terminal membrane-associated sialic acids contribute to the NP retention at the outer cell membrane of HPCs. This retention behavior is a unique characteristic of the HPCs and is independent of NP size.
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http://dx.doi.org/10.1186/s12951-019-0495-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513515PMC
May 2019

QCQuan: A Web Tool for the Automated Assessment of Protein Expression and Data Quality of Labeled Mass Spectrometry Experiments.

J Proteome Res 2019 05 11;18(5):2221-2227. Epub 2019 Apr 11.

VITO NV , Applied Bio & molecular Systems , Boeretang 200 , Mol 2400 , Belgium.

In the context of omics disciplines and especially proteomics and biomarker discovery, the analysis of a clinical sample using label-based tandem mass spectrometry (MS) can be affected by sample preparation effects or by the measurement process itself, resulting in an incorrect outcome. Detection and correction of these mistakes using state-of-the-art methods based on mixed models can use large amounts of (computing) time. MS-based proteomics laboratories are high-throughput and need to avoid a bottleneck in their quantitative pipeline by quickly discriminating between high- and low-quality data. To this end we developed an easy-to-use web-tool called QCQuan (available at qcquan.net ) which is built around the CONSTANd normalization algorithm. It automatically provides the user with exploratory and quality control information as well as a differential expression analysis based on conservative, simple statistics. In this document we describe in detail the scientifically relevant steps that constitute the workflow and assess its qualitative and quantitative performance on three reference data sets. We find that QCQuan provides clear and accurate indications about the scientific value of both a high- and a low-quality data set. Moreover, it performed quantitatively better on a third data set than a comparable workflow assembled using established, reliable software.
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http://dx.doi.org/10.1021/acs.jproteome.9b00072DOI Listing
May 2019

Molecular drug susceptibility testing and strain typing of tuberculosis by DNA hybridization.

PLoS One 2019 7;14(2):e0212064. Epub 2019 Feb 7.

Flemish Institute for Technological Research, VITO, Mol, Belgium.

In Mycobacterium tuberculosis (Mtb) the detection of single nucleotide polymorphisms (SNPs) is of high importance both for diagnostics, since drug resistance is primarily caused by the acquisition of SNPs in multiple drug targets, and for epidemiological studies in which strain typing is performed by SNP identification. To provide the necessary coverage of clinically relevant resistance profiles and strain types, nucleic acid-based measurement techniques must be able to detect a large number of potential SNPs. Since the Mtb problem is pressing in many resource-poor countries, requiring low-cost point-of-care biosensors, this is a non-trivial technological challenge. This paper presents a proof-of-concept in which we chose simple DNA-DNA hybridization as a sensing principle since this can be transferred to existing low-cost hardware platforms, and we pushed the multiplex boundaries of it. With a custom designed probe set and a physicochemical-driven data analysis it was possible to simultaneously detect the presence of SNPs associated with first- and second-line drug resistance and Mtb strain typing. We have demonstrated its use for the identification of drug resistance and strain type from a panel of phylogenetically diverse clinical strains. Furthermore, reliable detection of the presence of a minority population (<5%) of drug-resistant Mtb was possible.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212064PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366778PMC
November 2019

DNA capture into the ClyA nanopore: diffusion-limited versus reaction-limited processes.

J Phys Condens Matter 2018 08 12;30(30):304001. Epub 2018 Jun 12.

KU Leuven, Institute for Theoretical Physics, Celestijnenlaan 200D, 3001 Leuven, Belgium. Flemish Institute for Technological Research (VITO), Boeretang 200, B-2400 Mol, Belgium.

The capture and translocation of biomolecules through nanometer-scale pores are processes with a potentially large number of applications, and hence they have been intensively studied in recent years. The aim of this paper is to review existing models of the capture process by a nanopore, together with some recent experimental data of short single- and double-stranded DNA captured by the Cytolysin A (ClyA) nanopore. ClyA is a transmembrane protein of bacterial origin which has been recently engineered through site-specific mutations, to allow the translocation of double- and single-stranded DNA. A comparison between theoretical estimations and experiments suggests that for both cases the capture is a reaction-limited process. This is corroborated by the observed salt dependence of the capture rate, which we find to be in quantitative agreement with the theoretical predictions.
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http://dx.doi.org/10.1088/1361-648X/aacc01DOI Listing
August 2018

Thermodynamic framework to assess low abundance DNA mutation detection by hybridization.

