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1. Computational Image-based Stroke Assessment for Evaluation of Cerebroprotectants with Longitudinal and Multi-site Preclinical MRI

Author

Cabeen, Ryan P., Mandeville, Joseph, Hyder, Fahmeed, Sanganahalli, Basavaraju G., Thedens, Daniel R., Arbab, Ali, Huang, Shuning, Bibic, Adnan, Tarakci, Erendiz, Mihailovic, Jelena, Morais, Andreia, Lamb, Jessica, Nagarkatti, Karisma, Toga, Arthur W., Lyden, Patrick, and Ayata, Cenk

Subjects
Quantitative Biology - Quantitative Methods, Computer Science - Computer Vision and Pattern Recognition
Abstract

While ischemic stroke is a leading cause of death worldwide, there has been little success translating putative cerebroprotectants from rodent preclinical trials to human patients. We investigated computational image-based assessment tools for practical improvement of the quality, scalability, and outlook for large scale preclinical screening for potential therapeutic interventions in rodent models. We developed, evaluated, and deployed a pipeline for image-based stroke outcome quantification for the Stroke Preclinical Assessment Network (SPAN), a multi-site, multi-arm, multi-stage study evaluating a suite of cerebroprotectant interventions. Our fully automated pipeline combines state-of-the-art algorithmic and data analytic approaches to assess stroke outcomes from multi-parameter MRI data collected longitudinally from a rodent model of middle cerebral artery occlusion (MCAO), including measures of infarct volume, brain atrophy, midline shift, and data quality. We applied our approach to 1,368 scans and report population level results of lesion extent and longitudinal changes from injury. We validated our system by comparison with both manual annotations of coronal MRI slices and tissue sections from the same brain, using crowdsourcing from blinded stroke experts from the network. Our results demonstrate the efficacy and robustness of our image-based stroke assessments. The pipeline may provide a promising resource for ongoing rodent preclinical studies conducted by SPAN and other networks in the future.

Published
2022

2. Evaluating U-net Brain Extraction for Multi-site and Longitudinal Preclinical Stroke Imaging

Author

Tarakci, Erendiz, Mandeville, Joseph, Hyder, Fahmeed, Sanganahalli, Basavaraju G., Thedens, Daniel R., Arbab, Ali, Huang, Shuning, Bibic, Adnan, Mihailovic, Jelena, Morais, Andreia, Lamb, Jessica, Nagarkatti, Karisma, Dinitz, Marcio A., Rogatko, Andre, Toga, Arthur W., Lyden, Patrick, Ayata, Cenk, and Cabeen, Ryan P.

Subjects
Electrical Engineering and Systems Science - Image and Video Processing, Computer Science - Computer Vision and Pattern Recognition, Quantitative Biology - Quantitative Methods
Abstract

Rodent stroke models are important for evaluating treatments and understanding the pathophysiology and behavioral changes of brain ischemia, and magnetic resonance imaging (MRI) is a valuable tool for measuring outcome in preclinical studies. Brain extraction is an essential first step in most neuroimaging pipelines; however, it can be challenging in the presence of severe pathology and when dataset quality is highly variable. Convolutional neural networks (CNNs) can improve accuracy and reduce operator time, facilitating high throughput preclinical studies. As part of an ongoing preclinical stroke imaging study, we developed a deep-learning mouse brain extraction tool by using a U-net CNN. While previous studies have evaluated U-net architectures, we sought to evaluate their practical performance across data types. We ask how performance is affected with data across: six imaging centers, two time points after experimental stroke, and across four MRI contrasts. We trained, validated, and tested a typical U-net model on 240 multimodal MRI datasets including quantitative multi-echo T2 and apparent diffusivity coefficient (ADC) maps, and performed qualitative evaluation with a large preclinical stroke database (N=1,368). We describe the design and development of this system, and report our findings linking data characteristics to segmentation performance. We consistently found high accuracy and ability of the U-net architecture to generalize performance in a range of 95-97% accuracy, with only modest reductions in performance based on lower fidelity imaging hardware and brain pathology. This work can help inform the design of future preclinical rodent imaging studies and improve their scalability and reliability.

Published
2022

4. Anatomical and functional study of the musculus psoas major and nervus femoralis in correlation with pelvic diameters

Author

Vojvodić Aleksandar, Matić Aleksandar, Mihailović Jelena, Bjelogrlić Predrag, Puškaš Laslo, Stijak Lazar, Aleksić Dubravka, Filipović Branka, Vukomanović-Đurđević Biserka, and Kapor Slobodan

Subjects
anatomy, femoral nerve, pelvis, psoas muscles, sex factors, Medicine (General), R5-920
Abstract

Background/Aim. The iliopsoas muscle [musculus (m.) iliopsoas] originates from the Greek word psóa, which means loin and represents the only muscle in the body with anatomical preconditions to simultaneously and directly contribute to the stability and movement of the trunk, pelvis, and legs. M. iliopsoas belongs to the inner thigh muscle group and forms part of the posterior abdominal wall. This muscle is the major flexor of the hip joint, and it is functionally essential for proper posture, walking, running, and other physical activities. The aim of this study was to determine the relationship between the anatomical parameters of the pelvis and nervus (n.) femoralis, as well as the relationship between the same pelvic parameters and m. psoas major. Methods. The study was conducted at the Faculty of Medicine, University of Belgrade, on cadaveric material of the Institute of Anatomy “Niko Miljanić”. For measurement purposes, 14 cadavers were used, seven of which were male and seven female, aged 67–79 years. The measuring instruments used in this study were a ruler and an electronic digital caliper (measuring range 0–500 mm, resolution 0.01 mm). Statistical data processing was performed in the SPSS 11.0 program using the Mann-Whitney U test. Results. The results of this study indicate a significant statistical difference in pelvic width between male and female cadavers, which was observed in the reduction of the bituberal line in females, while the parameters of the bispinal line showed no significant difference between the two genders. The decrease of the bituberal line in females was followed by an increase in the width of the proximal origin with a statistically significant decrease in the length of the proximal origin of the m. psoas major. Furthermore, the vertical distance of n. femoralis from the exit point of the muscle to the bispinal line was significantly reduced in the male cadavers. Conclusion. Based on our results, we can assume that, in most cases, due to the smaller bituberal and bispinal line or narrower pelvis, a shorter proximal attachment of the m. psoas major will occur with greater width (L2–L5 level) in the female than in the male gender, resulting in a longer vertical distance of n. femoralis. Such results indicate a close correlation between the anatomical parameters of the m. psoas major, which may affect the distance of n. femoralis exit from the muscle.

Published
2023
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8. Advanced magnetic resonance techniques in early differentiation of pseudoprogression versus progression in the patients with glioblastoma multiforme

Author

Mihailović Jelena and Daković Marko

Subjects
glioblastoma multiforme, disease progression, drug therapy, radiotherapy, surgical procedures, operative, magnetic resonance imaging, spectrum analysis, diagnosis,differential, Medicine (General), R5-920
Abstract

Background/Aim. The diagnosis of glioblastoma multiforme progression may be confounded by a phenomena termed pseudoprogression (PSP) and pseudoresponse (RCT) which has become more common with the adoption of radiation therapy with concurrent and adjuvant application of temozolomide (CRT). Distinguishing of these phenomena is based on the follow-up scans since no single imaging method or technique is yet capable of performing their discrimination. In this study, we evaluated the dynamic susceptibility contrast (DSC perfusion) imaging and magnetic resonance (MR) spectroscopy to predict the prognosis and time to progression in the patients with glioblastoma multiforme. Methods. Fourty patients with primary glioblastoma multiforme were included in the analysis. The patients were examined in 3rd week after surgery and 10th week after the beginning of CRT. The MR exams were performed using the 1.5 T MR scanner (Avanto; Siemens, Erlangen, Germany). The maps of perfusion parameters and time-to-peak (TTP) parameter were calculated using the DPTools v3.79 software. The 3D CSI PRESS MR spectroscopy was performed in the area corresponding to the contrast enhancement on the T1W images. Results. Thirty-two of the 40 patients had progressive disease and 8 had pseudoprogression. Progressive disease showed the mean time of the peak values of 33 ± 7 s in 3rd and 30 ± 5 s in 10th week with no statistical significance between these two periods (p > 0.05). The patients with pseudoprogression showed the mean time of the peak values of 32 ± 8 s in 3rd week and 43 ± 9 s in 10th week; it was statistically significant difference (p < 0.05) was which favors better response to therapy. The spectroscopy results showed presence of glycine peak at 3.56 ppm in 6 patients with progressive disease which was not seen on spectra with pseudoprogression. Conclusion. The observed significant differences in the TTP values for PSP and RCT can provide basis for distinguishing two entities. The presence of glycine peak in the MR spectra could be a marker of RCT.

