Publications by authors named "Erik Goormaghtigh"

91 Publications

Macromolecular assembly and membrane activity of antimicrobial D,L-α-Cyclic peptides.

Colloids Surf B Biointerfaces 2021 Aug 31;208:112086. Epub 2021 Aug 31.

CIQUP, Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal. Electronic address:

Antimicrobial peptides are viewed as a promising alternative to conventional antibiotics, as their activity through membrane targeting makes them less prone to resistance development. Among them, antimicrobial D,L-α-cyclic peptides (CPs) have been proposed as an alternative, specially due to their cyclic nature and to the presence of D-α-amino acids that increases their resistance to proteases. In present work, second generation D,L-α-cyclic peptides with proven antimicrobial activity are shown to form complex macromolecular assemblies in the presence of membranes. We addressed the CPs:membrane interactions through a combination of experimental techniques (DSC and ATR-FTIR) with coarse-grained molecular dynamics (CG-MD) simulations, aiming at understanding their interactions, macromolecular assemblies and eventually unveil their mechanism of action. DSC shows that the interaction depends heavily on the negatively charge content of the membrane and on lipid/peptide ratio, suggesting different mechanisms for the different peptides and lipid systems. CG-MD proved that CPs can self-assemble at the lipid surface as nanotubes or micellar aggregates, depending on the peptide, in agreement with ATR-FTIR results. Finally, our results shed light into possible mechanisms of action of the peptides with pending hydrocarbon tail, namely membrane extensive segregation and/or membrane disintegration through the formation of disk-like lipid/peptide aggregates.
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http://dx.doi.org/10.1016/j.colsurfb.2021.112086DOI Listing
August 2021

Analysis of Glycoproteins by ATR-FTIR Spectroscopy: Comparative Assessment.

Methods Mol Biol 2021 ;2271:361-374

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles, Brussels, Belgium.

FTIR spectroscopy has been widely used to characterize biopharmaceuticals for many years, in particular to analyze protein structure. More recently, it was demonstrated to be a useful tool to study and compare protein samples in terms of glycosylation. Based on a spectral region specific to carbohydrate absorption, we present here a detailed protocol to compare the FTIR spectra of glycoproteins in terms of global glycosylation level and in terms of glycan composition. This FTIR information is compared to MS information. Both approaches yield consistent results but it appears FTIR analysis is easier and more rapid to perform comparisons.
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http://dx.doi.org/10.1007/978-1-0716-1241-5_25DOI Listing
June 2021

Attenuated total reflection-Fourier transform infrared spectroscopy: a tool to characterize antimicrobial cyclic peptide-membrane interactions.

Eur Biophys J 2021 May 20;50(3-4):629-639. Epub 2021 Mar 20.

Departamento de Química e Bioquímica, Centro de Investigação em Química, Faculdade de Ciências, CIQUP, Universidade do Porto, Porto, Portugal.

Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) has been used for the structural characterization of peptides and their interactions with membranes. Antimicrobial peptides (AMPs) are part of our immune system and widely studied in recent years. Many linear AMPs have been studied, but their cyclization was shown to enhance the peptide's activity. We have used cyclic peptides (CPs) of an even number of alternating D- and L-α-amino acids, an emerging class of potential AMPs. These CPs can adopt a flat-ring shape that can stack into an antiparallel structure, forming intermolecular hydrogen bonds between different units, creating a tubular β-sheet structure - self-assembled cyclic peptide nanotubes (SCPNs). To get the structural information on peptides in solution and/or in contact with membranes, Amide I and II absorptions are used as they can adopt frequency and shape band characteristics that are influenced by the strength of existing hydrogen bonds between the amide CO and NH involved in secondary structures such as helix, β-sheet or aperiodic structures. The combination of polarized lens with ATR-FTIR provides an important tool to study the orientation of peptides when interacting with lipid membranes as the information can be derived on the position relative to the membrane normal. This work shows how ATR-FTIR used together with polarized light was successfully used to characterize structurally two CPs (RSKSWPgKQ and RSKSWXKQ) in solution and upon interaction with negatively charged membranes of DMPG, assessing the formation and orientation of tubular structures (SCPNs) that were shown to be enhanced by the presence of the lipid membrane.
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http://dx.doi.org/10.1007/s00249-020-01495-0DOI Listing
May 2021

FTIR Imaging of Protein Microarrays for High Throughput Secondary Structure Determination.

Anal Chem 2021 03 12;93(8):3733-3741. Epub 2021 Feb 12.

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine, Université Libre de Bruxelles, CP206/2, B1050 Brussels, Belgium.

The paper introduces a new method designed for high-throughput protein structure determination. It is based on spotting proteins as microarrays at a density of ca. 2000-4000 samples per cm and recording Fourier transform infrared (FTIR) spectra by FTIR imaging. It also introduces a new protein library, called cSP92, which contains 92 well-characterized proteins. It has been designed to cover as well as possible the structural space, both in terms of secondary structures and higher level structures. Ascending stepwise linear regression (ASLR), partial least square (PLS) regression, and support vector machine (SVM) have been used to correlate spectral characteristics to secondary structure features. ASLR generally provides better results than PLS and SVM. The observation that secondary structure prediction is as good for protein microarray spectra as for the reference attenuated total reflection spectra recorded on the same samples validates the high throughput microarray approach. Repeated double cross-validation shows that the approach is suitable for the high accuracy determination of the protein secondary structure with root mean square standard error in the cross-validation of 4.9 ± 1.1% for α-helix, 4.6 ± 0.8% for β-sheet, and 6.3 ± 2.2% for the "other" structures when using ASLR.
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http://dx.doi.org/10.1021/acs.analchem.0c03677DOI Listing
March 2021

Amino acid side chain contribution to protein FTIR spectra: impact on secondary structure evaluation.