PLoS One 2017 25;12(5):e0177384. Epub 2017 May 25.

Flemish Institute for Technological Research, VITO, Mol, Belgium.

The knowledge of genomic DNA variations in patient samples has a high and increasing value for human diagnostics in its broadest sense. Although many methods and sensors to detect or quantify these variations are available or under development, the number of underlying physico-chemical detection principles is limited. One of these principles is the hybridization of sample target DNA versus nucleic acid probes. We introduce a novel thermodynamics approach and develop a framework to exploit the specific detection capabilities of nucleic acid hybridization, using generic principles applicable to any platform. As a case study, we detect point mutations in the KRAS oncogene on a microarray platform. For the given platform and hybridization conditions, we demonstrate the multiplex detection capability of hybridization and assess the detection limit using thermodynamic considerations; DNA containing point mutations in a background of wild type sequences can be identified down to at least 1% relative concentration. In order to show the clinical relevance, the detection capabilities are confirmed on challenging formalin-fixed paraffin-embedded clinical tumor samples. This enzyme-free detection framework contains the accuracy and efficiency to screen for hundreds of mutations in a single run with many potential applications in molecular diagnostics and the field of personalised medicine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0177384PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5444680PMC
September 2017

Direct detection of nano-scale extracellular vesicles derived from inflammation-triggered endothelial cells using surface plasmon resonance.

Nanomedicine 2017 07 30;13(5):1663-1671. Epub 2017 Mar 30.

Hasselt University, Biomedical Research Institute, Hasselt, Belgium. Electronic address:

A major conceptual breakthrough in cell signaling has been the finding of EV as new biomarker shuttles in body fluids. Now, one of the major challenges in using these nanometer-sized biological entities as diagnostic marker is the development of translational methodologies to profile them. SPR offers a promising label-free and real time platform with a high potential for biomarker detection. Therefore, we aimed to develop a uniform SPR methodology to detect specific surface markers on EV derived from patient with CHD. EVs having an approximate size range between 30 and 100 nm (~48.5%) and 100-300 nm (~51.5%) were successfully isolated. The biomarker profile of EV was verified using immunogold labeling, ELISA and SPR. Using SPR, we demonstrated an increased binding of EV derived from patients with CHD to anti-ICAM-1 antibodies as compared to EV from healthy donors. Our current findings open up novel opportunities for in-depth and label-free investigation of EV.
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http://dx.doi.org/10.1016/j.nano.2017.03.010DOI Listing
July 2017

Transient loading of CD34 hematopoietic progenitor cells with polystyrene nanoparticles.

Int J Nanomedicine 2017 12;12:459-472. Epub 2017 Jan 12.

VITO, Flemish Institute for Technological Research, Mol, Belgium; Theoretical Physics, Hasselt University, Diepenbeek, Belgium.

CD34 hematopoietic progenitor cells (HPCs) offer great opportunities to develop new treatments for numerous malignant and non-malignant diseases. Nanoparticle (NP)-based strategies can further enhance this potential, and therefore a thorough understanding of the loading behavior of HPCs towards NPs is essential for a successful application. The present study focusses on the interaction kinetics of 40 nm sized carboxylated polystyrene (PS) NPs with HPCs. Interestingly, a transient association of the NPs with HPCs is observed, reaching a maximum within 1 hour and declining afterwards. This behavior is not seen in dendritic cells (CD34-DCs) differentiated from HPCs, which display a monotonic increase in NP load. We demonstrate that this transient interaction requires an energy-dependent cellular process, suggesting active loading and release of NPs by HPCs. This novel observation offers a unique approach to transiently equip HPCs. A simple theoretical approach modeling the kinetics of NP loading and release is presented, contributing to a framework of describing this phenomenon.
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http://dx.doi.org/10.2147/IJN.S119407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5238761PMC
March 2017

Nonequilibrium Hybridization Enables Discrimination of a Point Mutation within 5-40 °C.

J Am Chem Soc 2016 Oct 4;138(41):13465-13468. Epub 2016 Oct 4.

Chemistry Department, University of Central Florida , Orlando, Florida 32816, United States.