Published
2019
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9. The application of the local histograms of apparent difusion coefficient in differentiation of brain astrocytomas

Author

Mihailović Jelena, Grujičić Danica, Lavrnić Slobodan, and Daković Marko

Subjects
astrocytoma, neoplasm staging, histology, diffusion magnetic resonance imaging, Medicine (General), R5-920
Abstract

Background/Aim. Microstructural diversity of brain astrocytomas makes their diagnostics and differentiation by using the diffusion weighted imaging (DWI) difficult. In this study we used the histogram-based positioning of regions of interests on the apparent diffusion coefficient (ADC) maps in order to restrict the determination of diffusion parameters to regions of interest (ROI) corresponding to maximum cellularity. Success of ADC standard deviation (ΔADC) and kurtosis (K) in differentiation of brain astrocytomas was evaluated. Methods. The thirtyone patients (16 women and 15 men, median age 37 years, age range 6–72 years) with suspected supratentorial astrocytomas were included in the retrospective study. The magnetic resonance imaging (MRI) examinations were performed using the 1.5 T MR system (Avanto; Siemens, Erlangen, Germany) and 8-channel phased array head coil. The DWI images were acquired in three orthogonal directions for the b-values 0, 500 and 1000 s mm-2. The histogram calculations and determination of diffusion parameters were performed using the MIPAV software package and the statistical analysis was done in the Openstat software. Results. The ADC values enabled differentiation of diffuse astrocytomas (DA) from a high-grade astrocytoma (HGA), but not between the classes of HGA. In addition, the ΔADC value provided discrimination between the anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) with 100% of sensitivity and 89% of specificity . The kurtosis value can also differentiate between the grades AA and GBM although with the lower sensitivity and specificity. Conclusion. The histogram analysis of tumor region on the ADC maps can provide a guidance for an appropriate choice of the ROIs. The parameters which characterize diffusion of such defined ROIs, as well as their combination can improve differentiation of brain astrocytomas. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. III41005]

Published
2019
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15. Proteomic Profiling of Major Peanut Allergens and Their Post-Translational Modifications Affected by Roasting

Author

Đukić, Teodora, primary, Smiljanić, Katarina, additional, Mihailović, Jelena, additional, Prodić, Ivana, additional, Apostolović, Danijela, additional, Liu, Shu-Hua, additional, Epstein, Michelle M., additional, van Hage, Marianne, additional, Stanić-Vučinić, Dragana, additional, and Ćirković Veličković, Tanja, additional

Published
2022
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16. Proteomic Profiling of Major Peanut Allergens and Their Post-Translational Modifications Affected by Roasting

Author

Đukić, Teodora, Smiljanić, Katarina, Mihailović, Jelena, Prodić, Ivana, Apostolović, Danijela, Liu, Shu-Hua, Epstein, Michelle M., van Hage, Marianne, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, Đukić, Teodora, Smiljanić, Katarina, Mihailović, Jelena, Prodić, Ivana, Apostolović, Danijela, Liu, Shu-Hua, Epstein, Michelle M., van Hage, Marianne, Stanić-Vučinić, Dragana, and Ćirković-Veličković, Tanja

Abstract

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.

Published
2022

17. Supplementary for: Đukić, T., Smiljanić, K., Mihailović, J., Prodić, I., Apostolović, D., Liu, S.-H., Epstein, M. M., van Hage, M., Stanić-Vučinić, D., & Ćirković Veličković, T. (2022). Proteomic Profiling of Major Peanut Allergens and Their Post-Translational Modifications Affected by Roasting. Foods, 11(24). https://doi.org/10.3390/foods11243993

Author

Đukić, Teodora, Smiljanić, Katarina, Mihailović, Jelena, Prodić, Ivana, Apostolović, Danijela, Liu, Shu-Hua, Epstein, Michelle M., van Hage, Marianne, Stanić-Vučinić, Dragana, Ćirković-Veličković, Tanja, Đukić, Teodora, Smiljanić, Katarina, Mihailović, Jelena, Prodić, Ivana, Apostolović, Danijela, Liu, Shu-Hua, Epstein, Michelle M., van Hage, Marianne, Stanić-Vučinić, Dragana, and Ćirković-Veličković, Tanja

Abstract

Post-translational modifications (PTMs) are covalent changes occurring on amino acid side chains of proteins and yet are neglected structural and functional aspects of protein architecture. The objective was to detect differences in PTM profiles that take place after roasting using open PTM search. We conducted a bottom-up proteomic study to investigate the impact of peanut roasting on readily soluble allergens and their PTM profiles. Proteomic PTM profiling of certain modifications was confirmed by Western blotting with a series of PTM-specific antibodies. In addition to inducing protein aggregation and denaturation, roasting may facilitate change in their PTM pattern and relative profiling. We have shown that Ara h 1 is the most modified major allergen in both samples in terms of modification versatility and extent. The most frequent PTM was methionine oxidation, especially in roasted samples. PTMs uniquely found in roasted samples were hydroxylation (Trp), formylation (Arg/Lys), and oxidation or hydroxylation (Asn). Raw and roasted peanut extracts did not differ in the binding of IgE from the serum of peanut-sensitised individuals done by ELISA. This study provides a better understanding of how roasting impacts the PTM profile of major peanut allergens and provides a good foundation for further exploration of PTMs.

Published
2022

20. Trypsin as a Proteomic Probe for Assessment of Food Protein Digestibility in Relation to Chemical and Post-translational Modifications

Author

Smiljanić, Katarina, Mihailović, Jelena, Prodić, Ivana, Đukić, Teodora, Vasović, Tamara, Jovanović, Vesna B., and Ćirković-Veličković, Tanja

Subjects
post-translational and chemical modifications, protein resistance in intestinal digestion, porcine and human trypsin, secondary trypsin specifity, bottom-up proteomics
Abstract

Post-translational modifications (PTMs) occur in many forms and shapes, widely influencing protein behavior. High-resolution tandem mass spectrometry (HRMS/MS), coupled with dedicated engines for the identification of unspecified PTMs, is a powerful method for their mapping. A majority of proteomic experiments utilize trypsin for digestion, which cleaves the C-terminal peptide bonds of arginine (Arg) and lysine (Lys) amino acids with high catalytic efficiency and selectivity, unless they are followed with proline. At the same time, Arg and Lys residues are frequently modified during food processing by heat and non-thermal treatments, causing oxidation, carbamylation, and various forms of side chain carbonylation, including the other common PTMs (methylation, acetylation, etc.). Consequently, we explored the possibility to re-assess already generated proteomic data (food protein/allergen tryptic peptides) with respect to the possible modulation of the tryptic intestinal digestion pattern caused by PTMs incorporated at Arg and Lys residues. However, most of the proteomic bottom-up experiments are run with porcine trypsin that has been reductively methylated to increase its stability and minimize autoproteolytic effects. Therefore, in this chapter, the utility of the aforementioned idea was explored, by reviewing the differences in structure, affinity, specificity, and catalytic efficiency of trypsin, primarily from porcine, bovine and human species. Porcine trypsin either from pancreas or in recombinant form showed superior performance compared to human and bovine tryptic counterparts. In addition, set of software tools for identification and analyses of PTMs was reviewed with the aim to isolate those capable of in-depth PTMs profiling and their simultaneous relative quantification, such as PEAKS PTM (PEAKS Studio, Bioinformatics Solution Inc., Ontario Canada). Based on our preliminary experimental results, conclusion is that the proposed idea is plausible, because if potential hindrance effects caused by PTMs are observed with porcine trypsin, then they can be just augmented within human intestinal digestion, with respect to inferior performance of human trypsin.