Eur Biophys J 2021 May 8;50(3-4):641-651. Epub 2021 Feb 8.

Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Campus Plaine CP206/02, 1050, Brussels, Belgium.

Prediction of protein secondary structure from FTIR spectra usually relies on the absorbance in the amide I-amide II region of the spectrum. It assumes that the absorbance in this spectral region, i.e., roughly 1700-1500 cm is solely arising from amide contributions. Yet, it is accepted that, on the average, about 20% of the absorbance is due to amino acid side chains. The present paper evaluates the contribution of amino acid side chains in this spectral region and the potential to improve secondary structure prediction after correcting for their contribution. We show that the β-sheet content prediction is improved upon subtraction of amino acid side chain contributions in the amide I-amide II spectral range. Improvement is relatively important, for instance, the error of prediction of β-sheet content decreases from 5.42 to 4.97% when evaluated by ascending stepwise regression. Other methods tested such as partial least square regression and support vector machine have also improved accuracy for β-sheet content evaluation. The other structures such as α-helix do not significantly benefit from side chain contribution subtraction, in some cases prediction is even degraded. We show that co-linearity between secondary structure content and amino acid composition is not a main limitation for improving secondary structure prediction. We also show that, even though based on different criteria, secondary structures defined by DSSP and XTLSSTR both arrive at the same conclusion: only the β-sheet structure clearly benefits from side chain subtraction. It must be concluded that side chain contribution subtraction benefit for the evaluation of other secondary structure contents is limited by the very rough description of side chain absorbance which does not take into account the variations related to their environment. The study was performed on a large protein set. To deal with the large number of proteins present, we worked on protein microarrays deposited on BaF slides and FTIR spectra were acquired with an imaging system.
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http://dx.doi.org/10.1007/s00249-021-01507-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189991PMC
May 2021

Evaluation of protein secondary structure from FTIR spectra improved after partial deuteration.

Eur Biophys J 2021 May 3;50(3-4):613-628. Epub 2021 Feb 3.

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles, B1050, Brussels, Belgium.

FTIR spectroscopy has become a major tool to determine protein secondary structure. One of the identified obstacle for reaching better predictions is the strong overlap of bands assigned to different secondary structures. Yet, while for instance disordered structures and α-helical structures absorb almost at the same wavenumber, the absorbance bands are differentially shifted upon deuteration, in part because exchange is much faster for disordered structures. We recorded the FTIR spectra of 85 proteins at different stages of hydrogen/deuterium exchange process using protein microarrays and infrared imaging for high throughput measurements. Several methods were used to relate spectral shape to secondary structure content. While in absolute terms, β-sheet is always better predicted than α-helix content, results consistently indicate an improvement of secondary structure predictions essentially for the α-helix and the category called "Others" (grouping random, turns, bends, etc.) after 15 min of exchange. On the contrary, the β-sheet fraction is better predicted in non-deuterated conditions. Using partial least square regression, the error of prediction for the α-helix content is reduced after 15-min deuteration. Further deuteration degrades the prediction. Error on the prediction for the "Others" structures also decreases after 15-min deuteration. Cross-validation or a single 25-protein test set result in the same overall conclusions.
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http://dx.doi.org/10.1007/s00249-021-01502-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189984PMC
May 2021

Searching for a Better Match between Protein Secondary Structure Definitions and Protein FTIR Spectra.

Anal Chem 2021 01 17;93(3):1561-1568. Epub 2020 Dec 17.

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Université Libre de Bruxelles, Campus Plaine CP206/2, B1050 Brussels, Belgium.

Obtaining protein secondary structure content from high-resolution structures requires definitions and thresholds for the various parameters involved, typically hydrogen bond energy or length/angle and backbone φ/ψ angles. Several definitions are currently used and can have a profound impact on secondary structure content. Fourier transform infrared (FTIR) spectroscopy has its own sensitivity to molecular geometry. It is, therefore, important to select a set of definitions that matches this sensitivity. Here, we used a new protein set consisting of 92 proteins designed for the calibration of spectroscopic methods. Spectra have been obtained from protein microarrays in a high throughput process. The potential for improving secondary structure predictions from FTIR spectra has been tested using 71 structures determined according to different definitions. The paper demonstrates that different secondary structure definitions result in large variations in secondary structure content that are not equivalent in view of the protein FTIR spectra. The prediction quality factor ζ can be improved by ca. 20-50% by selecting an adequate definition set. The results also indicate that the dictionary of secondary structure of proteins (DSSP) algorithm, which is currently widely used to evaluate protein secondary structure content, is a good choice when dealing with FTIR spectra.
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http://dx.doi.org/10.1021/acs.analchem.0c03943DOI Listing
January 2021

HER2 biosensing through SPR-envelope tracking in plasmonic optical fiber gratings.

Biomed Opt Express 2020 Sep 4;11(9):4862-4871. Epub 2020 Aug 4.

Electromagnetism and Telecommunication Department, University of Mons, 31 Bld Dolez, 7000 Mons, Belgium.