Detection of point mutations and single nucleotide polymorphisms in DNA and RNA has a growing importance in biology, biotechnology, and medicine. For the application at hand, hybridization assays are often used. Traditionally, they differentiate point mutations only at elevated temperatures (>40 °C) and in narrow intervals (ΔT = 1-10 °C). The current study demonstrates that a specially designed multistranded DNA probe can differentiate point mutations in the range of 5-40 °C. This unprecedentedly broad ambient-temperature range is enabled by a controlled combination of (i) nonequilibrium hybridization conditions and (ii) a mismatch-induced increase of equilibration time in respect to that of a fully matched complex, which we dub "kinetic inversion".
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http://dx.doi.org/10.1021/jacs.6b05628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645261PMC
October 2016

CONSTANd : A Normalization Method for Isobaric Labeled Spectra by Constrained Optimization.

Mol Cell Proteomics 2016 08 14;15(8):2779-90. Epub 2016 Jun 14.

From the: ‡Applied Bio & molecular Systems, VITO, Boeretang 200, 2400 Mol, Belgium; §Center for Proteomics, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium; ‖Center for Statistics, Hasselt University, Agoralaan 1, 3590 Diepenbeek, Belgium

In quantitative proteomics applications, the use of isobaric labels is a very popular concept as they allow for multiplexing, such that peptides from multiple biological samples are quantified simultaneously in one mass spectrometry experiment. Although this multiplexing allows that peptide intensities are affected by the same amount of instrument variability, systematic effects during sample preparation can also introduce a bias in the quantitation measurements. Therefore, normalization methods are required to remove this systematic error. At present, a few dedicated normalization methods for isobaric labeled data are at hand. Most of these normalization methods include a framework for statistical data analysis and rely on ANOVA or linear mixed models. However, for swift quality control of the samples or data visualization a simple normalization technique is sufficient. To this aim, we present a new and easy-to-use data-driven normalization method, named CONSTANd. The CONSTANd method employs constrained optimization and prior information about the labeling strategy to normalize the peptide intensities. Further, it allows maintaining the connection to any biological effect while reducing the systematic and technical errors. As a result, peptides can not only be compared directly within a multiplexed experiment, but are also comparable between other isobaric labeled datasets from multiple experimental designs that are normalized by the CONSTANd method, without the need to include a reference sample in every experimental setup. The latter property is especially useful when more than six, eight or ten (TMT/iTRAQ) biological samples are required to detect differential peptides with sufficient statistical power and to optimally make use of the multiplexing capacity of isobaric labels.
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http://dx.doi.org/10.1074/mcp.M115.056911DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974351PMC
August 2016

Designing biomedical proteomics experiments: state-of-the-art and future perspectives.

Expert Rev Proteomics 2016 05 25;13(5):495-511. Epub 2016 Apr 25.

a Applied Bio & molecular systems , VITO , Mol , Belgium.

With the current expanded technical capabilities to perform mass spectrometry-based biomedical proteomics experiments, an improved focus on the design of experiments is crucial. As it is clear that ignoring the importance of a good design leads to an unprecedented rate of false discoveries which would poison our results, more and more tools are developed to help researchers designing proteomic experiments. In this review, we apply statistical thinking to go through the entire proteomics workflow for biomarker discovery and validation and relate the considerations that should be made at the level of hypothesis building, technology selection, experimental design and the optimization of the experimental parameters.
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http://dx.doi.org/10.1586/14789450.2016.1172967DOI Listing
May 2016

Intracellular dynamics and fate of polystyrene nanoparticles in A549 Lung epithelial cells monitored by image (cross-) correlation spectroscopy and single particle tracking.

Biochim Biophys Acta 2015 Oct 9;1853(10 Pt A):2411-9. Epub 2015 Jul 9.

Biomedical Research Unit, Hasselt University, Agoralaan Building C, 3590 Diepenbeek, Belgium. Electronic address:

Novel insights in nanoparticle (NP) uptake routes of cells, their intracellular trafficking and subcellular targeting can be obtained through the investigation of their temporal and spatial behavior. In this work, we present the application of image (cross-) correlation spectroscopy (IC(C)S) and single particle tracking (SPT) to monitor the intracellular dynamics of polystyrene (PS) NPs in the human lung carcinoma A549 cell line. The ensemble kinetic behavior of NPs inside the cell was characterized by temporal and spatiotemporal image correlation spectroscopy (TICS and STICS). Moreover, a more direct interpretation of the diffusion and flow detected in the NP motion was obtained by SPT by monitoring individual NPs. Both techniques demonstrate that the PS NP transport in A549 cells is mainly dependent on microtubule-assisted transport. By applying spatiotemporal image cross-correlation spectroscopy (STICCS), the correlated motions of NPs with the early endosomes, late endosomes and lysosomes are identified. PS NPs were equally distributed among the endolysosomal compartment during the time interval of the experiments. The cotransport of the NPs with the lysosomes is significantly larger compared to the other cell organelles. In the present study we show that the complementarity of ICS-based techniques and SPT enables a consistent elaborate model of the complex behavior of NPs inside biological systems.
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http://dx.doi.org/10.1016/j.bbamcr.2015.07.004DOI Listing
October 2015

Coherent intensity fluctuation model for autocorrelation imaging spectroscopy with higher harmonic generating point scatterers-a comprehensive theoretical study.

Phys Chem Chem Phys 2015 Jul;17(29):18937-43

Biomed, Hasselt University, Agoralaan, Bldg C, B-3590 Diepenbeek, Belgium.

We present a general analytical model for the intensity fluctuation autocorrelation function for second and third harmonic generating point scatterers. Expressions are derived for a stationary laser beam and for scanning beam configurations for specific correlation methodologies. We discuss free translational diffusion in both three and two dimensions. At low particle concentrations, the expressions for fluorescence are retrieved, while at high particle concentrations a rescaling of the function parameters is required for a stationary illumination beam, provided that the phase shift per unit length of the beam equals zero.
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http://dx.doi.org/10.1039/c5cp02567bDOI Listing
July 2015

Gene expressions changes in bronchial epithelial cells: markers for respiratory sensitizers and exploration of the NRF2 pathway.

Toxicol In Vitro 2014 Mar 6;28(2):209-17. Epub 2013 Nov 6.

Flemish Institute for Technological Research (VITO), Unit Environmental Risk and Health, Boeretang 200, Mol, Belgium; Department of Biomedical Sciences, Antwerp University, Universiteitsplein 1, Antwerp, Belgium; Department of Environmental Medicine, University of Southern Denmark, Winslowsparken 17, Odense, Denmark. Electronic address:

For the classification of respiratory sensitizing chemicals, no validated in vivo nor in vitro tests are currently available. In this study, we evaluated whether respiratory sensitizers trigger specific signals in human bronchial epithelial (BEAS-2B) cells at the level of the transcriptome. The cells were exposed during 6, 10, and 24h to 4 respiratory sensitizers and 6 non-respiratory sensitizers (3 skin sensitizers and 3 respiratory irritants) at a concentration inducing 20% cell viability loss after 24h. Changes in gene expression were evaluated using Agilent Whole Human Genome 4×44K oligonucleotide arrays. A limited number of 11 transcripts could be identified as potential biomarkers to identify respiratory sensitizers. Three of these transcripts are associated to immune system processes (HSPA5, UPP1, and SEPRINE1). In addition, the transcriptome was screened for transcripts that are differentially expressed compared to vehicle control for each chemical. The results show that the NRF2-mediated oxidative stress response is activated in the cell line after stimulation with all of the chemicals that were selected in our study, and that - at the level of gene expression - this pathway shows no potential to discriminate between any of the three compound groups: respiratory sensitizers, skin sensitizers, or electrophilic respiratory irritants.
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http://dx.doi.org/10.1016/j.tiv.2013.10.017DOI Listing
March 2014

Targeted resequencing of HIV variants by microarray thermodynamics.

Nucleic Acids Res 2013 Oct 8;41(18):e173. Epub 2013 Aug 8.

Flemish Institute for Technological Research, VITO, Boeretang 200, B-2400 Mol, Belgium, Institute for Theoretical Physics, KULeuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium, Janssen Diagnostics bvba, Turnhoutseweg 30, B-2340 Beerse, Belgium and Theoretical Physics, Hasselt University, Campus Diepenbeek, Agoralaan - Building D, B-3590, Diepenbeek, Belgium.