Published
2020

21. Supplementary data for the article: Apostolovic, D.; Mihailovic, J.; Commins, S. P.; Wijnveld, M.; Kazimirova, M.; Starkhammar, M.; Stockinger, H.; Platts-Mills, T. A. E.; Cirkovic Velickovic, T.; Hamsten, C.; et al. Allergenomics of the Tick Ixodes Ricinus Reveals Important α-Gal–Carrying IgE-Binding Proteins in Red Meat Allergy. Allergy: European Journal of Allergy and Clinical Immunology 2020, 75 (1), 217–220. https://doi.org/10.1111/all.13978

Author

Apostolović, Danijela, Mihailović, Jelena, Commins, Scott P., Wijnveld, Michiel, Kazimirova, Maria, Starkhammar, Maria, Stockinger, Hannes, Platts-Mills, Thomas A. E., Ćirković-Veličković, Tanja, Hamsten, Carl, van Hage, Marianne, Apostolović, Danijela, Mihailović, Jelena, Commins, Scott P., Wijnveld, Michiel, Kazimirova, Maria, Starkhammar, Maria, Stockinger, Hannes, Platts-Mills, Thomas A. E., Ćirković-Veličković, Tanja, Hamsten, Carl, and van Hage, Marianne

Published
2020

22. Allergenomics of the tick Ixodes ricinus reveal important α-Gal-carrying IgE-binding proteins in red meat allergy

Author

Apostolović, Danijela, Mihailović, Jelena, Commins, Scott P., Wijnveld, Michiel, Kazimirova, Maria, Starkhammar, Maria, Stockinger, Hannes, Platts-Mills, Thomas A. E., Ćirković-Veličković, Tanja, Hamsten, Carl, van Hage, Marianne, Apostolović, Danijela, Mihailović, Jelena, Commins, Scott P., Wijnveld, Michiel, Kazimirova, Maria, Starkhammar, Maria, Stockinger, Hannes, Platts-Mills, Thomas A. E., Ćirković-Veličković, Tanja, Hamsten, Carl, and van Hage, Marianne

Published
2020

23. Discrete Hf18 Metal‐oxo Cluster as a Heterogeneous Nanozyme for Site‐Specific Proteolysis

Author

Moons, Jens, de Azambuja, Francisco, Mihailović, Jelena, Kozma, Karoly, Smiljanić, Katarina, Amiri, Mehran, Ćirković-Veličković, Tanja, Nyman, May, Parac-Vogt, Tatjana, Moons, Jens, de Azambuja, Francisco, Mihailović, Jelena, Kozma, Karoly, Smiljanić, Katarina, Amiri, Mehran, Ćirković-Veličković, Tanja, Nyman, May, and Parac-Vogt, Tatjana

Abstract

The selective hydrolysis of proteins by non‐enzymatic catalysis is difficult to achieve, yet it is crucial for applications in biotechnology and proteomics. Herein, we report that discrete hafnium metal‐oxo cluster [Hf18O10(OH)26(SO4)13⋅(H2O)33] (Hf18), which is centred by the same hexamer motif found in many MOFs, acts as a heterogeneous catalyst for the efficient hydrolysis of horse heart myoglobin (HHM) in low buffer concentrations. Among 154 amino acids present in the sequence of HHM, strictly selective cleavage at only 6 solvent accessible aspartate residues was observed. Mechanistic experiments suggest that the hydrolytic activity is likely derived from the actuation of HfIV Lewis acidic sites and the Brønsted acidic surface of Hf18. X‐ray scattering and ESI‐MS revealed that Hf18 is completely insoluble in these conditions, confirming the HHM hydrolysis is caused by a heterogeneous reaction of the solid Hf18 cluster, and not from smaller, soluble Hf species that could leach into solution.

Published
2020

24. Supplementary data for the article: Moons, J.; de Azambuja, F.; Mihailović, J.; Kozma, K.; Smiljanić, K.; Amiri, M.; Ćirković-Veličković, T.; Nyman, M.; Parac-Vogt, T. Discrete Hf18 Metal‐oxo Cluster as a Heterogeneous Nanozyme for Site‐Specific Proteolysis. Angewandte Chemie (International Edition) 2020, 59 (17), 1–9. https://doi.org/10.1002/anie.202001036.

Author

Moons, Jens, de Azambuja, Francisco, Mihailović, Jelena, Kozma, Karoly, Smiljanić, Katarina, Amiri, Mehran, Ćirković-Veličković, Tanja, Nyman, May, Parac-Vogt, Tatjana, Moons, Jens, de Azambuja, Francisco, Mihailović, Jelena, Kozma, Karoly, Smiljanić, Katarina, Amiri, Mehran, Ćirković-Veličković, Tanja, Nyman, May, and Parac-Vogt, Tatjana

Published
2020

25. Supplementary data for the article: Stanic-Vucinic, D.; Nikolic, S.; Vlajic, K.; Radomirovic, M.; Mihailovic, J.; Cirkovic Velickovic, T.; Grguric-Sipka, S. The Interactions of the Ruthenium(II)-Cymene Complexes with Lysozyme and Cytochrome c. Journal of Biological Inorganic Chemistry 2020. https://doi.org/10.1007/s00775-020-01758-3

Author

Stanić-Vučinić, Dragana, Nikolić, Stefan, Vlajić, Katarina, Radomirović, Mirjana Ž., Mihailović, Jelena, Ćirković-Veličković, Tanja, Grgurić-Šipka, Sanja, Stanić-Vučinić, Dragana, Nikolić, Stefan, Vlajić, Katarina, Radomirović, Mirjana Ž., Mihailović, Jelena, Ćirković-Veličković, Tanja, and Grgurić-Šipka, Sanja

Published
2020

26. The interactions of the ruthenium(II)-cymene complexes with lysozyme and cytochrome c

Author

Stanić-Vučinić, Dragana, Nikolić, Stefan, Vlajić, Katarina, Radomirović, Mirjana Ž., Mihailović, Jelena, Ćirković-Veličković, Tanja, Grgurić-Šipka, Sanja, Stanić-Vučinić, Dragana, Nikolić, Stefan, Vlajić, Katarina, Radomirović, Mirjana Ž., Mihailović, Jelena, Ćirković-Veličković, Tanja, and Grgurić-Šipka, Sanja

Abstract

The reactions of four cymene-capped ruthenium(II) compounds with pro-apaptotic protein, cytochrome c (Cyt), and anti-proliferating protein lysozyme (Ly) in carbonate buffer were investigated by ESI-MS, UV–Vis absorption and CD spectroscopy. The complexes with two chloride ligands (C2 and C3) were more reactive toward proteins than those with only one (C1 and C4), and the complex with S,N-chelating ligand (C4) was less reactive than one with O,N-chelating ligand (C1). Dehalogenated complexes are most likely species initially coordinating proteins for all tested complexes. During the time, protein adducts vividly exchanged non-arene organic ligand L with CO32- and OH-, while cymene moiety was retained. In water, only dehalogenated adducts were identified suggesting that in vivo, in the presence of various anions, dynamic ligand exchange could generate different intermediate protein species. Although all complexes reduced Cyt, the reduction was not dependent on their reactivity to protein, implying that initially noncovalent binding to Cyt occures, causing its reduction, followed by coordination to protein. Cyt reduction was accompanied with rupture of ferro-Met 80 and occupation of this hem coordination site by a histidine His-33/26. Therefore, in Cyt with C2 and C3 less intensive reduction of hem iron leave more unoccupied target residues for Ru coordination, leading to more efficient formation of covalent adducts, in comparison to C1 and C4. This study contribute to development of new protein-targeted Ru(II) cymene complexes, and to design of new cancer therapies based on targeted delivery of Ru(II) arene complexes bound on pro-apoptotic/anti-proliferating proteins as vehicles.

Published
2020

27. Digestomics of Cow's Milk: Short Digestion-Resistant Peptides of Casein Form Functional Complexes by Aggregation

Author

Radosavljević, Jelena, Apostolović, Danijela, Mihailović, Jelena, Atanasković-Marković, Marina, Burazer, Lidija, van Hage, Marianne, Ćirković-Veličković, Tanja, Radosavljević, Jelena, Apostolović, Danijela, Mihailović, Jelena, Atanasković-Marković, Marina, Burazer, Lidija, van Hage, Marianne, and Ćirković-Veličković, Tanja

Abstract

The aim of this study was to identify short digestion-resistant peptides (SDRPs) released by pepsin digestion of the whole cow's milk and examine their IgE reactivity and allergenicity. Raw milk was subjected to simulated gastric digestion. SDRPs were fractionated from the digests and identified by MS. Milk SDRPs were evaluated for aggregability, propensity to compete for IgE binding with individual milk allergens, and ability to bind IgG4 from allergic and milk-tolerant individuals. The majority of milk SDRPs originated from caseins (97% of peptides) and overlapped with the known IgE epitopes of cow's milk allergens. SDRPs competed with milk proteins for binding to human IgE and readily formed aggregates. The average peptide length was 10.6 +/- 3.5 amino acids. The ability to provoke allergenic in vivo responses was confirmed by skin-prick testing (SPT) in five milk-allergic subjects. This was attributed to the peptide ability to aggregate into non-covalent complexes. SDRPs are able to induce response in SPT, but only in 50% of the sera SDRPs were able to inhibit IgG4 binding to caseins. Hence, SDRPs corresponding to the mainly continuous epitopes of milk proteins induce allergenic in vivo responses in milk-allergic subjects due to aggregation.