In the biomedical detection context, plasmonic tilted fiber Bragg gratings (TFBGs) have been demonstrated to be a very accurate and sensitive sensing tool, especially well-adapted for biochemical detection. In this work, we have developed an aptasensor following a triple strategy to improve the overall sensing performances and robustness. Single polarization fiber (SPF) is used as biosensor substrate while the demodulation is based on tracking a peculiar feature of the lower envelope of the cladding mode resonances spectrum. This method is highly sensitive and yields wavelength shifts several tens of times higher than the ones reported so far based on the tracking of individual modes of the spectrum. An amplification of the response is further performed through a sandwich assay by the use of specific antibodies. These improvements have been achieved on a biosensor developed for the detection of the HER2 () protein, a relevant breast cancer biomarker. These advanced developments can be very interesting for point-of-care biomedical measurements in a convenient practical way.
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http://dx.doi.org/10.1364/BOE.401200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510885PMC
September 2020

Membrane targeting antimicrobial cyclic peptide nanotubes - an experimental and computational study.

Colloids Surf B Biointerfaces 2020 Dec 9;196:111349. Epub 2020 Sep 9.

CIQUP, Centro de Investigação em Química, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal. Electronic address:

The search of new antibiotics, particularly with new mechanisms of action, is nowadays a very important public health issue, due to the worldwide increase of resistant pathogens. Within this effort, much research has been done on antimicrobial peptides, because having the membrane as a target, they represent a new antibiotic paradigm. Among these, cyclic peptides (CPs) made of sequences of D- and L-amino acids have emerged as a new class of potential antimicrobial peptides, due to their expected higher resistance to protease degradation. These CPs are planar structures that can form Self-assembled Cyclic Peptide Nanotubes (SCPNs), in particular in the presence of lipid membranes. Aiming at understanding their mechanism of action, we used biophysical experimental techniques (DSC and ATR-FTIR) together with Coarse-grained molecular dynamics (CG-MD) simulations, to characterize the interaction of these CPs with model membranes of different electrostatic charges' contents. DSC results revealed that the CPs show a strong interaction with negatively charged membranes, with differences in the strength of interactions depending on peptide and on membrane charge content, at odds with no or mild interactions with zwitterionic membranes. ATR-FTIR suggested that the peptides self-assemble at the membrane surface, adopting mainly a β-structure. The experiments with polarized light showed that in most cases they lie parallel to the membrane surface, but other forms and orientations are also apparent, depending on peptide structure and lipid:peptide ratio. The nanotube formation and orientation, as well as the dependence on membrane charge were also confirmed by the CG-MD simulations. These provide detail on the position and interactions, in agreement with the experimental results. Based on the findings reported here, we could proceed to the design and synthesis of a second-generation CPs, based on CP2 (soluble peptide), with increased activity and reduced toxicity.
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http://dx.doi.org/10.1016/j.colsurfb.2020.111349DOI Listing
December 2020

A convenient protein library for spectroscopic calibrations.

Comput Struct Biotechnol J 2020 10;18:1864-1876. Epub 2020 Jul 10.

Université Libre de Bruxelles CP206/2, B1050 Brussels, Belgium.

While several Raman, CD or FTIR spectral libraries are available for well-characterized proteins of known structure, proteins themselves are usually very difficult to acquire, preventing a convenient calibration of new instruments and new recording methods. The problem is particularly critical in the field of FTIR spectroscopy where numerous new methods are becoming available on the market. The present papers reports the construction of a protein library (cSP92) including commercially available products, that are well characterized experimentally for their purity and solubility in conditions compatible with the recording of FTIR spectra and whose high-resolution structure is available. Overall, 92 proteins were selected. These proteins cover well the CATH space at the level of classes and architectures. In terms of secondary structure content, an analysis of their high-resolution structure by DSSP shows that the mean content in the different secondary structures present in cSP92 is very similar to the mean content found in the PDB. The 92-protein set is analyzed in details for the distribution of helix length, number of strands in β- sheets, length of β-strands and amino acid content, all features that may be important for the interpretation of FTIR spectra.
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http://dx.doi.org/10.1016/j.csbj.2020.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369421PMC
July 2020

FTIR spectroscopy as an analytical tool to compare glycosylation in therapeutic monoclonal antibodies.

Anal Chim Acta 2020 May 31;1112:62-71. Epub 2020 Mar 31.

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles, Bld Du Triomphe 2, CP206/2, B1050, Brussels, Belgium. Electronic address:

Glycosylation is the most common protein post-translational modification (PTM), especially in biopharmaceuticals. It is a critical quality attribute as it impacts product solubility, stability, half-life, pharmacokinetics and pharmacodynamics (PK/PD), bioactivity and safety (e.g. immunogenicity). Yet, current glycan analysis methods involve multiple and lengthy sample preparation steps which can affect the robustness of the analyses. The development of orthogonal, direct and simple method is therefore desirable. In this study, we suggest use of FTIR spectroscopy to address this challenge. Use of this technique, combined with statistical tools, to compare samples or batches in terms of glycosylation or monosaccharide profile, has three potential applications: to compare glycosylation of a biosimilar and the original (innovator) molecule, for monitoring of batch-to-batch consistency, and for in-process control. Fourteen therapeutic monoclonal antibodies (mAbs), one Fc-fusion protein and several other common glycoproteins have been used to demonstrate that FTIR spectra of glycoproteins display spectral variations according to their glycan and monosaccharide compositions. We show that FTIR spectra of glycoproteins provide a global but accurate fingerprint of the glycosylation profile. This fingerprint is not only sensitive to large differences such as the presence or absence of several monosaccharides but also to smaller modifications of the glycan and monosaccharide content.
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http://dx.doi.org/10.1016/j.aca.2020.03.038DOI Listing
May 2020

Multimodal plasmonic optical fiber grating aptasensor.