Within a single infected individual, a virus population can have a high genomic variability. In the case of HIV, several mutations can be present even in a small genomic window of 20-30 nucleotides. For diagnostics purposes, it is often needed to resequence genomic subsets where crucial mutations are known to occur. In this article, we address this issue using DNA microarrays and inputs from hybridization thermodynamics. Hybridization signals from multiple probes are analysed, including strong signals from perfectly matching (PM) probes and a large amount of weaker cross-hybridization signals from mismatching (MM) probes. The latter are crucial in the data analysis. Seven coded clinical samples (HIV-1) are analyzed, and the microarray results are in full concordance with Sanger sequencing data. Moreover, the thermodynamic analysis of microarray signals resolves inherent ambiguities in Sanger data of mixed samples and provides additional clinically relevant information. These results show the reliability and added value of DNA microarrays for point-of-care diagnostic purposes.
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http://dx.doi.org/10.1093/nar/gkt682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794611PMC
October 2013

Screening for potential hazard effects from four nitramines on human eye and skin.

Toxicol In Vitro 2013 Jun 14;27(4):1205-10. Epub 2013 Feb 14.

Health Effects Laboratory, Department of Environmental Chemistry, NILU-Norwegian Institute for Air Research, Instituttveien 18, Kjeller, Norway.

Amines have potential to be used in CO2 capture and storage (CCS) technology, but as they can be released into the environment and be degraded into more toxic compounds, such as nitrosamines and nitramines, there have been concerns about their negative impact on human health. We investigated the potential toxic effects from acute exposure to dimethylnitramine (DMA-NO2), methylnitramine (MA-NO2), ethanolnitramine (MEA-NO2) and 2-methyl-2-(nitroamino)-1-propanol (AMP-NO2). The eye irritation, and skin sensitization, irritation and corrosion potential of these substances have been evaluated in vitro using the Bovine Corneal Opacity and Permeability (BCOP) assay, VITOSENS® assay, Reconstructed Human Epidermis (RHE) skin irritation test and Corrositex Skin corrosion test, respectively. Exposure to DMA-NO2 induced a mild eye irritation response, while MA-NO2, MEA-NO2 and AMP-NO2 were shown to be very severe eye irritants. MA-NO2 and MEA-NO2 were tested for skin sensitization and found to be non-sensitizers to the skin. In addition, none of the four test substances was irritant or corrosive to the skin.
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http://dx.doi.org/10.1016/j.tiv.2013.02.004DOI Listing
June 2013

Assessment of the developmental neurotoxicity of compounds by measuring locomotor activity in zebrafish embryos and larvae.

Neurotoxicol Teratol 2013 May-Jun;37:44-56. Epub 2013 Jan 26.

VITO NV, Flemish Institute for Technological Research, Environmental Risk and Health, Boeretang 200, Mol, Belgium.

The developmental neurotoxic potential of the majority of environmental chemicals and drugs is currently undetermined. Specific in vivo studies provide useful data for hazard assessment but are not amenable to screen thousands of untested compounds. In this study, methods which use zebrafish embryos, eleutheroembryos and larvae as model organisms, were proposed as alternatives for developmental neurotoxicity (DNT) testing. The evaluation of spontaneous tail coilings in zebrafish embryos aged 24-26 hours post fertilization (hpf) and the swimming activity of eleutheroembryos at 120 and larvae at 144 hpf, i.e. parameters for locomotor activity, were investigated as potential endpoints for DNT testing, according to available standard protocols. The overall performance and predictive value of these methods was then examined by testing a training set of 10 compounds, including known developmental neurotoxicants and compounds not considered to be neurotoxic. The classification of the selected compounds as either neurotoxic or non-neurotoxic, based on the effects observed in zebrafish embryos and larvae, was compared to available mammalian data and an overall concordance of 90% was achieved. Furthermore, the specificity of the selected endpoints for DNT was evaluated as well as the potential similarities between zebrafish and mammals with regard to mechanisms of action for the selected compounds. Although further studies, including the screening of a large testing set of compounds are required, we suggest that the proposed methods with zebrafish embryos and larvae might be valuable alternatives for animal testing for the screening and prioritization of compounds for DNT.
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http://dx.doi.org/10.1016/j.ntt.2013.01.003DOI Listing
December 2013

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments.

Nucleic Acids Res 2013 Mar 9;41(5):2779-96. Epub 2013 Jan 9.

University of Essex-Mathematical Sciences, Colchester CO4 3SQ, Essex, United Kingdom.

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.
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http://dx.doi.org/10.1093/nar/gks1358DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597649PMC
March 2013

Functionality and specificity of gene markers for skin sensitization in dendritic cells.

Toxicol Lett 2011 Jun 26;203(2):106-10. Epub 2011 Feb 26.