Published
2020

28. Supplementary information for the article: Radosavljević, J.; Apostolović, D.; Mihailović, J.; Atanasković-Marković, M.; Burazer, L.; van Hage, M.; Ćirković Veličković, T. Digestomics of Cow’s Milk: Short Digestion-Resistant Peptides of Casein Form Functional Complexes by Aggregation. Foods 2020, 9 (11), 1576. https://doi.org/10.3390/foods9111576.

Author

Radosavljević, Jelena, Apostolović, Danijela, Mihailović, Jelena, Atanasković-Marković, Marina, Burazer, Lidija, van Hage, Marianne, Ćirković-Veličković, Tanja, Radosavljević, Jelena, Apostolović, Danijela, Mihailović, Jelena, Atanasković-Marković, Marina, Burazer, Lidija, van Hage, Marianne, and Ćirković-Veličković, Tanja

Abstract

Figure S1-S3: Figure S1: Digestion of BLG at pH 1.2, 2.5 and 4.0; Figure S2: Digestion of ALA at pH 1.2, 2.5 and 4.0; Figure S3: MALDI spectra of peptides used in the study Table S1. IgE levels of patients used in the study determined by ImmunoCAP Methods: 1.1 Detection of ALA and BLG by immunoblot; 1.2 Mass spectrometry analysis; 1.3 Size-exclusion chromatography; 1.4 IgG4-binding properties of peptides obtained by digestion; 1.5 Digestion of purified ALA and BLG at different pH; 1.6 MALDI-TOF MS.

Published
2020

29. Proteomika posttranslacionih i hemijskih modifikacija proteina i interakcije proteina od značaja u alergiji na hranu

Author

Mihailović, Jelena, Ćirković Veličković, Tanja, Beškoski, Vladimir, Radosavljević, Jelena, Smiljanić, Katarina, Apostolović, Danijela, and Ćirković-Veličković, Tanja

Subjects
red meat, α-Gal, chemical modifications, crveno meso, Alergija na hranu, kikiriki, protein-ligand interactions, protein-ligand interakcije, food allergens, proteomics, hemijske modifikacije, Food allergy, proteomika, posttranslational modifications, peanut, posttranslacione modifikacije, alergeni hrane, EGCG
Abstract

Alergija na hranu je reakcija preosetljivosti imunskog sistema na hranu koja dovodi do stvaranja IgE antitela. Ne postoji efikasan tretman i izbegavanje namirnica koje izazivaju simptome je jedini način za sprečavanje ozbiljnih posledica. Cilj ove teze je izučavanje posttranslacionih i hemijskih modifikacija (PTM i HM) i interakcija alergena hrane sa biološki aktivnim polifenolom biljnog porekla. Alergija na crveno meso je novootkriveni tip odložene reakcije, sa PTM galaktozil-α-1,3- galaktozom (α-Gal) kao epitopom. Ispitani su podložnost model proteina - tiroglobulina (TG) digestiji pepsinom, prisustvo α-Gal na rezultujućim peptidima i pretpostavljeno mesto vezivanja α-Gal za TG. Pokazano je da je α-Gal celim tokom simulirane želudačne digestije vezan za nastale peptide TG, koji zadržavaju sposobnost da vežu IgE. Alergija na kikiriki je među najopasnijim zbog učestalosti, postojanosti i ozbiljnosti simptoma. Postoje razlike u alergenosti između sirovog i pečenog kikirikija, koje mogu poticati od razlika u modifikacijama alergena. U ovoj disertaciji je proučena razlika u modifikacijama glavnih alergena iz sirovog i pečenog kikirikija. Detektovano je 27 modifikacija, od kojih 4 isključivo na proteinima pečenog kikirikija. Pokazane su razlike u profilima modifikacija proteina između ekstrakata, koje mogu uticati na alergenost. 2S albumini kikirikija su zbog kompaktne strukture otporni na delovanje digestivnih enzima, što se povezuje sa njihovom alergenošću. Ispitane su interakcije između njih i epigalokatehin-3-galata (EGCG), katehina zelenog čaja sa imunomodulatornim svojstvima koji pospešuje digestiju proteina pepsinom. Vezivanje EGCG-a za alergene izaziva promene u njihovoj strukturi (entalpijski povoljan proces), sa konstantama vezivanja reda veličine 104 M-1. Food allergy is an immune system reaction to certain foods that results in IgE antibody generation. Efficient treatment does not exist, so avoidance of trigger foods is the only way to prevent serious consequences. The aim of this thesis was to study posttranslational and chemical modifications (PTM and CM) of food allergens and their interactions with biologically active plant polyphenol. Red meat allergy is a newly discovered type of allergy with delayed symptoms and a PTM - galactose-α-1,3-galactose (α-Gal) as an epitope. Digestibility of a model protein - thyroglobulin (TG) by pepsin and presence of α-Gal on resulting peptides was studied, and the position of α-Gal containing glycan on TG was proposed. It was shown that throughout simulated gastric digestion α-Gal remains bound to IgE reactive TG peptides. Peanut allergy is among most dangerous types of food allergies due to prevalence, persistence and symptom severity. Differences in allergenicity between raw and roasted peanuts were previously shown and might stem from differences in protein modifications. Differences in major allergen modifications between raw and roasted peanut were studied. Twenty-seven modifications were detected, 4 of which exclusively on allergens from roasted peanuts. Differences in modification profiling were found between the two protein preparations that can affect their allergenicity. Peanut 2S albumins are compact digestion-resistant proteins, which attributes to their allergenicity. Their interactions with epigallocatechin-3-gallate (EGCG), an immunomodulatory green tea catechin that facilitates pepsin digestion of proteins were studied. The results show that binding of EGCG to these allergens induces conformational changes, and is enthalpy favorable with binding constants of 104 M-1.

Published
2019

33. Advanced magnetic resonance techniques in early differentiation of pseudoprogression versus progression in the patients with glioblastoma multiforme . [Napredne tehnike magnetne rezonance u ranom razdvajanju pseudoprogresije od progresije kod bolesnika sa glioblastomom multiforme]

Author

Mihailović Jelena and Daković Marko

Subjects
operative, glioblastoma multiforme, disease progression, differential, diagnosis, magnetic resonance imaging, spectrum analysis, radiotherapy, drug therapy, surgical procedures
Abstract

Background/Aim. The diagnosis of glioblastoma multiforme progression may be confounded by a phenomena termed pseudoprogression (PSP) and pseudoresponse (RCT) which has become more common with the adoption of radiation therapy with concurrent and adjuvant application of temozolomide (CRT). Distinguishing of these phenomena is based on the follow-up scans since no single imaging method or technique is yet capable of performing their discrimination. In this study, we evaluated the dynamic susceptibility contrast (DSC perfusion) imaging and magnetic resonance (MR) spectroscopy to predict the prognosis and time to progression in the patients with glioblastoma multiforme. Methods. Fourty patients with primary glioblastoma multiforme were included in the analysis. The patients were examined in 3rd week after surgery and 10th week after the beginning of CRT. The MR exams were performed using the 1.5 T MR scanner (Avanto; Siemens, Erlangen, Germany). The maps of perfusion parameters and time-to-peak (TTP) parameter were calculated using the DPTools v3.79 software. The 3D CSI PRESS MR spectroscopy was performed in the area corresponding to the contrast enhancement on the T1W images. Results. Thirty-two of the 40 patients had progressive disease and 8 had pseudoprogression. Progressive disease showed the mean time of the peak values of 33 ± 7 s in 3rd and 30 ± 5 s in 10th week with no statistical significance between these two periods (p > 0.05). The patients with pseudoprogression showed the mean time of the peak values of 32 ± 8 s in 3rd week and 43 ± 9 s in 10th week; it was statistically significant difference (p < 0.05) was which favors better response to therapy. The spectroscopy results showed presence of glycine peak at 3.56 ppm in 6 patients with progressive disease which was not seen on spectra with pseudoprogression. Conclusion. The observed significant differences in the TTP values for PSP and RCT can provide basis for distinguishing two entities. The presence of glycine peak in the MR spectra could be a marker of RCT.