Opt Express 2020 Mar;28(5):7539-7551

Tilted fiber Bragg gratings (TFBGs) are now a well-established technology in the scientific literature, bringing numerous advantages, especially for biodetection. Significant sensitivity improvements are achieved by exciting plasmon waves on their metal-coated surface. Nowadays, a large part of advances in this topic relies on new strategies aimed at providing sensitivity enhancements. In this work, TFBGs are produced in both single-mode and multimode telecommunication-grade optical fibers, and their relative performances are evaluated for refractometry and biosensing purposes. TFBGs are biofunctionalized with aptamers oriented against HER2 (Human Epidermal Growth Factor Receptor-2), a relevant protein biomarker for breast cancer diagnosis. In vitro assays confirm that the sensing performances of TFBGs in multimode fiber are higher or identical to those of their counterparts in single-mode fiber, respectively, when bulk refractometry or surface biosensing is considered. These observations are confirmed by numerical simulations. TFBGs in multimode fiber bring valuable practical assets, featuring a reduced spectral bandwidth for improved multiplexing possibilities enabling the detection of several biomarkers.
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http://dx.doi.org/10.1364/OE.385747DOI Listing
March 2020

Intertwined metal homeostasis, oxidative and biotic stress responses in the Arabidopsis frd3 mutant.

Plant J 2020 04 4;102(1):34-52. Epub 2020 Jan 4.

InBioS-PhytoSystems, Functional Genomics and Plant Molecular Imaging, University of Liège, 4000, Liège, Belgium.

FRD3 (FERRIC REDUCTASE DEFECTIVE 3) plays a major role in iron (Fe) and zinc (Zn) homeostasis in Arabidopsis. It transports citrate, which enables metal distribution in the plant. An frd3 mutant is dwarf and chlorotic and displays a constitutive Fe-deficiency response and strongly altered metal distribution in tissues. Here, we have examined the interaction between Fe and Zn homeostasis in an frd3 mutant exposed to varying Zn supply. Detailed phenotyping using transcriptomic, ionomic, histochemical and spectroscopic approaches revealed the full complexity of the frd3 mutant phenotype, which resulted from altered transition metal homeostasis, manganese toxicity, and oxidative and biotic stress responses. The cell wall played a key role in these processes, as a site for Fe and hydrogen peroxide accumulation, and displayed modified structure in the mutant. Finally, we showed that Zn excess interfered with these mechanisms and partially restored root growth of the mutant, without reverting the Fe-deficiency response. In conclusion, the frd3 mutant molecular phenotype is more complex than previously described and illustrates how the response to metal imbalance depends on multiple signaling pathways.
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http://dx.doi.org/10.1111/tpj.14610DOI Listing
April 2020

An Innovative Platform Merging Elemental Analysis and Ftir Imaging for Breast Tissue Analysis.

Sci Rep 2019 07 8;9(1):9854. Epub 2019 Jul 8.

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles CP206/2, B1050, Brussels, Belgium.

Histopathology and immunohistology remain the gold standard for breast cancer diagnostic. Yet, these approaches do not usually provide a sufficiently detailed characterization of the pathology. The purpose of this work is to demonstrate for the first time that elemental analysis and Fourier transform infrared spectroscopy microscopic examination of breast tissue sections can be merged into one dataset to provide a single set of markers based on both organic molecules and inorganic trace elements. For illustrating the method, 6 mammary tissue sections were used. Fourier transform infrared (FTIR) spectroscopy images reported a fingerprint of the organic molecules present in the tissue section and laser ablation elemental analysis (LA-ICP-MS) images brought inorganic element profiles. The 6 tissue sections provided 31 10 and 150,000 spectra for FTIR and LA-ICP-MS spectra respectively. The results bring the proof of concept that breast tissue can be analyzed simultaneously by FTIR spectroscopy and laser ablation elemental analysis (LA-ICP-MS) to provide in both case reasonably high resolution images. We show how to bring the images obtained by the two methods to a same spatial resolution and how to use image registration to analyze the data originating from both techniques as one block of data. We finally demonstrates the elemental analysis is orthogonal to all FTIR markers as no significant correlation is found between FTIR and LA-ICP-MS data. Combining FTIR and LA-ICP-MS imaging becomes possible, providing two orthogonal methods which can bring an unprecedented diversity of information on the tissue. This opens a new avenue of tissue section analyses providing unprecedented diagnostic potential.
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http://dx.doi.org/10.1038/s41598-019-46056-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614471PMC
July 2019

Discrimination of breast cancer from benign tumours using Raman spectroscopy.

PLoS One 2019 14;14(2):e0212376. Epub 2019 Feb 14.

Qatar Biomedical Research Institute, Doha, Qatar.