Flemish Institute for Technological Research (VITO N.V.), Environmental Risk and Health Unit, Toxicology, Belgium.

Transcriptomic analyses revealed a discriminating gene expression profile in human CD34+ progenitor-derived dendritic cells (DC) after exposure to skin sensitizers versus non-sensitizers. Starting from the differential expression in a small set of genes, a preliminary classification model (VITOSENS®) has been developed to identify chemicals as (non-)sensitizing. The objective of the current study is to gain knowledge on the role of the VITOSENS® markers in the DC maturation process, as well as to investigate their activation by a skin sensitizer versus a non-sensitizing danger molecule. To evaluate the functional relevance of VITOSENS® biomarkers in DC maturation, their response induced by the sensitizer dinitrofluorobenzene (DNFB) was pharmacologically counteracted. Flow cytometry analyses revealed that CD86 was down-regulated after COX2 inhibition, whereas expression of HLA-DR was reduced by stimulating CCR2. When exposing DC to DNFB versus lipopolysaccharide S (LPS), expression of most discriminating genes CREM and CCR2 was not altered by LPS as opposed to DNFB. To summarize, the observations in this research indicate that a selection of the VITOSENS® genes may be functionally involved in sensitizer-induced DC activation. By comparing their responsiveness towards a non-sensitizing danger signal and a sensitizer, VITOSENS® gene markers CREM and CCR2 appear to display a specific response.
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http://dx.doi.org/10.1016/j.toxlet.2011.02.015DOI Listing
June 2011

Hybridisation thermodynamic parameters allow accurate detection of point mutations with DNA microarrays.

Biosens Bioelectron 2010 Dec 16;26(4):1692-5. Epub 2010 Aug 16.

Flemish Institute for Technological Research (VITO), Boeretang 200, B-2400 Mol, Belgium.

We consider mixtures of two DNA sequences t and t' differing by a single nucleotide, which are analyzed by an Agilent custom DNA microarray. In particular we focus on the case in which t, the "wild type", is predominantly abundant and t' the "mutant" is at very low concentrations compared to t. We show that by using appropriately designed arrays it is possible to accurately quantify the presence of t' even at low relative concentrations (≈1%). The detection method is based on thermodynamic models of DNA hybridisation and on the analysis of a large number of hybridisation intensities from probes containing one or two mismatches with respect to t and t'.
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http://dx.doi.org/10.1016/j.bios.2010.07.039DOI Listing
December 2010

Gene markers in dendritic cells unravel pieces of the skin sensitization puzzle.

Toxicol Lett 2010 Jul 10;196(2):95-103. Epub 2010 Apr 10.

Flemish Institute for Technological Research (VITO N.V.), Environmental Risk and Health Unit, Toxicology, Belgium.

The underlying events of how dendritic cells (DC) are capable of evoking an antigen-specific skin sensitization response are not yet understood. Recently, we revealed a set of genes in human cord blood CD34(+) DC (CD34-DC) that show a discriminating behaviour after skin sensitizing exposure. Based on their differential expression, an in vitro assay was developed to identify chemicals as sensitizing or not. This study was designed to investigate the genes' involvement in the DC response to skin sensitizers and as such gain insights in the sensitization cascade. Functional connection of the marker genes was inquired by constructing a molecular network using Ingenuity software. By real-time RT-qPCR, we established the effective expression of 3 additional gene transcripts in the generated network in CD34-DC, of which CREB1 and TNF-alpha were significantly altered in expression by sensitizing versus non-sensitizing exposure. Next, it was tested whether the discriminating response of CCR2 and COX2 marker genes was translated at the protein level in CD34-DC exposed to 3 sensitizers versus 3 non-sensitizers. Significantly differential protein expression of CCR2 and COX2 was confirmed using flow cytometry. Our results indicate that the marker genes may be functionally relevant in DC mediated skin sensitization.
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http://dx.doi.org/10.1016/j.toxlet.2010.04.003DOI Listing
July 2010

Assessment of chemical skin-sensitizing potency by an in vitro assay based on human dendritic cells.

Toxicol Sci 2010 Jul 7;116(1):122-9. Epub 2010 Apr 7.

Unit Environmental Risk and Health, Toxicology, Flemish Institute for Technological Research (VITO NV), Mol, Belgium.