Published
2019

34. Digestomics of raw and roasted hazelnut according to harmonized static digestion method suitable for solid food and characterization of gastric-phase products

Author

Prodić, Ivana, Smiljanić, Katarina, Hoffmann-Sommergruber, Karin, Ćirković-Veličković, Tanja, and Mihailović, Jelena

Subjects
digestion, food matrix, Hazelnut allergens
Abstract

Introduction Stability to gastric digestion represents a very important parameter of food protein allergenicity. Usu- ally digestion experiments are carried out on purified proteins or protein extracts; however, use of solid food is far closer to the in vivo situation, taking into account food protein interactions with other food components, such as polyphenols and lipids. Objective The aim of this study was to investigate and compare digestion stability and allergenicity of large and small peptides released after pepsin digestion of whole raw and roasted hazelnut kernels under standardized and physiologically relevant in vitro conditions. Methodology In vitro simulated oral and gastric phase digestion was carried out with ground raw and roasted hazelnut kernels. Digested proteins were extracted from the mixture and analyzed by SDS-PAGE, 2D-PAGE, and compared with Image Master 2D Platinum 7.0. Western blot probed with allergic patients’ sera and specific antibodies for Cor a 8. Main findings Several important hazelnut seed storage digestion resistant proteins and peptides have been identi- fied and characterized. Most abundant hazelnut allergens were resolved on a 2DE map, for instance, acidic and basic chains of Cor a 9, and Cor a 11. Digestion-resistant peptides of Cor a 11 and Cor a 9 were able to bind IgE. Lipid transfer protein (Cor a 8) was highly resistant to gastric proteolysis. Conclusion To conclude, roasted hazelnut is more prone to gastric digestion than raw, and cause milder IgE response in patients. Gastric phase digestion of raw and roasted hazelnut kernels results in partial extraction and digestion of Cor a 11 and Cor a 9 into digestion-resistant peptides with preserved IgE-binding epitopes. These results demonstrate substantial resistance of raw and roasted hazelnut allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) digestion and retained their allergenicity.

Published
2019

35. Comparative study of raw and thermally treated peanut major allergen post- translational modifications (PTMs)

Author

Mihailović, Jelena, Prodić, Ivana, Smiljanić, Katarina, and Ćirković-Veličković, Tanja

Subjects
PTMs, mass spectrometry based bottom-up proteomics, relative quantification, allergens, peanut, LFQ
Abstract

Introduction. Peanut allergy affects a large portion of world population causing reactions ranging from mild to severe. Major peanut allergen IgE epitopes are well characterized but little is known about their post-translational modifications (PTM) and how they are affected by thermal treatment. PTM profile may differ between raw and thermally treated peanut, which could affect its allergic potential depending on type, size and position of modifications. Objective. Our aim was to analyse and compare PTM profiles of 4 major peanut allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as well as their amounts in raw and roasted samples using bottom-up proteomics methods. Methodology. Full peanut protein extracts (both thermally treated and non-treated) were digested in gel and in solution, and analysed by a Top10 nLC-MS/MS method by LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Germany). Within the extracts major allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were identified, label free quantified (LFQ) and searched for PTMs by Peaks X software (Bioinformatics solutions Inc.I, Canada). Epitope sequences were acquired from the Immune Epitope Database (IEDB www.iedb.org). Main findings. LFQ results show that there is no significant change in the amountsof any of the studied allergens between raw and roasted extracts.Out of the 4 allergens Ara h 6 is modified in the highest portion, with respect to the protein size: 15% and 12% of its positions are modified in raw and roasted sample, respectively. Total of 21 modifications were quantified between the two preparations, with oxidation (M), methylation (K,R) and dethiomethylation affecting the largest number of peptides. Conclusions. Peanut allergen epitopes are indeed carriers of PTMs that differ in pattern and quantity between treated and non-treated extracts. The in silico discovered PTMs could affect protein digestibility and allergenicity. Further investigation is necessary in order to fully understand the impact protein modifications could have on their allergenic potential.

Published
2019

37. Digestomics of raw and roasted hazelnut according to Infogest protocol and characterization of gastric-phase products

Author

Prodić, Ivana, Smiljanić, Katarina, Mihailović, Jelena, Hoffmann-Sommergruber, Karin, and Ćirković-Veličković, Tanja

Subjects
hazelnut allergens, Cor a 9, Cor a 8, digestion, lipid transfer proteins, food matrix
Abstract

Brief introduction: Stability to gastric digestion represents a very important parameter of food protein allergenicity. Usually digestion experiments are carried out on purified proteins or protein extracts; however, use of solid food is far closer to the in vivo situation, taking into account food protein interactions with other food components, such as polyphenols and lipids. Objective: The aim of this study was to investigate and compare digestion stability and allergenicity of large and small peptides released after pepsin digestion of whole raw and roasted hazelnut kernels under standardized and physiologically relevant in vitro conditions. Methodology: In vitro simulated oral and gastric phase digestion was carried out with ground raw and roasted hazelnut kernels. Digested proteins were extracted from the mixture and analyzed by SDS-PAGE, 2D-PAGE, and compared with Image Master 2D Platinum 7.0. Western blot probed with allergic patients’ sera and specific antibodies for Cor a 8. Main findings: Several important hazelnut seed storage digestion resistant proteins and peptides have been identified and characterized. Most abundant hazelnut allergens were resolved on a 2DE map, for instance acidic and basic chains of Cor a 9, and Cor a 11. Digestion-resistant peptides of Cor a 11 and Cor a 9 were able to bind IgE. Lipid transfer protein (Cor a 8) was highly resistant to gastric proteolysis. Conclusion: To conclude, roasted hazelnut is more prone to gastric digestion than raw, and cause milder IgE response in patients. Gastric phase digestion of raw and roasted hazelnut kernels results in partial extraction and digestion of Cor a 11 and Cor a 9 into digestion- resistant peptides with preserved IgE-binding epitopes. These results demonstrate substantial resistance of raw and roasted hazelnut allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) digestion and retained their allergenicity.

Published
2019

38. Proteomika posttranslacionih i hemijskih modifikacija proteina i interakcije proteina od značaja u alergiji na hranu

Author

Ćirković-Veličković, Tanja, Beškoski, Vladimir, Radosavljević, Jelena, Smiljanić, Katarina, Apostolović, Danijela, Mihailović, Jelena, Ćirković-Veličković, Tanja, Beškoski, Vladimir, Radosavljević, Jelena, Smiljanić, Katarina, Apostolović, Danijela, and Mihailović, Jelena