Breast cancer is the most common cancer among women worldwide, with an estimated 1.7 million cases and 522,000 deaths in 2012. Breast cancer is diagnosed by histopathological examination of breast biopsy material but this is subjective and relies on morphological changes in the tissue. Raman spectroscopy uses incident radiation to induce vibrations in the molecules of a sample and the scattered radiation can be used to characterise the sample. This technique is rapid and non-destructive and is sensitive to subtle biochemical changes occurring at the molecular level. This allows spectral variations corresponding to disease onset to be detected. The aim of this work was to use Raman spectroscopy to discriminate between benign lesions (fibrocystic, fibroadenoma, intraductal papilloma) and cancer (invasive ductal carcinoma and lobular carcinoma) using formalin fixed paraffin preserved (FFPP) tissue. Haematoxylin and Eosin stained sections from the patient biopsies were marked by a pathologist. Raman maps were recorded from parallel unstained tissue sections. Immunohistochemical staining for estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2/neu) was performed on a further set of parallel sections. Both benign and cancer cases were positive for ER while only the cancer cases were positive for HER2. Significant spectral differences were observed between the benign and cancer cases and the benign cases could be differentiated from the cancer cases with good sensitivity and specificity. This study has shown the potential of Raman spectroscopy as an aid to histopathological diagnosis of breast cancer, in particular in the discrimination between benign and malignant tumours.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0212376PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6375635PMC
November 2019

Grafting of Oligo(ethylene glycol)-Functionalized Calix[4]arene-Tetradiazonium Salts for Antifouling Germanium and Gold Surfaces.

Langmuir 2018 05 15;34(21):6021-6027. Epub 2018 May 15.

Laboratoire de Chimie Organique , Université Libre de Bruxelles (ULB) , avenue F. D. Roosevelt 50 , CP160/06, B-1050 Brussels , Belgium.

Biosensors that can determine protein concentration and structure are highly desired for biomedical applications. For the development of such biosensors, the use of Fourier transform infrared (FTIR) spectroscopy with the attenuated internal total reflection (ATR) configuration is particularly attractive, but it requires appropriate surface functionalization of the ATR optical element. Indeed, the surface has to specifically interact with a target protein in close contact with the optical element and must display antifouling properties to prevent nonspecific adsorption of other proteins. Here, we report robust monolayers of calix[4]arenes bearing oligo(ethylene glycol) (oEG) chains, which were grafted on germanium and gold surfaces via their tetradiazonium salts. The formation of monolayers of oEGylated calix[4]arenes was confirmed by AFM, IR, and contact angle measurements. The antifouling properties of these modified surfaces were studied by ATR-FTIR spectroscopy and fluorescence microscopy, and the nonspecific absorption of bovine serum albumin was found to be reduced by 85% compared to that of unmodified germanium. In other words, the organic coating by oEGylated calix[4]arenes provides remarkable antifouling properties, opening the way for the design of germanium- or gold-based biosensors.
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http://dx.doi.org/10.1021/acs.langmuir.8b00464DOI Listing
May 2018

Gene expression data and FTIR spectra provide a similar phenotypic description of breast cancer cell lines in 2D and 3D cultures.

Analyst 2018 May;143(11):2520-2530

Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Brussels, Belgium.

Thirteen breast cancer cell lines were grown in traditional two-dimensional (2D) monolayer and three-dimensional (3D) laminin-rich extracellular matrix (lrECM) culture models. Microarray-based transcriptional profiling data were published for these cell lines under both culture conditions. Colonies embedded in Matrigel matrix were fixed in formalin, embedded in paraffin and cut into 4 μm thick sections. The sections were mounted onto infrared-transparent barium fluoride windows and deparaffinized for Fourier transform infrared (FTIR) imaging. Samples consisting of Matrigel-coated 2D-grown cells followed the same processing procedure, simplifying comparison with 3D-cultured cells as well as with routinely prepared formalin-fixed, paraffin-embedded tissue specimens. Gene expression was found to be dominated by the cell line genome. Cluster analysis first groups the same cell line samples, independent of whether cells have been grown in 2D or 3D cultures. FTIR spectroscopy first groups by culture conditions when considering the full spectrum length. The paper reports two important results. First, both gene expression level and FTIR spectroscopy are multivariate techniques that contain sufficient information to identify uniquely both any cell line (among thirteen breast cancer cell lines) and phenotype induced by growing the cells in 2D or 3D lrECM cultures. Second, we established the presence of a strong correlation between gene expression patterns and FTIR spectral data for the thirteen breast cancer cell lines grown in both 2D and 3D lrECM cultures. These results suggest that, although based on completely different principles, the two approaches describe similarly the patterns of variations in cells.
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http://dx.doi.org/10.1039/c8an00145fDOI Listing
May 2018

Deciphering the biochemical similarities and differences among mouse embryonic stem cells, somatic and cancer cells using ATR-FTIR spectroscopy.

Analyst 2018 Mar;143(7):1624-1634

Center for Drug Research & Development and Pharmacokinetic Applications (ARGEFAR), Ege University, 35100, Izmir, Turkey.