The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemicals as skin (non-)sensitizing. However, skin sensitization is not an all-or-none phenomenon, and up to now, the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study, we analyzed the feasibility to predict the sensitizing potency, i.e., the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto, we extended the in vitro-generated gene expression data set by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes but that it does correlate with LLNA EC3 values. In a further analysis, we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of four orders of magnitude, i.e., from weak to extreme sensitizers.
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http://dx.doi.org/10.1093/toxsci/kfq108DOI Listing
July 2010

Locomotor activity in zebrafish embryos: a new method to assess developmental neurotoxicity.

Neurotoxicol Teratol 2010 Jul-Aug;32(4):460-71. Epub 2010 Mar 6.

VITO, Flemish Institute for Technological Research, Unit of Environmental Risk and Health, Division of Toxicology, Boeretang 200, 2400 Mol, Belgium.

Currently, neurotoxicity testing defined by OECD and FDA is based solely on in vivo experiments, using large numbers of animals, being expensive, time-consuming and unsuitable for screening numerous chemicals. The great demand for thousands of chemicals yet to be evaluated, urges the development of alternative test methods which are cheaper, faster and highly predictive for developmental neurotoxicity. In this study, we developed a new method to assess locomotor activity in early life stage of zebrafish at 24 h post fertilization (hpf), in comparison to locomotor activity of zebrafish larvae at 96 to 192 hpf. We hypothesized that this endpoint at early life stages could be used to predict the developmental neurotoxic potential of chemicals and performed exposure studies with chlorpyrifos to demonstrate this. Furthermore, the case study with chlorpyrifos was used to critically evaluate behavioral data analysis and improve method sensitivity. The approach for data analysis using distribution plots for parameters on locomotor activity, next to mean values allowed to obtain more accurate information from the same set of behavioral data, both for embryos and larvae. Embryos exposed to chlorpyrifos, within the range 0.039 to 10 mg/l, exhibited a significant concentration-dependent increase in the frequency and total duration of their spontaneous tail coilings at 24-26 hpf. Larvae exhibited altered swimming activity, as evidenced by a significant decrease in the total duration of movement and an increase in mean turn angle in the range 0.18 to 0.75 mg/l chlorpyrifos. Methodological evaluation showed that locomotor effects in larvae were most pronounced and reproducible at 96 hpf, compared to older individuals (120, 144, 168 and 192 hpf). These new methods based on locomotor activity at early life stages of zebrafish allowed to classify chlorpyrifos as a developmental neurotoxicant. Further research to judge the validity of these alternative methods is currently performed with an extended set of expected positive or negative chemicals for developmental neurotoxicity.
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http://dx.doi.org/10.1016/j.ntt.2010.03.002DOI Listing
September 2010

Gene profiles of THP-1 macrophages after in vitro exposure to respiratory (non-)sensitizing chemicals: identification of discriminating genetic markers and pathway analysis.

Toxicol In Vitro 2009 Sep 13;23(6):1151-62. Epub 2009 Jun 13.

Unit Environmental Risk and Health, Flemish Institute for Technological Research (VITO NV), Boeretang 200, 2400 Mol, Belgium.

It is recognized that respiratory sensitization is a hazard of high concern. Despite international regulatory requirements there is no established protocol for the identification of chemical respiratory sensitizers. New tests should be based on mechanistic understanding and should be preferentially restricted to in vitro assays. The major goal of this study was to investigate the genetic response of human THP-1 macrophages after contact with respiratory (non-)sensitizers, and to identify genes that are able to discriminate between both groups. THP-1 macrophages were exposed during different time points to 3 respiratory sensitizers, 2 irritants, and 1 skin sensitizer. Gene expression changes were evaluated using Agilent Whole Human Genome arrays. Fisher Linear Discriminant Analysis was used to obtain a ranking of genes that reflects their potential to discriminate between respiratory (non-)sensitizing chemicals. Among the 20 most discriminating genes which were categorized into molecular and biological Gene Ontology (GO) terms, EIF4E, PDGFRB, SEMA7A, and ZFP36L2 could be associated with respiratory sensitization. When categorizing the top-1000 genes into biological GO terms, 24 genes were associated with immune function. Using a pathway analysis tool, platelet-derived growth factor signaling was observed to be activated in THP-1 macrophages in the context of respiratory sensitization.
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http://dx.doi.org/10.1016/j.tiv.2009.06.007DOI Listing
September 2009