Abstract

Alergija na hranu je reakcija preosetljivosti imunskog sistema na hranu koja dovodi dostvaranja IgE antitela. Ne postoji efikasan tretman i izbegavanje namirnica koje izazivajusimptome je jedini način za sprečavanje ozbiljnih posledica. Cilj ove teze je izučavanjeposttranslacionih i hemijskih modifikacija (PTM i HM) i interakcija alergena hrane sabiološki aktivnim polifenolom biljnog porekla.Alergija na crveno meso je novootkriveni tip odložene reakcije, sa PTM galaktozil-α-1,3-galaktozom (α-Gal) kao epitopom. Ispitani su podložnost model proteina - tiroglobulina(TG) digestiji pepsinom, prisustvo α-Gal na rezultujućim peptidima i pretpostavljenomesto vezivanja α-Gal za TG. Pokazano je da je α-Gal celim tokom simulirane želudačnedigestije vezan za nastale peptide TG, koji zadržavaju sposobnost da vežu IgE.Alergija na kikiriki je među najopasnijim zbog učestalosti, postojanosti i ozbiljnostisimptoma. Postoje razlike u alergenosti između sirovog i pečenog kikirikija, koje mogupoticati od razlika u modifikacijama alergena. U ovoj disertaciji je proučena razlika umodifikacijama glavnih alergena iz sirovog i pečenog kikirikija. Detektovano je 27modifikacija, od kojih 4 isključivo na proteinima pečenog kikirikija. Pokazane su razlikeu profilima modifikacija proteina između ekstrakata, koje mogu uticati na alergenost.2S albumini kikirikija su zbog kompaktne strukture otporni na delovanje digestivnihenzima, što se povezuje sa njihovom alergenošću. Ispitane su interakcije između njih iepigalokatehin-3-galata (EGCG), katehina zelenog čaja sa imunomodulatornimsvojstvima koji pospešuje digestiju proteina pepsinom. Vezivanje EGCG-a za alergeneizaziva promene u njihovoj strukturi (entalpijski povoljan proces), sa konstantamavezivanja reda veličine 104 M-1., Food allergy is an immune system reaction to certain foods that results in IgE antibodygeneration. Efficient treatment does not exist, so avoidance of trigger foods is the onlyway to prevent serious consequences. The aim of this thesis was to study posttranslationaland chemical modifications (PTM and CM) of food allergens and their interactions withbiologically active plant polyphenol.Red meat allergy is a newly discovered type of allergy with delayed symptoms and aPTM - galactose-α-1,3-galactose (α-Gal) as an epitope. Digestibility of a model protein -thyroglobulin (TG) by pepsin and presence of α-Gal on resulting peptides was studied,and the position of α-Gal containing glycan on TG was proposed. It was shown thatthroughout simulated gastric digestion α-Gal remains bound to IgE reactive TG peptides.Peanut allergy is among most dangerous types of food allergies due to prevalence,persistence and symptom severity. Differences in allergenicity between raw and roastedpeanuts were previously shown and might stem from differences in protein modifications.Differences in major allergen modifications between raw and roasted peanut werestudied. Twenty-seven modifications were detected, 4 of which exclusively on allergensfrom roasted peanuts. Differences in modification profiling were found between the twoprotein preparations that can affect their allergenicity.Peanut 2S albumins are compact digestion-resistant proteins, which attributes to theirallergenicity. Their interactions with epigallocatechin-3-gallate (EGCG), animmunomodulatory green tea catechin that facilitates pepsin digestion of proteins werestudied. The results show that binding of EGCG to these allergens induces conformationalchanges, and is enthalpy favorable with binding constants of 104 M-1.

Published
2019

39. Proteomika posttranslacionih i hemijskih modifikacija proteina i interakcije proteina od značaja u alergiji na hranu

Author

Ćirković Veličković, Tanja, Beškoski, Vladimir, Radosavljević, Jelena, Smiljanić, Katarina, Apostolović, Danijela, Mihailović, Jelena, Ćirković Veličković, Tanja, Beškoski, Vladimir, Radosavljević, Jelena, Smiljanić, Katarina, Apostolović, Danijela, and Mihailović, Jelena

Abstract

Alergija na hranu je reakcija preosetljivosti imunskog sistema na hranu koja dovodi do stvaranja IgE antitela. Ne postoji efikasan tretman i izbegavanje namirnica koje izazivaju simptome je jedini način za sprečavanje ozbiljnih posledica. Cilj ove teze je izučavanje posttranslacionih i hemijskih modifikacija (PTM i HM) i interakcija alergena hrane sa biološki aktivnim polifenolom biljnog porekla. Alergija na crveno meso je novootkriveni tip odložene reakcije, sa PTM galaktozil-α-1,3- galaktozom (α-Gal) kao epitopom. Ispitani su podložnost model proteina - tiroglobulina (TG) digestiji pepsinom, prisustvo α-Gal na rezultujućim peptidima i pretpostavljeno mesto vezivanja α-Gal za TG. Pokazano je da je α-Gal celim tokom simulirane želudačne digestije vezan za nastale peptide TG, koji zadržavaju sposobnost da vežu IgE. Alergija na kikiriki je među najopasnijim zbog učestalosti, postojanosti i ozbiljnosti simptoma. Postoje razlike u alergenosti između sirovog i pečenog kikirikija, koje mogu poticati od razlika u modifikacijama alergena. U ovoj disertaciji je proučena razlika u modifikacijama glavnih alergena iz sirovog i pečenog kikirikija. Detektovano je 27 modifikacija, od kojih 4 isključivo na proteinima pečenog kikirikija. Pokazane su razlike u profilima modifikacija proteina između ekstrakata, koje mogu uticati na alergenost. 2S albumini kikirikija su zbog kompaktne strukture otporni na delovanje digestivnih enzima, što se povezuje sa njihovom alergenošću. Ispitane su interakcije između njih i epigalokatehin-3-galata (EGCG), katehina zelenog čaja sa imunomodulatornim svojstvima koji pospešuje digestiju proteina pepsinom. Vezivanje EGCG-a za alergene izaziva promene u njihovoj strukturi (entalpijski povoljan proces), sa konstantama vezivanja reda veličine 104 M-1., Food allergy is an immune system reaction to certain foods that results in IgE antibody generation. Efficient treatment does not exist, so avoidance of trigger foods is the only way to prevent serious consequences. The aim of this thesis was to study posttranslational and chemical modifications (PTM and CM) of food allergens and their interactions with biologically active plant polyphenol. Red meat allergy is a newly discovered type of allergy with delayed symptoms and a PTM - galactose-α-1,3-galactose (α-Gal) as an epitope. Digestibility of a model protein - thyroglobulin (TG) by pepsin and presence of α-Gal on resulting peptides was studied, and the position of α-Gal containing glycan on TG was proposed. It was shown that throughout simulated gastric digestion α-Gal remains bound to IgE reactive TG peptides. Peanut allergy is among most dangerous types of food allergies due to prevalence, persistence and symptom severity. Differences in allergenicity between raw and roasted peanuts were previously shown and might stem from differences in protein modifications. Differences in major allergen modifications between raw and roasted peanut were studied. Twenty-seven modifications were detected, 4 of which exclusively on allergens from roasted peanuts. Differences in modification profiling were found between the two protein preparations that can affect their allergenicity. Peanut 2S albumins are compact digestion-resistant proteins, which attributes to their allergenicity. Their interactions with epigallocatechin-3-gallate (EGCG), an immunomodulatory green tea catechin that facilitates pepsin digestion of proteins were studied. The results show that binding of EGCG to these allergens induces conformational changes, and is enthalpy favorable with binding constants of 104 M-1.

Published
2019

40. Lysozyme and Cytochrome C adducts of ruthenium(II)-cymene complexes

Author

Radomirović, Mirjana Ž., Stanić-Vučinić, Dragana, Nikolić, Stefan, Mihailović, Jelena, Ćirković-Veličković, Tanja, Grgurić Šipka, Sanja, Radomirović, Mirjana Ž., Stanić-Vučinić, Dragana, Nikolić, Stefan, Mihailović, Jelena, Ćirković-Veličković, Tanja, and Grgurić Šipka, Sanja

Abstract

Objective. In contrast to platinum-based antitumour compounds, the mode of action of ruthenium-based compounds is via protein targets involved in cellular signaling pathways and the histone proteins. As proteins are the first potential binding targets for the complexes in the bloodstream after their intravenous administration, interactions of anticancer therapeutics with proteins are very important to be investigated with aim to elucidate their pharmacokinetic pathways. Four half-sandwich ruthenium(II)-cymene complexes, developed earlier and with promising cytotoxic activity, are investigated for their interactions with proteins, cytochrome c and lysozyme. Material and Methods. Ruthenium(II)-cymene complexes were incubated for 24 and 48 h at 37 °C in the presence of cytochrome c and lysozyme, both in 20 mM ammonium hydrogen carbonate pH 7.4 and water. High-resolution LTQ-Orbitrap ESI MS was used to monitor the adducts formed between ruthenium complexes and proteins. Results. The complexes with two Cl- ligands have shown higher reactivity to proteins than those with only one, and were more reactive toward cytochrome c in comparison to lysozyme. The complex with S,N-chelating ligand was less reactive to proteins than one with O,N-chelating ligand. Species initially coordinating the proteins are most likely dehalogenated complexes. During the time, vivid ligand exchange of non-arene organic ligand L with CO32- and OH- takes place after initial formation of protein adducts. In water, only dehalogenated adducts were identified suggesting that in vivo, in the presence of various anions, dynamic ligand exchange could be expected generating different intermediate protein species. Conclusions. Protein reactivity toward Ru(II) complexes is determined by protein structure and ligands in Ru(II) coordination sphere, but this reactivity should be described from both kinetics, as well as stability aspect. In extracellular or intracellular milieu, ability of metal binding ligands (