Cellular macromolecules play important roles in cellular behaviors and biological processes. In the current work, cancer (KLN205), normal (MSFs) and mouse embryonic stem cells (mESCs) are compared using ATR-FTIR spectroscopy. Modifications in the composition, concentration, structure and function-related changes in the cellular components were deciphered using the infrared spectra. Our results revealed that cancer and embryonic stem cells are very similar but highly different from the normal cells based on the spectral variations in the protein, lipid, carbohydrate and nucleic acid components. The longest lipid acyl chains exist in mESCs, while cancer cells harbor the lowest lipid amount, short lipid acyl chains, a high content of branched fatty acids and thin cell membranes. The highest cellular growth rate and accelerated cell divisions were observed in the cancer cells. However, the normal cells harbor low nucleic acid and glycogen amounts but have a higher lipid composition. Any defect in the signaling pathways and/or biosynthesis of these cellular parameters during the embryonic-to-somatic cell transition may lead to physiological and molecular events that promote cancer initiation, progression and drug resistance. We conclude that an improved understanding of both similarities and differences in the cellular mechanisms among the cancer, normal and mESCs is crucial to develop a potential clinical relevance, and ATR-FITR can be successfully used as a novel approach to gain new insights into the stem cell and cancer research. We suggest that targeting the cellular metabolisms (glycogen and lipid) can provide new strategies for cancer treatment.
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http://dx.doi.org/10.1039/c8an00017dDOI Listing
March 2018

Insights into Biochemical Alteration in Cancer-Associated Fibroblasts by using Novel Correlative Spectroscopy.

ChemistryOpen 2017 02 9;6(1):149-157. Epub 2017 Jan 9.

Berzelii Technology Centre for Neurodiagnostics Department of Engineering Science Uppsala University Uppsala 75105 Sweden.

The microenvironment of a tumor changes chemically and morphologically during cancer progression. Cancer-stimulated fibroblasts promote tumor growth, however, the mechanism of the transition to a cancer-stimulated fibroblast remains elusive. Here, the multi-modal spectroscopic methods Fourier transform infrared imaging (FTIRI), X-ray absorption spectroscopy (XAS) and X-ray fluorescence imaging (XFI) are used to characterize molecular and atomic alterations that occur in cancer-stimulated fibroblasts. In addition to chemical changes in lipids (olefinic and acyl chain) and protein aggregation observed with FTIRI, a new infrared biomarker for oxidative stress in stimulated fibroblasts is reported. Oxidative stress is observed to cause lipid peroxidation, which leads to the appearance of a new band at 1721 cm, assigned to 4-hydroxynonenal. Complementary to FTIRI, XFI is well suited to determining atom concentrations and XAS can reveal the speciation of individual elements. XFI reveals increased concentrations of P, S, K, Ca within stimulated fibroblasts. Furthermore, XAS studies reveal alterations in the speciation of S and Ca in stimulated fibroblasts, which might provide insight into the mechanisms of cancer progression. Using XFI, not only is the concentration change of individual elements observed, but also the subcellular localization. This study demonstrates the wealth of biochemical information provided by a multi-modal imaging approach and highlights new avenues for future research into the microenvironment of breast tumors.
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http://dx.doi.org/10.1002/open.201600102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288759PMC
February 2017

Infrared imaging of high density protein arrays.

Analyst 2017 Apr;142(8):1371-1380

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles CP206/2, B1050 Brussels, Belgium.

We propose in this paper that protein microarrays could be analysed by infrared imaging in place of enzymatic or fluorescence labelling. This label-free method reports simultaneously a large series of data on the spotted sample (protein secondary structure, phosphorylation, glycosylation, presence of impurities, etc.). In the present work, 100 μm protein spots each containing about 100 pg protein were deposited to form high density regular arrays. Using arrays of infrared detectors, high resolution images could be obtained where each pixel of the image is in fact a full infrared spectrum. With microarrays, hundreds of experimental conditions can be tested easily and quickly, with no further labelling or chemistry of any kind. We describe how the noise present in the infrared spectra can be split into image noise and detector noise. We also detail how both types of noise can be most conveniently dealt with to generate very high quality spectra of less than 100 pg protein. Finally, the results suggest that the protein secondary structure is preserved during microarray building.
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http://dx.doi.org/10.1039/c6an02048hDOI Listing
April 2017

Biophysical characterization data of the artificial protein Octarellin V.1 and binding test with its X-ray helpers.

Data Brief 2016 Sep 26;8:1221-6. Epub 2016 Jul 26.

GIGA-Research, Molecular Biomimetics and Protein Engineering, University of Liège, Liège, Belgium.

The artificial protein Octarellin V.1 (http://dx.doi.org/10.1016/j.jsb.2016.05.004[1]) was obtained through a direct evolution process over the de novo designed Octarellin V (http://dx.doi.org/10.1016/S0022-2836(02)01206-8[2]). The protein has been characterized by circular dichroism and fluorescence techniques, in order to obtain data related to its thermo and chemical stability. Moreover, the data for the secondary structure content studied by circular dichroism and infra red techniques is reported for the Octarellin V and V.1. Two crystallization helpers, nanobodies (http://dx.doi.org/10.1038/nprot.2014.039[3]) and αRep (http://dx.doi.org/10.1016/j.jmb.2010.09.048[4]), have been used to create stable complexes. Here we present the data obtained of the binding characterization of the Octarellin V.1 with the crystallization helpers by isothermal titration calorimetry.
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http://dx.doi.org/10.1016/j.dib.2016.07.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4982917PMC
September 2016

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.

J Struct Biol 2016 07 12;195(1):19-30. Epub 2016 May 12.

GIGA-Research, Molecular Biomimetics and Protein Engineering, University of Liège, Liège, Belgium. Electronic address:

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features.
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http://dx.doi.org/10.1016/j.jsb.2016.05.004DOI Listing
July 2016

Structural and Functional Investigation of the Ag(+)/Cu(+) Binding Domains of the Periplasmic Adaptor Protein SilB from Cupriavidus metallidurans CH34.