Published
2019

41. Interactions of ruthenium(II)-cymene complexes with cytochrome c

Author

Radomirović, Mirjana Ž., Stanić-Vučinić, Dragana, Nikolić, Stefan, Mihailović, Jelena, Ćirković-Veličković, Tanja, Grgurić Šipka, Sanja, Radomirović, Mirjana Ž., Stanić-Vučinić, Dragana, Nikolić, Stefan, Mihailović, Jelena, Ćirković-Veličković, Tanja, and Grgurić Šipka, Sanja

Abstract

The ruthenium-based antitumour compounds act more via protein targets involved in carcinogenesis, in contrast to platinum-based compounds. Also, after intravenous administration of antitumour complexes proteins are the first binding targets in circulation. Therefore, interactions of anticancer compounds with proteins are important for elucidation of their pharmacokinetic pathways. Four half-sandwich ruthenium(II)-cymene complexes (C1, C2, C3 and C4), developed earlier and with promising cytotoxic activity, are investigated for their interactions with cytochrome c (Cyt). Complexes were incubated with Cyt for 48 h at 37 °C and high-resolution LTQ-Orbitrap ESI MS was used to monitor the formed adducts. The changes in heme state and tertiary structure around the heme were monitored by CD and UV-VIS spectra in the presence of oxygen. The complexes containing two chloride ligands (C2 and C3) were more reactive toward Cyt than those with only one (C1 and C4). The complex with S,N-chelating ligand (C4) was less reactive than one with O,N-chelating ligand (C1). All complexes reduced heme iron of Cyt, but the extent of reduction was inverse to the order of their reactivity to Cyt (C1>C4>>C2>C3). CD spectra in Soret region indicated that Cyt reduction was accompanied with slight tertiary structure change, the rupture of ferro-Met-80 and occupation of this heme coordination site by His-33/His-26. Extent of heme reduction by complexes inverse with respect to their reactivity implies that initially noncovalent binding of complexes occures, causing heme reduction, followed by comlex coordination to protein. In the presence of less reactive complexes more intensive reduction of heme leaves less available histidine residues (main targets for Ru coordination), leading to less efficient formation of adducts.

Published
2019

42. Investigation of raw and thermally treated peanut major allergen post- translational modifications (PTMs)

Author

Mihailović, Jelena, Prodić, Ivana, Smiljanić, Katarina, Ćirković-Veličković, Tanja, Mihailović, Jelena, Prodić, Ivana, Smiljanić, Katarina, and Ćirković-Veličković, Tanja

Abstract

Introduction. Peanut allergy affects a large portion of world population causing reactions ranging from mild to severe. Major peanut allergen IgE epitopes are well characterized but little is known about their post-translational modifications (PTM) and how they are affected by thermal treatment. PTM profile may differ between raw and thermally treated peanut, which could affect its allergic potential depending on type, size and position of modifications. Objective. Our aim was to analyse and compare PTM profiles of 4 major peanut allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6, as well as their amounts in raw and roasted samples using bottom-up proteomics methods. Methodology. Full peanut protein extracts (both thermally treated and non-treated) were digested in gel and in solution, and analysed by a Top10 nLC-MS/MS method by LTQ Orbitrap XL (Thermo Fisher Scientific Inc., Germany). Within the extracts major allergens - Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were identified, label free quantified (LFQ) and searched for PTMs by Peaks X software (Bioinformatics solutions Inc.I, Canada). Epitope sequences were acquired from the Immune Epitope Database (IEDB www.iedb.org). Main findings. LFQ results show that there is no significant change in the amounts of any of the studied allergens between raw and roasted extracts. Out of the 4 allergens Ara h 6 is modified in the highest portion, with respect to the protein size: 15% and 12% of its positions are modified in raw and roasted sample, respectively. Total of 21 modifications were quantified between the two preparations, with oxidation (M), methylation (K,R) and dethiomethylation affecting the largest number of peptides. Conclusions. Peanut allergen epitopes are indeed carriers of PTMs that differ in pattern and quantity between treated and non-treated extracts. The in silico discovered PTMs could affect protein digestibility and allergenicity. Further investigation is necessary in order to fully understand the impact p

Published
2019

43. Characterisation of peanut allergens and possible post-translational modifications (PTMs)

Author

Liu, Shu-hua, Mihailović, Jelena, Smiljanić, Katarina, Epstein, Michelle M., and Ćirković-Veličković, Tanja

Subjects
chemical modifications, Ara h 1, post-translational modifications, peanut allergens
Abstract

Background Peanut allergy is the most common type of food allergy causing severe reactions or even fatal anaphylaxis in sensitised individuals. The major peanut allergens are Ara h 1, Ara h 2, Ara h 3, and Ara h 6 which cause the most severe responses. Their molecular properties have been characterised but possible post-translational modifications (PTMs) that might explain their severe allergenicity are not well understood. The goal of this study was to utilize a combination of nanoLC-Mass Spectrometry (MS)/MS methods and PEAKS Studio 8.0 (Bioinformatics Solutions Inc., Ontario, Canada) program to evaluate PTMs in the major peanut allergens. Method Acquired MS data of purified peanut allergens, Ara h 1, Ara h 2, Ara h 3, and Ara h 6 were analysed and identified via hybridized databases obtained from UniProt (www.uniprot.org). More than 1200 reviewed (Swiss-Prot) and unreviewed (TrEMBL) entries from peanut were combined with common MS contaminants, the Repository of Adventitious Proteins (cRAP), to create a hybridized database. We then focused on Ara h 2 (Conglutin-7) and Ara h 6 (Conglutin) because of their propensity to cause severe anaphylactic reactions. Epitopes found in the Immune Epitope Database (www.iedb.org) were analysed for possible PTMs by matching PEAKS PTM results with mapped positions of epitope sequences. Results We identified 37 proteins from the purified peanut allergens. There were 33 peanut proteins and 4 contaminants originating from human keratin and pig trypsin. Ara h 2 had 242 epitopes, 29 potential PTMs and 4 mutations. Eight of the epitopes had up to 8 possible PTMs. Several relevant PTMs were discovered, including tryptophan oxidation to oxolactone in position 25, sulfonation of N-terminus of cysteine in position 116 and oxidation of methionine in position 50 and 125. Notably, all had either a “NNQRCMCEALQ” or “QQIMENQSD” motif, which are linked to Th2 cytokines and T cell proliferation. We observed 8 epitopes, 9 likely PTMs and no mutations for Ara h 6 and half of the epitopes had possible PTMs and a maximum of 4 PTMs was found on one epitope. Conclusion The analysis of relevant peanut allergens by nanoLC-MS/MS methods and PEAKS Studio 8.0 program revealed several PTMs, which might have important ramifications due to their influence on allergenicity and digestibility resulting from modification properties by trypsin and other food protein enzymes. These data suggest that PTMs on certain peanut epitopes could be involved in the pathogenesis of severe food allergy to peanuts.

Published
2018

44. Post-translational modifications (PTMs) of major peanut allergen epitopes occurring as a result of purification process

Author

Mihailović, Jelena, Apostolović, Danijela, Smiljanić, Katarina, and Ćirković-Veličković, Tanja

Subjects
PTMs, major peanut allergens, mass spectrometry based proteomics, peanut allergy, IgE epitopes
Abstract

Peanut allergy affects a large portion of world population causing reactions ranging from mild to severe. Seed storage proteins Ara h 1, Ara h 2, Ara h 3 and Ara h 6 are peanut allergens, with well characterized IgE epitopes but little is known about PTMs effect on their properties. Our aim was to investigate PTMs present on known epitopes of said purified proteins using bottom-up proteomcs methods. Out of the 4 allergens, Ara h 2 has the highest number of PTMs (14), while the most common are hydroxylation Pro, dehydration and deamidation (N, Q). Peanut allergen epitopes are indeed carriers of PTMs that could affect protein allergenicity and digestibility. Further investigation on peanut extracts prepared in different ways is necessary in order to fully understand the impact protein modifications could have on their allergenic potential.

Published
2018

46. Digestomics of walnut and its nsLTPs allergens reveals their ultimate resistance to gastric digestion

Author

Prodić, Ivana, Dubiela, Pawel, Mihailović, Jelena, Stanić-Vučinić, Dragana, Smiljanić, Katarina, Hoffmann-Sommergruber, Karin, and Ćirković-Veličković, Tanja

Subjects
panallergens, gastric digestion, INFOGEST protocol, walnut, Specific lipid transfer proteins
Abstract

Background: Sensitization to non-specific lipid transfer protein (ns-LTPs) in plant foods is regarded as a risk factor for generalized allergic reactions. Stability to gastric digestion represents very important parameter of food proteins allergenicity. Usually studies of digestion were carried out on purified proteins, but has never been examined the influence of different food matrices on different allergens. Allergens from the nsLTP family are known to share a characteristic structure which is highly resistant to proteolysis, and therefore, IgE cross-reactivity of nsLTPs needs to be investigated in the environment such as complex food matrix. Objective: The aim of this research project is to reveal how proteins are digested (by Minekus protocol) within the natural food matrix and possible consequences on their allergenicity, with the special focus on ns-LTP. Methods: Pure nsLTPs from walnut were labelled with Alexa 633 and added to whole grain of grounded raw walnuts, incubated with human α-amylase, and pepsin, therefore mimicking the effects of oral and gastric digestion, in total duration of 2h. Proteins extracted from the mixture were analyzed by one-dimensional (1D) and two-dimensional SDS-PAGe, and respective 1D and 2D immunoblots. Results: Most proteins from pepsin digested walnut sample were more resistant to digestion according to 1D SDS PAGE. Pepsin digested raw walnut sample with nsLTP were assessed by 2D PAGE to compare profiles of the digested and control sample (no pepsin added). 2D SDSPAGE of digested and control walnut samples showed almost identical profiles, especially in the context of fluorescently labelled nsLTP allergens. These results demonstrate substantial resistance of nsLTP allergens to gastric digestion since they remained mostly intact after 2 h of gastric (pepsin) digestion. Conclusion: Further research is needed to be able to grade stability/resistance of selected food allergens to gastric digestion as a consequence of food matrix modulating effects. We propose that certain combinations of foods and allergens could provide additional protection or on the contrary ease the digestion, by comparing trends between control and digested samples and between different digested combinations as well.

Published
2018

47. Major peanut allergen Ara h1 and Ara h 3 epitope post-translational modifications (PTMs)

Author

Mihailović, Jelena, Apostolović, Danijela, Smiljanić, Katarina, and Ćirković-Veličković, Tanja

Subjects
food allergy, proteomics, PTMs, peanut allergens, post translational modifications
Abstract

Objective: Peanuts are widely used for the preparation of a variety of foods and are also relied on as a protein extender. Peanut allergies affect a large portion of world population causing reactions ranging from mild to severe that can lead to anaphylaxis and even death. Seed storage proteins Ara h 1 and Ara h 3 are known as major peanut allergens. IgE epitopes of these allergens have been characterized, but little is known about how post-translational modifications (PTMs) affect their allergenicity and digestibility. Our aim was to investigate PTMs present on known epitopes of said proteins using bottom-up proteomcs methods. Material and Methods: Purified 2S albumins (Ara h 1 and Ara h 3) were analysed by a Top5 nLC- MS/MS method by LTQ Orbitrap XL (Thermo Fischer Scientific, Germany). Spectra were compared to Uniprot derived Peanut protein database, hybridized with the Repository of Adventitious Proteins (cRAP), using Peaks 8.5 software package (BSI, Canada). Epitopes were searched for possible PTMs by matching PEAKS PTM results with mapped positions of epitope sequences (found in the Immune Epitope Database – IEDB www.iedb.org). Results: According to IEDB Ara h 1 contains 327 peptide epitopes, within which we detected 8 likely PTMs. Hydroxylation Pro and pyro-glu from Q were found as most common in Ara h 1 epitopes. Ara h 3 has only 110 epitopes, according to IEDB with 10 likely PTMs. Hydroxylation Pro, dehydration and methylation (KR) were found as most frequent in Ara h 3 epitopes. PTMs could be found in the vicinity of trypsin cleavage sites, which could have an impact on digestibility. Conclusions: Peanut allergen epitopes are indeed carriers of PTMs. These results show promise in revealing a possible role PTMs could have on protein allergenicity and digestibility. Further investigation is necessary in order to fully understand the impact protein modifications could have on their allergenic potential Book of Abstracts

Published
2018

48. Digestomics of Japanese abalone in real food matrix

Author

Prodić, Ivana, Khulal, Urmila, Mutić, Jelena, Mihailović, Jelena, Smiljanić, Katarina, and Ćirković-Veličković, Tanja

Subjects
tropomyosin, Abalon, food allergy, seashells, food digestion
Abstract

Objective: Haliotis discus (Japanese abalone), mollusks among various shellfish, is a highly nutritive food resource in the world, but also among the eight allergic food groups accounting for approximately 90% of all immunoglobulin E food allergies worldwide. The general objective of our research is to comprehensively investigate stability and structures of pepsin-resistant allergens, of their larger fragments, and of short digestion resistant peptides (SDRPs) released by pepsin digestion of whole raw and extract of shellfish, under standardized and physiologically relevant gastric conditions. Materials and Methods: Extract of raw whole shellfish (eRSS) and whole raw shellfish (wRSS), were pepsin digested according to standardized static digestion protocol. Controls were treated in a same manner without adding pepsin. Supernatant of samples and its counterpart controls were precipitated with TCA/acetone. Obtained proteins were assessed by 2D SDS PAGE and 1D SDS- PAGE, under reducing and non-reducing conditions. 1D SDS-PAGE of RSS were analyzed by ncLC-MS/MS (Orbitrap LTQ) shot-gun proteomics. Relative quantification was performed by LFQ algorithm within Peaks 8.5 software package Bioinformatics Solutions Inc. (BSI), Waterloo, Canada. Results and Conclusion: 1D SDS-PAGE analysis of eRSS and wRSS, and its controls showed a range of proteins in varied concentrations between 10-250 kDa. In extracted and whole raw shellfish, approximately 22 prominent protein bands were observed including the distinct bands corresponding with the molecular weights of recognized shellfish allergen, tropomyosin (37- 39kDa). Fewer high molecular weight proteins were observed followed by protein smearing, specifically around the low molecular weight protein bands. The smearing could possibly be due to the breakdown products and the glycation. There were slight differences between the protein profiles under reducing and non-reducing conditions as well. Nevertheless, there was the retention of a band in the 37kDa molecular weight marker in all 4 samples, likely consistent with heat stable tropomyosin (TM). Mass spectrometry showed allergens that are characterized (Hal d 1 and Hal di 1), with 90% of sequence homology with main tropomyosin allergens from seafood. Scientific impact and relevance: The results will highlight effects of food matrix on shellfish allergens digestibility proving its relevancy in molecular allergology. Moreover, an insight will be obtained on the differences in digestibility of allergenic versus non-allergenic tropomyosins in the real food matrix. Book of Abstracts

Published
2018

49. Gastric digestome of whole peanut grains from the aspect of immunoproteomics: Characterization of digested allergens in the real food matrix

Author

Prodić, Ivana, Stanić-Vučinić, Dragana, Apostolović, Danijela, Radosavljević, Jelena, Mihailović, Jelena, Smiljanić, Katarina, and Ćirković-Veličković, Tanja

Subjects
gastric digestion, digestomics, peanut allergens, INFOGEST 1.0
Abstract

Objective: Major peanut allergens, Ara h 2 and Ara h 6, are known to be resistant to pepsin digestion, and they sensitize individual via the gastrointestinal tract. Mikenus et al. published a standardized static digestion method for food, based on physiological conditions emphasizing the impact of food matrices. Immunoreactive proteins (large fragments) and peptides (short digestion resistant peptides SDRPs

Published
2018

50. Дифузијски наглашено снимање и магнетно-резонантна спектроскопија у разликовању тумора мозга

Author

Mihailović, Jelena M., Daković, Marko, Mojović, Miloš, and Kačar, Katarina

Subjects
мултиваријантна анализа, фрактална анализа, multivariate analysis, дифузијски наглашено снимање, глијални тумори, магнетно- резонантна спектроскопија, diffusion weighted imaging, метилација, fractal analysis, magnetic resonance spectroscopy, methilation, glial tumors
Abstract

Тумори који захватају централи нервни систем, првенствено ендокранијум, представљају посебан изазов у дијагностици магнетном резонанцијом. Класични преглед овом техником често не даје довољно информација неопходних, како за њихово међусобно разликовање, тако и за разликовање од других не-туморских патологија... Central nervous system tumours, especially brain, make their diagnostics and differentiation by use of magnetic resonance imaging difficult. Usualyy it doesn’t give enough information for their differentiation...

Published
2017

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