Biochemistry 2016 05 13;55(20):2883-97. Epub 2016 May 13.

Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles , B-1050 Bruxelles, Belgium.

Silver ion resistance in bacteria mainly relies on efflux systems, and notably on tripartite efflux complexes involving a transporter from the resistance-nodulation-cell division (RND) superfamily, such as the SilCBA system from Cupriavidus metallidurans CH34. The periplasmic adaptor protein SilB hosts two specific metal coordination sites, located in the N-terminal and C-terminal domains, respectively, that are believed to play a different role in the efflux mechanism and the trafficking of metal ions from the periplasm to the RND transporter. On the basis of the known domain structure of periplasmic adaptor proteins, we designed different protein constructs derived from SilB domains with either one or two metal binding sites per protein chain. ITC data acquired on proteins with single metal sites suggest a slightly higher affinity of Ag(+) for the N-terminal metal site, compared to that for the C-terminal one. Remarkably, via the study of a protein construct featuring both metal sites, nuclear magnetic resonance (NMR) and fluorescence spectroscopies concordantly show that the C-terminal site is saturated prior to the N-terminal one. The C-terminal binding site is supposed to transfer the metal ions to the RND protein, while the transport driven by this latter is activated upon binding of the metal ion to the N-terminal site. Our results suggest that the filling of the C-terminal metal site is a key prerequisite for preventing futile activation of the transport system. Exhaustive NMR studies reveal for the first time the structure and dynamics of the functionally important N-terminal domain connected to the membrane proximal domain as well as of its Ag(+) binding site.
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http://dx.doi.org/10.1021/acs.biochem.6b00022DOI Listing
May 2016

FTIR imaging of the 3D extracellular matrix used to grow colonies of breast cancer cell lines.

Analyst 2016 Jan;141(2):620-9

Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles (ULB), Bld du Triomphe, CP206/02, B-1050 Brussels, Belgium.

Infrared imaging was applied to investigate a reconstituted basement membrane, known as Matrigel, in three-dimensional cell cultures. Matrigel, in the vicinity of the colonies, was examined for four breast cancer cell lines presenting different 3D colony morphologies. MCF-7 and T-47D present mass colonies, SKBR-3 grape-like colonies and MDA-MB-231 stellate colonies associated with a more invasive phenotype. The edge of the cell colonies was found to be significantly depleted in Matrigel. Except in a limited number of cases, Matrigel appeared to be thinner at the edges of the colonies but not completely destroyed or torn off as it would be for a purely mechanical effect. When a PCA was run on the spectra of one or several colonies, the score images on PC#3 and PC#4 presented structures in the Matrigel areas which appeared as fringes, lines, dots or regular patterns. This effect represents a very small fraction of the total variance but is reproducible for all the 4 cell lines. PC#4 presents systematically a maximum near 1624 cm(-1) and a minimum around 1700 cm(-1). When spectra are normalized, the effect is less marked but does not disappear. The nature of the variations that exist in the Matrigel layer is therefore not solely related to thickness but also to the chemical composition. At this stage, the weakness of the effect prevents a thorough investigation.
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http://dx.doi.org/10.1039/c5an01997dDOI Listing
January 2016

FTIR spectral signature of anticancer drugs. Can drug mode of action be identified?

Biochim Biophys Acta 2016 Jan 29;1864(1):85-101. Epub 2015 Aug 29.

Center for Structural Biology and Bioinformatics, Laboratory for the Structure and Function of Biological Membranes, Campus Plaine CP206/02, Université Libre de Bruxelles, CP206/2, B1050 Brussels, Belgium. Electronic address:

Infrared spectroscopy has brought invaluable information about proteins and about the mechanism of action of enzymes. These achievements are difficult to transpose to living organisms as all biological molecules absorb in the mid infrared, with usually a high degree of overlap. Deciphering the contribution of each enzyme is therefore almost impossible. On the other hand, small changes in the infrared spectra of cells induced by environmental conditions or drugs may provide an accurate signature of the metabolic shift experienced by the cell as a response to a change in the growth medium. The present paper aims at reviewing the contribution of infrared spectroscopy to the description of small chemical changes that occur in cells when they are exposed to a drug. In particular, this review will focus on cancer cells and anti-cancer drugs. Results accumulated so far tend to demonstrate that infrared spectroscopy could be a very accurate descriptor of the mode of action of anticancer drugs. If confirmed, such a segmentation of potential drugs according to their "mode of action" will be invaluable for the discovery of new therapeutic molecules. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.
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http://dx.doi.org/10.1016/j.bbapap.2015.08.010DOI Listing
January 2016

Simultaneous Fitting of Absorption Spectra and Their Second Derivatives for an Improved Analysis of Protein Infrared Spectra.

Molecules 2015 Jul 10;20(7):12599-622. Epub 2015 Jul 10.

Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden.

Infrared spectroscopy is a powerful tool in protein science due to its sensitivity to changes in secondary structure or conformation. In order to take advantage of the full power of infrared spectroscopy in structural studies of proteins, complex band contours, such as the amide I band, have to be decomposed into their main component bands, a process referred to as curve fitting. In this paper, we report on an improved curve fitting approach in which absorption spectra and second derivative spectra are fitted simultaneously. Our approach, which we name co-fitting, leads to a more reliable modelling of the experimental data because it uses more spectral information than the standard approach of fitting only the absorption spectrum. It also avoids that the fitting routine becomes trapped in local minima. We have tested the proposed approach using infrared absorption spectra of three mixed α/β proteins with different degrees of spectral overlap in the amide I region: ribonuclease A, pyruvate kinase, and aconitase.
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http://dx.doi.org/10.3390/molecules200712599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6331840PMC
July 2015

Intraspecific variability of cadmium tolerance and accumulation, and cadmium-induced cell wall modifications in the metal hyperaccumulator Arabidopsis halleri.

J Exp Bot 2015 Jun 6;66(11):3215-27. Epub 2015 Apr 6.

Laboratory of Plant Physiology and Molecular Genetics, Université Libre de Bruxelles, 1050 Brussels, Belgium

Certain molecular mechanisms of Cd tolerance and accumulation have been identified in the model species Arabidopsis halleri, while intraspecific variability of these traits and the mechanisms of shoot detoxification were little addressed. The Cd tolerance and accumulation of metallicolous and non-metallicolous A. halleri populations from different genetic units were tested in controlled conditions. In addition, changes in shoot cell wall composition were investigated using Fourier transform infrared spectroscopy. Indeed, recent works on A. halleri suggest Cd sequestration both inside cells and in the cell wall/apoplast. All A. halleri populations tested were hypertolerant to Cd, and the metallicolous populations were on average the most tolerant. Accumulation was highly variable between and within populations, and populations that were non-accumulators of Cd were identified. The effect of Cd on the cell wall composition was quite similar in the sensitive species A. lyrata and in A. halleri individuals; the pectin/polysaccharide content of cell walls seems to increase after Cd treatment. Nevertheless, the changes induced by Cd were more pronounced in the less tolerant individuals, leading to a correlation between the level of tolerance and the extent of modifications. This work demonstrated that Cd tolerance and accumulation are highly variable traits in A. halleri, suggesting adaptation at the local scale and involvement of various molecular mechanisms. While in non-metallicolous populations drastic modifications of the cell wall occur due to higher Cd toxicity and/or Cd immobilization in this compartment, the increased tolerance of metallicolous populations probably involves other mechanisms such as vacuolar sequestration.
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http://dx.doi.org/10.1093/jxb/erv144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4449548PMC
June 2015

Infrared imaging of MDA-MB-231 breast cancer cell line phenotypes in 2D and 3D cultures.

Analyst 2015 Apr;140(7):2336-43

Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles (ULB), Bld du Triomphe 2, CP206/02, B-1050 Brussels, Belgium.

One current challenge in the field of breast cancer infrared imaging is the identification of carcinoma cell subtypes in the tissue. Neither sequencing nor immunochemistry is currently able to provide a cell by cell thorough classification. The latter is needed to build accurate statistical models capable of recognizing the diversity of breast cancer cell lines that may be present in a tissue section. One possible approach for overcoming this problem is to obtain the IR spectral signature of well-characterized tumor cell lines in culture. Cultures in three-dimensional matrices appear to generate an environment that mimics better the in vivo environment. There are, at present, series of breast cancer cell lines that have been thoroughly characterized in two- and three-dimensional (2D and 3D) cultures by full transcriptomics analyses. In this work, we describe the methods used to grow, to process, and to characterize a triple-negative breast cancer cell line, MDA-MB-231, in 3D laminin-rich extracellular matrix (lrECM) culture and compare it with traditional monolayer cultures and tissue sections. While unsupervised analyses did not completely separate spectra of cells grown in 2D from 3D lrECM cultures, a supervised statistical analysis resulted in an almost perfect separation. When IR spectral responses of epithelial tumor cells from clinical triple-negative breast carcinoma samples were added to these data, a principal component analysis indicated that they cluster closer to the spectra of 3D culture cells than to the spectra of cells grown on a flat plastic substrata. This result is encouraging because of correlating well-characterized cell line features with clinical biopsies.
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http://dx.doi.org/10.1039/c4an01833hDOI Listing
April 2015

A FTIR imaging characterization of fibroblasts stimulated by various breast cancer cell lines.

PLoS One 2014 12;9(11):e111137. Epub 2014 Nov 12.

Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Brussels, Belgium.

It is well known that the microenvironment plays a major role in breast cancer progression. Yet, the mechanism explaining the transition from normal fibroblasts to cancer-stimulated fibroblasts remains to be elucidated. Here we report a FTIR imaging study of the effects of three different breast cancer cell lines on normal fibroblasts in culture. Fibroblast activation process was monitored by FTIR imaging and spectra compared by multivariate statistical analyses. Principal component analysis evidenced that the fibroblasts stimulated by these cancer cell lines grouped together and remained distinctly separated from normal fibroblasts indicating a modified different chemical composition in the cancer-stimulated fibroblasts. Similar changes in fibroblasts were induced by the various breast cancer cell lines belonging to different sub-types. Most significant changes were observed in the region of 2950 and 1230 cm(-1), possibly related to changes in lipids and in the 1230 cm(-1) area assigned to phosphate vibrations (nucleotides). Interestingly, the cancer-cell induced changes in the fibroblasts also occurred when there was no possible direct contact between the two cell lines in the co-culture. When contact was possible, the spectral changes were similar, suggesting that soluble factors but not direct cell-cell interactions were responsible for fibroblast activation. Overall, the results indicate that IR imaging could be used in the future for analyzing the microenvironment of breast tumors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111137PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4229076PMC
July 2015
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